Neuro-mesenchymal units regulate ILC2 and obesity through brain-adipose circuits

Abstract

The present disclosure provides a neural-mesenchymal signaling axis that controls group 2 innate lymphoid cells (ILC2s), adipose tissue physiology, metabolism, and obesity. This signaling axis involves ILC2 receptor reconstituted during transfection (RET), mesenchymal stromal cells (MSCs) bearing the beta2 adrenergic receptor (ADRB2), and higher brain regions including the paraventricular nucleus of the hypothalamus (PVH).

Description

[Background technology]

[0001] background Sympathetic neurons interact with adipocytes and immune cells contribute to adipose tissue biology. Interactions between the nervous and immune systems have recently emerged as major regulators of host defense and inflammation. 1-4 Nevertheless, it remains unclear whether neural and immune cells cooperate to coordinate metabolism in the brain-body axis. Summary of the Invention

[0002] overview The present disclosure is based on the discovery of a neuro-mesenchymal signaling axis that controls group 2 innate lymphoid cells (ILC2s), adipose tissue physiology, metabolism, and obesity through brain-adipose tissue circuits. Sympathetic neurons in adipose tissue act on adjacent adipose mesenchymal stromal cells (MSCs) through the beta2 adrenergic receptor (ADRB2) to control the expression of glial-derived neurotrophic factor (GDNF) and the activity of gonadal adipose tissue (GAT) ILC2s. The neuro-mesenchymal signaling axis also regulates gonadal adipose tissue (GAT) ILC2s by connecting to higher brain regions including the paraventricular nucleus of the hypothalamus (PVH). Thus, the present disclosure provides methods for manipulating ILC2 signaling that results in energy expenditure, insulin resistance, and obesity propensity.

[0003] In some aspects, the disclosure provides a method of increasing the activity or proliferation of group 2 innate lymphoid cells (ILC2s), comprising contacting an ILC2 with a rearranged during transfection (RET) agonist. In some aspects, the disclosure provides a method of increasing the activity of ILC2s, comprising contacting a mesenchymal stromal cell (MSC) with a beta2 adrenergic receptor (ADRB2) agonist.

[0004] In some aspects, the disclosure provides methods for increasing production of interleukin-5 (IL-5), interleukin-13 (IL-13), and / or Met-enkephalin (Met-Enk) by ILC2s comprising contacting adipose ILC2s with a RET agonist and / or contacting MSCs with an ADRB2 agonist.

[0005] In some aspects, the disclosure provides methods for reducing susceptibility to obesity and / or increasing fat homeostasis comprising (a) administering to a subject a RET agonist that contacts ILC2s in adipose tissue, (b) administering to a subject an ADRB2 agonist that contacts MSCs in adipose tissue, or (c) a combination thereof. In some embodiments, the increase in fat homeostasis is an increase in glucose tolerance and / or a decrease in gonadal adipose tissue (GAT) fat mass.

[0006] In some embodiments, the disclosure provides a method of treating a disorder associated with decreased ILC2 activity or proliferation, comprising: (a) administering to a subject a RET agonist that contacts ILC2s in adipose tissue; (b) administering to a subject an ADRB2 agonist that contacts MSCs in adipose tissue; or a combination thereof.

[0007] In some aspects, the disclosure provides a method of treating cold exposure comprising: (a) administering to a subject a RET agonist that contacts ILC2s, (b) administering to a subject an ADRB2 agonist, or (c) a combination thereof. In some embodiments, administering the RET agonist and / or the ADRB2 agonist increases the subject's body temperature.

[0008] In some embodiments, the RET agonist includes: (1) a combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand (GFL) or an analog or mimetic thereof; or (2) an antibody or antigen-binding fragment thereof that specifically binds to RET and increases RET tyrosine kinase activity.

[0009] In some embodiments, the combination of soluble GFRα and GFRα ligand or an analog mimetic thereof is: (1) a combination of (a) soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) soluble GFRα4 and persephin (PSP N) or an analog or mimetic thereof; (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to and dimerizes GFRα; or (2) a combination of two or more of (a), (b), (c), (d), (e), (f) and (g).

[0010] In some embodiments, the ADRB2 agonist is clenbuterol, bitolterol, fenoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, albuterol, terbutaline, albuterol, aformoterol, bambuterol, formoterol, salmeterol, abesiterol, carmoterol, indacaterol, olodaterol, valanterol, isoxsuprine, mabuterol, zilpaterol, or a combination thereof.

[0011] In some aspects, the disclosure provides a method of treating a disorder associated with increased ILC2 activity or proliferation, comprising (a) administering to a subject a RET antagonist that contacts ILC2s in adipose tissue, (b) administering to a subject an ADRB2 antagonist that contacts MSCs in adipose tissue, or (c) a combination of (a) and (b). In some embodiments, the disorder is hypothermia, cachexia, allergy, helminth infection, allergic asthma, atopic dermatitis, intestinal inflammatory disease, or a combination thereof.

[0012] In some embodiments, the RET antagonist is (1) an antibody that specifically binds and inhibits (a) RET tyrosine kinase activity, (b) GDNF family binding receptor alpha (GFRα), or (c) a GFRα ligand, or an antigen-binding fragment thereof; (2) an inhibitory nucleic acid molecule that reduces the expression, transcription, or translation of RET, GFRα, or a GFRα ligand; or (3) a RET tyrosine kinase inhibitor, optionally AST487, motesanib, cabozantinib, vandetanib, ponatinib, sunitinib, sorafenib, or alectinib. In some embodiments, the GFRα is GFRα1, GFRα2, GFRα3, or GFRα4, or the GFRα ligand is glial cell line-derived neurotrophic factor (GDNF), neurturin (NTRN), artemin (ARTN), or persephin (PSPN). In some embodiments, the inhibitory nucleic acid molecule is an sRNA, shRNA, or an antisense nucleic acid molecule.

[0013] In some embodiments, the ADRB2 antagonist is butoxamine, ICI-118,551, propranolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, or a combination thereof.

[0014] In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the RET agonist, ADRB2 agonist, RET antagonist and / or ADRB2 antagonist is administered to a subject. In some embodiments, the subject is a human.

[0015] In some embodiments, the ILC2s and / or MSCs are in or derived from adipose tissue, hi some embodiments, the adipose tissue is gonadal adipose tissue (GAT). [Brief description of the drawings]

[0016] [Figure 1A-1C] Figure 1A-1L. Sympathetic-mesenchymal interactions regulate ILC2 in gonadal adipose tissue (GAT). Figure 1A shows GAT stained sympathetic fibers for tyrosine hydroxylase (TH, top panel) and endothelial cells (CD31, bottom panel). Scale bar: 300 μm. Figure 1B shows ILC2 function after 6-hydroxydopamine (6-OHDA) administration. n=5. Figure 1C shows ILC2 function after pegylated diphtheria toxin (PegDT) treatment. R26 / DTRfl is the diphtheria toxin receptor (DTR) inserted into the ROSA26 locus in floxed mice (Fl), and R26 / DTRTh is the DTR inserted into the ROSA26 locus in sympathetic neurons (Th). n=4. [Fig. 1D-1H]FIG. 1D shows ILC2 function after Clenbuterol administration. n=5. FIG. 1E shows ILC2 function after Clozapine N-oxide (CNO) administration. R26 / 3Dfl is a DREADD-bearing adeno-associated virus inserted into the ROSA26 locus of floxed mice, n=5, and R26 / 3DΔTh is a DREADD-bearing adeno-associated virus inserted into the ROSA26 locus of TH knockout mice, n=4. FIG. 1F shows GAT ILC2 activity in cells with wild-type β2 adrenergic receptor (Adrb2WT), n=7, and in lymphoid cells with ADRB2 deletion (Adrb2ΔIl7ra), n=8. FIG. 1G shows ILC2 function after 6-OHDA administration. Adrb2WT n=13; Adrb2ΔIl7ra n=15. FIG. 1H shows GAT cell population. n=6. PDGFRA+MSCs are platelet-derived growth factor receptor alpha positive mesenchymal stem cells, and PDGFRA-MSCs are platelet-derived growth factor receptor alpha negative mesenchymal stem cells. [Fig. 1I-1L] Figure 1I shows sympathetic fibers (TH, green), glial cells (GFAP, red) and cell nuclei (DAPI, blue). Scale bar: 50 μm. Figure 1J shows sympathetic fibers (TH, green) and MSCs (platelet-derived growth factor receptor alpha, PDGFRA). Scale bar: 20 μm. Figure 1K shows GAT ILC2s in ADRB2 wild-type (Adrb2fl) cells (n=6) and cells with ADRB2 deletion in glial cells (Adrb2ΔGfap) (n=8). Figure 1L shows GAT ILC2s in ADRB2 wild-type (Adrb2fl) cells (n=10) and cells with ADRB2 deletion in MSCs (Adrb2ΔPdgfra) (n=8). Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: standard error of the mean (sem) unpaired two-tailed Student's t test (bg). One-way ANOVA (h). *P<0.05; **P<0.01; ***P<0.005; ****p<0.001; ns not significant. [Figures 2A-2E]Figures 2A-2O. Sympathetic cues integrate mesenchymal-derived glial cell line-derived neurotrophic factor (GDNF) and innate type 2 cytokines. Figures 2A-2C show the effect of 6-OHDA treatment. Figure 2A shows RNAseq of platelet-derived growth factor receptor alpha positive mesenchymal stem cells (PDGFRA+MSCs). Top: Mean difference plot of vehicle vs. 6-OHDA; bottom: Heatmap of downregulated genes. Vehicle n=4, 6-OHDA n=5. Figure 2B shows Gdnf expression in total GAT RNA (n=5). Figure 2C shows Gdnf expression in GAT cell populations (n=4). Figures 2D-2E show clenbuterol administration. Figure 2D shows Gdnf expression in total GAT RNA (n=5). Figure 2E shows Gdnf expression in GAT cell populations (n=5). [Fig. 2F-2I] Figure 2F shows Gdnf expression in total GAT RNA in ADRB2 wild-type cells (Adrb2fl, n=9) and cells with ADRB2 deletion in MSCs (Adrb2ΔPdgfra). Figure 2G shows Gdnf expression in GAT cell populations. Adrb2fl n=6, Adrb2ΔPdgfra n=4. Figure 2H shows GDNF median fluorescence intensity (MFI) in MSCs. n=4. Figure 2I shows GAT staining for PDGFRA (left) and GDNF (right). Scale bar: 50 μm. [Fig. 2J-2O]Figure 2J shows transfection-reconstituted (RET) expression in ILC2 cells, T cells (T), natural killer cells (NK), B cells (B) and macrophages (Mφ). n=7. Figure 2K shows ILC2 activity in GAT in mice expressing wild-type RET (Retfl) and Ret deleted from hematopoietic stem cells (RetΔVav1). n=5. Figure 2L shows ILC2 activity in GAT in Rag1 knockout (Rag1- / -) knockout mice expressing wild-type Ret (RetWT) or mice with Ret knocked out from IL-5 positive cells (RetΔIl5). n=6. Figures 2M-2N show in vitro stimulation with GDNF. Figure 2M shows cytokine expression in GAT ILC2. n=4. Figure 2N shows MFI innate type 2 cytokines. UN is untreated and GDNF is stimulated with GDNF; n=6. FIG. 2O shows ILC2 activity in GAT derived bone marrow (BM) chimeras. Rag1 knockout, RET wild type = Rag1- / -, RetWT (n=5), Rag1 knockout, RET gain of function = Rag1- / -, RetMEN2B; n=4. Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: sem unpaired two-tailed Student's t test (bh, ko). One-way ANOVA (j). *P<0.05; **P<0.01; ***P<0.005; ns not significant. [Figure 3A-3F]Figures 3A-3O. ILC2-specific RET cues control adipose tissue physiology and obesity. Figures 3A-M show measurements after 16 weeks on a high-fat diet (HFD) regimen in Ret wild-type (Retfl; RetWT), Ret knockout (RetΔVav1; RetΔ) or Ret gain-of-function (RetMEN2B) mice. Figure 3A shows weight gain, n=6. Figure 3B shows glucose tolerance test, n=6. Figure 3C shows GAT weight, n=6. Figures 3D-H show ILC2 chimeras with ILC2 RET wild-type (RetWT) or RET knockout (RetΔ) transplants. Figures 3I-M show ILC2 RetWT or RetMEN2B transplants. Figure 3D shows weight gain in RetWT mice (n=5) and RetΔ mice (n=6). Figure 3E shows glucose tolerance test in RetWT (n=4) and RetΔ (n=6). Figure 3F shows GAT weight in RetWT (n=4) and RetΔ (n=6). [Figure 3G-3K] FIG. 3G shows adipocyte area in RetWT (n=4), RetΔ (n=6), background: white 200 μm2 range interval, grey 1000 μm2 range interval. FIG. 3H shows GAT. Scale bar: 100 μm. FIG. 3I shows GAT weight. n=5. FIG. 3J shows glucose tolerance test. n=5. FIG. 3K shows GAT weight. n=5. [Fig. 3L-3O]Figure 3L shows adipocyte area. Background: white 300 μm2 coverage interval; grey 1000 μm2. n=5. Figure 3M shows GAT. Scale bar: 100 μm. Figure 3N shows uncoupling protein 1 expression (Ucp1), cytochrome c oxidase subunit 8B (Cox8b), and cell death-inducing DFFA like effector A (Cidea) expression in GAT. Rag1- / -RetMEN2B BM chimeras. Rag1- / -,RetWT n=4, Rag1- / -,RetMEN2B n=5. Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: s.e.m. Repeated measures ANOVA (Figures 3A, 3B, 3D, 3E, 3I, and 3J) and tests of interactions (Int), time, and genotype (Gen) are reported (Figures 3A, 3D, 3I). Unpaired two-tailed Student's t test (Figures 3C, 3F, 3K); Mann-Whitney test (Figures 3N and 3O). *P<0.05; **P<0.01; ***P<0.005; ****p<0.001; ns not significant. [Figure 4A-4D] Figures 4A-4N. The aortic-renal-adipose circuit connects to the brain and regulates ILC2s. Figures 4A-4F show viral tracings (VT, right panels) and tyrosine hydroxylase (TH, left panels). Scale bars: 50 μm. Figure 4A shows GAT. Figure 4B shows GAT sympathetic nerve fibers. Figure 4C shows genitofemoral (GF) nerve fibers (arrows). Figure 4D shows TH-positive fibers in the genitofemoral nerve. [Fig. 4E-4H] Figure 4E shows the aortorenal ganglion (ARG, circle). Figure 4F shows TH-positive neuronal cell bodies in the aortorenal ganglion. Figure 4G shows, left: brain atlas scheme of a coronal section and, right: PRV-RFP viral tracing from the GAT corresponding to the highlighted region on the left. PVH is the paraventricular nucleus of the hypothalamus. Figure 4H shows, left: brain atlas scheme of a coronal section and, right: PRV-RFP viral tracing from the aortorenal ganglion (ARG) corresponding to the highlighted region on the left. Scale bar 200 μm (Figure 4G, 4H). [Fig. 4I-4N]Figure 4I shows the surgical genitofemoral nerve (GF) ablation (GFx) scheme (left) and GAT Gdnf expression (right). n=4. Figure 4J shows GAT ILC2 activity in control (sham) and genitofemoral nerve ablation (GFx). n=5. Figure 4K shows left: chemogenetic inhibition scheme using adeno-associated virus 4D (AAV 4D) and right: GAT Gdnf expression. n=5. Figure 4L shows GAT ILC2 activity in mice with Gdnf expression inhibited by AAV 4D. n=5. Figure 4M shows left: chemogenetic activation scheme using adeno-associated virus 3D (AAV 3D) and right: GAT Gdnf expression (right). n=4. Figure 4N, GAT ILC2 activity in mice with Gdnf expression activated by AAV 3D. n=4. Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: sem unpaired two-tailed Student's t test. *P<0.05; **P<0.01; ***P<0.005; ns not significant. [Figure 5A-5D] Figures 5A-5D. Sympathetic nervous system in GAT and ILC2 function. Figure 5A shows GAT with stained sympathetic fibers (TH) and endothelial cells (CD31). Scale bar: 300 μm. Figure 5B shows GAT ILC2-derived Met-Enk production after 6-OHDA administration. n=5. Figure 5C shows CD4 T cells and TH-positive CD4 T cells after 6-OHDA administration. n-4. Figure 5D shows GAT ILC2-derived Met-Enk after clenbuterol administration. n=5. Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: sem unpaired two-tailed Student's t-test. *P<0.05; ***P<0.005. [Figure 6A] Sympathetic regulation of GAT mesenchymal stem cells (MSCs). Figure 6A shows a heatmap of up- and down-regulated genes in MSCs upon 6-OHDA administration. Vehicle n=4, 6-OHDA n=5. [Figure 6B-6E]Figure 6B shows total GAT Il33 expression after 6-OHDA treatment. n=5. Figure 6C shows GAT Il33 expression after Clenbuterol administration. n=5. Figure 6D shows MSC-derived Il33 expression in PDGFRA+MSCs after 6-OHDA and Clenbuterol administration. n=6. Figure 6E shows MSC-derived Il25 expression in PDGFRA+MSCs after 6-OHDA and Clenbuterol administration. n=6. Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: sem unpaired two-tailed Student's t test. ns not significant. [Figure 7A-7F] Figure 7A-7L. ILC2-autonomous RET signaling controls type 2 innate cytokines in GAT. Figure 7A-7C show GAT ILC2 function in GDNF family receptor alpha (GFRa) mice. Figure 7A shows GAT ILC2 function in GFR1α positive (Gfra1+ / +) and knockout (Gfra1- / -) fetal liver chimeras. n=5. Figure 7B shows GFR2α positive (Gfra2+ / +, n=10) and knockout (Gfra2- / - n=5) mice. Figure 7C shows GFR3α positive (Gfra3+ / + n=8); and knockout (Gfra3- / -. n=8) mice. Figure 7D shows a scheme for generating mixed bone marrow (BM) chimeras of Rag1 knockout mice, interleukin 2 receptor gamma knockout (Rag1- / -, Il2rg- / -) mice and RetΔVav1 mice. Figure 7E shows GAT ILC2 activity from Ret floxed (Retfl), Rag1- / -, Il2rg- / - mixed BM chimeras and Ret knockout (RetΔVav1), Rag1- / -, Il2rg- / - mixed BM chimeras. Retfl n=6; RetΔVav1 n=7. Figure 7F shows ILC2 activity in mice with Rag1 knockout, Ret wild type (Rag1- / -, RetWT n=6) and Rag1 knockout, Ret knockout (Rag1- / -.RetΔIl5 n=6) in interleukin-5 cells. [Fig. 7G-7L]Figure 7G shows ILC2 activity in mice. RetWT n=10 and RetΔIl5 (n=8). Figure 7H shows a scheme for generating mixed bone marrow (BM) chimeras of Rag1 knockout, interleukin 2 receptor gamma knockout (Rag1- / -, Il2rg- / -) and RetΔIl5 mice. Figure 7I shows ILC2 activity in Ret wild type (RetWT, n=4) mixed BM chimeras and Ret knockout IL5 cells (RetΔIl5 n=4). Figure 7J shows GAT ILC2 in Rag1- / -, Ret wild type (RetWT) and Ret gain of function (RetMEN2B) mixed bone marrow (BM) chimeras. Rag1- / -, RetWT n=5, Rag1- / -.RetMEN2B n=6. Figure 7K shows the mixed bone marrow (BM) chimera scheme to generate RetMEN2B mixed BM chimeras. Figure 7L shows ILC2 activity in mixed BM chimeras in Rag1 knockout and Ret wild-type mice (Rag1- / -, RetWT; n=6) and (Rag1- / -.RetMEN2B; n=7). Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: sem unpaired two-tailed Student's t test. *P<0.05; **P<0.01; ***P<0.005; ns not significant. [Figure 8A-8C] Figures 8A-8E. ILC2-specific RET signaling is sufficient to control adipocyte physiology and obesity. Figure 8A shows GAT ILC2 function after 6-OHDA administration. RetWT n=8 and RetΔIl5 n=7. Figures 8B, 8C show measurements after 16 weeks on a high-fat diet (HFD) regimen in Ret wild type (Rag1- / -.RetWT;RetWT) and Ret knockout (Rag1- / -.RetΔIl5;RetΔIl5). Figure 8B shows weight gain during the 16-week HFD regimen. Rag1- / -.RetWT n=4, Rag1- / -.RetΔIl5 n=5. Figure 8C shows weight gain during the 16-week HFD regimen. RetWT n=5, RetΔIl5 n=5. [Fig. 8D-8E]Figure 8D shows total GAT RNA expression of Ucp1, Cox8b and Cidea. n=5. Figure 8E shows GAT RNA expression of Ucp1 in GAT explant co-cultures with RetWT and Ret knockout (RetΔ) cells stimulated with glial-derived neurotrophic factor (GDNF). Mean and error bars: sem two-tailed unpaired Student's t-test (Figure 8A); repeated measures ANOVA (Figure 8B, 8C); Mann-Whitney test (Figure 8D, 8E). *P<0.05; **P<0.01; ns not significant; unstimulated not stimulated with GDNF. [Figure 9A] Figures 9A-9G. The aortic-renal-adipose circuit connects to the brain. Figure 9A shows dorsal root ganglion (DRG) T13 viral tracing (VT) and tyrosine hydroxylase (TH) staining. Scale bar: 100 μm. [Figure 9B-9C] Figure 9B: Left: Brain atlas scheme of a coronal section. Right: Polysynaptic tracings from the GAT corresponding to the highlighted region on the left. Figure 9C: Left: Brain atlas scheme of a coronal section. Right: Polysynaptic tracings from the aortorenal ganglion (ARG) corresponding to the highlighted region on the left. Figures 9B-9C: Central amygdala (CA), zona incerta (ZI), periaqueductal gray (PAG) and locus coeruleus (SubCD). [Fig. 9D-9G] FIG. 9D shows electrolytic lesions (electroablation) of the PVH. Scale bar 500 μm. FIG. 9E shows GAT ILC2 in control (sham) and PVH ablated (Abl) mice. Sham n=5; PVH ablation n=6. FIG. 9F shows GAT Il33 expression in AAV(4D) compared to contralateral control after CNO administration. n=5. FIG. 9G shows GAT Il33 expression in AAV(3D) compared to contralateral control after CNO administration. n=4. Data are representative of three independent experiments. n represents biologically independent animals. Mean and error bars: sem unpaired two-tailed Student's t test. *P<0.05; **P<0.01. [Figure 10]Figure 10. Sympathetic aortorenal-adipose circuits connect to the brain and regulate ILC2s. GAT neuro-mesenchymal units convert sympathetic cues into neurotrophic factor expression. Neurotrophic factors then control adipose ILC2s through the neuromodulatory receptor RET, shaping host metabolism, energy expenditure and obesity. PVH-paraventricular nucleus of the hypothalamus; SNS-sympathetic nervous system; ARG-aortorenal ganglion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description Obesity results from the excessive accumulation of lipid reservoirs, and these lipid reservoirs can be used as a high-energy source during periods of food restriction. Sympathetic neuronal cues promote lipolysis during food restriction. 5-7 , and ILC2s contribute to visceral adipose tissue metabolism via production of type 2 innate cytokines and Met-Enkephalin (Met-Enk) 8-13 This raises the hypothesis that the nervous system and ILC2s cooperate by previously unrecognized mechanisms to sense metabolic stress and drive adipose physiology through higher-order brain interactions.

[0018] A newly discovered neuro-mesenchymal unit is described herein that controls ILC2, adipose tissue physiology, metabolism, and obesity via the brain-adipose circuit. Sympathetic neurons in this brain-adipose circuit act on adjacent adipose mesenchymal cells via β2-adrenergic receptors (ADRB2) to control the expression of glial-derived neurotrophic factor (GDNF) and the activity of gonadal adipose ILC2. Thus, ILC2-autonomous manipulation of GDNF receptor mechanisms results in alterations of ILC2 function, energy expenditure, insulin resistance, and obesity propensity. Retrograde tracing, chemical, surgical, and chemical manipulations have identified an unrecognized sympathetic aortorenal circuit that regulates gonadal adipose ILC2 and connects to higher brain regions, including the paraventricular nucleus of the hypothalamus (PVH).

[0019] Thus, the methods provided herein transduce long-range neural circuit cues into adipose-resident ILC2 function, manipulating a newly discovered neural-mesenchymal unit that shapes host metabolism and obesity.

[0020] How to use In some embodiments, the methods provided herein increase the activity, proliferation, or activity and proliferation of group 2 innate lymphoid cells (ILC2) by contacting the ILC2 with a reconstituted during transfection (RET) agonist, contacting mesenchymal stromal cells (MSCs) with a beta-2 adrenergic receptor (ADRB2) agonist, or contacting the ILC2 with a RET agonist and contacting the MSCs with an ADRB2 agonist.

[0021] In some embodiments, the present disclosure provides a method for increasing the activity or proliferation of ILC2. ILC2 is a subset of innate lymphoid cells that is important for maintaining tissue homeostasis and regulating lymphoid tissue development, tissue repair and fat metabolism. ILC2 are abundant in the mucosal barriers of adipose tissue, lung, small intestine, large intestine, mesenteric lymph nodes, bone marrow, spleen, liver and kidney, and act as important initiators of type 2 inflammation and tissue repair. They are activated by cytokines including interleukin-25 (IL-25), interleukin-33 (IL-33) and thymic stromal lymphopoietin.

[0022] Any activity of ILC2 may be increased by the methods provided herein. Non-limiting examples of ILC2 activities that may be increased include adipose tissue metabolism, tissue homeostasis, defense against parasites, tissue repair, inflammation, and immunopathology related to type 2 immunity. ILC2 activity may be measured by any method known in the art, including but not limited to quantitative PCR measurement and fluorescent quantification of proteins produced by ILC2 cells (e.g., cytokines). In some embodiments, the methods provided herein increase the activity of ILC2 in adipose tissue metabolism. ILC2 activity (e.g., adipose tissue metabolism) may be increased by 5% to 50%, 10% to 100%, 25% to 150%, 50% to 200%, 75% to 250%, 100% to 300%, 150% to 350%, 200% to 400%, 250% to 450%, 300% to 500%, 350% to 550% or more compared to a control. In some embodiments, ILC2 activity is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. The control may be ILC2 not contacted with a RET agonist, or the same ILC2 before contact with a RET agonist.

[0023] In some embodiments, ILC2 proliferation is increased after contact with a RET agonist. ILC2 proliferation refers to the proliferation and replication of ILC2. ILC2 proliferation may be measured by any method known in the art, including but not limited to immunohistochemistry of ILC2 surface proteins and quantitative PCR of ILC2 specific proteins (e.g., RET receptor, neuropeptide receptor Nmur1, interleukin-33 receptor ST2, IL-17A / IL-17B receptor). ILC2 proliferation may be increased by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, ILC2 proliferation is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control, which can be ILC2 not contacted with a RET agonist, or the same ILC2 before contact with a RET agonist.

[0024] RET agonists In some embodiments, the method provided herein comprises contacting ILC2 with a reconstituted (RET) agonist during transfection. RET is a receptor tyrosine kinase for members of the glial cell line-derived neurotrophic factor (GDNF) family of extracellular signaling molecules. RET loss of function mutations are associated with the development of Hillspring disease, and RET gain of function mutations are associated with the development of various types of human cancers, including medullary thyroid carcinoma, multiple endocrine neoplasia type 2A and type 2B, pheochromocytoma, and parathyroid hyperplasia.

[0025] RET is also known as cadherin family member 12, cadherin-related family member 16, CDHF12, CDHR16, HSCR1, hydroxyaryl-protein kinase, MEN2A, MEN2B, MTC1, PTC, ret proto-oncogene, RET-ELE1, RET / PTC, RET51, and RET-HUMAN. The amino acid sequence of RET can be found, for example, in UniProtKB P07949, which has two isoforms, P07949-1 (isoform 1) and P07949-2 (isoform 2). The nucleotide sequence can be found, for example, in AK291807 (mRNA / cDNA sequence).

[0026] A RET agonist is a compound that binds to and increases the activity of a RET protein compared to a control. The control can be a measurement taken from an ILC2 before contact with a RET agonist, a measurement taken from an ILC2 in the same sample (e.g., in vitro or in vivo) that has not been contacted with a RET agonist, or a sample that has not been contacted with a RET agonist. A RET agonist can increase the activity of a RET protein by at least 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to a control.

[0027] The RET agonist herein can be any RET agonist known in the art.Non-limiting examples of RET agonist include: (1) soluble glial-derived neurotrophic factor (GDNF) family binding receptor alpha (GFRα) and GFRα ligand (GFL) combination or its analog or mimetic; or (2) antibody or its antigen-binding fragment that specifically binds to RET and increases RET tyrosine kinase activity.

[0028] RET agonist may be completely specific to RET, or may preferentially agonize RET compared to other tyrosine kinases, or may agonize both RET and other tyrosine kinases.These agonists may be useful even if RET is less agonized than other tyrosine kinases, but it is preferred that the agonist used in the methods described herein agonize RET more than other tyrosine kinases.As used herein, preferentially agonize RET (compared to other tyrosine kinases) means that the agonist agonizes RET at least 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more than other tyrosine kinases.

[0029] The combination of soluble GFRα and GFL or an analog or mimetic thereof may include any soluble GFRα or GFL (or an analog or mimetic thereof) known in the art. Non-limiting examples of soluble GFRα and GFL include the combination of soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) soluble GFRα4 and persephin (PSPN) or an analog or mimetic thereof. (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to GFRα and dimerizes GFRα; or (2) a combination of two or more of (a), (b), (c), (d), (e), (f) and (g).

[0030] Soluble GFRα molecules and GFLs include any GFRα and GFLs known in the art and described herein, for example, GFRα1, GFRα2, GFRα3 and GFRα4.And their respective ligands GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN).GFRα and GFL analogs, mimetics, derivatives and conjugates include GFRα and GFL analogs that have mutations in amino acid sequence compared to natural (for example, endogenous) GFRα and GFL sequence but retain the function of activating RET.

[0031] In some embodiments, the soluble GFRα molecule is GFRα1. GFRα1 is also known as GDNF receptor, GDNFR, GDNFRA, GFR-alpha-1, RETIL, RETL1, TRNR1, and GDNF family receptor alpha1. The amino acid sequence of GFRα1 can be found, for example, in UniProtKB P56159, which has two isoforms, P56159-1 (isoform 1) and P56159-2 (isoform 2). The nucleotide sequence can be found, for example, in AF042080.1 (mRNA / cDNA sequence).

[0032] In some embodiments, the soluble GFRα molecule is GFRα2. GFRα2 is also known as Neurturin receptor, GFRA2, GDNFRB, NRTNR-ALPHA, NTNRA, RETL2, TRNR2, and GDNF family receptor alpha 2. The amino acid sequence of GFRα2 can be found, for example, in UniProtKB O00451, which has three isoforms, O00451-1 (isoform 1), O00451-2 (isoform 2), and O00451-3 (isoform 3). The nucleotide sequence can be found, for example, in AY326396 (mRNA / cDNA sequence).

[0033] In some embodiments, the soluble GFRα molecule is GFRα3. GFRα3 is also known as Artemin receptor, GFRA3, GDNFR3 and GDNF family receptor alpha. The amino acid sequence of GFRα3 can be found for example in UniProtKB O60609, which has two isoforms, O60609-1 (isoform 1) and O60609-2 (isoform 2). The nucleotide sequence can be found for example in AK297693 (mRNA / cDNA sequence).

[0034] In some embodiments, the soluble GFRα molecule is GFRα4. GFRα4 is also known as Persephin receptor and GFRA4. The amino acid sequence of GFRα4 can be found, for example, in UniProtKB Q9GZZ7, which has three isoforms: Q9GZZ7-1 (isoform GFRα4b), Q9GZZ7-2 (isoform GFRα4a), and Q9GZZ703 (isoform GFRα4c). The nucleotide sequence can be found, for example, in AF253318.

[0035] In some embodiments, the GFL is glial cell line-derived neurotrophic factor (GDNF). GDNF is also known as ATF1, ATF2, HFB1-HSCR3, and glial cell line-derived neurotrophic factor. The amino acid sequence can be found, for example, in UniProtKB P39905. It has three isoforms, P39905-1 (isoform 1), P39905-2 (isoform 2), P39905-3 (isoform 3), P39905-4 (isoform 4), and P39905-5 (isoform 5). The nucleotide sequence can be found, for example, in CR541923 (mRNA / cDNA sequence).

[0036] In some embodiments, the GFL is neurturin (NRTN). The amino acid sequence can be found, for example, in UniProtKB Q99748. The nucleotide sequence can be found, for example, in BC137399 (mRNA / cDNA sequence).

[0037] In some embodiments, the GFL is Artemin (ARTN), also known as Enovin, Neublastin, EVN and NBN. The amino acid sequence can be found, for example, in UniProtKB Q5T4W7, which has three isoforms, Q5T4W7-1 (isoform 1), Q5T4W7-2 (isoform 2) and Q5T4W7-3 (isoform 3). The nucleotide sequence can be found, for example, in AF109401 (mRNA / cDNA sequence).

[0038] In some embodiments, the GFL is persephin (PSPN). The amino acid sequence can be found, for example, in UniProtKB O60542. The nucleotide sequence can be found, for example, in AF040962 (mRNA / cDNA sequence).

[0039] Examples of analogs, derivatives and conjugates of GFLs include mutants of GDNF that retain GDNF receptor agonist function, as described in U.S. Pat. No. 9,133,441; mutants of GDNF, as described in U.S. Pat. No. 9,243,046; GFL mutants that efficiently activate RET but lack the heparin binding site and do not interact with HSPGs in the extracellular matrix, as described in U.S. Pat. No. 8,034,572 (e.g., ΔN-GDNF); mutants of GFLs that have reduced heparin, heparan sulfate and heparan sulfate proteoglycan binding ability, as described in U.S. Pat. No. 8,034,572 (e.g., ΔN-GDNF); Nos. 4,445,432, 9,127,083 and 9,469,679; GDNF-derived peptides described in U.S. Pat. No. 8,138,148; neublastin molecules and dimerizing proteins described in U.S. Pat. Nos. 7,276,580, 7,598,059 and 7,655,463; and chimeric GDNF family ligands that activate GFRα / RET described in U.S. Pat. No. 6,866,851.

[0040] Other examples of analogs, derivatives and conjugates of GFLs include GDNF analogs, GDNF isoforms, precursors, fragments and splice variants described in WO 2012 / 151476, EP 2440581 and other patent publications referenced therein, such as those described in WO 2009 / 053536, U.S. Patent Application Publication No. 2009 / 0069230, WO 2008 / 069876, WO 2007 / 019860 and U.S. Patent Application Publication No. 2006 / 0258576.

[0041] Still other agonists of RET include GDNF family ligands (GFLs) and mimetics or RET signaling pathway activators and direct RET activators, which are described in US Pat. No. 8,901,129.

[0042] Another agonist of RET is soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035). As shown by Tokugawa et al. (Neurochem Int. 2003 Jan;42(1):81-6), XIB4035 induced RET autophosphorylation, similar to GDNF. The chemical structure of XIB4035 is shown below: [ka]

[0043] Another agonist of RET is soluble GFRα and the BT compounds, which are described in WO 2011 / 070177.

[0044] Another agonist of RET is soluble GFRα and an antibody that specifically binds and dimerizes GFRα.An antibody that specifically binds and dimerizes GFRα can be obtained by screening this activity among a set of GFRα-binding antibodies.

[0045] Further agonists of RET are antibodies or antigen-binding fragments of such antibodies that specifically bind to RET and increase RET tyrosine kinase activity.RET-binding antibodies are known in the art, such as those described in U.S. Patent No. 6,861,509, and various commercially available antibodies are known.Antibodies that specifically bind to RET and increase RET tyrosine kinase activity can be obtained by screening for this activity among a set of RET-binding antibodies.

[0046] Further agonists of RET include multikinase inhibitors, including, but not limited to, cabozantinib, levatinib, sunitinib and alectinib. Further agonists of RET include selective RET inhibitors selpercatinib (LOXO-292), pralsetinib (BLU-667), BOS 172738 (Boston Pharmaceuticals), HM06 (Helsinn), TPX-0046 (Turning Point Therapeutics), LOX-18228 (Eli Lilly), osimertinib, RXDX-105 (Hoffmann-La Roche), regorafenib, RPI 1 and GSK 3352589.

[0047] The ILC2 cells may be contacted with two or more RET agonists. In some embodiments, the ILC2 cells are contacted with 1-10, 2-9, 3-8, 4-7, or 5-6 RET agonists. In some embodiments, the ILC2 cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RET agonists. In embodiments where the ILC2 cells are contacted with multiple (e.g., two or more) RET agonists, the ILC2 may be contacted with the multiple RET agonists simultaneously or sequentially.

[0048] Mesenchymal stromal cells In some embodiments, the method of the present disclosure provided herein comprises contacting mesenchymal stromal cells (MSCs) with β2-adrenergic receptor (ADRB2) agonist.MSCs are spindle-shaped fibroblast-like cells isolated from bone marrow, adipose and other tissue sources, with in vitro multipotent differentiation potential.MSCs can differentiate into chondrocytes, osteoblasts, adipocytes, myoblasts and other cell types.MSCs express adrenergic receptors (α1A, α1B, α2A, α2B, β1, β2, β3), CD90, CD105 and CD73 on their surface, but do not express CD45, CD34, CD14, CD11b, CD79α, CD19 and HLA-DR.

[0049] In some embodiments, the methods of the disclosure provided herein include increasing the activity of MSCs, increasing the proliferation of MSCs, or increasing the activity and proliferation of MSCs. Any activity of MSCs may be increased by the methods provided herein. Non-limiting examples of MSC activities that may be increased include differentiation into other cell types (e.g., adipocytes, chondrocytes, osteoblasts, adipocytes, myoblasts, and other cell types), extracellular collagen production, and alkaline phosphatase activity. MSC activity may be measured by any method known in the art, including, but not limited to, quantitative PCR measurement of proteins produced by MSCs (e.g., adipogenic proteins, including, but not limited to, AP-1, KLF4, KLF6, C / EBPα, C / EBPβ, C / EBPδ, PPARγ, STAT5A, SREBP-1), cell morphology changes during differentiation (e.g., spindle-shaped MSCs changing into round adipocytes), and cytoskeletal reorganization during differentiation. In some embodiments, the methods provided herein increase the activity of MSCs in adipocyte differentiation. The MSC activity may be increased by 5% to 50%, 10% to 100%, 25% to 150%, 50% to 200%, 75% to 250%, 100% to 300%, 150% to 350%, 200% to 400%, 250% to 450%, 300% to 500%, 350% to 550% or more compared to a control. In some embodiments, the MSC activity is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. The control may be MSCs not contacted with an ADRB2 agonist or the same MSCs prior to contact with an ADRB2 agonist.

[0050] In some embodiments, MSC proliferation is increased after contact with an ADRB2 agonist. MSC proliferation may be measured by any method known in the art, including but not limited to immunohistochemistry of MSC surface proteins (e.g., adrenergic receptors (α1A, α1B, α2A, α2B, β1, β2, β3), CD90, CD105, and CD73), quantitative PCR of MSC-specific proteins (e.g., adrenergic receptors (α1A, α1B, α2A, α2B, β1, β2, β3), CD90, CD105, and CD73), and quantification of cell proliferation markers (e.g., Ki67, PCNA). MSC proliferation may be increased by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, MSC proliferation is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. The control may be MSCs not contacted with an ADRB2 agonist or the same MSCs prior to contact with an ADRB2 agonist.

[0051] β2-adrenergic receptor (ADRB2) agonists In some embodiments, the method provided herein comprises contacting MSCs with a β2-adrenergic receptor (ADRB2) agonist.ADRB2 is a cell membrane-spanning β-adrenergic receptor that binds epinephrine and mediates downstream physiological responses such as smooth muscle relaxation and bronchodilation.ADRB2 functions in human muscular, circulatory, optical, digestive, immune, and respiratory systems.ADRB2 is believed to be associated with the risk of Parkinson's disease, and different polymorphic forms, point mutations, and / or downregulation of this gene are associated with nocturnal asthma, obesity, type 2 diabetes, and cardiovascular disease.

[0052] ADRB2 is also known as adrenergic receptor beta 2, B2AR, beta 2 adrenergic receptor, beta 2 adrenergic receptor, and catecholamine receptor. The amino acid sequence of ADRB2 can be found, for example, in UniProtKB P07550-1. The nucleotide sequence can be found, for example, in X04827 (mRNA / cDNA sequence).

[0053] An ADRB2 agonist is a compound that binds to and increases the activity of ADRB2 protein compared to a control. The control can be a measurement taken from MSCs prior to contact with an ADRB2 agonist, a measurement taken from MSCs in the same sample (e.g., in vitro or in vivo) that has not been contacted with an ADRB2 agonist, or a sample that has not been contacted with an ADRB2 agonist. An ADRB2 agonist can increase the activity of MSC protein by at least 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more compared to a control.

[0054] In some embodiments, the ADRB2 agonist is a short-acting beta agonist (SABA). SABAs have effects lasting 4-6 hours depending on the agonist. SABAs are the first-line drug for acute treatment and are generally used in combination with other compounds (e.g., long-acting beta agonists (LABAs), corticosteroids). Non-limiting examples of SABAs include bitolterol (Tolnarate), fenoterol (Berotec), isopreoterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine (Yutopar), albuterol (Ventolin / Proventil) and terbutaline (Bricanil).

[0055] In some embodiments, the ADRB2 agonist is a long-acting beta agonist (LABA). LABAs are most commonly used in combination with steroids and have effects lasting 12-24 hours depending on the agonist. Non-limiting examples of LABAs include arfomotrol (Brovana), bambuterol (Bambec / Oxeol), clenbuterol (Dilaterol / Spiropent), formoterol (Foradil / Oxis / Perforomist), and salmeterol (Serevent).

[0056] In some embodiments, the ADRB2 agonist is an ultra-long acting beta agonist (ULABA). ULABA has an effect that lasts longer than 24 hours, and the duration of their effect depends on the agonist. Non-limiting examples of ULABA include abesiterol, carmoterol, indacaterol (Arcapta Neohaler), olodaterol (Striverdi Respimat), and vilanterol.

[0057] In some embodiments, the ADRB2 agonist has an unknown duration of action. Non-limiting examples of ADRB2 agonists with unknown duration of action include isoxsuprine, mabuterol, and zilpaterol.

[0058] The MSCs may be contacted with two or more ADRB2 agonists. In some embodiments, the MSCs are contacted with 1-10, 2-9, 3-8, 4-7, or 5-6 ADRB2 agonists. In some embodiments, the MSCs are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ADRB2 agonists. In embodiments where the MSCs are contacted with multiple (e.g., two or more) ADRB2 agonists, the MSCs may be contacted with the multiple ADRB2 agonists simultaneously or sequentially.

[0059] Cytokine production In some embodiments, methods for increasing cytokine production are provided herein. Cytokine production can be from ILC2, MSC, or any other cell that produces cytokine (e.g., T cell, B cell, macrophage, mast cell, endothelial cell, fibroblast). In some embodiments, RET agonist provided herein increases cytokine production from ILC2 cells. In some embodiments, ADRB2 agonist provided herein increases cytokine production from MSC. In some embodiments, RET agonist provided herein increases cytokine production from ILC2 cells, and ADRB2 agonist provided herein increases cytokine production from MSC.

[0060] Cytokines are small (approximately 5-20 kilodalton) peptides used in cell signaling by binding to receptors on target cells. Cytokines are important in the host immune response to infection, inflammation, trauma, sepsis, cancer and reproduction. Cytokines may be type 1 cytokines (e.g., TNFα, IFNγ) that enhance cellular responses and type 2 cytokines (e.g., TGF-β, IL-4, IL-10, IL-13) that enhance antibody responses. Cytokine production may be measured by any method known in the art, including but not limited to immunofluorescent staining of cytokines, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunosorbent spot (ELIspot) assays, antibody array assays, and bead-based assays.

[0061] Non-limiting examples of cytokines that may be increased by the methods disclosed herein include interleukin-5 (IL-5), interleukin-13 (IL-13), Met-enkephalin (Met-Enk), amphiregulin, interleukin-4 (IL-4), interleukin-9 (IL-9), eotaxin, interferon gamma-inducible protein 10 (IP-10), vascular endothelial growth factor (VEGF), TIMP metallopeptidase inhibitor 1 (TIMP1), adipocyte lipid-binding protein (ALBP), and fatty acid translocase (FAT / CD36).

[0062] In some embodiments, the methods provided herein increase cytokine production. Cytokine production may be increased by 5% to 50%, 10% to 100%, 25% to 150%, 50% to 200%, 75% to 250%, 100% to 300%, 150% to 350%, 200% to 400%, 250% to 450%, 300% to 500%, 350% to 550% or more compared to a control. In some embodiments, cytokine production is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. The controls may be ILC2s not contacted with a RET agonist, MSCs not contacted with an ADRB2 agonist, or ILC2s not contacted with a RET agonist, as well as MSCs not contacted with an ADRB2 agonist or the same ICL2s prior to contact with a RET agonist, and / or MSCs not contacted with an ADRB2 agonist.

[0063] Treatment method Obesity and Fat Homeostasis In some embodiments, the method of reducing susceptibility to obesity, increasing fat homeostasis, or reducing susceptibility to obesity and increasing fat homeostasis is further provided herein in a subject in need thereof.The method of reducing susceptibility to obesity and / or increasing fat homeostasis may comprise contacting ILC2 (for example, in a subject) with any RET agonist provided herein, contacting MSC with any ADRB2 agonist provided herein, or combinations thereof, compared to a control.The control may be a subject with ILC2 that is not contacted with a RET agonist, a subject with MSC that is contacted with an ADRB2 agonist, or the same subject with ICL2 before contacting with a RET agonist and / or MSC before contacting with an ADRB2 agonist.

[0064] The subject in need thereof may be any subject with obesity or increased susceptibility to obesity. Obesity is a disorder involving excess body fat that increases the risk of health problems, including but not limited to cardiovascular disease, diabetes, high blood pressure and high cholesterol. Obesity occurs when a person's body mass index (BMI) is 30 or higher. BMI is calculated by dividing weight (kilograms) by height (meters) squared (United States Center for Disease Control) for adults aged 18 years or older, and for children under 18 years old, taking into account gender and age. Obesity is generally self-diagnosed by calculating BMI, and subjects with obesity may have any symptoms, including but not limited to back or joint pain, overeating, fatigue, sleep apnea, and excess body fat. Conventional treatments for obesity include but are not limited to exercise, low-fat diet (daily calorie consumption less than 30%), and behavioral therapy.

[0065] In some embodiments, the method provided herein reduces the susceptibility of obesity in subjects.Susceptibility to obesity may be determined by any metric known in the art, including behavioral, environmental, and genetic factors assessment.Behavioral risk factors that increase susceptibility to obesity include consuming foods that are high in saturated fat and trans fat (more than 30% of daily calorie consumption) and being inactive (no physical activity beyond the physical activity of daily living).Environmental risk factors that increase susceptibility to obesity include the availability of foods that are low in saturated fat and trans fat (less than 30% of daily calorie consumption), inability to be physically active, and prenatal and postnatal (within the first year of life) maternal influences.Genetic risk factors that increase susceptibility to obesity include mutations in genes encoding the hormones leptin, leptin receptor, proopiomelanocortin, and melanocortin-4 receptor; fat mass; and genes encoding obesity-associated protein (FTO), transmembrane protein 18 (TMEM18), glucosamine 6-phosphate deaminase 2 (GNPDA2), brain-derived neurotrophic factor (BDNF), nerve growth regulator 1 (NEGR1), SH2B adaptor protein 1 (SH2B1), ETS variant transcription factor 5 (ETV5), mitochondrial carrier 2 (MTCH2), potassium channel tetramerization domain containing 15 (KCTD15), Fas inhibitor of apoptosis molecule 2 (FAIM2), SEC homology B (SEC16B), and TNNI3-interacting kinase. These include mutations in genes encoding (TNNI3K), leucine-rich repeat protein, neuronal 6C (LRRN6C), 3-hydroxy-3-methylglutaryl-coA reductase (HMGCR), protein kinase D1 (PRKD1), gastric inhibitory polypeptide receptor (RBJ / GIPR), solute carrier family 39 member 8 (SLC39A8), transmembrane protein 160 (TMEM160), Fanconi anemia complementation group L (FANCL), cell adhesion molecule 2 (CADM2), LDL receptor protein 1B (LRP1B), polypyrimidine tract binding protein 2 (PTBP2), mitochondrial translation initiation factor 3 (MTIF3), zinc finger 608 (ZNF608), and protein homolog (TUB).

[0066] In some embodiments, the methods provided herein reduce susceptibility to obesity. Susceptibility to obesity may be measured by any method known in the art, including, but not limited to, body fat (e.g., adipose tissue) reduction, reduced saturated and trans fat consumption, and increased physical activity. Susceptibility to obesity may be reduced by 5% to 50%, 10% to 100%, 25% to 150%, 50% to 200%, 75% to 250%, 100% to 300%, 150% to 350%, 200% to 400%, 250% to 450%, 300% to 500%, 350% to 550% or more compared to a control. In some embodiments, susceptibility to obesity is reduced by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. A control can be a subject not treated with the methods provided herein.

[0067] In some embodiments, the method provided herein increases the fat homeostasis of a subject in need thereof.As used herein, adipose tissue homeostasis refers to the balance between storing excess calories as triglycerides in white adipocytes and utilizing excess calories stored from white adipocytes during calorie withdrawal.Adipose homeostasis imbalance occurs when excess calories are stored as triglycerides more than necessary and calorie withdrawal does not occur, or when excess calories are stored as triglycerides more than necessary and calorie withdrawal does not occur.Non-limiting symptoms of fat homeostasis imbalance include decreased glucose tolerance, decreased gonadal adipose tissue mass, and increased hyperglycemia.

[0068] In some embodiments, the subject herein with reduced fat homeostasis has reduced glucose tolerance. Reduced glucose tolerance means that the subject has a glucose level of 140 to 199 mg / deciliter (mg / dL) (7.8 to 11.0 mmol) after 2 hours in a 75 g glucose tolerance test. Untreated reduced glucose tolerance is also known as prediabetes and is likely to progress to type 2 diabetes without intervention.

[0069] In some embodiments, the methods provided herein increase glucose tolerance. Glucose tolerance may be measured by any method known in the art, including but not limited to the 75-gram glucose tolerance test. Glucose tolerance may be increased by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, glucose tolerance is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. A control can be a subject not treated with the methods provided herein.

[0070] In some embodiments, the subject herein with decreased fat homeostasis has increased adipose tissue mass. The adipose tissue may be subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT). VAT surrounds the subject's internal organs and may be gonadal adipose tissue (GAT), omental adipose tissue (OAT), retroperitoneal adipose tissue (RAT), mesenteric adipose tissue (MAT) or pericardial adipose tissue (PAT). In some embodiments, the increased adipose tissue mass is an increased GAT mass. GAT mass is found around the testis (epididymis) in males and around the ovary (periovarian) in females. GAT expresses more PPARγ and SREBP1C and adipogenic transcription factor CCAAT enhancer binding protein alpha (C / EBP-alpha) genes compared to SAT.

[0071] In some embodiments, the methods provided herein reduce adipose tissue (e.g., GAT) mass. Adipose tissue mass may be measured by any method known in the art, including, but not limited to, measuring the weight of a subject, measuring the expression of adipose tissue-specific genes (e.g., PPARγ, SREBP1C, and / or CEBP-α), and immunofluorescence staining of adipose tissue-specific proteins (e.g., PPARγ, SREBP1C, and / or CEBP-α). Adipose tissue mass may be reduced by 5% to 50%, 10% to 100%, 25% to 150%, 50% to 200%, 75% to 250%, 100% to 300%, 150% to 350%, 200% to 400%, 250% to 450%, 300% to 500%, 350% to 550% or more compared to a control. In some embodiments, the adipose tissue mass is reduced by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, or more compared to a control. A control can be a subject not treated with the methods provided herein.

[0072] ILC2 and / or MSC cells may be contacted with two or more RET agonists and / or ADRB2 agonists. In some embodiments, ILC2 and / or MSC are contacted with 1-10, 2-9, 3-8, 4-7, or 5-6 RET / ADRB2 agonists. In some embodiments, ILC2 and / or MSC are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RET / ADRB2 agonists. In embodiments where ILC2 and / or MSC are contacted with multiple (e.g., two or more) RET / ADRB2 agonists, ILC2 and / or MSC may be contacted with the multiple RET / ADRB2 agonists simultaneously or sequentially.

[0073] Disorders Associated with Decreased ILC2 Expression or Activity In some embodiments, the method of treating disorder associated with decreased ILC2 activity or proliferation in a subject is also provided herein.The method of treating disorder associated with decreased ILC2 activity or proliferation may include contacting ILC2 (for example, in a subject) with any RET agonist provided herein, contacting MSC with any ADRB2 agonist provided herein, or combinations thereof, compared to a control.The control may be a subject with ILC2 that is not contacted with a RET agonist, a subject with MSC that is contacted with an ADRB2 agonist, or the same subject with ILC2 before contacting with a RET agonist and / or MSC before contacting with an ADRB2 agonist.

[0074] The disorder associated with decreased ILC2 activity or proliferation in a subject may be any disorder associated with decreased ILC2 activity or proliferation. Non-limiting examples of disorders associated with decreased ILC2 activity or proliferation include weight gain, obesity, diabetes, metabolic syndrome, or a combination thereof.

[0075] In some embodiments, the disorder associated with reduced ILC2 activity or proliferation is weight gain. Weight gain can be caused by an increase in adipose tissue, body fluids, or muscle mass. Increased adipose tissue occurs when a subject regularly consumes more calories than are consumed by daily physical activity. Increased body fluids can be due to drugs, fluid and salt retention, intravenous fluids, renal failure, or heart failure. Increased muscle mass is commonly seen during exercise. In some embodiments, the disorder associated with reduced ILC2 activity or proliferation is weight gain due to increased adipose tissue (e.g., GAT). Conventional treatments for weight gain (e.g., due to increased adipose tissue) include, but are not limited to, increased daily physical activity, increased consumption of foods low in saturated fat and trans fat, and treatment with diuretics (e.g., furosemide, bumetanide, torsemide, hydrochlorothiazide, metolazone, spironolactone).

[0076] In some embodiments, the disorder associated with decreased ILC2 activity or proliferation is obesity. Obesity may be diagnosed by any method and may occur with any of the conditions described herein.

[0077] In some embodiments, the disorder associated with decreased ILC2 activity or proliferation is diabetes. Diabetes is a disorder in which the subject's ability to produce or respond to insulin is impaired, resulting in blood and urinary glucose levels of 200 mg / dL or higher. Diabetes may be type 1 diabetes (juvenile diabetes) or type 2 diabetes (adult-onset diabetes). In some embodiments, diabetes is type 2 diabetes. In type 2 diabetes, the subject does not produce enough insulin or does not respond to insulin, resulting in hyperglycemia. Symptoms of type 2 diabetes include, but are not limited to, increased thirst, frequent urination, hunger, fatigue, and blurred vision. Conventional treatments for diabetes include, but are not limited to, increased daily physical activity, increased consumption of foods low in saturated and trans fats, monitoring blood glucose levels, treatment with antidiabetic drugs (e.g., metformin, sulfonylureas, glinides, thiazolidinediones, DDP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors), and insulin therapy.

[0078] In some embodiments, the disorder associated with decreased ILC2 activity or proliferation is metabolic syndrome (also known as insulin resistance syndrome). Metabolic syndrome is a group of conditions that increase the risk of heart disease, stroke, and diabetes (e.g., type 2 diabetes) compared to subjects without metabolic syndrome. Metabolic syndrome is typically diagnosed when a subject has three or more of the following: high blood pressure (e.g., systolic ≥ 135mmHg and diastolic ≥ 85mmHg), hyperglycemia (e.g., fasting blood glucose ≥ 100mg / dL), excess body fat around the waist (e.g., waist circumference > 40 inches for men and > 35 inches for women), and abnormal cholesterol levels (total cholesterol > 200mg / dL, non-high density lipoprotein > 130mg / dL, low density lipoprotein > 100mg / dL, and / or high density lipoprotein < 50mg / dL). Conventional treatments for metabolic syndrome include, but are not limited to, increased daily physical activity, increased consumption of foods low in saturated and trans fats, smoking cessation, stress reduction, hypertension medications (e.g., ACE inhibitors, angiotensin II receptor ablation drugs, diuretics, beta ablation drugs), cholesterol medications (e.g., statins, niacin, bile acid resins), diabetes medications (metformin, pioglitazone, rosiglitazone), and low-dose aspirin.

[0079] ILC2 activity may be measured by any method known in the art, including but not limited to quantitative PCR measurement and fluorescent quantification of proteins produced by ILC2 cells (e.g., cytokines). ILC2 proliferation may be measured by any method known in the art, including but not limited to immunohistochemistry of ILC2 surface proteins and quantitative PCR of ILC2-specific proteins (e.g., RET receptor, neuropeptide receptor Nmur1, interleukin-33 receptor ST2, IL-17A / IL-17B receptor).

[0080] ILC2 activity may be increased by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, ILC2 activity is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. ILC2 proliferation may be increased by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, ILC2 proliferation is increased by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. The control can be ILC2s not contacted with a RET agonist, or the same ILC2s before contact with a RET agonist, ILC2s in cells that are not contacted with an ADRB2 agonist, or the same ILC2s in cells before contact with an ADRB2 agonist.

[0081] ILC2 and / or MSC cells may be contacted with two or more RET agonists and / or ADRB2 agonists. In some embodiments, ILC2 and / or MSC are contacted with 1-10, 2-9, 3-8, 4-7, or 5-6 RET / ADRB2 agonists. In some embodiments, ILC2 and / or MSC are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RET / ADRB2 agonists. In embodiments where ILC2 and / or MSC are contacted with multiple (e.g., two or more) RET / ADRB2 agonists, ILC2 and / or MSC may be contacted with the multiple RET / ADRB2 agonists simultaneously or sequentially.

[0082] Disorders associated with increased ILC2 activity or proliferation In some embodiments, methods for treating disorders associated with increased ILC2 activity or proliferation in a subject are also provided herein.Methods for treating disorders associated with increased ILC2 activity or proliferation may include contacting ILC2 (e.g., in a subject) with a RET antagonist, contacting MSCs with an ADRB2 antagonist, or a combination thereof, compared to a control.The control may be a subject with ILC2 that is not contacted with a RET antagonist, a subject with MSCs that is contacted with an ADRB2 antagonist, or the same subject with ILC2 before contacting with a RET antagonist and / or MSCs before contacting with an ADRB2 antagonist.

[0083] The disorder associated with increased ILC2 activity or proliferation in a subject may be any disorder associated with increased ILC2 activity or proliferation. Non-limiting examples of disorders associated with increased ILC2 activity or proliferation include hyperthermia, cachexia, allergy, helminth infection, allergic asthma, atopic dermatitis, intestinal inflammatory disease, or a combination thereof.

[0084] In some embodiments, the disorder associated with increased activity or proliferation of ILC2 is hypothermia. As used herein, hypothermia refers to a significant and potentially dangerous reduction in body temperature. The normal body temperature of a human subject is about 98.6°F (37°C), and hypothermia occurs when the body temperature of a human subject falls below 95°F (35°C). Hypothermia is often caused by exposure to cold or immersion in cold water. Conventional treatments for hypothermia include methods to return a subject (e.g., a human) to normal body temperature.

[0085] In some embodiments, the disorder associated with increased ILC2 activity or proliferation is cachexia. Cachexia is a loss of more than 5% of body weight over 12 months or less if the subject is not trying to lose weight and has a known disease or disorder, and is accompanied by at least three of the following symptoms compared to control: muscle weakness, fatigue, loss of appetite, decreased lean mass index, elevated inflammation as determined by blood test, anemia, or low levels of protein albumin. Cachexia occurs in diseases such as cancer, congestive heart failure, chronic obstructive pulmonary disease (COPD), chronic kidney disease, cystic fibrosis, and rheumatoid arthritis. Conventional treatments for alleviating the symptoms of cachexia include appetite stimulants (e.g., megestrol acetate, Megace); drugs such as dronabinol (Marinol) to improve nausea, appetite, and mood; agents that reduce inflammation; dietary changes; dietary supplements; and adaptive exercise.

[0086] In some embodiments, the disorder associated with increased ILC2 activity or proliferation is allergy. Allergy is an immune system response to non-harmful exogenous substances. Categories of allergy include but are not limited to food (e.g. milk, soybean, egg, wheat, peanut, nut, fish, shellfish), seasonal (e.g. pollen, mold, ragweed), latex, drug (e.g. penicillin), insect sting or bite (e.g. wasp, bee, hornet, ant, mosquito, tick) and toxin (e.g. poison ivy, poison oak, poison sumac, poison sumac). Symptoms of allergy include but are not limited to bloodshot eyes, itchy rash, sneezing, runny nose, shortness of breath, swelling, and hives. Conventional treatments for allergy relief include drug therapy (e.g., antihistamines, glucocorticoids, epinephrine, mast cell stabilizers, anti-leukotrienes, anticholinergics, decongestants), immunotherapy (e.g., injectable immunotherapy, sublingual immunotherapy), and alternative medicines (e.g., saline nasal irrigation, butterbur).

[0087] In some embodiments, the disorder associated with increased ILC2 activity or proliferation is a helminth infection. Helminths are parasitic worms that live and feed on living hosts. Helminth categories include, but are not limited to, annelids (e.g., ringworms, segmented worms), flatworms (e.g., tapeworms, flukes, blood flukes), nematodes (e.g., roundworms), and thorny heads (e.g., wireworms). Symptoms of helminth infection include, but are not limited to, abdominal pain, weight loss, nausea, vomiting, fever, cough, dyspnea, urticaria, myalgia, pneumonitis, lymphadenopathy, hepatosplenomegaly, and convulsions. Conventional treatments for helminth infection include mebendazole, albendazole, niclosamide, praziquantel, and steroids (e.g., dexamethasone, prednisolone).

[0088] In some embodiments, the disorder associated with increased activity or proliferation of ILC2 is allergic asthma.Allergic asthma is a long-term inflammatory disease of the airways of the lungs.Allergic asthma can be caused by exposure to any known allergen, including but not limited to dust mites, cockroaches, animal dander and mold.Non-limiting symptoms of allergic asthma include wheezing, coughing, chest tightness and shortness of breath.Conventional treatment of allergic asthma includes avoidance of allergens, inhaled corticosteroids, and anti-leukotrienes.

[0089] In some embodiments, the disorder associated with increased ILC2 activity or proliferation is atopic dermatitis. Atopic dermatitis (atopic eczema) is a long-term inflammation of the skin, resulting in itching, redness, swelling, and cracking. Atopic dermatitis is typically diagnosed when a subject has three or more of the following: skin folds are involved (e.g., flexural dermatitis of the skin in front of the feet, elbow fossae, popliteal fossae, around the eyes, neck, and cheeks), a history of asthma or allergic rhinitis (or a family history if the subject is under 4 years old), symptoms beginning before 2 years of age, a history of dry skin (within the past year), and dermatitis visible on flexural or cheek, forehead, and extensor surfaces. A clear fluid may come from the affected skin area. The cause of atopic dermatitis is unknown, but it is believed to involve genetics, immune system dysfunction, environmental exposure, and skin permeability problems. Conventional treatments for atopic dermatitis include avoiding triggers (such as woolen clothing, soap, perfume, chlorine, dust, and cigarette smoke), bathing daily followed by applying moisturizing creams, steroid creams, and medications to relieve itch.

[0090] In some embodiments, the disorder associated with increased ILC2 activity or proliferation is intestinal inflammatory disease. Intestinal inflammatory disease (also known as inflammatory bowel disease (IBD)) is an ongoing inflammation of all or part of the intestinal tract. Intestinal inflammatory disease may occur in the small intestine or large intestine (bowel). Intestinal inflammatory disease is an umbrella term that includes any intestinal inflammatory disease, including but not limited to ulcerative colitis (UC) and Crohn's disease (CD). Symptoms of intestinal inflammatory disease include diarrhea, fatigue, abdominal pain and cramps, bloody stools, loss of appetite, and unintentional weight loss. Conventional treatments for intestinal inflammatory diseases include, but are not limited to, anti-inflammatory drugs (e.g., corticosteroids, aminosalicylates), immune system suppressants (e.g., azathioprine, mercaptopurine, methotrexate), biologics (e.g., infliximab, adalimumab, golimumab, certolizumab, vedolizumab, ustekinumab), antibiotics (e.g., ciprofloxacin, metronidazole), antidiarrheal drugs (e.g., psyllium powder, methylcellulose, loperamide), painkillers (e.g., acetaminophen, ibuprofen, naproxen sodium, diclofenac sodium), and vitamins.

[0091] RET antagonists may be any RET antagonists known in the art. RET antagonists include peptide antagonists (including modified peptides and conjugates), inhibitory antibody molecules, inhibitory nucleic acid molecules and small molecules. Some RET antagonists may be completely specific to RET, may preferentially antagonize RET (compared to other tyrosine kinases), or may antagonize both RET and other tyrosine kinases (such as some of the small molecule RET tyrosine kinase inhibitors described below). Such antagonists may be useful even when RET is antagonized weaker than other tyrosine kinases, but it is preferred that the antagonists used in the methods described herein antagonize RET more than other tyrosine kinases. As used herein, preferentially antagonizing RET (relative to other tyrosine kinases) means that the antagonist antagonizes RET by at least 10%, 25%, 50%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more than other tyrosine kinases.

[0092] Antagonists of RET include antibodies that specifically bind and inhibit (a) RET tyrosine kinase activity, (b) GDNF family binding receptor alpha (GFRα), or (c) GFRα ligand, or antigen-binding fragments thereof. Examples include the antibodies described in U.S. Pat. No. 8,968,736, U.S. Pat. No. 9,522,185, and U.S. Patent Application Publication No. 2017 / 0096488 that bind to human GFRα3. RET binding antibodies are known in the art, such as those described in U.S. Pat. No. 6,861,509, and various commercially available antibodies are known. Antibodies that specifically bind and inhibit (a) RET tyrosine kinase activity, (b) GDNF family binding receptor alpha (GFRα), or (c) GFRα ligand can be obtained by screening one of these activities among a set of antibodies that bind to RET, GFRα, or GFRα ligand.

[0093] Antagonists of RET include inhibitory nucleic acid molecules that reduce the expression, transcription or translation of RET, GFRα or GFRα ligand. Suitable inhibitory nucleic acid molecules include RET-specific, GFRα-specific or GFRα ligand-specific inhibitory nucleic acids, such as siRNA, antisense, aptamers or ribozymes specifically targeted to RET, GFRα or GFRα ligand.

[0094] RET antagonists include RET tyrosine kinase inhibitors. Exemplary RET tyrosine kinase inhibitors include AST487, motesanib, cabozantinib, vandetanib, ponatinib, sunitinib, sorafenib and alectinib.

[0095] AST 487 (NVP-AST487; 630124-46-8; also known as UNII-W34UO2M4T6; IUPAC name: 1-[4-[(4-ethylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[4-[6-(methylamino)pyrimidin-4-yl]oxyphenyl]urea) is an inhibitor of RET, receptor tyrosine-protein kinase FLT 3, kinase insert domain receptor (KDR; VEGFR2), Abelson murine leukemia viral oncogene homolog 1 (c-ABL), and stem cell factor receptor (c-KIT) that has been shown to inhibit RET autophosphorylation and activation of downstream effectors (Akeno-Stuart et al., Cancer Res. 2007 Jul 15;67(14):6956-64). The chemical structure of AST 487 is shown below: [ka]

[0096] Motesanib (also known as AMG-706; IUPAC name: N-(3,3-dimethyl-2,3-dihydro-1H-indol-6-yl)-2-[(pyridin-4-ylmethyl)amino]pyridine-3-carboxamide) is an inhibitor of RET, VEGFR, platelet-derived growth factor receptor (PDGFR) and c-KIT. The chemical structure of motesanib is shown below: [ka]

[0097] Cabozantinib (also known as CABOMETYX; COMETRIQ; XL-184; BMS-907351; IUPAC name: N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)phenyl)-N'-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide) is an inhibitor of vascular endothelial growth factor receptors (VEGFRs), including RET, hepatocyte growth factor receptor (MET), AXL receptor tyrosine kinase (AXL; tyrosine-protein kinase receptor UFO), and VEGFR2. The chemical structure of cabozantinib is shown below: [ka]

[0098] Vandetanib (also known as CAPRELSA; ZACTIMA; ZD-6474; IUPAC name: N-(4-bromo-2-fluorophenyl)-6-methoxy-7-((1-methylpiperidin-4-yl)methoxy)quinazolin-4-amine) is an inhibitor of RET, VEGFR, including VEGFR2, and epidermal growth factor receptor (EGFR). The chemical structure of vandetanib is shown below: [ka]

[0099] Ponatinib (ICLUSIG; also known as AP24534; IUPAC name: 3-(2-imidazo[1,2-b]pyridazin-3-ylethynyl)-4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]benzamide) is an inhibitor of RET and fibroblast growth factor receptors (FGFR). The chemical structure of ponatinib is shown below: [ka]

[0100] Sunitinib (also known as SUTENT; SU11248; IUPAC name: N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1 H-indol-3-ylidene)methyl]-2,4-dimethyl-1 H-pyrrole-3-carboxamide) is an inhibitor of RET, PGFR, VEGFR, c-KIT, granulocyte colony-stimulating factor receptor (GCSFR) and FLT 3. The chemical structure of sunitinib is shown below: [ka]

[0101] Sorafenib (also known as NEXAVAR; IUPAC name: 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide) is an inhibitor of RET, VEGFR, PDGFR and Raf family kinases. The chemical structure of sorafenib is shown below: [ka]

[0102] Alectinib (also known as ALECENSA; IUPAC name: 9-ethyl-6,6-dimethyl-8-[4-(morpholin-4-yl)piperidin-1-yl]-11-oxo-6,11 dihydro-5 H-benzo[b]carbazole-3-carbonitrile) is an inhibitor of RET and anaplastic lymphoma kinase (ALK). The chemical structure of alectinib is shown below: [ka]

[0103] Other suitable RET antagonists are disclosed in U.S. Pat. Nos. 6,235,769, 7,504,509, 8,067,434, 8,426,437, 8,629,135, 8,937,071, 8,999,973, 9,035,063, 9,382,238, 9,297,0 No. 11, U.S. Patent Application Publication No. 2015 / 0238477, U.S. Patent Application Publication No. 2015 / 0272958, U.S. Patent Application Publication No. 2016 / 0271123, U.S. Patent Application Publication No. 20160354377, U.S. Patent Application Publication No. 2017 / 0096425, and U.S. Patent Application Publication No. 2017 / 0121312, and related patent applications worldwide.

[0104] The ADRB2 antagonist may be any ADRB2 antagonist known in the art. The ADRB2 agonist may be a non-selective beta-adrenergic receptor antagonist that binds to both beta1 and beta2 adrenergic receptors, including propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol and timolol. The ADRB2 antagonist may be specific to ADRB2 (compared to other beta-adrenergic receptors). Non-limiting examples of ADRB2 specific antagonists include butaxamine and ICI-118,551. Other possible ADRB2 antagonists for use in the methods herein include acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, metoprolol, nebivolol, esmolol and SR 59230A.

[0105] ILC2 activity may be measured by any method known in the art, including but not limited to quantitative PCR measurement and fluorescent quantification of proteins produced by ILC2 cells (e.g., cytokines). ILC2 proliferation may be measured by any method known in the art, including but not limited to immunohistochemistry of ILC2 surface proteins and quantitative PCR of ILC2-specific proteins (e.g., RET receptor, neuropeptide receptor Nmur1, interleukin-33 receptor ST2, IL-17A / IL-17B receptor).

[0106] ILC2 activity may be reduced by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, ILC2 activity is reduced by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. ILC2 proliferation may be reduced by 5%-50%, 10%-100%, 25%-150%, 50%-200%, 75%-250%, 100%-300%, 150%-350%, 200%-400%, 250%-450%, 300%-500%, 350%-550% or more compared to a control. In some embodiments, ILC2 proliferation is reduced by at least 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550% or more compared to a control. The control can be ILC2s not contacted with a RET antagonist, or the same ILC2s before contacting with a RET antagonist, ILC2s in cells that are not contacted with MSCs with an ADRB2 antagonist, or the same ILC2s in cells before contacting with an ADRB2 antagonist.

[0107] ILC2 and / or MSC cells may be contacted with two or more RET antagonists and / or ADRB2 antagonists. In some embodiments, ILC2 and / or MSC are contacted with 1-10, 2-9, 3-8, 4-7, or 5-6 RET / ADRB2 antagonists. In some embodiments, ILC2 and / or MSC are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RET / ADRB2 antagonists. In embodiments where ILC2 and / or MSC are contacted with multiple (e.g., two or more) RET / ADRB2 antagonists, ILC2 and / or MSC may be contacted with the multiple RET / ADRB2 antagonists simultaneously or sequentially.

[0108] cold exposure In some embodiments, the method of treating cold exposure is also provided herein.The method of treating cold exposure may include contacting ILC2 (for example, in a subject) with RET agonist, contacting MSC with ADRB2 agonist, or combinations thereof, compared with control.The control may be the subject with ILC2 that is not contacted with RET agonist, the subject with MSC that is contacted with ADRB2 agonist, or the same subject with ILC2 before contacting with RET agonist and / or MSC before contacting with ADRB2 agonist.

[0109] Cold exposure can occur outdoors in damp, windy, and / or cold weather, or indoors in a residence that is not heated sufficiently to prevent cold exposure. If left untreated, cold exposure can result in damage including, but not limited to, mild frostbite, frostbite, trench foot, frostbite, and hypothermia. Mild frostbite causes numbness and pale skin for a short time, but returns to normal feel and color upon warming. Frostbite is the freezing of the skin and tissues below the skin that do not return to normal feel or color upon warming. Trench foot is damage that develops gradually over several days of exposure to low temperatures that do not actually freeze the skin, and is characterized by reddened skin, numbness or burning pain, leg cramps, and the development of blisters or ulcers after 2-7 days. Chilblains (pernio) is a reaction to cold temperatures and is characterized by localized redness and swelling, raised skin, altered sensation, soft blue bumps that develop after rewarming, and blisters and ulcers. Hypothermia has been discussed above.

[0110] Risk factors for cold exposure injury include, but are not limited to, being an infant (under 1 year old); being elderly (over 65 years old); consuming alcohol, being outdoors at high altitude, strong winds, wet weather, or immersion in cold water; being fatigued or dehydrated and exposed to cold temperatures at work; having a condition such as diabetes, HIV, cancer, or heart disease; taking certain medicines such as anticoagulants or immunosuppressants; and having a recent health event such as surgery or injury.

[0111] A subject's body temperature may be increased by 1°F to 45°F, 5°F to 40°F, 10°F to 35°F, 15°F to 30°F, 20°F to 25°F, or 1°C to 25°C, 5°C to 20°C, 10°C to 15°C, or more compared to a control by the methods of treating cold exposure provided herein. In some embodiments, the subject's temperature is increased by at least 1°F, 2°F, 3°F, 4°F, 5°F, 6°F, 7°F, 8°F, 9°F, 10°F, 11°F, 12°F, 13°F, 14°F, 15°F, 16°F, 17°F, 18°F, 19°F, 20°F, 21°F, 22°F, 23°F, 24°F, 25°F, 26°F, 27°F, 28°F, 29°F, 30°F, 31°F, 32°F, 33°F, 34°F, 35°F, 36°F, 37°F, 38°F, 39°F, 40°F, 41°F, 42°F, 43°F, 44°F, 45°F, or more compared to a control. In some embodiments, the temperature of the subject is increased by at least 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., or 25° C. The control can be ILC2s not contacted with a RET agonist, or the same ILC2s before contacting with a RET agonist, ILC2s in cells without contacting MSCs with an ADRB2 agonist, or the same ILC2s in cells before contacting with an ADRB2 agonist.

[0112] ILC2 and / or MSC cells may be contacted with two or more RET agonists and / or ADRB2 agonists. In some embodiments, ILC2 and / or MSC are contacted with 1-10, 2-9, 3-8, 4-7, or 5-6 RET / ADRB2 agonists. In some embodiments, ILC2 and / or MSC are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RET / ADRB2 agonists. In embodiments where ILC2 and / or MSC are contacted with multiple (e.g., two or more) RET / ADRB2 agonists, ILC2 and / or MSC may be contacted with the multiple RET / ADRB2 agonists simultaneously or sequentially.

[0113] Method of administration In some embodiments, the methods provided herein include contacting a cell (e.g., ILC2, MSC) with a RET agonist, a RET antagonist, an ADRB2 agonist, or an ADRB2 antagonist. Contacting refers to providing a compound (e.g., a RET agonist, a RET antagonist, an ADRB2 agonist, an ADRB2 antagonist) or a pharmaceutical composition comprising a compound to the cell.

[0114] The cells contacted in the methods provided herein may be in vitro or in vivo. In some embodiments, the cells contacted in the methods provided herein are in vitro. In vitro cells may be maintained under conditions that mimic the in vivo environment (e.g., in cell culture). In vitro cells may be single or part of a population of cells. A population of cells may include 2 cells, 1,000 cells, 500 cells to 10,000 cells, 5,000 cells to 100,000 cells, 50,000 cells to 1,000,000 cells, 500,000 cells to 10,000,000 cells, or more than 10,000,000 cells.

[0115] In some embodiments, the cell contacted in the method provided herein is in vivo.When the cell is contacted in vivo, RET agonist, RET antagonist, ADRB2 agonist, ADRB2 antagonist, or combination thereof may be administered to a subject.The subject may be any subject that requires it, including but not limited to human, rodent (e.g. mouse, rat, hamster), non-human primate (e.g. chimpanzee, gorilla, or orangutan), domestic pet (e.g. dog, cat, rabbit), or livestock animal (e.g. horse, cow, chicken, pig, goat, sheep, donkey).In some embodiments, the subject is human.

[0116] In some embodiments, the in vitro cells (e.g., ILC2, MSC) are derived from a tissue of a subject. The tissue in a subject may be any tissue occurring in the subject. Derived from a tissue refers to the isolation of the in vitro cells from the tissue. Derivation of the in vitro cells from a tissue may use any method known in the art, including but not limited to chemical digestion (e.g., trypsin) or mechanical tissue digestion (e.g., homogenization). Non-limiting examples of possible tissues from which the in vitro cells may be derived include adipose, skeletal muscle, smooth muscle, cardiac muscle, nerve, blood, kidney, pancreas, stomach, small intestine, large intestine, rectum, brain, spinal cord, bone, cartilage, skin, hair, liver, ovary, uterus, testis, prostate, heart, lung, trachea, tongue, and salivary gland. In some embodiments, the in vitro cells are derived from adipose tissue.

[0117] In some embodiments, an agonist (e.g., a RET agonist, an ADRB2 agonist), an antagonist (e.g., a RET antagonist, an ADRB2 antagonist), or a combination thereof is administered to a subject in need thereof. Administration may be by any method known in the art, including, but not limited to, injection (e.g., intravenous, intramuscular, intraarterial, intracerebroventricular), inhalation, and ingestion (e.g., oral, rectal).

[0118] When agonist (e.g., RET agonist, ADRB2 agonist), antagonist (e.g., RET antagonist, ADRB2 antagonist) or combination thereof is administered to a subject, a dose of each of agonist and / or antagonist is administered. The absolute amount depends on various factors, including concurrent treatment, number of administrations, and individual patient parameters, including age, physical condition, size and weight. These are factors well known to those skilled in the art and can be addressed with only routine experimentation. In general, it is preferred to use the maximum dose, i.e., the highest safe dose according to sound medical judgment. Multiple doses may be administered to a subject in need thereof.

[0119] The active agent of the present invention (e.g., the compounds and cells described herein) is administered to a subject in an effective amount to treat the disease. According to some aspects of the present invention, the effective amount is the amount of agonist (e.g., RET agonist, ADRB2 agonist), antagonist (e.g., RET antagonist, ADRB2 antagonist) alone or in combination with another drug, which, depending on the disease being treated, produces a therapeutic response against the disease. The biological effect may be the amelioration and / or absolute elimination of the disease or symptoms caused by the disease. In another embodiment, the biological effect is the complete suppression of the disease, for example, as evidenced by the absence of symptoms of the disease.

[0120] The effective amount of the compound (i.e., either agonist, antagonist, or combination thereof) used in the method of the present invention in treating the diseases described herein may vary depending on the specific compound used, the mode of delivery of the compound, and whether it is used alone or in combination. The effective amount for any particular application may also vary depending on factors such as the disease being treated, the specific compound being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular molecule of the present invention using routine and accepted methods known in the art without undue experimentation. In combination with the teachings provided herein, by selecting from among various active compounds and considering important factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred mode of administration, an effective treatment treatment regimen that does not cause substantial toxicity and is effective for treating a particular subject can be designed.

[0121] In other embodiments, a compound may be isolated. As used herein, isolated means that the material referred to is removed from its natural environment, e.g., a cell. Thus, an isolated biological material may be free of some or all cellular components, i.e., components of the cell in which the natural material naturally occurs (e.g., cytoplasm or membrane components). In the case of nucleic acid molecules, isolated nucleic acids include PCR products, isolated RNA, synthetically (e.g., chemically) produced RNA, e.g., siRNA, antisense nucleic acid, aptamers, etc. Isolated nucleic acid molecules include sequences inserted into a plasmid, cosmid, or other vector to form part of a chimeric recombinant nucleic acid construct, or produced by expression of a nucleic acid encoding it. Thus, in certain embodiments, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids associated within the cell, or both, or may be associated with a cell membrane if it is a membrane-bound protein, or may be produced synthetically (e.g., chemically), or may be produced by expression of a nucleic acid encoding it. An isolated cell (such as an ILC2 cell or MSC) may be removed from the anatomical site in which it is found in an organism or may be produced by in vitro expansion of an isolated cell or cell population. An isolated material may, but need not, be purified.

[0122] Purified refers to a protein, nucleic acid, or cell or cell population, and refers to the separation of a desired material from contaminants to a sufficient extent to allow the practitioner to use the purified material for a desired purpose.Preferably, this means that at least one order of purification of the starting material or natural material is achieved, more preferably two or three orders of purification, and most preferably four or five orders of purification.In certain embodiments, the purified RET agonist, RET antagonist, ADRB2 agonist, ADRB2 antagonist, or combination thereof may be at least 60%, at least 80%, or at least 90% by weight of the total protein or nucleic acid or cell population, as the case may be.In certain embodiments, the purified RET agonist, RET antagonist, ADRB2 agonist, ADRB2 antagonist, or combination thereof is purified to homogeneity when assayed by standard relevant laboratory protocols.

[0123] In some embodiments, the agonist (e.g., RET agonist, ADRB2 agonist), antagonist (e.g., RET antagonist, ADRB2 antagonist) or combination thereof is administered to a subject in a pharmaceutical composition. In some embodiments, the pharmaceutical composition is sterile. The pharmaceutical composition of the present invention comprises an effective amount of one or more agents dissolved or dispersed in a pharma- ceutically acceptable carrier. Pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not produce adverse allergic or other untoward reactions when administered to an animal, such as a human, as appropriate. Furthermore, it will be understood that for animal (e.g., human) administration, the preparation should meet the standards of sterility, pyrogenicity, general safety and purity required by the relevant government regulatory agency. The compound is generally suitable for administration to humans. This term requires that the compound or composition is non-toxic and sufficiently pure such that no further manipulation of the compound or composition is required prior to administration to humans.

[0124] Pharmaceutically acceptable carriers, as known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences (1990), incorporated herein by reference), include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and similar materials and combinations thereof. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0125] example Example 1: Neural-mesenchymal signals regulate GAT ILC2 cells Analysis of gonadal adipose tissue (GAT) of mice revealed the presence of a dense network of sympathetic neuronal fibers (Figures 1A, 5A). To examine whether adrenergic cues affect local ILC2 cells (also called "ILC2" and "ILC2s"), we ablated dopaminergic and noradrenergic neurons using 6-hydroxydopamine (6-OHDA). Systemic ablation of these neurons resulted in a marked reduction in ILC2-derived interleukin (IL-)5, IL-13 and Met-Enk (Figures 1B, 5B) and no change in the percentage of CD4 T cells (Figure 5C). Subsequently, ROSA26.DTR (diphtheria toxin receptor) mice were bred to tyrosine hydroxylase-Cre (Th-Cre, R26 / DTR Th ) mice, and subsequently pegylated diphtheria toxin (PegDTR) was obtained. Th By administering this drug to animals, peripheral sympathetic neurons were selectively ablated. 14Selective ablation of peripheral sympathetic neurons resulted in impaired ILC2 activity (Figure 1C). In contrast, systemic activation of β2-adrenergic receptors (ADRB2) with clenbuterol resulted in an increase in ILC2-derived cytokines in GAT (Figures 1D, 5D). Consistently, mice with designer receptors exclusively activated by designer drugs (DREADD) on sympathetic neurons (R26 / 3D Th Chemogenetic activation of neurons in adipose tissue led to increased ILC2 function (Figure 1E). To investigate how sympathetic tone regulates adipose ILC2s, we transduced Il7ra-Cre mice with Adrb2 fl / fl Mouse (Adrb2 ΔIl7ra ) to delete Adrb2 in lymphoid cells. Adipose ILC2 function is determined by Adrb2 ΔIl7ra In mice, this was not perturbed (Figure 1F), suggesting that sympathetic cues indirectly regulate adipose ILC2s. Supporting this hypothesis, Adrb2 ΔIl7ra Chemical sympathetic ablation in mice still impaired ILC2 cytokine production (Figure 1G). To elucidate the cellular link between sympathetic neuronal cues and ILC2 activity, the expression of Adrb2 in non-immune adipose-resident cell types was investigated. Mesenchymal stromal cells (MSCs) expressing platelet-derived growth factor receptor alpha (PDGFRA) showed the highest levels of Adrb2, followed by glial cells, endothelial cells, adipocytes and other mesenchymal counterparts (Figure 1H). Interestingly, glial cells and MSCs are in close proximity to GAT sympathetic neurons (Figure 1I, 1J). Thus, we investigated whether Gfap-Cre or Pdgfra-Cre mice upregulate Adrb2. fl / fl Expression of ADRB2 was disrupted in glial cells and MSCs by crossing into mice (Adrb2 ΔGfap and Adrb2 ΔPdgfra ) Mice with glia-autonomous deletion of ADRB2 had unperturbed ILC2 function, but Adrb2 ΔPdgfraMice showed reduced adipose ILC2-derived cytokines when compared to their littermate controls (Figures 1K, 1L). Collectively, these data indicate that neural-mesenchymal signals regulate GAT ILC2s.

[0126] Example 2: Neural-mesenchymal interactions regulate adipose ILC2s via the neurotrophin receptor RET To investigate how neural-mesenchymal interactions lead to activation of GAT-resident ILC2s, genome-wide transcriptional profiling of PDGFRA-positive MSCs from 6-OHDA-treated mice was compared with their littermate controls. This analysis revealed 227 regulated transcripts, of which 35 were downregulated (Figures 2A, 6A). Of note, expression of the ILC2-activating alarmins IL-33 and IL-25 was not perturbed in mice treated with either 6-OHDA or clenbuterol (Figures 2A, 6A–6E). Among the altered genes, glial-derived neurotrophic factor (GDNF) was highly expressed by PDGFRA-positive MSCs, and 6-OHDA treatment significantly reduced its expression (Figure 2A). Importantly, upon chemical sympathetic ablation or ADRB2 stimulation, Gdnf was regulated in GAT and PDGFRA + was selectively regulated in MSCs but not in their other mesenchymal counterparts, adipocytes and endothelial cells (Figures 2B-2E). ΔPdgfra GAT and MSCs purified from IL-1 had reduced Gdnf expression (Figures 2F, 2G). Consistent with these findings, stimulation of ADRB2 in purified MSCs increased MSC-derived GDNF and PDGFRA, which colocalized with GDNF. + Resulted in an increase in GAT cells 15,16 (Figures 2H, 2I). GDNF family ligands and their preferred co-receptors (GFRa) have been shown to activate the reconstituted tyrosine kinase receptor (RET) during transfection in a subset of nervous, renal and hematopoietic cells. 16~20Analysis of GAT immune cell subsets revealed that GAT ILC2s express high levels of Ret (Figure 2J). To investigate the role of the kinase receptor RET in adipose ILC2s, we first transduced Vav1-Cre into Ret fl / fl Mouse (Ret ΔVav1 ) to delete Ret in hematopoietic cells. ΔVav1 Mice showed reduced ILC2-derived IL-5 and IL-13 in the GAT (Figure 2K). Consistent with this finding, analysis of RET coreceptor single knockouts revealed that the preferential GDNF coreceptor, Gfra1, was selectively required for ILC2 function (Figures 7A-7C). Subsequent analysis of Ret-competent (Ret fl ) or Ret deficiency (Ret ΔVav1 ) We utilized mixed bone marrow (BM) chimeras by transferring BM into lymphoid hosts (Figure 7D). Analysis of these chimeric mice suggested that cell-intrinsic RET signaling is required for innate type 2 cytokines in GAT (Figure 7E). To further confirm the ILC2-autonomous effect of RET, we transformed Il5-Cre mice into Ret fl / fl Mouse (Ret ΔIl5 ), followed by mating with Ret ΔIl5 Rag1 mice - / - mice to rule out putative T helper cell effects. - / - Ret ΔIl5 Analysis of mice confirmed that RET operates in an ILC2-autonomous manner to regulate innate type 2 cytokines and Met-Enk in GAT (Figure 2L, Figure 7F, Figure 7I). To further evaluate ILC2 activity in mice, we used Ret WT Mouse and Ret ΔIl5 Mouse and Rag1 - / - , Il2rg - / - Mixed bone marrow (BM) chimeras of Ret knockout mice were generated (Figure 7H). ΔIl5 Rag1 - / - Mouse, Il2rg - / - The mouse is Ret WTRag1 - / - Mouse, Il2rg - / - In contrast, ILC2 activity was reduced in mice compared to controls (Figure 7I). Consistent with this, in vitro activation of purified ILC2s with GDNF family ligands led to increased innate cytokine production and expression of gain-of-function Ret MEN2B Analysis of bone marrow chimeras revealed increased ILC2-derived IL-5, IL-13 and Met-Enk (Figures 2M-2O, 7J-7L). ΔIl5 Chemical sympathetic ablation in mice resulted in unperturbed ILC2 cytokine production, indicating that ILC2-autonomous RET signaling is required to consolidate sympathetic tone (Figure 8A). Collectively, these data indicate that neural-mesenchymal interactions consolidate adipose ILC2s via the neurotrophin receptor RET.

[0127] Example 3: ILC2-endogenous neurotrophic factor cues are required to control fat homeostasis and obesity To determine whether ILC2-specific RET signaling regulates adipose tissue physiology, various degrees of RET signaling were tested in the setting of obesity proneness and associated glucose tolerance dysfunction. 21 First, Ret ΔVav1 Mice were fed a high-fat diet (HFD). When compared to their littermate controls, Ret ΔVav1 and Ret. ΔIl5 ILC2-chimeras had increased susceptibility to HFD-induced obesity, decreased glucose tolerance and increased GAT weight (Figures 3A-3C, 8B, 8C). To more specifically define the link between ILC2, GDNF-RET signaling and obesity propensity, RET-sufficient and RET-deficient ILC2-chimeras were generated in aerobic host mice. RET-deficient ILC2-chimeras had increased susceptibility to HFD-induced obesity, decreased glucose tolerance and altered frequency of adipocyte size (Figures 3D-3H). In contrast, gain-of-function Ret MEN2BChimeras generated with ILC2s from mice showed resistance to HFD-induced obesity, improved glucose tolerance and increased frequency of small adipocytes (Figures 3I-3M). Type 2 cytokines and Met-Enk have been shown to promote energy expenditure via adipose tissue. 9、11、12、22 To define the contribution of ILC2s and neuromodulatory cues in this process, expression of uncoupling protein 1 (UCP1) was assessed in RET loss- and gain-of-function models. ΔVav1 Mouse and Ret ΔIl5 Mice have reduced Ucp1, Cox8b and Cidea expression in GAT, but not Ret MEN2B BM chimeras showed increased Ucp1 levels (Figures 3N, 3O, and 8D). Further evidence that ILC2-autonomous GDNF-RET cues are required for Ucp1 expression was provided by the expression of Rag1 - / - Il2rg - / - The ILC2 complementation of mouse-derived GAT explant cultures was provided by the ILC2 (Figure 3P, Figure 8E). Notably, addition of GDNF to GAT explant / ILC2 cocultures efficiently induced Ucp1 expression in a RET-dependent manner (Figure 3Q, Figure 8E). Collectively, these data indicate that ILC2 endogenous neurotrophic factor cues are required to control fat homeostasis and obesity.

[0128] Example 4: A novel adrenal adipose circuit connects distinct brain regions and regulates GAT ILC2 function To investigate how local sympathetic fibers integrate local and higher-order circuits, we characterized the connectivity of GAT neurons. First, we performed viral tracing (VT) by injecting retrograde green fluorescent protein (GFP)-labeled adeno-associated virus (AAV) into the GAT. Analysis of such mice revealed infection of tyrosine hydroxylase (TH)-positive neurons in adipose tissue, as well as sympathetic TH-positive fibers in the genitofemoral nerve, which runs longitudinally ventral to the psoas muscle (Figures 4A-4D). Importantly, distinct neuronal cell bodies were traced to the prevertebral aortorenal ganglion (ARG) and dorsal root ganglion (DRG) (Figures 4E, 4F, 9A). Nevertheless, GFP-labeled cell bodies in the aortorenal ganglion were dopaminergic, whereas their DRG counterparts were TH-negative, indicating that the renal ganglion harbors cell bodies of efferent sympathetic innervation of the GAT (Figures 4E, 4F, 9A). To investigate whether the GAT-aortorenal axis connects to higher-order circuits, polysynaptic tracing was performed using fluorescent protein-producing pseudorabies virus (PRV). Retrograde tracing with PRV from the GAT or aortorenal ganglion revealed polysynaptic connections to overlapping distinct brain regions in the brainstem, midbrain, amygdala and hypothalamus (Figures 4G, 4H, 9B, 9C). Notably, the hypothalamic paraventricular nucleus (PVH) was consistently traced from the GAT and aortorenal ganglion, indicating that the GAT connects polysynaptically to this hypothalamic nucleus (Figures 4G, 4H). Interestingly, the PVH has previously been reported to regulate brain sympathetic outflow to peripheral somatic tissues, 23、24 , which is consistent with the adipose congenital type 2 cytokine defect observed in mice with surgical stereotactic PVH ablation (Figures 9D, 9E).

[0129] To further analyze the influence of aortorenal neural circuits on GAT ILC2s, unilateral surgical ablation was performed on the genitofemoral nerve (GFx). Compared to sham contralateral controls, GFx GAT harbored ILC2s with impaired function associated with reduced GDNF expression (Figure 4I, 4J). To better clarify the link between GAT innervating sympathetic aortorenal neurons and ILC2 function, the activity of these neurons was modulated using a chemical genetic approach. Thus, GAT was unilaterally injected with inhibitor or activator DREADD-carrying adeno-associated viruses (AAVs) (AAV(4D) and AAV(3D) respectively). Subsequently, a designer drug (clozapine-N-oxide (CNO)) was injected, which results in neuronal inhibition or stimulation of DREADD-bearing neurons. When compared to the respective sham contralateral controls, inhibition of AAV(4D)-expressing neurons resulted in a decrease in GDNF and innate type 2 cytokines, whereas activation of AAV(3D)-bearing neurons resulted in an increase in GDNF and ILC2 function (Figure 4K-4N). In contrast, inhibition of AAV(4D)-expressing neurons and activation of AAV(3)D-expressing neurons did not affect interleukin-33 (Il33) expression (Figure 9F, 9G). Together, these data reveal a novel aortic-renal-adipose circuit that connects distinct brain regions and controls GAT ILC2 function.

[0130] Example 5: Discussion Defining whether neural circuits and immune cells work together to drive inter-organ communication is critical for understanding organismal physiology and systemic disease. This study establishes an unrecognized inter-organ and multi-tissue communication circuit that integrates neural and mesenchymal derived signals to orchestrate ILC2 function and obesity. There exists a brain-body axis that transmits sympathetic nerves to the aorta-kidney-adipose interface that regulates ILC2. In particular, the neural-mesenchymal unit converts sympathetic tone into neurotrophin expression in GAT. In turn, neurotrophins control adipose ILC2s through the neuromodulatory receptor RET to shape host metabolism, energy expenditure and obesity (Figure 10).

[0131] Adipose mesenchymal cells were shown to regulate local immune cell homeostasis through expression of IL-33 25-27 Here, we demonstrate that mesenchymal cells link neural cues to adipose ILC2 function through production of GDNF. Although sympathetic neural cues have been shown to directly inhibit pulmonary ILC2s during infection, 28 , adrenergic signals indirectly activate GAT ILC2s, indicating that sympathetic signals may encompass dual mechanisms by which ILC2s are activated or inhibited in a context- and organ-dependent manner.

[0132] Neurosympathetic cue signals directly mediate lipolysis in the context of neuroadipose connections 7 This study indicates that sympathetic cues indirectly regulate energy expenditure through neuro-mesenchymal interactions that result in ILC2-derived cytokine production. Thus, combining these direct and indirect sympathetic effects to regulate energy homeostasis may ensure an efficient and integrated multi-tissue response to dietary challenge. The importance of brain PVH in integrating systemic metabolic cues 24 , sympathetic outflow 23、24 Considering the brain PVH in regulating the aortorenal adipose circuit (Figures 4, 10), we are led to hypothesize that the PVH acts as a central hub translating metabolic body status into peripheral immune functions that ensure energy homeostasis. Finally, this data may also give a better knowledge of how abnormal neuronal and immune functions are associated with obesity and metabolic disorders in humans. 9、29、30 .

[0133] Example 6: Materials and Methods and References material and method Mice: C57BL / 6J mice were purchased from Charles River and crossed with C57BL / 6J Ly5.1 to obtain C57BL / 6 Ly5.1 / Ly5.2 (CD45.1 / CD45.2). 31 , Pdgfra-Cre 32 , Vav1-Cre33 , Il7ra-Cre 34 , Il5-Cre 35 , Th-Cre 36 , Adrb2 fl / fl37 , Rag1 - / -38、 Il2rg - / -39 , Ret. MEN2B40 , ROSA26.RFP 41 , ROSA26.3D 42 , ROSA26.DTR 43 , Ret. fl / fl44 , Gfra1 - / -45 , Gfra2 - / -46 , and Gfra3 - / -47 The mice were on a full C57BL / 6J background. Mice were bred and maintained under specific pathogen-free conditions at the Champalimaud Centre for the Unknown (CCU) animal facility. Mice were systematically compared to co-housed littermate controls unless otherwise stated. Females aged 8–9 weeks were used in this study. A power analysis was performed to estimate the number of experimental mice. All animal experiments were approved by the national and institutional ethical committees, respectively, the Direcao Geral de Veterinaria and the ethical committee of the CCU. Randomization and blinding were not used unless otherwise stated.

[0134] Cell isolation: For adipose tissue cell isolation, tissue was collected in PBS, cut into small pieces, and incubated with Liberase™ (2.5 μg / ml, Roche) and DNase I (20 U / ml; Roche) for 1 h at 37°C with gentle agitation. Single cell suspensions were obtained by passing through a 100 μm cell strainer (Thermo Fisher Scientific) and centrifugation was used to separate the stromal vascular fraction from the adipocyte fraction. Red blood cells were lysed with red blood cell lysis buffer (eBioscience) and removed by centrifugation.

[0135] Flow cytometry and cell sorting: For ex vivo cytokine analysis, cells were incubated with PMA (50 ng / ml), ionomycin (500 ng / ml) (Sigma) and brefeldin A (eBioscience) in complete RPMI (supplemented with 10% fetal bovine serum (FBS), 1% HEPES, sodium pyruvate, glutamine, streptomycin and penicillin (Corning)) for 4 h prior to intracellular staining, unless otherwise stated. Intracellular staining was performed using the IC fixation / permeabilization kit (eBioscience). Cell suspensions were purified with 100% IgG antibodies (anti-CD45 (30-F11; 1:200); anti-CD45.1 (A20; 1:200); anti-CD45.2 (104; 1:200); anti-CD11c (N418; 1:200); anti-CD11b (Mi / 70; 1:400); anti-CD8α (53-6.7; 1:200); anti-CD19 (eBio1D3; 1:200); anti-NK1.1 (PK136; 1:100); anti-CD3ε (eBio500A2; 1:200); anti-TER119 (TER-119; 1:200); anti-Gr1 (RB6-8C5; 1:400) from eBiosciences. );Anti-CD4(RM4-5;1:200);Anti-CD90.2(Thy1.2;53-2.1;1:200);Anti-TCRβ(H57-595;1:200);Anti-TCRγδ(GL3;1:200);Anti-B220(RA3-6B2;1:200);Anti-KLRG1(2F1 / KL RG1;1:200);antiLy-6A / E(Sca1;D7;1:200);anti-CD16 / CD32(93;1:50);anti-gp38(eBio8.1.1;1:100);anti-F4 / 80(BM8;1:200);anti-IL-4(11B11;1:100). Anti-PDGFRA (APA 5; 1:400) and anti-CD31 (MEC 13.3; 1:200) from Biolegend. Anti-IL-5 (TRFK5; 1:200) from BD Biosciences. Anti-GDNF (B-8; 1:200) was purchased from Santa Cruz biotechnology. Anti-Met-Enk (bs-1759 R-A680; 1:400) from Bioss.LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (1:50) and anti-IL-13 (eBio13A; 1:200) were purchased from Invitrogen. Cell populations were gated on live cells and ILC2:CD45. + Lin - Thy1.2 + Sca-1 + KLRG1 + The lineages were defined as CD3ε, CD8α, TCRβ, TCRγδ, CD19, Gr1, CD11c, CD11b and TER119; glial cells: CD45-CD31-GFAP+, MSCs: CD45-CD31-PDGFRA+gp38+, endothelial cells: CD45-CD31+. Flow cytometry analysis and cell sorting were performed using FACSFusion, LSRFortessa and LSRFortessa X-20 (BD Biosciences). Sorted populations were >95% pure. Data analysis was performed using FlowJo v10 software (Tristar).

[0136] Sympathetic manipulation: Chemical sympathetic ablation was performed by intraperitoneal injection of 200 mg / kg 6-OHDA (Sigma) 3 days and 1 day before analysis. Control mice were injected on the same days with PBS 0.4% ascorbic acid (Sigma), which was used as a vehicle for 6-OHDA. PEGylated diphtheria toxin was injected into the R26 / DTR mice as previously described. Th Sympathetic nerve ablation was also performed in mice by administering 14 For activation of ADRB2, its agonist clenbuterol (Sigma) was administered in drinking water containing 4% sucrose to a final concentration of 10 mg / kg / day for 8 days. Control animals received water containing 4% sucrose for 8 days. R26 / 3 D Th For chemogenetic activation of sympathetic neurons in mice, 4 mg / kg CNO (Sigma), 2 mg / kg in drinking water containing 4% sucrose, and 2 mg / kg in PBS were administered intraperitoneally. 48 .

[0137] Quantitative RT-PCR: Total RNA from sorted or cultured cells was extracted using RNeasy micro kit or RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Where indicated, total adipose tissue or adipocyte fractions were collected in Trizol (Invitrogen), followed by chloroform and isopropanol RNA extraction according to the manufacturer's protocol. RNA concentrations were determined using a Nanodrop Spectrophotometer (Nanodrop Technologies). Quantitative real-time PCR was performed on a StepOne and QuantStudio 5 real-time PCR system (Applied Biosystems) using Hprt and Gapdh as housekeeping genes. Briefly, RNA was reverse transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems), followed by preamplification PCR using TaqMan PreAmp Master Mix (Applied Biosystems). TaqMan Gene Expression Master Mix (Applied Biosystems) was used for real-time PCR. TaqMan gene expression assays (Applied Biosystems) were as follows: Hprt Mm00446968_m1; Gapdh Mm99999915_g1; Il5 Mm00439646_m1; Il13 Mm00434204_m1; Areg Mm01354339_m1; Penk Mm01212875_m1; Ret Mm00436304_m1; Gdnf Mm00599849_m1; Ucp1 Mm01244861_m1; Adrb2 Mm02524224_s1; Il25 Mm00499822_m1; and Il33 Mm00505403_m1. Analysis was performed using the comparative CT method (2-ΔCT). If comparison between samples or fold changes are required, the comparative ΔCT method (2 -ΔΔCT ) was applied.

[0138] RNA Sequencing and Data Analysis: PDGFRA+MSCs from mice treated with 6-OHDA or vehicle were isolated and RNA was extracted and purified as previously described. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Sequencing was performed on a HiSeq4000 platform (PE100, Illumina). FastQC (version 0.11.9) was used to analyze the overall quality of FASTQ files with raw RNA sequencing reads. 49 The first 10 base pairs (HEADCROP=10) were removed using Trimmomatic-0.39. 50 Alignment and read processing were performed using STAR 2.7.3a (https: / / github.com / alexdobin / STAR / releases) 51 The sequence was aligned to the reference file of the Mus musculus genome assembly GRCm38.p6 and the corresponding genome annotation file (http: / / www.ensembl.org / Mus_musculus / Info / Index). Volcano plots of differentially expressed genes were obtained using EdgeR (version 3.30.3) (https: / / bioconductor.org / packages / release / bioc / html / edgeR.html). 52 Statistics for all genes were obtained using DeSeq2 (version 1.28.1) (https: / / bioconductor.org / packages / release / bioc / html / DESeq2.html). 53Genes with an average read less than 10, a false discovery rate (FDR) greater than 0.05, and a log2 (difference between groups) greater than -2 were excluded from further analysis. In this way, a list of differentially expressed genes was obtained, including 227 upregulated genes and 35 downregulated genes in the 6-OHDA group. A heatmap of regulated genes was obtained by plotting z-scores (normalized read counts per gene) using GraphPad Prism software (GraphPad Software, La Jolla, CA).

[0139] In vitro and in vivo MSC activation: For in vitro experiments, purified GAT PDGFRA + MSCs were cultured in complete DMEM supplemented with 10% FBS, 1% HEPES, sodium pyruvate, glutamine, streptomycin and penicillin (Corning) at 37°C. After 2 h in complete DMEM without FBS, MSCs were stimulated with 10 μg / ml clenbuterol for 16 h. For RNA analysis, MSCs were lysed using RLT buffer (Qiagen). For GDNF protein analysis, MSCs were incubated with brefeldin A (eBioscience) during the period of clenbuterol stimulation prior to intracellular staining.

[0140] In vitro and in vivo ILC2 activation: For in vitro experiments, purified GAT ILC2s were cultured in complete RPMI at 37°C. After 2 h in RPMI without FBS, ILC2s were stimulated with 50 ng / ml GDNF family ligand (R&D Systems) for 3 h. For RNA analysis, ILC2s were lysed using RLT buffer (Qiagen). For ex vivo cytokine protein analysis, stimulated ILC2s were incubated with PMA (50 ng / mL), ionomycin (500 ng / mL) (Sigma) and brefeldin A (eBioscience) for 4 h, followed by intracellular staining.

[0141] Bone marrow and fetal liver chimeras: Rag1 - / - .Ret MEN2B and Ret. DVav1 Bone marrow cells extracted from femurs and tibias of mice and their respective littermate controls. Fetal livers were cultured at E13.0 Gfra1 - / - Bone marrow and fetal liver cells were obtained from mice and their respective littermate controls. Bone marrow and fetal liver cells were CD3-depleted using Dynabeads Biotin Binder (Invitrogen) according to the manufacturer's instructions. 106 cells of each genotype (CD45.2) were incubated with non-lethally irradiated (3 Gy) Rag1 cells alone or in direct competition with a third party WT competitor (CD45.1 / CD45.2) at a 1:1 ratio. - / - Il2rg - / - Mice (CD45.1) were injected intravenously with IgG4A and IgG5A. Mice were analyzed 10 to 12 weeks after transplantation.

[0142] High-fat diet: Unless otherwise stated, animals were placed on a HFD (60Kj% fat (lard) E15742-3407, Ssniff GmbH) for 16 weeks. Glucose tolerance tests were performed 14 weeks after the start of HFD administration. Mice were fasted for 8 hours and administered glucose (Sigma) in PBS at 2 mg / kg, and glucose was measured using an ACCU-CHECK Aviva glucometer (Roche).

[0143] ILC2 adoptive transfer:Ret MEN2B ,Ret D ILC2s from mice and their respective littermate controls were purified from visceral adipose tissue for adoptive transfer. Purified ILC2s were expanded in vitro in supplemented RPMI in the presence of recombinant mouse IL-2, IL-7 (10 ng / mL; Peprotech) and IL-33 (10 ng / mL; R&D Systems) for 8 days. 2 × 10 5 ILC2 to Rag1 - / - Il2rg - / - Recipients were injected intraperitoneally. Two weeks after adoptive transfer, mice were placed on a HFD.

[0144] Explant culture: GAT to Rag1- / - Il2rg - / - The cells were then cut into 2 mm pieces and incubated in complete RPMI at 37° C. for 4 hours. WT Mouse or Ret Δ Approximately 10 minutes from the mouse 4 Isolated GAT ILC2s were co-cultured with GAT explants and 50 ng / mL of GDNF family ligands for 16 h. Explants were harvested in Trizol (Invitrogen) and disrupted by sonication for RNA analysis.

[0145] Virus administration: Virus tracing experiments were performed using a Hamilton® syringe (Hamilton) by injecting 10 μl of pseudorabies virus (PRV)-614 (PRV-Bartha containing a CMV-mRFP reporter gene cassette inserted into the gG locus of the viral genome) or pAAV-Ef1a-mCherry-IRES-Cre (Addgene viral prep#55632-AAVrg) into the gonadal fat pad. Adrenal cortex renal ganglion injections of 1 μl (PRV)-614-RFP were performed using a Nanoject III Programmable Nanoliter Injector (Drummond Scientific). Six days after PRV injection, brains were collected after perfusion with PBS and 4% PFA for fixation and further processing. For AAV tracing, GAT and aortorenal ganglion were collected 3 weeks after injection and fixed in 4% PFA for further processing. For neuronal function manipulation, rAAV-PGA-hM3DqDREADD-GFP (AAV(3D)) or rAAV-PGA-hM4DqDREADD-GFP (AAV(4D)) (Addgene) was injected into one gonadal fat pad, and the contralateral fat pad was injected with PBS, which served as the contralateral control. 4 mg / kg CNO was administered as above. For local neuronal activation, CNO was administered 4 weeks after injection, and for local neuronal inactivation, CNO was administered 6 weeks after injection.

[0146] PVH electroablation: Bilateral ablation of the PVH was performed by electrical injury in 9- to 12-week-old C57BL / 6J mice using stereotactic surgery as previously described. 54 Mice were kept under deep anesthesia using a mixture of isoflurane and oxygen (1–3% isoflurane at 1 l / min). Surgery was performed using a stereotaxic apparatus (Kopf). After identification of the bregma, a hole was made for the insertion of the lesion electrode into the brain. The electrode was made by isolating a 0.25 mm stainless steel insect pin with heat-shrinkable polyester tubing, except for 0.5 mm at the tip. The electrode tip was aimed at the paraventricular hypothalamic nucleus, −0.35 mm anterior to the bregma, 0.25 mm lateral to the midline, and 5.8 mm ventral to the skull top (Paxinos Mouse Brain Atlas, Franklin 2001). Bilateral lesions were made by passing a current of 0.75 mA through the electrodes for 3 s, separately on the left and right. Sham-lesioned mice underwent the same procedure, but no current was passed through the electrodes. After surgery, animals were allowed free access to food and water and allowed to recover for 1 week. Successfully lesioned mice were selected based on histopathological analysis. Mice were analyzed after 10-12 weeks.

[0147] Immunofluorescence and microscopy: Brains from animals injected with PRV-614-RFP were cut into 50 μm slices using a microtome and mounted in Mowiol (Sigma). Brain images and H&E stained adipose tissue images were obtained with a Zeiss AxioScan Z1 slide scanner (20x Plan Apochromat dry 0.80 0.55 objective). R26 / RFP DGfap , R26 / RFP DPdgfraGonadal adipose tissue was obtained from 100% gonadal fat pads and C57BL / 6J mice. For ultramicroscopic imaging, whole gonadal fat pads were collected, and for confocal imaging, sections of approximately 1 × 1 mm were obtained from tissue fixed overnight at 4 °C with 4% PFA. Samples were blocked and permeabilized with PBS containing 0.6% Triton X-100 (Sigma) and 2% BSA (Sigma) and incubated for 1–2 days at room temperature with the following antibodies: anti-TH (P40101; Pel-Freez); anti-GDNF (B-8; Santa-Cruz); or CD31 (390, FITC; Abcam). Alexa Fluor 568 goat anti-rabbit and Alexa Fluor 488 goat anti-rabbit (Invitrogen) were used as secondary antibodies overnight at room temperature. Removal of the gonadal tissue began with dehydration in a series of increasing concentrations of ethanol solutions (20%, 40%, 60%, 80% and 100%) for 24 h each, followed by immersion in ethyl cinnamate and staining. 55 For cleared whole tissue imaging, samples were mounted in ethyl cinnamate and acquired with a LightSheet Zeiss Z.1 (Plan Apochromat 20x / 1.0, 2.4 objective). For confocal imaging, samples were mounted in Mowiol and acquired with a Zeiss LSM 710 confocal microscope using a Pl-Apochromat 25x / 0.8 M27 immersion objective and a Pl-Apochromat 63x / 1.4 oil immersion objective. For confirmation of viral tracing infection, a Leica M205 stereomicroscope coupled to a Leica DFC 7000 T camera (Leica Microsystems, Wetzlar, Germany) was used. Images were processed using ImageJ 1.53 (NIH), Zen Blue 3.0 (Zeiss) and Imaris 9.6 (Oxford Instruments).

[0148] Statistics: Results are presented as mean ± sem. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, La Jolla, Calif). Student's t test was performed for homoscedastic populations. Welch's corrected t test was applied to samples with different variances unless otherwise stated. Paired t test was applied to contralateral control samples. Results were considered significant at P < 0.05.

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[0150] Equivalent Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure relate to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, kits, and / or methods is encompassed within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0151] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0152] The indefinite articles "a" and "an," as used herein and in the claims, unless clearly indicated otherwise, should be understood to mean "at least one."

[0153] The term "and / or" as used herein and in the claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), in yet another embodiment to both A and B (optionally including other elements), etc.

[0154] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be judged to be inclusive, i.e., including at least one of, but also including more than one, and optionally including additional unlisted items, of a number or list of elements. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein, when used in the claims, when preceded by terms of exclusivity, such as "either," "one of," "only one of," "exactly one of," "consisting essentially of," etc., shall be judged only as indicating exclusive alternatives (i.e., "one or the other but not both") and shall have its ordinary meaning as used in the field of patent law.

[0155] As used herein and in the claims, the phrase "at least one" in reference to a list of one or more elements means at least one element selected from any one or more of the elements in the list of elements, but it should be understood that it does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally inclusive of more than one, A, with no B present (and optionally including elements other than B); in another embodiment to at least one, optionally inclusive of more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment to at least one, optionally inclusive of more than one, A, and at least one, optionally inclusive of more than one, B (optionally including other elements), etc.

[0156] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0157] In the claims and the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean inclusive but not exclusive. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures Section 2111.03.

Claims

1. 1. A composition comprising a rearranged during transfection (RET) agonist and / or a β2-adrenergic receptor (ADRB2) agonist for use in a method for increasing the activity or proliferation of group 2 innate lymphoid cells (ILC2s), the method comprising contacting ILC2s with the rearranged during transfection (RET) agonist and / or contacting mesenchymal stromal cells (MSCs) with the β2-adrenergic receptor (ADRB2) agonist.

2. RET agonists, (1) a combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand (GFL) or an analog or mimetic thereof; or (2) An antibody or an antigen-binding fragment thereof that specifically binds to RET and increases RET tyrosine kinase activity The composition of claim 1 comprising:

3. The combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand or an analog or mimetic thereof, (1) (a) a combination of soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) a combination of soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) a combination of soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) a combination of soluble GFRα4 and persephin (P SPN) or an analog or mimetic thereof; (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to and dimerizes GFRα; or (2) A combination of two or more of (a), (b), (c), (d), (e), (f), and (g). The composition of claim 2 comprising:

4. 2. The composition of claim 1, wherein the ADRB2 agonist is clenbuterol, bitolterol, fenoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, albuterol, terbutaline, aformoterol, bambuterol, formoterol, salmeterol, abesiterol, carmoterol, indacaterol, olodaterol, valanterol, isoxsuprine, mabuterol, zilpaterol, or a combination thereof.

5. The composition of claim 1 , wherein the contacting is in vitro.

6. The composition of claim 1 , wherein the contacting is in vivo.

7. The composition of claim 1 , wherein the method comprises administering to the subject a RET agonist and / or an ADRB2 agonist.

8. The composition of claim 7 , wherein the subject is a human.

9. The composition of claim 1, wherein the ILC2s and / or MSCs are in or derived from adipose tissue.

10. 10. The composition of claim 9, wherein the adipose tissue is gonadal adipose tissue (GAT).

11. A composition comprising a rearranged during transfection (RET) agonist and / or a β2-adrenergic receptor (ADRB2) agonist for use in a method for increasing production of interleukin-5 (IL-5), interleukin-13 (IL-13), and / or Met-enkephalin (Met-Enk) by group 2 innate lymphoid cells (ILC2), the method comprising contacting adipose ILC2s with the rearranged during transfection (RET) agonist and / or contacting mesenchymal stromal cells (MSCs) with the β2-adrenergic receptor (ADRB2) agonist.

12. RET agonists, (1) a combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand (GFL) or an analog or mimetic thereof; or (2) An antibody or an antigen-binding fragment thereof that specifically binds to RET and increases RET tyrosine kinase activity The composition of claim 11 comprising:

13. The combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand or an analog or mimetic thereof, (1) (a) a combination of soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) a combination of soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) a combination of soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) a combination of soluble GFRα4 and persephin (P SPN) or an analog or mimetic thereof; (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to and dimerizes GFRα; or (2) A combination of two or more of (a), (b), (c), (d), (e), (f), and (g). The composition of claim 12 comprising:

14. 12. The composition of claim 11, wherein the ADRB2 agonist is clenbuterol, bitolterol, fenoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, albuterol, terbutaline, aformoterol, bambuterol, formoterol, salmeterol, abesiterol, carmoterol, indacaterol, olodaterol, valanterol, isoxsuprine, mabuterol, zilpaterol, or a combination thereof.

15. The composition of claim 11 , wherein the contacting is in vitro.

16. The composition of claim 11 , wherein the contacting is in vivo.

17. The composition of claim 11 , wherein a RET agonist and / or an ADRB2 agonist is administered to the subject.

18. 18. The composition of claim 17, wherein the subject is a human.

19. The composition of claim 11, wherein the ILC2s and / or MSCs are in or derived from adipose tissue.

20. 20. The composition of claim 19, wherein the adipose tissue is gonadal adipose tissue (GAT).

21. 1. A composition comprising a rearranged during transfection (RET) agonist in contact with group 2 innate lymphoid cells (ILC2s) in adipose tissue, a beta-2 adrenergic receptor (ADRB2) agonist in contact with mesenchymal stromal cells (MSCs) in adipose tissue, or a combination thereof, for use in a method for reducing susceptibility to obesity and / or increasing fat homeostasis, wherein said method (a) administering to a subject a reconstituted during transfection (RET) agonist that contacts group 2 innate lymphoid cells (ILC2s) in adipose tissue; (b) administering to the subject a beta-2 adrenergic receptor (ADRB2) agonist that contacts mesenchymal stromal cells (MSCs) in the adipose tissue; or (c) Combinations thereof The composition comprising:

22. 22. The composition of claim 21, wherein the increased fat homeostasis is increased glucose tolerance and / or decreased gonadal adipose tissue (GAT) fat mass.

23. RET agonists, (1) a combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand (GFL) or an analog or mimetic thereof; or (2) An antibody or an antigen-binding fragment thereof that specifically binds to RET and increases RET tyrosine kinase activity 22. The composition of claim 21, comprising:

24. The combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand or an analog or mimetic thereof, (1) (a) a combination of soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) a combination of soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) a combination of soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) a combination of soluble GFRα4 and persephin (P SPN) or an analog or mimetic thereof; (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to and dimerizes GFRα; or (2) A combination of two or more of (a), (b), (c), (d), (e), (f), and (g).

24. The composition of claim 23, comprising:

25. 22. The composition of claim 21, wherein the ADRB2 agonist is clenbuterol, bitolterol, fenoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, albuterol, terbutaline, aformoterol, bambuterol, formoterol, salmeterol, abesiterol, carmoterol, indacaterol, olodaterol, valanterol, isoxsuprine, mabuterol, zilpaterol, or a combination thereof.

26. 22. The composition of claim 21, wherein the subject is a human.

27. 1. A composition comprising a rearranged during transfection (RET) agonist contacting ILC2s in adipose tissue, a beta2 adrenergic receptor (ADRB2) agonist contacting mesenchymal stromal cells (MSCs) in adipose tissue, or a combination thereof, for use in a method for treating a disorder associated with decreased activity or proliferation of Group 2 innate lymphoid cells (ILC2s), the composition comprising: (a) administering to a subject a reconstituted during transfection (RET) agonist that contacts ILC2s in adipose tissue; (b) administering to the subject a beta-2 adrenergic receptor (ADRB2) agonist that contacts mesenchymal stromal cells (MSCs) in the adipose tissue; or (c) Combinations thereof The composition comprising:

28. 28. The composition of claim 27, wherein the disorder is weight gain, obesity, diabetes, metabolic syndrome, or a combination thereof.

29. RET agonists, (1) a combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand (GFL) or an analog or mimetic thereof; or (2) An antibody or an antigen-binding fragment thereof that specifically binds to RET and increases RET tyrosine kinase activity 28. The composition of claim 27, comprising:

30. The combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand or an analog or mimetic thereof, (1) (a) a combination of soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) a combination of soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) a combination of soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) a combination of soluble GFRα4 and persephin (P SPN) or an analog or mimetic thereof; (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to and dimerizes GFRα; or (2) A combination of two or more of (a), (b), (c), (d), (e), (f), and (g).

30. The composition of claim 29, comprising:

31. 28. The composition of claim 27, wherein the ADRB2 agonist is clenbuterol, bitolterol, fenoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, albuterol, terbutaline, aformoterol, bambuterol, formoterol, salmeterol, abesiterol, carmoterol, indacaterol, olodaterol, valanterol, isoxsuprine, mabuterol, zilpaterol, or a combination thereof.

32. 28. The composition of claim 27, wherein the subject is a human.

33. 1. A composition comprising a rearranged during transfection (RET) agonist in contact with ILC2s in adipose tissue, a beta2 adrenergic receptor (ADRB2) agonist in contact with mesenchymal stromal cells (MSCs) in adipose tissue, or a combination thereof, for use in a method of treating a disorder associated with increased activity or proliferation of Group 2 innate lymphoid cells (ILC2s), the composition comprising: (a) administering to a subject a reconstituted during transfection (RET) antagonist that contacts ILC2s in adipose tissue; (b) administering to the subject a beta-2 adrenergic receptor (ADRB2) antagonist that contacts mesenchymal stromal cells (MSCs) in the adipose tissue; or (c) A combination of (a) and (b) A composition comprising:

34. 34. The composition of claim 33, wherein the disorder is hypothermia, cachexia, allergy, helminth infection, allergic asthma, atopic dermatitis, bowel inflammatory disease, or a combination thereof.

35. The composition of claim 33, wherein the RET antagonist is (1) an antibody that specifically binds to and inhibits (a) RET tyrosine kinase activity, (b) GDNF family binding receptor alpha (GFRα), or (c) a GFRα ligand, or an antigen-binding fragment thereof; (2) an inhibitory nucleic acid molecule that reduces the expression, transcription or translation of RET, GFRα, or a GFRα ligand; or (3) a RET tyrosine kinase inhibitor, optionally AST487, motesanib, cabozantinib, vandetanib, ponatinib, sunitinib, sorafenib, or alectinib.

36. The composition of claim 35, wherein the GFRα is GFRα1, GFRα2, GFRα3 or GFRα4; or the GFRα ligand is glial cell line-derived neurotrophic factor (GDNF), neurturin (NTRN), artemin (ARTN) or persephin (PSPN).

37. 36. The composition of claim 35, wherein the inhibitory nucleic acid molecule is an sRNA, shRNA, or antisense nucleic acid molecule.

38. 34. The composition of claim 33, wherein the ADRB2 antagonist is butoxamine, ICI-118,551, propranolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, or a combination thereof.

39. 34. The composition of claim 33, wherein the subject is a human.

40. 1. A composition comprising a rearranged during transfection (RET) agonist contacting ILC2s in adipose tissue, a β2-adrenergic receptor (ADRB2) agonist contacting ILC2s, or a combination thereof, for use in a method of treating cold exposure, the composition comprising: (a) administering to a subject a reconstituted during transfection (RET) agonist in contact with Group 2 innate lymphoid cells (ILC2s); (b) administering to the subject a beta-2 adrenergic receptor (ADRB2) agonist that contacts ILC2s; or (c) Combinations thereof The composition comprising:

41. RET agonists, (1) a combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand (GFL) or an analog or mimetic thereof; or (2) An antibody or an antigen-binding fragment thereof that specifically binds to RET and increases RET tyrosine kinase activity 41. The composition of claim 40, comprising:

42. The combination of a soluble GDNF family binding receptor alpha (GFRα) and a GFRα ligand or an analog or mimetic thereof, (1) (a) a combination of soluble GDNF family binding receptor alpha 1 (GFRα1) and glial cell line-derived neurotrophic factor (GDNF) or an analog or mimetic thereof; (b) a combination of soluble GFRα2 and neurturin (NTRN) or an analog or mimetic thereof; (c) a combination of soluble GFRα3 and artemin (ARTN) or an analog or mimetic thereof; (d) a combination of soluble GFRα4 and persephin (P SPN) or an analog or mimetic thereof; (e) soluble GFRα and N(4)-(7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl)-N(1),N(1)-diethyl-pentane-1,4-diamine (XIB4035); (f) soluble GFRα and a BT compound; (g) soluble GFRα and an antibody that specifically binds to and dimerizes GFRα; or (2) A combination of two or more of (a), (b), (c), (d), (e), (f), and (g).

42. The composition of claim 41, comprising:

43. 41. The composition of claim 40, wherein the ADRB2 agonist is clenbuterol, bitolterol, fenoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, procaterol, ritodrine, albuterol, terbutaline, aformoterol, bambuterol, formoterol, salmeterol, abesiterol, carmoterol, indacaterol, olodaterol, valanterol, isoxsuprine, mabuterol, zilpaterol, or a combination thereof.

44. 41. The composition of claim 40, wherein the subject is a human.

45. 41. The composition of claim 40, wherein administering the RET agonist and / or the ADRB2 agonist increases the body temperature of the subject.

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