Methods and compositions for modulating glucagon secretion

Abstract

The present invention relates to methods and compositions for regulating the migration and activity of type 2 innate lymphoid cells (ILC2) in order to modulate pancreatic glucagon secretion. The activation of sympathetic neuronal pathways innervating the intestine induces a reduction of ILC2 in the small intestine and promotes their rapid accumulation in pancreatic tissue. In the pancreas, the ILC2 secrete IL-5 and IL-13, which act directly on pancreatic α-cells expressing IL-5Rα and IL-13Rα to increase glucagon secretion.

Description

METHODS AND COMPOSITIONS FOR MODULATING GLUCAGON SECRETION

[0001] The present application claims the benefit of priority to the Portuguese provisional patent application no. 119868, filed on November 29, 2024, the contents of which is incorporated herein by reference.

[0002] The present invention relates to the field of metabolic regulation and immunometabolic interactions, specifically to methods and compositions for modulating glucagon secretion through neuronal pathways and type 2 innate lymphoid cells (ILC2).

[0003] Glucose homeostasis relies on the coordinated secretion of pancreatic hormones that regulate systemic energy availability. During periods of nutrient deprivation, the counter-regulatory hormone glucagon, produced by pancreatic α-cells, plays a central physiological role in maintaining euglycemia by stimulating hepatic glucose production.

[0004] Defects in glucagon secretion are strongly implicated in a range of metabolic disorders, including type 2 diabetes, insulin resistance, hypoglycemia unawareness, and non-alcoholic fatty liver disease (NAFLD). Despite its importance, the upstream mechanisms controlling glucagon output remain incompletely understood, particularly those that integrate signals originating outside the pancreas.

[0005] Immune cell signals contribute to the regulation of metabolism and have been implicated in metabolic diseases. Neuronal and immune cell interactions contribute to host physiology and defense. Nevertheless, whether neuronal and immune cells establish inter-organ communication axes to orchestrate endocrine function remains unexplored.

[0006] Type 2 innate lymphoid cells (ILC2) are tissue-resident cells enriched at barrier surfaces, including the intestinal mucosa. They are known to respond rapidly to environmental cues and to secrete type 2 cytokines such as IL-5 and IL-13, which shape both immune and metabolic processes.

[0007] In the present invention, it is disclosed methods and compositions for modulating glucagon secretion through neuronal pathways and type 2 innate lymphoid cells (ILC2), particularly focusing on the cells’ migration to the pancreas and their influence on glucagon levels during fasting.

[0008] Although various publications and patent documents have explored the roles of ILC2 in adipose tissue biology, allergic inflammation, and tissue-resident immune regulation, none of these disclosures teach or suggest the mechanisms uncovered in the present invention.

[0009] WO2023178169A2 discloses a sympathetic-to-mesenchymal stromal cell (MSC) axis in adipose tissue, where β2-adrenergic signals induce MSC-derived neurotrophic factors that act on local adipose-resident ILC2 to regulate energy expenditure and obesity. This document, however, does not describe ILC2 migration between organs, involvement of the intestine or pancreas, nor any regulation of pancreatic endocrine function. The prior art is limited to adipose-tissue homeostasis, while the present invention reveals an entirely distinct neuro-immune-endocrine pathway controlling glucagon biology and glucose homeostasis.

[0010] Patent application US20240270855A1 describes a sympathetic-driven neuro-mesenchymal axis operating within adipose tissue, in which β2-adrenergic signaling activates adipose mesenchymal stromal cells to release neurotrophic factors that act on local adipose-resident ILC2s to regulate energy expenditure, insulin sensitivity and obesity. In contrast, the present invention concerns a completely different biological axis: a gut-pancreas neuronal-ILC2 circuit in which fasting-induced sympathetic activity drives migration of intestinal ILC2s to the pancreas, where ILC2-derived IL-5 and IL-13 directly stimulate glucagon secretion by pancreatic α-cells. Thus, unlike the prior art, the invention uniquely identifies inter-organ ILC2 trafficking, pancreatic endocrine regulation, and a neuro-immune mechanism controlling glucagon biology.

[0011] The article “Interactions between adipose tissue and the immune system in health and disease” (Wensveen et al., 2016) reviews how adipose tissue is not just a fat-storage organ but also an immunologically active site: it describes interactions between adipocytes, resident macrophages, T-cells, type 2 innate lymphoid cells (ILC2), and the sympathetic nervous system within the context of metabolic homeostasis and inflammation. The key differences with the present invention lie in the following: while the review is confined to local adipose tissue interactions and focuses on ILC2 activity within that tissue for energy homeostasis and inflammation, the present invention describes a distinct inter-organ migration of ILC2 from the intestine to the pancreas, driven by sympathetic neuronal signaling, where the ILC2 then directly regulate pancreatic α-cells’ glucagon secretion via IL-5 / IL-13.

[0012] In the review article “Aging and Immunometabolic Adaptations to Thermogenesis” (Lettieri-Barbato and Aquilano, 2020), the authors examine the diverse roles of innate lymphoid cells (ILCs) across different tissue environments, highlighting how ILCs are largely tissue-resident, acquire organ-specific phenotypes, and engage in local interactions with stromal, epithelial, neuronal and vascular cells to regulate immunity, tissue repair, metabolism and homeostasis. However, this document does not present a mechanism in which ILC2 migrate between distinct organs (such as intestine to pancreas) in response to metabolic cues, nor does it describe ILC2-derived cytokines acting directly on pancreatic α-cells to regulate glucagon secretion.

[0013] Article “Glucagon-like peptide 1 signaling inhibits allergen-induced lung IL-33 release and reduces group 2 innate lymphoid cell cytokine productionin vivo” (Toki et al., 2018) reviews the biology of innate lymphoid cells (ILCs) in allergic and inflammatory diseases, focusing on their development, classification, tissue-residency, activation by alarmins such as IL-33 / IL-25 / TSLP, and roles in conditions like asthma, chronic rhinosinusitis, and atopic dermatitis. It emphasizes ILC2 involvement in type 2 immunity via cytokines IL-5 and IL-13, their interactions with other immune cells, and potential therapeutic strategies targeting ILC2 in allergic inflammation. The key differences compared to the present invention are that this prior art addresses ILC2’s role in local tissue-resident immune responses (especially mucosal / airway barriers) rather than migration between organs. Also, it neither describes a gut-pancreas axis or any role in endocrine / hormonal regulation not it implicates sympathetic neuronal signaling and inter-organ ILC2 relocation as a mechanism to modulate α-cell endocrine output.

[0014] As discussed above, the prior art is limited to local, tissue-restricted ILC2 activity and does not describe inter-organ migration of ILC2, nor their recruitment to the pancreas in response to metabolic cues. Likewise, the cited neuro-immune pathways relate only to adipose stromal circuits and do not extend to pancreatic endocrine regulation.

[0015] Crucially, none of the prior art documents herein identify or imply a sympathetic neuron-driven gut-to-pancreas axis in which ILC2-derived IL-5 and IL-13 directly stimulate pancreatic α-cells to modulate glucagon secretion. Accordingly, the prior art neither anticipates nor renders obvious the unique neuro-immune-endocrine mechanism and therapeutic strategies described herein.

[0016] The invention discloses, in a first aspect, a non-therapeutic method for inducing accumulation of type 2 innate lymphoid cells (ILC2) in pancreatic tissue, comprising the steps of (i) activating a sympathetic neuronal pathway that innervates the intestine; (ii) reducing the number of ILC2 in the small intestine; and (iii) promoting migration of ILC2 expressing ADRB2 from the small intestine to the pancreas.

[0017] In a second aspect, the present invention disclosed a non-therapeutic method for modulating glucagon secretion from pancreatic α-cells, comprising contacting pancreatic α-cells that express IL-5Rα and IL-13Rα with IL-5 and / or IL-13 secreted by ILC2 that have migrated from the intestinal tissue.

[0018] Fasting induces a sympathetic neuronal response that triggers migration of intestinal ILC2 to the pancreas. ILC2 produce IL-5 and IL-13, which directly stimulate α-cells to increase glucagon output. The therapeutic modulation of this axis can be achieved by intervening at multiple points, including adrenergic signaling, cytokine signaling, lymphocyte trafficking, and neuronal activation.

[0019] Thus, another aspect of the present invention covers the agents that promote, inhibit, or redirect ILC2 migration from the intestine to the pancreas and the therapeutic uses thereof.

[0020] Glucagon regulation is essential for maintaining glucose homeostasis. While insulin lowers blood glucose by promoting uptake and storage, glucagon acts as the essential counterbalance, raising blood glucose when levels fall, particularly during fasting, exercise, or hypoglycemia.

[0021] When blood glucose drops, glucagon triggers the liver to release glucose through glycogen breakdown and gluconeogenesis, preventing dangerous hypoglycemia. If glucagon is insufficient or dysregulated, the body cannot properly defend against falling glucose, leading to impaired fasting tolerance, hypoglycemia unawareness, and broad metabolic instability.

[0022] Conversely, excessive glucagon contributes to hyperglycemia and insulin resistance. Thus, precise glucagon control is fundamental to stable metabolic homeostasis, ensuring continuous energy supply to vital organs across changing nutritional states.

[0023] Thus, the technical problem to be solved by the present invention is how to therapeutically modulate glucagon secretion by controlling extra-pancreatic cellular and neuronal pathways that regulate pancreatic α-cell function.

[0024] The present invention is based on the surprising discovery that group 2 innate lymphoid cells (ILC2) control glucose homeostasis via the regulation of the hormone glucagon.

[0025] Fasting activates a sympathetic neuronal circuit that triggers migration of intestinal ILC2 to the pancreas, where ILC2 induce glucagon secretion via IL-5 and IL-13. Retrograde tracing, chemical, genetic and chemogenetic manipulations showed that intestinal sympathetic neurons control gut-pancreatic ILC2 migration and connect to high-order brain areas.

[0026] Thus, by modulating neuronal signals, ILC2 behavior, or their cytokines, glucagon secretion can be modulated.

[0027] The present invention provides a previously unrecognized mechanism for maintaining glucose homeostasis by establishing a functional gut-pancreas communication axis that links sympathetic neuronal activity to the migration and endocrine function of type 2 innate lymphoid cells (ILC2).

[0028] Upon fasting or reduced energy availability, sympathetic neurons in the small intestine initiate a coordinated biological program in ILC2, triggering their mobilization from the gut and promoting their accumulation within pancreatic tissue. This targeted repositioning of ILC2 enables a rapid and physiologically appropriate increase in glucagon secretion by pancreatic α-cells, thereby supporting glycogenolysis and gluconeogenesis.

[0029] This neuroimmune mechanism offers a precise and dynamic means of modulating endogenous glucose production, representing a significant advantage over existing approaches that primarily target insulin pathways.

[0030] A further advantage of the invention lies in the discovery that pancreatic ILC2 directly stimulate α-cells to produce glucagon through the release of type 2 cytokines, in particular interleukin-5 (IL-5) and interleukin-13 (IL-13). Pancreatic α-cells uniquely express the corresponding cytokine receptors IL-5Rα and IL-13Rα1, enabling them to respond directly to ILC2-derived signals.

[0031] Also, experimental results demonstrate that activation of the ILC2-glucagon axis markedly improves pathological features of non-alcoholic fatty liver disease (NAFLD), reducing hepatic steatosis and normalizing triglyceride accumulation under high-fat / high-sugar dietary conditions. These technical effects confirm that the invention not only provides mechanistic insights into inter-organ metabolic regulation but also offers a promising strategy for correcting metabolic dysfunctions that are poorly addressed by current insulin-centric interventions.

[0032] With the purpose of promoting an understanding of the principles in accordance with the embodiments of the present invention, reference will be made to the embodiments illustrated in the figures and to the language used to describe the same.

[0033] It must be understood that there is no intention of limiting the scope of the present invention to the contents of the figures. Any alterations or later changes of the inventive features illustrated herein, as well as any additional application of the principles and embodiments of the invention shown, which would occur normally for one skilled in the art when reading this description, are considered as being within the scope of the claimed invention.Fig.1

[0034]

[0035] presents experimental data comparing metabolic parameters in different mouse models under fasting conditions. Individual panels illustrate:Afasting plasma glucose after a 6-hour fast,Bhepatic glycogen following a 6-hour fast,Cpyruvate tolerance test after a 6-hour fast,Dfasting plasma glucose after a 16-hour fast,Epyruvate tolerance test after a 16-hour fast,Fcirculating triglyceride levels,Ghepatic expression of gluconeogenic genes after a 16-hour fast,Hpancreatic insulin transcript levels,Icirculating insulin levels,Jinsulin tolerance test and corresponding glucagon measurements,Kglucose tolerance test and its area-under-curve analysis,Lpancreatic glucagon (Gcg) transcript levels,Mcirculating glucagon levels andNfasting plasma glucose obtained from an independent experiment.

[0036]

[0037] shows additional metabolic measurements under varied fasting intervals and includes results from antibiotic-treated animals. Data cover, respectively,Afasting plasma glucose after a 6-hour fast (ZT0),Bhepatic glycogen after a 6-hour fast,Cpyruvate tolerance test AUC (6-hour fast),Dpyruvate tolerance test AUC (16-hour fast),Ecirculating glucagon in antibiotic-treated and control mice,Fpyruvate tolerance test and AUC in antibiotic-treated and control mice,Gblood glucose levels,Hglucose infusion rate in clamp,Iglucose levels during clamping,Jinsulin-suppressed endogenous glucose production,Kglucose uptake,Lglucagon tolerance test.

[0038]

[0039] displays analyses of immune cell populations in the pancreas, including:Aflow-cytometric analysis of pancreatic ILC subsets,Bquantification of pancreatic ILC subsets,Ccytokine-producing ILC2,Dtranscriptional analysis of pancreatic ILC2,Echimera generation scheme,Fcirculating glucagon,Gpyruvate tolerance test,Hfasting plasma glucose,Ihepatic glycogen,Jcirculating triglycerides,Kblood glucagon in vehicle controls,LPTT in vehicle controls,Mfasting plasma glucose in vehicle controls,Nfasting glucose in Nmur1-Cre.R26-DTR mice,Ocirculating glucagon in Nmur1-Cre.R26-DTR mice,Ppyruvate tolerance test in Nmur1-Cre.R26-DTR mice,Qglucagon secretion in alpha-cell cultures,Rstimulated glucagon secretion,Sglucagon secretion under different conditions,Tglucagon secretion in WT and Stat6- / - alpha cells,Uglucagon and Gcg transcript levels after cytokine neutralization.

[0040]

[0041] provides the flow cytometry gating strategy used to identify pancreatic immune cell populations.

[0042]

[0043] includes additional analyses of pancreatic ILC2 populations under fasting or refeeding conditions, includingAfasting-induced ILC2 changes (flow cytometry),BGATA3+ and RORγt+ cell numbers,Cpancreatic ILC2 numbers,Dpancreatic ILC2 numbers (independent),Epancreatic immune cells,Fpancreatic ILC2 at ZT03 vs ZT15,Gpancreatic ILC2 after fasting-refeeding cycles,Hblood glucose in cycles,Iblood glucagon in cycles,Jblood glucose in cycles,Kblood glucagon in cycles,Lcytokine-producing ILC2,Mheatmap of differentially expressed genes,Nheatmap of ILC2 identity genes,OILC2 subset percentages,PIL-17RB expression in ILC2.

[0044]

[0045] presents data from adoptive transfer and chimera experiments, showingAILC2 repopulation after adoptive transfer,Bpancreatic Gcg transcripts,Cpancreatic glucagon protein,DGcg transcripts in WT vs RAG-deficient ILC2 chimeras,Eblood glucagon in chimeras,Fpyruvate tolerance test and AUC,Gfasting glucose (16h),Hhepatic glycogen (16h),Itriglycerides (16h),JPTT AUC summary,Kpancreatic Gcg after PF,LPTT AUC after PF,MPTT AUC in Nmur1-Cre.R26-DTR mice.

[0046]

[0047] illustrates the gating and characterization of pancreatic α-cells, includingAalpha-cell gating and hormone gene expression,Bpurity of alpha-cell sorting,Cheatmap of alpha- and beta-cell expression,Dreceptor expression in alpha cells,EIl13ra1 and Il5ra transcripts,Fhuman islet receptor expression (scRNA-seq).

[0048]

[0049] provides data onAepithelial cell gating,BIL-5RA expression on alpha cells,Cglucagon secretion under multiple stimuli,Dlive alpha-cell counts,Eglucagon secretion with STAT3 / 6 inhibition,Fco-culture glucagon secretion with ILC2,Gglucagon secretion from whole islets,Hcytokine production by ILC2 and CD4 T cells,IIl5 and Il13 transcripts,JIl5-reporter+ cells,KIl13-reporter+ cells,Lglucagon secretion in cultured alpha cells.

[0050]

[0051] showsAimmunostaining of islets,BRNA in situ hybridization (Gcg and Il5-tdTomato),CKi67+ ILC2,DBrdU+ ILC2,EILC2 numbers across tissues,Fphotoconversion and KikRed+ ILC2,Gsingle-cell profiling of KikGreen+ and KikRed+ cells,Henteric and pancreatic ILC2 distribution,Iquantification of photoconverted ILC2,Jpancreatic ILC2 after FTY720, K KikRed+ ILC2 in mLN, L pancreatic ILC2 after mLN ablation.

[0052]

[0053] includes imaging, flow cytometry, and phenotypic analyses of ILC2 populations in different tissues under fasting conditions, whereinAislet immunostaining,BRNA in situ hybridization of Gcg,Cpancreatic ILC2 flow plots,Dliver ILC2 flow plots,Eliver ILC2 percentage,Fcytokine-producing ILC2,Gliver ILC2 phenotype.

[0054]

[0055] shows photoconversion-based tracking of ILC2 across multiple organs several days after labeling, including quantification of labeled cell distribution, whereinAphotoconversion flow plots,BKikGreen+ and KikRed+ ILC2 across organs,CKikRed+ ILC2 quantification.

[0056]

[0057] presents single-cell transcriptional analysis of ILC2 populations and representative flow cytometry plots from blood samples, whereinAis a ILC2 gene-expression heatmap andBare blood flow-cytometry plots.

[0058]

[0059] shows experimental schemes and results involving neuromodulation, includingAchemogenic activation scheme,Bpancreatic ILC2 after activation,Ccirculating glucagon after activation,Dpyruvate tolerance test,Eblood glucose,Fpolysynaptic tracing images,Gpancreatic ILC2 after systemic 6-OHDA,Hglucagon after systemic 6-OHDA,Ipancreatic ILC2 after intestinal 6-OHDA,Jglucagon after intestinal 6-OHDA,KPTT after intestinal 6-OHDA,Lfasting glucose after intestinal 6-OHDA,Mpancreatic ILC2 in Th-Cre model,Nglucagon in Th-Cre model,Opancreatic ILC2 in Adrb2 mutants,PILC2 distribution in Adrb2 mutants,Qglucagon in Adrb2 mutants,Rpyruvate tolerance in Adrb2 mutants,Sfasting glucose in Adrb2 mutants,Tpancreatic ILC2 in Rag1- / - Adrb2 mutants,Uglucagon in Rag1- / - Adrb2 mutants,Vfasting glucose in Rag1- / - Adrb2 mutants,WILC2 after clenbuterol,Xgenetic and chemogenic scheme,Ypancreatic ILC2 in Rag1- / - Adrb2 mutants after activation,Zglucagon and glucose in Rag1- / - Adrb2 mutants.

[0060]

[0061] includesAnorepinephrine after chemogenic activation,Bpolysynaptic retrograde tracing,Cduodenal norepinephrine after fasting,Dserum epinephrine and norepinephrine.

[0062]

[0063] provides liver histological images and pathology scoring in animals subjected to dietary models of fatty liver disease, whereinAexperimental scheme for diet-induced fatty liver,Bliver histology,Cpathology and ORO score,Dliver histology,Epathology and ORO score,Fliver histology,Gpathology and ORO score with IL-33, ILC2 and PF.

[0064]

[0065] presents additional analyses of adrenergic signaling, includingAnorepinephrine after local 6-OHDA,Bimmunostaining of Th and AAV2(3D),CILC2 after clenbuterol.

[0066]

[0067] includesAILC2 in NCD or HFD / HSD with IL-33,Bcirculating glucagon in NCD or HFD / HSD with IL-33,CILC2 under IL-33 and PF,Dserum glucagon under IL-33 and PF,Eliver histology,Fpathology score.

[0068]

[0069] provides a schematic representation of a proposed physiological pathway involving neuronal signals, ILC2 migration, and glucagon production.

[0070] The invention will now be described by reference to illustrative embodiments. The embodiments below are provided to support and exemplify the subject-matter of the claims and should not be interpreted as limiting the scope of protection. Unless otherwise specified, references to ILC2 refer to type 2 innate lymphoid cells naturally present within mammalian intestinal tissues.

[0071] The present invention discloses methods and compositions for modulating glucagon secretion through neuronal pathways and ILC2.

[0072] In one embodiment, the invention provides a non-therapeutic method for inducing accumulation of ILC2 in pancreatic tissue, comprising the steps of:

[0073] (i) activating a sympathetic neuronal pathway that innervates the intestine;

[0074] (ii) reducing the number of ILC2 in the small intestine; and

[0075] (iii) promoting migration of ILC2 expressing ADRB2 from the small intestine to the pancreas.

[0076] The activation of the sympathetic pathway initiates a cascade of neuroimmune interactions that alter the spatial distribution of ILC2 within the organism. Intestinal sympathetic neurons release catecholaminergic mediators that are sensed by ILC2 residing in the small intestine, which express adrenergic receptors including ADRB2. The ILC2 reduce their residency within the intestinal lamina propria and begin migrating away from the gut toward pancreatic tissue. This redistribution can be observed by a decrease in ILC2 numbers in the small intestine accompanied by an increase in ILC2 presence within the pancreas.

[0077] In one embodiment of the invention, the activation of the sympathetic pathway occurs naturally during fasting. Fasting is known to enhance sympathetic outflow to the gastrointestinal tract, and the present invention demonstrates that such enhancement serves as an endogenous trigger for ILC2 migration. Under fasting conditions, the number of ILC2 in the intestinal lamina propria decreases, and ILC2 relocate to pancreatic tissue where they become detectable in close proximity to islets of Langerhans. This fasting-induced response represents a physiologically relevant and naturally occurring embodiment of the invention, providing an example of a non-therapeutic condition capable of initiating neuroimmune-driven ILC2 migration.

[0078] In another embodiment of the invention, the activation of the sympathetic pathway is achieved exogenously using adrenergic agonists. In preferred embodiments of the present invention, the adrenergic agonists are selected from the group consisting of clenbuterol, salbutamol, terbutaline, isoproterenol, salmeterol, formoterol and mixtures thereof. In a more preferred embodiment, the adrenergic agonist is clenbuterol.

[0079] Since ILC2 express adrenergic receptors such as ADRB2, the administration of an adrenergic agonist leads to their activation, reduction in numbers within the intestine, and subsequent trafficking toward the pancreas. The use of an adrenergic agonist is particularly advantageous for experimental settings where controlled induction of ILC2 migration is desired. In these embodiments, the adrenergic agonist is administered at doses sufficient to stimulate sympathetic activity in intestinal neurons without imposing a therapeutic intent on the organism.

[0080] In further embodiments, the migration of ILC2 from the intestine to the pancreas is facilitated by modulation of sphingosine-1-phosphate (S1P)-mediated retention pathways. ILC2 also express S1P receptors, including S1PR1, which ordinarily maintain the cells within the intestinal mucosa by responding to S1P gradients. When S1P receptor signaling is inhibited, the retention signals that prevent ILC2 egress are reduced, and the cells more readily migrate out of the gut.

[0081] When ILC2 that express both ADRB2 and S1PR1 are exposed simultaneously to adrenergic stimulation and S1P inhibition, their migration from the small intestine to the pancreas becomes more pronounced and occurs on a shortened timescale.

[0082] In some embodiments, the ILC2 that migrate to the pancreas express marker proteins characteristic of activated ILC2, including GATA3 and KLRG1. These markers identify ILC2 with the transcriptional and functional properties required for responding to neuronal activation and relocating to distal tissues.

[0083] The migrating ILC2 may also express IL-5Rα and IL-13Rα, which are associated with type 2 cytokine production. In particular embodiments, the accumulation of ILC2 in the pancreas occurs within 2 to 12 hours following neuronal activation, illustrating the rapid responsiveness of these cells to neuronal signals. This temporal window is supported by observations showing measurable enrichment of ILC2 in the pancreas within this timeframe following sympathetic stimulation.

[0084] In another embodiment, the present invention provides a non-therapeutic method for modulating glucagon secretion from pancreatic α-cells. This modulation is achieved through the interaction of the pancreatic α-cells with IL-5 and / or IL-13 produced by the ILC2 that have migrated from the intestine tissue.

[0085] Pancreatic α-cells express IL-5Rα and IL-13Rα, and when exposed to their respective ligands, these α-cells upregulate glucagon secretion. Because migrated ILC2 in the pancreas naturally secretes IL-5 and IL-13, their presence in the pancreatic microenvironment provides an endogenous mechanism for increasing glucagon output. In some embodiments, ILC2 are activated by IL-33 prior to secreting IL-5 and IL-13, since IL-33 stimulation enhances type 2 cytokine production and further strengthens the effect on α-cell glucagon secretion.

[0086] In embodiments involving co-secretion, IL-5 and IL-13 are released together by ILC2 and act synergistically to stimulate glucagon expression and secretion. In such embodiments, the combined effect of IL-5 and IL-13 is greater than the effect induced by either cytokine alone.

[0087] In certain embodiments, the pancreatic α-cells contacted by IL-5 and IL-13 are derived from fasted animals, reflecting a physiological condition under which the neuroimmune regulatory pathway described herein is naturally engaged. This embodiment demonstrates that the invention is compatible with bothinvivoandex vivocontexts involving isolated pancreatic α-cells.

[0088] In further embodiments, adrenergic agonists are provided for use in modulating glucagon secretion in subjects experiencing hypoglycemia unawareness.

[0089] Additional embodiments provide IL-13 for use in modulating glucagon secretion in subjects with reduced fasting tolerance. IL-13 directly stimulates pancreatic α-cells through IL-13Rα, resulting in increased glucagon secretion. Such stimulation enhances the metabolic adaptation to fasting by restoring appropriate glucagon output.

[0090] In some embodiments, IL-13 is used in subjects affected by non-alcoholic fatty liver disease (NAFLD), where glucagon-induced hepatic lipid metabolism is impaired. In further embodiments, IL-13 is administered in combination with IL-5 to enhance the glucagon-modulatory effects of both cytokines.

[0091] Although the invention primarily describes non-therapeutic mechanistic pathways, when presented as a substance for a specific therapeutic use, adrenergic agonists and interleukins may be utilized to augment glucagon secretion in subjects where restoring counter-regulatory responses is medically advantageous. In related embodiments, adrenergic agonists are particularly beneficial for subjects exhibiting impaired counter-regulatory glucagon responses, as improving glucagon responses enhances metabolic safety.

[0092] The present invention also provides a population of ILC2 cells expressing ADRB2, S1PR1, GATA3, KLRG1, and IL-Rα, and exhibiting the capacity to migrate from the intestine to the pancreas following activation of intestinal sympathetic neurons.

[0093] These ILC2 populations represent a specialized subset primed for adrenergic responsiveness and capable of regulating pancreatic hormone secretion. In certain embodiments, the ILC2 further express ST2, the receptor for IL-33, enabling greater activation and cytokine production upon IL-33 stimulation. In additional embodiments, the ILC2 populations produce both IL-5 and IL-13 following adrenergic stimulation, thereby providing the cytokine signals necessary for enhancing glucagon secretion once they have accumulated in pancreatic tissue.

[0094] Overall, the present invention underscores the significant interplay between the immune system and metabolic regulation, particularly through ILC2 pathways and fasting-induced physiological responses.Examples

[0095] Hereinafter, reference is made to the examples of the present invention, which aim to describe it in more details as regards tests that have been conducted and experimental results that have been achieved.

[0096] Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as commonly understood by someone skilled in the art to which this invention belongs.

[0097] Methods and materials are described in this document for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and are not intended to be limiting.

[0098] Innatelymphoidcellsregulateglucosehomeostasis

[0099] To test whether lymphocytes impact glucose homeostasis, the fasting plasma glucose levels between wild-type mice (C57BL / 6j),Rag1- / -animals, which lack adaptive lymphocytes, andRag2- / -.Il2rg- / -mice that lack adaptive and innate lymphocytes were compared.

[0100] AlymphoidRag2- / -.Il2rg- / -mice selectively displayed reduced fasting plasma glucose levels upon short-term fasting, and impaired mobilization of hepatic glycogen, which is a dominant glucose source obtained via glycogenolysis upon short-term fasting (A-C;A-C).

[0101] During longer fasting periods, glucose homeostasis largely depends on the hepatic transformation of non-carbohydrate substrates into glucose, i.e., gluconeogenesis. After long-term fasting,Rag2- / -.Il2rg- / -mice displayed reduced fasting plasma glucose, which associated with impaired gluconeogenesis, as measured by pyruvate tolerance test (PTT), and increased triglyceride blood levels (D-F andD).

[0102] SinceRag2- / -.Il2rg- / -mice may have different intestinal bacterial communities that could impact glucose homeostasis, antibiotic treatedRag2- / -.Il2rg- / -mice were compared to their untreated littermates upon fasting. Importantly, antibiotic treatedRag2- / -.Il2rg- / -mice and their littermate controls displayed equally reduced FPG, glucagon and gluconeogenic activity when compared to C567BL / 6j andRag1- / -animals (E-G).

[0103] Gluconeogenesis occurs in hepatocytes via gluconeogenic enzymes in response to fasting and pancreatic hormones. Analysis of fastedRag2- / -.Il2rg- / -mice revealed that the expression of their hepatic gluconeogenic genes was unperturbed (G), suggesting that innate lymphocytes regulate pancreatic endocrine function.

[0104] Plasma glucose levels depend on the balanced secretion of glucose-depositing and -mobilizing pancreatic hormones, respectively insulin and glucagon. The latter promotes hepatic glycogenolysis and gluconeogenesis in response to hypoglycemia. When compared to wild-type (WT) andRag1- / -mice,Rag2- / -.Il2rg- / -mice displayed normal levels of the insulin genesIns1andIns2, unperturbed concentration of blood insulin, unperturbed insulin sensitivity despite reduced glucagon levels, and normal glucose sensitivity as measured by glucose tolerance test (GTT) (H-K).

[0105] In agreement, hyperinsulinemic-euglycemic clamp experiments confirmed thatRag2- / -.Il2rg- / -animals are not insulin resistant (H-K). Notably, in this rather extreme condition,Rag2- / -.Il2rg- / -mice showed unperturbed endogenous glucose production, while displaying increased glucose infusion rate, confirming thatRag2- / -.Il2rg- / -mice are not insulin resistant (H-K).

[0106] Strikingly, and in contrast to their normal insulin levels,Rag2- / -.Il2rg- / -mice showed a marked reduction of preproglucagon gene (Gcg) expression and low levels of the glucose-mobilizing hormone glucagon (L,M). Importantly, administration of exogenous glucagon increased glycemia inRag2- / -.Il2rg- / -mice (N), which was in line with the finding thatRag2- / -.Il2rg- / -mice are not glucagon resistant as measured by glucagon tolerance test (L). Taken together, these results indicate that innate lymphoid cells (ILC) regulate glucose homeostasis by promoting glucose-mobilizing processes.

[0107] ILC2controlthepancreatichormoneglucagon

[0108] To gain insight on the ILC subsets that regulate pancreatic endocrine function, the impact of fasting on pancreas resident ILC was studied. NK cells and ILC2 were the most abundant innate pancreas resident ILC in steady state (A,B and). When compared to their control counterparts fedadlibitum, fasted animals selectively displayed increased percentage and numbers of pancreatic type 2 ILC (ILC2), while group 1 (NK cells and ILC1) and group 3 ILC were unperturbed (A,B andA-K). In agreement, while the frequency of interleukin (IL)-5 and IL-13 producing ILC2 was stable upon fasting, the total number of pancreatic cytokine- producing ILC2 was increased (C andL). Next, a genome-wide transcriptional profiling was employed to define whether fasting impacts pancreatic ILC2 more broadly. Analysis of pancreatic ILC2 before and after fasting revealed that their genetic signatures are similar (D andM,N). Notably, analysis of genes associated with ILC2 identity and function confirmed that these genes were unperturbed upon fasting (M-P). Together, these data indicate that fasting promotes selective increase of pancreatic ILC2 that associate with increased glucagon levels.

[0109] To establish the causal link between fasting, ILC2 and pancreatic glucagon, ILC2 chimeras were generated by transplantingRagcompetent or deficient ILC2 intoRag2- / -.Il2rg- / -alymphoid mice, thus creating intestinal and pancreatic ILC2 pools (E andA-J). In contrast toRag2- / -.Il2rg- / -controls, ILC2 chimeras efficiently increased the expression of the preproglucagon geneGcgand blood glucagon levels upon fasting (F andB,E). In agreement, transplantation of ILC2 efficiently restored gluconeogenesis and fasting plasma glucose levels, which associated with normal hepatic glycogen and triglyceride levels (G-J andF-J). To confirm that glucagon is the link between ILC2 and fasting plasma glucagon levels, ILC2 chimeras were fasted in the present or absence of PF-06291874 (PF), a very selective glucagon receptor antagonist. While PF treatment maintained intact glucagon levels, it efficiently impaired gluconeogenesis and fasting plasma glucose levels in ILC2 chimeras (K-M andK,L). Thus, glucagon signaling is a necessary link between ILC2 and normal fasting plasma glucose levels. To probe the link between fasting, ILC2 and pancreatic glucagon in a more controllable manner,Nmur1-CretoR26-DTRmice were bred, thus generating a conditional and inducible ILC2-deletor mouse upon Diphtheria Toxin (DT) administration. DT treatedNmur1-Cre.R26-DTRmice displayed reduced glucagon, reduced fasting plasma glucose levels, and impaired gluconeogenesis when compared to their littermate controls (N-P andM). Taken together, these results indicate that ILC2 regulate glucose homeostasis and gluconeogenesis by increasing glucagon levels.

[0110] Glucagon is predominantly secreted from alpha cells in the islets of Langerhans. To define whether ILC2-derived cytokines play a role in this process, the expression of type 2 cytokine receptors in pancreatic alpha cells was interrogated. Transcriptomic analysis revealed that purified alpha cells expressIl13ra1,Il5raandCsf2ra, which was also observed in human alpha cells (A-F). Interestingly, the expression of type 2 cytokine receptors was similar between pancreatic alpha cells and intestinal epithelial cells, which are known to integrate IL-13 signals efficiently (E andA) (16). Accordingly, stimulation of purified alpha cells or isolated islets with IL-5 and IL-13 selectively promoted glucagon expression, while addition of STAT3 and STAT6 (components of the IL-5 and IL-13 signaling cascades) inhibitors to these cultures significantly impaired glucagon levels (Q,R andB-G). Aligned with these findings, ILC2 were the major source of pancreatic type 2 cytokines as scored by protein, transcript levels andIl5reporter mice (H-K).

[0111] Further evidence that ILC2-derived cytokines are required for glucagon control was provided by co-cultures of purified alpha cells with ILC2 (Q). Notably, addition of ILC2 to alpha cell cultures efficiently induced glucagon release in an IL-5 and IL-13 dependent manner (S). In agreement, ILC2 co-cultures withStat6- / -alpha cells resulted in unperturbed glucagon levels (T andL) and neutralization of IL-5 and IL-13invivoled to decreased plasma Glucagon and preproglucagon (Gcg) transcript levels upon fasting (U). In aggregate, these data indicate that ILC2 regulate glucose metabolism through induction of the pancreatic hormone glucagon.

[0112] FastinginducesentericILC2migrationviaagut-pancreaticaxis

[0113] Given the impact of pancreatic ILC2 on glucagon levels, first we investigated whether ILC2 could be found in pancreatic islets of Langerhans, where glucagon is produced by alpha cells. Using ILC2 chimeras, we identified ST2+KLRG1+ILC2 within the pancreatic islet (A and0 A). Next, we validated this finding through an independent RNAin situhybridization approach. Thus, we generated reporterIl5-TomatoILC2 chimeras and probedIl5andGcgexpression. This approach confirmed the presence ofIl5-expressing ILC2 in the islet and increased alpha-cell derivedGcgexpression upon fasting (B and0 B). Sequentially, we interrogated the mechanism underlying pancreatic accumulation of ILC2 upon fasting. Analysis of Ki67 expression and Bromodeoxyuridine (BrdU) incorporation revealed that pancreatic ILC2 from animals that were fasted or fedad libitumhad identical proliferative profiles (C,D and0 C,D). This data suggested that ILC2 migrate to the pancreas upon fasting. To investigate this hypothesis, fasted or fedadlibitumanimals were used to quantify ILC2 pools in different organs. When compared to theirad libitumcounterparts, fasted animals displayed unperturbed ILC2 numbers in the bone marrow, spleen, lungs, visceral adipose tissue, and liver (E and0 E-G). In contrast, fasted animals had a significant reduction of ILC2 in the lamina propria of the small intestine (E), suggesting that ILC2 migrate from the gut to the pancreas upon fasting. To validate that ILC2 follow a gut-pancreatic migration route, we traced ILC2 using genetic and organ-specific photoconversion. To this end, the small intestine of mice carrying Kikume-Green / Red (KikGR) was selectively irradiated to promote photoconversion (green to red fluorescent protein) of enteric cells (F andA). Seven days after photoconversion, analysis of spleen, liver, lungs, blood, and pancreas of ad libitum fed mice revealed that only a minute number of intestinal KikRed positive ILC2 were present in these organs (B).

[0114] Importantly, when compared to theirad libitumcontrols, fasted animals displayed increased photoconverted ILC2 selectively in the pancreas, indicating that fasting induces a gut-pancreatic migration axis for ILC2 (F andC). Finally, we employed single-cell transcriptional profiling to define whether recent enteric emigrant (KikRed) ILC2 differ from their pancreas-resident (KikGreen) counterparts upon fasting. Single-cell RNA-seq demonstrated that recent enteric emigrant and pancreas-resident ILC2 share similar genetic signatures (G andA). Taken together, these data indicate that the intestine is a source of pancreatic ILC2 upon fasting.

[0115] Migration of inflammatory ILC2, notably to the blood and lung, have been previously described after intestinal helminth infection. These migratory ILC2 are IL-25 responsive, KLRG1highand ST2low. However, our sequencing and flow cytometry data revealed thatIl17rbis unperturbed in pancreatic ILC2 upon fasting (N,P andA), suggesting that fasting- induced migration of intestinal ILC2 may differ from that associated with helminth infection. To test this hypothesis, we initially analyzed adhesion and chemokine receptor molecules known to mediate ILC migration and intestinal homing / retention. When compared to enteric ILC2 upon fasting, recent enteric emigrant ILC2 showed unperturbed levels for most tested molecules, while displaying a marked reduction ofItga4,Itgb7andCcr9expression (H). In agreement, more recent enteric emigrant displayed similar or even lower levels of those intestinal postcode integrins when compared to pancreas resident ILC2 (H). ILC2 trafficking into the blood upon infection was previously shown to be controlled by the molecular pair S1P-S1PR1. Importantly, expression ofS1pr1was unperturbed in recent enteric emigrant ILC2 in the pancreas (I) and blocking S1PR1 pharmacologically (FTY720) had no impact on the accumulation of pancreatic ILC2 upon fasting (J andB). Thus, while inflammatory ILC2 migration relies on IL- 25 responsive cells via a S1PR1-dependend mechanism, fasting induces migration of ILC2 that downregulate the enteric anchoring molecules ITGA4, ITGA7 and CCR9.

[0116] Given that fasting induces the loss of the ILC2 enteric postcode signature, that KikRed positive ILC2 were not found in the blood, and that their migration is not blocked by S1PR1 inhibition (H,J andB,C), enteric ILC2 might initially exit the intestine to the draining lymph nodes. Interestingly, we observed increased KikRed positive ILC2 in the mesenteric lymph nodes (LN) upon fasting (K), and mesenteric LN ablated mice failed to increase pancreatic ILC2 number upon fasting (L). Nevertheless, the release of ILC2 into circulation from the gut or mesenteric LN might be also very dynamic, thus falling below detection of current methods. Altogether, these data indicate that upon fasting, enteric ILC2 downregulate their enteric postcode molecules and selective migrate to the pancreas.

[0117] SympatheticneuronalsignalstriggerILC2migrationtothepancreas

[0118] Neuronal activity has been previously linked to the regulation of glucose homeostasis and to impact ILC function. Thus, we hypothesized that neuronal signals shape the gut-pancreatic migratory route of ILC2. To test this hypothesis, we modulated the activity of neurons connecting to the intestine using designer receptor exclusively activated by designer drugs (DREADD). Thus, activator DREADD-carrying adeno-associated virus (AAV(3D)), were injected in the proximal small intestine. Sequentially, we administered the designer drug Clozapine-N-oxide (CNO) that leads to neuronal stimulation of DREADD-carrying neurons (A). When compared to their controls (AAV(GFP)) injected with CNO, activation of intestinal AAV(3D)-carrying neurons resulted in increased intestinal norepinephrine, decreased ILC2 in the proximal small intestine and increased pancreatic ILC2, which associated with higher blood glucagon levels, and increased gluconeogenesis and plasma glucose levels (B-E andA). Interestingly, retrograde polysynaptic tracing with fluorescent protein-producing pseudorabies virus (PRV) revealed polysynaptic connections from the proximal small intestine to discrete brain areas that were previously associated with systemic glucose sensing, including the dorsomedial nucleus of the hypothalamus, the lateral hypothalamus and the basomedial nucleus of the amygdala (F andB).

[0119] Previous studies have identified immune cell subsets that integrate sympathetic neuronal signals, while sympathetic neurons were shown to shape adipose metabolism via indirect control of ILC2. Thus, we hypothesized that sympathetic signals control ILC2 migration to the pancreas. To address this hypothesis, initially we confirmed that lymphocyte deficient mice have normal intestinal and serum norepinephrine levels after fasting (C,D). Sequentially, we eliminated peripheral catecholaminergic neurons using systemic 6-hydroxydopamine (6-OHDA). Ablation of these neurons prevented the accumulation of ILC2 in the pancreas and led to impaired glucagon levels upon fasting (G,H). Akin to the effects of systemic administration, micro injections of 6-OHDA in the proximal small intestine led to reduced intestinal norepinephrine upon fasting, impaired ILC2 in the pancreas, reduced fasting plasma glucagon, impaired gluconeogenesis, and impaired fasting plasma glucose levels (I-L andA). In agreement, injection of Cre- dependent activator DREADD virus (AAV(3Dfl)) in the gut of tyrosine hydroxylase-Cre (Th-Cre) mice, followed by administration of CNO that leads to stimulation of DREADD-carrying sympathetic neurons, led to increased numbers of pancreatic ILC2 that associate with increased glucagon levels (M-N andB). Next, to establish the link between fasting, sympathetic innervation and ILC2 migration along an intestinal pancreatic axis, we eliminated peripheral catecholaminergic neurons by administering 6-OHDA to KikGR mice that were selectively irradiated in the proximal small intestine. While fasting led to increase of pancreatic KikRed positive ILC2 in untreated control animals, mice treated with 6-OHDA failed to accumulate KikRed positive ILC2 in the pancreas upon fasting (O).

[0120] To explore if sympathetic signals directly impact pancreatic ILC2 accumulation, we initially deleted the adrenergic beta 2 receptor (Adrb2) in lymphoid cells by breedingIl7ra-Cremice toAdrb2fl / flmice (Adrb2ΔIl7ra). Strikingly, when compared to their littermateIl7ra-Crecontrols, ILC2 fromAdrb2ΔIl7ramice failed to accumulate in the pancreas and to promote increased glucagon, gluconeogenesis, and glucose levels upon fasting (P-S). To confirm the link between ILC2- intrinsic sympathetic signals and migration of ILC2 to the pancreas more selectively, we bredIl5- CretoAdrb2fl / flmice (Adrb2ΔIl5), andAdrb2ΔIl5mice were subsequently bred toRag1- / -mice to exclude putative T helper cell effects (T-V). When compared to their littermate controls, ILC2 fromRag1- / -.Adrb2ΔIl5mice failed to accumulate in the pancreas and to promote increased fasting plasma glucagon and glucose levels upon fasting (T-V). Sequentially, we tested the impact of ADRB2 signaling in the expression of adhesion and chemokine receptor molecules on enteric ILC2 (W andC). In line with the impact of fasting on these cells, incubation of intestinal ILC2 with the adrenergic receptor agonist Clenbuterol resulted on downregulation of the enteric anchoring genesItga4,Itgb7andCcr9, whileS1pr1levels were unperturbed (W andC). Together, these data indicate that ILC2-autonomous ADRB2 signals control the migration of enteric ILC2 to the pancreas.

[0121] Finally, to establish the link between intestinal neuronal activation, ILC2-intrinsic sympathetic signals, and accumulation of ILC2 to the pancreas, we employed a combined genetic and chemogenetic approach. Thus,Rag1- / -.Adrb2ΔIl5mice and their littermate controls were injected in the proximal small intestine with activator DREADD virus (AAV(3D)), followed by administration of the designer drug CNO that leads to stimulation of DREADD-carrying neurons (X). When compared to their littermate controls,Rag1- / -.Adrb2ΔIl5displayed reduced ILC2 numbers in the pancreas, which associated with decreased glucagon and glucose levels upon CNO administration (X-Z). Collectively, these results indicate that sympathetic neurons control the migration of ILC2 through a gut-pancreatic axis, via ILC2-autonomous ADRB2 signals.

[0122] ILC2improvenon-alcoholicfattyliverviaglucagoninduction

[0123] Glucagon promotes lipid catabolism, notably by enhancing the break-down of fatty acids in the liver. To define whether the ILC2-glucagon axis can be harnessed to regulate hepatic lipid-associated pathology, we tested how ILC2 activation impacts on non-alcoholic fatty liver (NAFL). To this end, WT animals were initially placed on normal chow diet (NCD) or high fat / high sugar diet (HFD / HSD) for 12 weeks. When compared to NCD animals, the livers of HFD / HSD mice showed extensive steatosis (A-C). Notably, HFD / HSD livers displayed increased pathology score and increased hepatic lipid depots as revealed by Oil red O (ORO) staining (B, C). Sequentially, HFD / HSD mice were treated with the alarmin IL-33, which induces the activation of ILC2, and increased pancreatic ILC2 and blood glucagon levels (A, B). Strikingly, when compared to untreated HFD / HSD controls, IL-33 treated HFD / HSD mice displayed a marked decrease in liver pathology score and hepatic steatosis (B, C). To better define the link between IL-33, ILC2 and NAFL, we also placedRag1- / -andRag2- / -.Il2rg- / -animals on HFD / HSD followed by IL-33 administration (A). While IL-33 treatment induced NAFL improvement in WT andRag1- / -animals,Rag2- / -.Il2rg- / -animals failed to reduce liver pathology and hepatic steatosis, indicating that ILC are required to recover from NAFL upon IL-33 treatment (B-G). Sequentially, we generated ILC2 chimaeras by transplanting pancreatic ILC2 intoRag2- / -.Il2rg- / -that were placed in HFD / HSD. Treatment of HFD / HSD ILC2 chimaeras with IL-33 led to a significant improvement of NAFL (F, G). Collectively, these data demonstrate that ILC2 are required for NAFL recovery upon IL-33 treatment. Next, we tested whether glucagon is necessary for ILC2-dependent NAFL improvement. To this end, we initially treated HFD / HSD WT animals with IL-33 and with PF-06291874 (PF), a potent and selective glucagon receptor antagonist. PF treatment efficiently blocked improvements in NAFL pathology (E, F). In agreement, treatment of HFD / HSD ILC2 chimaeras with IL-33 and PF failed to improve NAFL, while their IL-33 treated chimaera counterparts efficiently improved fatty liver pathology (F, G). Altogether, these data indicate that ILC2 improve metabolic associated fatty liver disease via glucagon induction.

[0124] Discussion

[0125] Exploring how neuronal and immune cells cooperate to control endocrine function is critical to understand organismic physiology and metabolic diseases. Our work deciphers an unappreciated inter-organ communication circuitry at the interface of the nervous, immune, and endocrine systems. We found that sympathetic neuronal signals reduce the expression of gut ILC2 postcode receptors and promote ILC2 migration to the pancreas, where these cells stimulate the production of the glucose mobilizing hormone glucagon. Notably, in response to low energy levels, sympathetic neurons promote the migration of intestinal ILC2 to the pancreas via cell-intrinsic ADRB2 signals. In turn, ILC2-derived cytokines induce pancreatic alpha cells to produce glucagon, thus shaping glucose mobilization and metabolism ().

[0126] Sympathetic cues were previously shown to directly inhibit pulmonary ILC2 in the context of week-long helminth infections, while these adrenergic inputs were reported to indirectly control adipose ILC2 via mesenchyme-derived signals. Here we show that sympathetic neurons directly promote the fast migration of intestinal ILC2 to the pancreas, indicating that sympathetic signals can trigger distinct ILC2 programs in a time-, space- and context-dependent manner. ILC2 were previously reported to indirectly regulate insulin production via macrophages and dendritic cell-derived retinoic acid in conditions of high glucose, beta cell stress and increased inflammatory mediators. A more recent study showed that IL-33 activated ILC2 in the liver reduce gluconeogenic gene expression. Our study reveals that pancreatic ILC2 selectively control glucagon production in response to low glucose levels, suggesting that ILC2 may regulate endocrine function distinctively in response to diverse metabolic statuses of the host. Given that T lymphocytes were previously shown to regulate exogenous glucose absorption, we propose that adaptive and innate lymphocytes calibrate glucose levels by a division of labor: adaptive T cells promote exogenous glucose uptake, while ILC2 control endogenous glucose production at times of low carbohydrate intake.

[0127] Sympathetic neurons directly mediate glucagon levels in the context of neuro-pancreatic connections. Our work indicates that sympathetic cues promote migration of gut ILC2 to the pancreas, where they promote glucagon expression via ILC2-derived cytokines. As such, coupling direct and indirect sympathetic signals to induce fast and efficient glucagon production and glucose mobilization may have ensured efficient life-saving responses to hypoglycemia, notably by ensuring brain function and muscle activity in the context of fight or flight responses. Here we describe an effector neuroimmune hub that responds to low energy levels, suggesting that brain areas that harbor glucose sensor circuits may regulate the small intestine, translating energy body states into peripheral immune functions that promote adequate endocrine responses. Finally, our data may also grant better knowledge on how neuronal and immune functions might be harnessed in the context of endocrine and metabolic disorders in humans.

[0128] The citation list is as follows:

[0129] WO2023178169A2

[0130] US20240270855A1

[0131] Wensveen FM, Valentić S, Šestan M, Wensveen TT, Polić B. Interactions between adipose tissue and the immune system in health and malnutrition. Semin Immunol. 2015 Sep;27(5):322-33. doi: 10.1016 / j.smim.2015.10.006. Epub 2015 Nov 18. PMID: 26603491.

[0132] Lettieri-Barbato D, Aquilano K. Aging and Immunometabolic Adaptations to Thermogenesis. Ageing Res Rev. 2020 Nov;63:101143. doi: 10.1016 / j.arr.2020.101143. Epub 2020 Aug 15. PMID: 32810648.

[0133] Toki S, Goleniewska K, Reiss S, Zhang J, Bloodworth MH, Stier MT, Zhou W, Newcomb DC, Ware LB, Stanwood GD, Galli A, Boyd KL, Niswender KD, Peebles RS Jr. Glucagon-like peptide 1 signaling inhibits allergen-induced lung IL-33 release and reduces group 2 innate lymphoid cell cytokine production in vivo. J Allergy Clin Immunol. 2018 Nov;142(5):1515-1528.e8. doi: 10.1016 / j.jaci.2017.11.043. Epub 2018 Jan 10. PMID: 29331643; PMCID: PMC9639650.

Claims

A non-therapeutic method for inducing accumulation of type 2 innate lymphoid cells (ILC2) in pancreatic tissue, the method characterized by comprising:(i) activating a sympathetic neuronal pathway that innervates the intestine;(ii) reducing the number of ILC2 in the small intestine; and(iii) promoting migration of ILC2 expressing ADRB2 from the small intestine to the pancreas.The method of claim 1, wherein, in step (i), activation of the sympathetic neuronal pathway is induced during fasting.The method of claim 1, wherein, in step (i), activation of the sympathetic neuronal pathway is induced by using adrenergic agonists, wherein the adrenergic agonists are selected from the group consisting of clenbuterol, salbutamol, terbutaline, isoproterenol, salmeterol, formoterol and mixtures thereof.The method of claim 1, wherein the ILC2 further expresses GATA3 and KLRG1.The method of claim 1, wherein the ILC2 accumulates in the pancreas within 2 to 12 hours following neuronal activation.A non-therapeutic method for modulating glucagon secretion from pancreatic α-cells, the method characterized by comprising contacting pancreatic α-cells that express IL-5Rα and IL-13Rα with IL-5 and / or IL-13 secreted by ILC2 that have migrated from the intestinal tissue.The method of claim 6, wherein the ILC2 are activated by IL-33 prior to secretion of IL-5 or IL-13.The method of any of claims 6 or 7, wherein IL-5 and IL-13 are co-secreted.The method of any of claims 6 or 7, wherein the pancreatic α-cells are derived from fasted animals.Adrenergic agonists for use in modulating glucagon secretion in a subject having impaired counter-regulatory glucagon responses.The adrenergic agonists for use according to claim 10, wherein the subject is experiencing hypoglycemia unawareness.IL-13 for use in modulating glucagon secretion in a subject with reduced fasting tolerance.The IL-13 for use according to claim 12, wherein the subject is affected by non-alcoholic fatty liver disease.The IL-13 for use according to claim 12 and 13, wherein the IL-13 is administered in combination with IL-5.A population of ILC2 cells characterized by expression of ADRB2, S1PR1, GATA3, KLRG1, and IL-Rα, and exhibiting the capacity to migrate from the intestine to the pancreas upon activation of intestinal sympathetic neurons.The population of claim 15, wherein the ILC2 further express ST2.The population of claim 15, wherein the ILC2 produce both IL-5 and IL-13 following adrenergic stimulation.

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