Treatment and diagnosis of inflammatory disorders

Abstract

To provide a method of treating an inflammatory disorder in a subject, and a composition for treating the inflammatory disorder.SOLUTION: The treatment method comprises administering to a subject a nucleic acid molecule for inhibiting the expression of Hom-1. In particular, the nucleic acid molecule is an RNAi agent or an antisense morpholino oligonucleotide. Further disclosed is a method of selecting a therapeutic for an inflammatory disorder in a subject or monitoring the efficacy of a therapeutic for an inflammatory disorder in a subject, the method comprising: detecting an expression level of Hom-1 in an inflamed tissue sample obtained from the subject.SELECTED DRAWING: None

Description

[Background technology]

[0001] Tissue macrophages play a key role in host defense against pathogen invasion and immune homeostasis. Plasticity is a hallmark of macrophages. Macrophages can be activated to display a proinflammatory (M1) or anti-inflammatory (M2) phenotype by being present in a microenvironment replete with signals from host cells and microbes. Aberrant differentiation and activation of macrophages play a key role in the pathogenesis of inflammation. For example, IBD patients show an increase in mucosal CD14+ macrophages that display the M1 proinflammatory phenotype. Due to their role as central enforcers of both innate and adaptive immunity, macrophages are considered an ideal target for the control of autoimmune and inflammatory disorders. However, how macrophage plasticity is regulated remains incompletely understood, and intracellular factors that can be manipulated to modulate macrophage function remain largely unknown. Summary of the Invention

[0002] In one aspect, a method of treating an inflammatory disorder in a subject is described comprising administering to a subject in need thereof a nucleic acid molecule that inhibits expression of Hom-1.

[0003] In one embodiment, the nucleic acid molecule is an RNAi agent or an antisense oligonucleotide. The nucleic acid molecule can be administered topically, orally, rectally, nasally, intravenously, intraarticularly, conjunctivally, intracranially, intraperitoneally, intrapleurally, intramuscularly, intrathecally or subcutaneously. In one embodiment, the nucleic acid molecule is administered as is.

[0004] In another aspect, described herein is a composition for treating an inflammatory disorder, comprising a nucleic acid molecule that inhibits expression of Hom-1 and a pharma- ceutically acceptable carrier. In one embodiment, the nucleic acid molecule is a morpholino oligonucleotide having the sequence of SEQ ID NO: 3, 4, 5, or 6. The composition can be formulated for topical, oral, rectal, nasal, intravenous, intra-articular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, or subcutaneous administration routes. In one embodiment, the nucleic acid molecule (e.g., a morpholino oligonucleotide having the sequence of SEQ ID NO: 3, 4, 5, or 6) is a intact nucleic acid molecule.

[0005] In yet another aspect, a method for identifying a therapeutic agent for an inflammatory disorder is described, comprising contacting an inflamed tissue sample with a test therapeutic agent and detecting an expression level of Hom-1 in the tissue sample. If the expression level is equal to or less than a control level, the test therapeutic agent is a candidate therapeutic agent for the inflammatory disorder.

[0006] In one aspect, a method is described for selecting a therapeutic agent for an inflammatory disorder in a subject in need thereof, comprising contacting an inflamed tissue sample obtained from the subject with a therapeutic agent, detecting an expression level of Hom-1 in the tissue sample that is lower or the same as a control level, and administering the therapeutic agent to the subject.

[0007] In another embodiment, a method for monitoring the effectiveness of a therapeutic agent for an inflammatory disorder in a subject in need thereof is described. The method includes detecting an expression level of Hom-1 in an inflamed tissue sample obtained from the subject after administering the therapeutic agent to the subject, comparing the detected level with a control level, and making a treatment decision based on the comparison, and when the detected level is higher than the control level, administering the therapeutic agent to the subject is determined to be effective. Or, the administration of a different therapeutic agent is continued.

[0008] In another aspect, a method of treating an inflammatory disorder in a subject in need thereof is described. The method comprises providing modified macrophages, monocytes or dendritic cells treated with a Hom-1 inhibitor or containing an expression construct expressing a Hom-1 inhibitor, wherein the modified macrophages, monocytes or dendritic cells express lower levels of Hom-1 compared to control levels, and administering to the subject an effective amount of the modified macrophages, monocytes or dendritic cells. In one embodiment, the method comprises detecting expression of Hom-1 in an inflamed tissue sample obtained from the subject that is higher than control levels prior to the providing step. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The details of one or more embodiments are set forth in the detailed description below. Other features, objects, and advantages of the embodiments will be apparent from the description and the claims.

[0010] Unexpectedly, it was discovered that knockdown of Hom-1 expression in tissue macrophages could reduce tissue inflammation and preserve the viability of mucosal epithelial cells.

[0011] Hom-1, a human homeobox transcription factor, is an antagonist of canonical Wnt signaling. The nucleic acid sequence of Hom-1 (SEQ ID NO:1) and the amino acid sequence encoded by Hom-1 (SEQ ID NO:2) are shown below: acctggccgc atgcgcctc tcctcctccc cacccgtgg cccgcagcag ctctccagct ttggctccgt ggactggctc tcccagagca gctgctcagg gccgacccac acccccaggc ctgccgactt ctccctgggg agcctccctg gcccaggcca gacatccggc gcccctcgc aaggaggccg ccgggtcctc aaatctgcct gcgccggaga ggaccatggc cgggttgagt aaggagccaa ataccttgcg ggccccccgt gtccgcacag ccttcaccat ggagcaggtc cgcaccttgg agggcgtctt ccagcaccac cagtacct gggagc agatgcagct ctcagaggtc cagataaaaa cctggtttca gaatcgccgc atgaaacaca aacggcaaat gcaggacccc cagctgcaca gccccttctc ggggtctctc catgcgcccc cagctttcta ctcaacgtct tctggccttg ccaatggcct gcagctgctg tgcccttggg cacccctgtc cgggccccag gctctgatgc tgccccctgg ctccttctgg ggtctctgcc aagtggcaca agaggccctg gcatctgcgg gagctctcg cgcccctcgcgc taccccaggc cggccttcgc tgggaccagc cctgtccacg gggccccggg gcctgtgtgc tatgccacag acgggggatg cattttgagg aggcacctct gactcccaca ctcgcggtct tgctgatcgc acctggctcc tacctggagg actcagttgt tctgtttaca tcctggtggc acctctcacc ctgacccaca caaaggttct ggagattact ggagaatata tataaatata tatatgtacg tatatatgta aatacacata tacgtatata taaatatata tatacatatg tgtgtgtata tatatatata tttttttttt tttttttttt tttgagacgg agtgttgctc tgtcacccag gctggagtgc aatgacgcaa tctcggctca ctgcaacctc cgcctcctgg gttcaagcga ttctccagcc tcagcctccc gagtagctgg gattacagac acccgccacc acgcccggct aattttttct atttttagta gaaatggggt ttcaccatgt tagccaggct ggtctcaaac tcctgaccct gtgatccgcc cgcctcggcc tcccaaagtg ctgggattac aggcatgagc cactgcaccc ggccctgaga atatatttat taaagccacc tcttcactga aagttaccga aagagtcggt ttaggaagga aacgaagggt cagtgaacag agtcaaatgc agaagtgggc ttgtcatggg tagggctttc ggcgtacgat aaaaggatca tttgtttttt aaaaggggtt ggaaaaactg gttttccagt tggaaacagt aaaggttgta agctttgtgt gtacaaaaga aaacagggaa tgcaggtgtg tttatagcgt tgtggttcaa gtccctctta acaagaactc caaagctgga aagcaggagg gaacaaaggt gaacatgaag gcgaggatgc tggggccctg cagtgcgctc taggctgtgc gtgagccggg actgtaccca cagcttgctg agggctgctc ttcttgggcc agggaaagca gggcagccgg gacctgcggc tgtgcctgga ctgaagctgt cccgcaggtc cccaccctcc aacacgtgct cacctgtccc cctcctcgca gcagcctcgg gacaaaacaa tgactcaagg acagcacttc tcgcagaagg tctggaagtg cccagaatgg gaggcacgga agcccctccc ggggaggact cccgcgttga tggaccgttc ttggtgcaga ctcctgactg cgtgcatgaa acctgagaca agtgcaattc cttccatgtc gccccagagt gcccaggagg caggcagtgc ggggtgccca ggcagacggg ttcagcctgc agaactggag gcgacctgtg aaacccaccc gggcacccca acaggaacag aagcgtggtc ctgcggctgc gtccccagcg agtttcactt tccccttgct cgtttctccc ttgttgtaag tgtttacaac tggcatgtgc ttttaaacgt caggtaagag gggaacagct gctgtacatc gtcctggcga gtgacaatgt gacagaagcc tgggcgaggc cctcggaggg cagcagctgg acaggggcta ctgggtttgg cctggacagc actgatttgt ggatgtggat gggggcacgt tgtccgtgat aaaagtacaa gtgcccctca caaaaaaaaa aaaaaaa (SEQ ID NO: 1, underlined: coding sequence) mrlssspprg pqqlssfgsv dwlsqsscsg pthtprpadf slgslpgpgq tsgareppqa vsikeaagss nlpapertma glskepntlr aprvrtaftm eqvrtlegvf qhhqylsple rkrlaremql sevqiktwfq nrrmxkrqm q dpqlhspfs gslhappafy stssglangl qllcpwapls gpqalmlppg sfwglcqvaq ealasagasc cgqplashpp tpgrpslgpa lstgprglca mpqtgdaf (SEQ ID NO: 2, underlined: amino acids 91-1 51 / Homeodomain)

[0012] Described herein are methods of treating an inflammatory disorder in a subject in need thereof by administering to the subject a Hom-1 inhibitor, e.g., a nucleic acid molecule that inhibits expression of Hom-1.

[0013] The nucleic acid molecule can be an RNAi agent or an antisense oligonucleotide. In one embodiment, the nucleic acid molecule is a morpholino oligonucleotide. Morpholino oligonucleotides have standard DNA bases (A, C, G, T), but the bases are bonded to a morpholine ring and linked via a phosphorodiamidate group. Anti-Hom-1 morpholino oligonucleotides are: 5'-TACTCAACCCTGACATAGAGGGTAA-3' (SEQ ID NO: 3), 5'-GAGCCCGGTTTGCATACACGGCTAA-3' (SEQ ID NO: 4), 5'-GCCCAGATAAGCAGCGCCTAATTGC-3' (SEQ ID NO: 5), and It may have a sequence selected from 5'-CTGTAGGAAAAGCAAGATCAGAACA-3' (SEQ ID NO: 6) .

[0014] The term "RNAi agent" refers to an RNA (or its analogue) that has sufficient sequence complementarity with a target RNA to direct RNA interference. In general, an interfering RNA ("iRNA") is a double-stranded small interfering RNA (siRNA) or small hairpin RNA (shRNA) that causes catalytic degradation of a specific mRNA. Antisense oligonucleotides are usually single-stranded DNA, RNA or its analogues that have a sequence that can bind to a target nucleic acid molecule.

[0015] The anti-Hom-1 nucleic acid molecule can be administered to a subject by any route of administration, such as topical, oral, rectal, nasal, intravenous, intraarticular, conjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, or subcutaneous. The route can be selected based on the site of inflammation. The pharmaceutical composition containing the anti-Hom-1 nucleic acid molecule can be formulated for any route of administration, such as an injection solution, pill, capsule, eye drop, spray, inhalant, topical cream or gel, or aerosol.

[0016] In one embodiment, the anti-Hom-1 nucleic acid molecule is administered neat, that is, no delivery vehicle such as a liposome or viral vector is used in conjunction with the nucleic acid molecule.

[0017] Prior to administering any Hom-1 inhibitor to a subject, the subject can be tested to determine whether the subject has an elevated expression level of Hom-1 and / or elevated expression level of a proinflammatory cytokine compared to a control level. In one embodiment, the expression level is detected in an inflamed tissue sample obtained from the subject. A subject with an increased expression level of Hom-1 can be treated with a Hom-1 inhibitor. The control level can be a level representative of Hom-1 expression levels in non-inflamed tissue or a subject not having an inflammatory disorder, or a level found in non-inflamed tissue of the subject to be treated.

[0018] "Subject" refers to humans and non-human animals. "Treating" or "treatment" refers to the administration of a compound or agent to a subject having a disorder for the purpose of curing, alleviating, ameliorating, correcting, delaying the onset of, or ameliorating the disorder, symptoms of the disorder, pathology associated with the disorder, or predisposition to the disorder. An "effective amount" refers to an amount of a compound capable of producing a medically desirable result in the treated subject. Treatment methods can be performed alone or in conjunction with other drugs or therapies.

[0019] Also described herein is a screening method for identifying a therapeutic agent for inflammatory disorders. The method includes contacting an inflamed tissue sample with a test therapeutic agent and detecting the expression level of Hom-1 in the tissue sample. If the expression level is equal to or lower than the control level, the test therapeutic agent is a candidate therapeutic agent for inflammatory disorders.

[0020] Also described is a method for selecting a therapeutic agent for an inflammatory disorder in a subject in need thereof, comprising contacting an inflamed tissue sample obtained from the subject with the therapeutic agent, detecting a lower or equal expression level of Hom-1 in the tissue sample compared to a control level, and administering the therapeutic agent to the subject.

[0021] Further described is a method for monitoring the effectiveness of a therapeutic agent for inflammatory disorders in a subject in need thereof.The method includes: detecting the expression level of Hom-1 in an inflamed tissue sample obtained from a subject after administering the therapeutic agent to the subject; comparing the detected level with a control level; and making a therapeutic decision based on the comparison.If the detected level is lower than the control level, it indicates that the therapeutic agent is effective in treating inflammation in the subject.If the detected level is equal to or higher than the control level, it can be decided to continue to give the same therapeutic agent or to try a different therapeutic agent.

[0022] The therapeutic or test therapeutic agent may be a protein, peptide, peptidomimetic, peptoid, cell, antibody or fragment thereof, small molecule compound, nucleic acid molecule, or plant extract. In one embodiment, the therapeutic or test therapeutic agent may be a steroidal, non-steroidal anti-inflammatory drug, or an immunosuppressant.

[0023] In the above screening, selection or monitoring methods, the control level can be a representative level of expression level of Hom-1 in non-inflamed tissue. The control level can also be the expression level of Hom-1 in an inflamed tissue sample before contact with a therapeutic agent or test therapeutic agent. A person skilled in the art can determine a suitable control level.

[0024] In one aspect, a method of treating an inflammatory disorder using modified macrophages, monocytes, or dendritic cells is described. The method includes providing modified macrophages, monocytes, or dendritic cells that have been treated with a Hom-1 inhibitor or that contain an expression construct that expresses a Hom-1 inhibitor. The modified macrophages, monocytes, or dendritic cells express lower levels of Hom-1 compared to control levels. An effective amount of the modified macrophages, monocytes, or dendritic cells is administered to a subject with an inflammatory disorder.

[0025] The Hom-1 inhibitor can be a protein, peptide, peptidomimetic, peptoid, cell, antibody or fragment thereof, small molecule compound, nucleic acid molecule, or plant extract. In one embodiment, the inhibitor is an RNAi agent or an antisense oligonucleotide (e.g., morpholino oligonucleotide).

[0026] Prior to administering the modified cells to the subject, the expression level of Hom-1 can be determined in a sample (e.g., an inflamed tissue sample) obtained from the subject. If the level is higher than the threshold, the subject is considered suitable for treatment. The control level can be a level representative of the level in non-inflamed tissue or a level detected in a non-inflamed tissue sample obtained from the subject to be treated. Again, a suitable control level can be determined by one of skill in the art.

[0027] Hom-1 expression levels can be determined either at the mRNA level or at the protein level. Methods for measuring mRNA and protein levels are known in the art.

[0028] In any of the methods described herein, in addition to or alternatively to detecting expression levels of Hom-1 as an indicator of inflammation (e.g., the presence or extent of inflammation), inflammation can also be measured using detection of expression and / or secretion of pro-inflammatory cytokines, expression and / or secretion of anti-inflammatory cytokines, expression of markers of M1 or M2 macrophages, expression of markers of DC differentiation and activation.

[0029] Inflammatory disorders are characterized by local or systemic acute or chronic inflammation. Inflammatory disorders include inflammatory skin diseases (e.g. dermatitis, psoriasis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, dermatomyositis or eosinophilic fasciitis), inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis), acute respiratory distress syndrome, fulminant hepatitis, pancreatitis, hypersensitivity lung disease (e.g. hypersensitivity pneumonitis, eosinophilic pneumonia, delayed hypersensitivity, interstitial lung disease or ILD, idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis), asthma, COPD, allergic rhinitis, rheumatoid arthritis, psoriatic arthritis, These include, but are not limited to, systemic lupus erythematosus, myasthenia gravis, juvenile onset diabetes, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, multiple sclerosis, primary lateral sclerosis, amyotrophic lateral sclerosis, anaphylaxis, systemic anaphylaxis, hypersensitivity responses, systemic inflammatory conditions, drug allergies, insect sting allergies, allograft rejection, graft versus host disease, Sjogren's syndrome, human immunodeficiency, viral infections, atherosclerosis, hypertension, diabetes, as well as chronic kidney disease, ocular inflammatory diseases, uveitis and conjunctivitis, neuritis.

[0030] Those skilled in the art can determine whether an individual has an inflammatory disorder. The expression level of Hom-1 in a sample (e.g., a tissue, cell or body fluid sample) obtained from a subject suspected of having an inflammatory disorder can also be used as a diagnostic tool.

[0031] The following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. EXAMPLES

[0032] Macrophages are key regulators of both innate and adaptive immunity. How macrophage plasticity is regulated by intracellular factors is not fully understood. The data below demonstrate that the human homeobox transcription factor Hom-1 plays a pivotal role in directing macrophage polarization toward the M1 phenotype. Hom-1 expression is abnormally elevated in tissue macrophages isolated from inflamed mucosa of IBD patients. Using an en bloc culture model, we show that knockdown of Hom-1 expression in tissue macrophages with morpholino oligonucleotides can reduce tissue inflammation and protect the viability of mucosal epithelial cells. Taken together, our data suggest that Hom-1 can be a novel target for the management of inflammatory disorders.

[0033] Hom-1 expression is upregulated in macrophages isolated from inflamed gastrointestinal mucosa Using an in vitro monocyte-derived macrophage model, Hom-1 was shown to regulate monocyte-to-macrophage differentiation and proinflammatory activation. To examine the potential role of Hom-1 in tissue macrophage differentiation and activation, Hom-1 expression was examined in mucosal macrophages isolated from the mucosa of IBD patients. Hom-1 expression was found to be significantly elevated in macrophages isolated from inflamed mucosa compared to control macrophages isolated from normal mucosa of the same patients. Using FACAS and ELISA analysis, it was found that the expression of M1 surface markers such as CD40, CD80, and CD86, as well as the expression and secretion of M1 proinflammatory cytokines, were elevated in macrophages isolated from inflamed mucosa, in conjunction with the elevated expression of Hom-1. In addition, the expression of reactive oxygen species (ROS) and nitric oxide (NO) was also found to be elevated in macrophages isolated from inflamed mucosa.

[0034] Hom-1 regulates mucosal macrophage plasticity and polarizes mucosal macrophages toward the M1 phenotype Plasticity is a hallmark of macrophages. In response to environmental cues, macrophages display a wide range of phenotypes, ranging from the classical proinflammatory M1 phenotype to a variety of M2 phenotypes with distinctive features. Corticosteroids are widely used to manage inflammatory disorders and have been shown to induce the M2 phenotype in macrophages. To determine whether Hom-1 plays a role in regulating the plasticity of mucosal macrophages, the effect of corticosteroids on the expression of Hom-1 was examined. Incubation of mucosal CD14 macrophages with prednisolone resulted in a significant reduction in Hom-1 expression levels and This resulted in a characteristic reduction in the secretion of the M1 cytokine IL12, but increased secretion of the M2 cytokine IL10. Consistent with a potential role for Hom-1 in regulating macrophage plasticity, we found that the morphology of GFP-transfected, but not GFP-Hom-1-transfected, mucosal macrophages could be induced to exhibit the roundup phenotype characteristic of PD. FACS analysis of cell surface expression of CD80 and ELISA analysis of IL12 secretion in the culture medium of macrophages expressing either GFP or GFP-Hom-1 indicated that Hom-1 rendered macrophages resistant to PD-induced CD80 reduction and IL12 secretion. To further examine whether Hom-1 regulates macrophage polarization, Hom-1 expression upon induction of M2-to-M1 switch was examined using the in vitro macrophage differentiation model described above. Hom-1 expression was found to be elevated upon induction of M2-to-M1 polarization. To clarify the primary regulatory role of Hom-1 in macrophage polarization, the effect of knockdown of Hom-1 on LPS-induced M2-to-M1 phenotypic switch was examined. Downregulation of Hom-1 was found to render macrophages resistant to LPS-induced M1 polarization, suggesting a primary regulatory role of Hom-1 in the process. To further define the function of Hom-1 in macrophage polarization, we examined the effect of ectopic expression of Hom-1 in M2 macrophages and found that overexpression of Hom-1 led to a significant increase in the surface expression of the M1 marker CD80, as well as increased secretion of the M1 cytokines IL1b, IL12, and TNFa. Collectively, the data suggested that Hom-1 plays an important role in regulating macrophage plasticity and polarizes macrophages toward the M1 phenotype.

[0035] Hom-1 differentially regulates the expression of M1 and M2 genes To examine the potential mechanism of macrophage polarization regulated by Hom-1, we examined the effect of ectopic expression of Hom-1 on the characteristic expression of M1 and M2 genes. We found that Hom-1 expression promoted the expression of M1 genes, such as IL1, IL12, and TNFα, but suppressed the expression of M2 genes, such as IL10 and TGFβ. Combined with our previous observation that Hom-1 expression is required for expression of M1 genes but not of the M2 genes tested, our data suggest that Hom-1 regulates macrophage plasticity by polarizing expression of M1 and M2 genes.

[0036] Targeting Hom-1-regulated macrophage plasticity in the pathogenesis of tissue inflammation To further determine whether Hom-1-regulated macrophages can be targeted to reduce tissue inflammation, en bloc cultures of tissues obtained from inflamed or normal mucosa of ulcerative colitis (UC) patients were used. Consistent with clinical findings, secretion of inflammatory cytokines such as TNFα, IL1β, and nitrate was found to be significantly elevated in the inflamed tissue cultures. Anti-Hom-1 morpholino oligonucleotides (MOs) were then added to the tissue cultures to examine whether they could downregulate the expression of Hom-1 in mucosal macrophages. It was found that Hom-1 MOs efficiently inhibited Hom-1 expression in macrophages in en bloc tissue cultures. To further verify the effect of Hom-1 MOs on tissue inflammation, the concentration of TNFα upon incubation of en bloc tissues with Hom-1 was examined using an ELISA assay. It was found that Hom-1 MOs dose-dependently reduced the amount of TNFα in the cultures. To further examine the effect of Hom-1 MO on the secretion of other proinflammatory cytokines, we examined the effect of Hom-1 MO on the secretion of IL1 and nitrate, and found that, similar to TNFα, Hom-1 MO strongly inhibited these proinflammatory cytokines in the en bloc culture system. Because ex vivo en bloc cultures can reflect the in vivo tissue microenvironment, our data suggested that tissue macrophages could be targeted by Hom-1 MO to reduce tissue inflammation.

[0037] Hom-1 MO rescues epithelial cell viability during inflammation in inflamed tissues Apoptosis of epithelial cells leading to mucosal ulceration is a hallmark of IBD. Tissue inflammation was thought to be a major factor in mucosal epithelial cell apoptosis. Using en bloc tissue culture, it was found that tissue isolated from inflamed mucosa exhibited a higher rate of mucosal epithelial cell apoptosis compared to that of epithelial cells in normal control tissue. When Hom-1 MO was added to en bloc tissue culture, it was found that Hom-1 MO, but not control MO, exerted a strong inhibitory effect on epithelial cell apoptosis in tissue culture. Combined with our finding that Hom-1 MO could reduce tissue inflammation, the data suggested that Hom-1 MO could function as an agent to manage inflammation.

[0038] Isolation and culture of monocytes Peripheral blood mononuclear cells (PBMCs) from healthy adult donors at Boston Children's Hospital were isolated by Ficoll density gradient centrifugation. Experiments with human material were performed in accordance with guidelines approved by the Institutional Review Board at Brigham and Women's Hospital. CD14 + Monocytes were cultured using anti-CD14 coated microbeads (Miltenyi Biotec). Purified from BMC. Freshly isolated CD14 + The purity of monocytes was >95% as analyzed by flow cytometry. Monocytes were cultured at 1 × 10 in 12-well plates using RPMI 1640 medium containing 10% fetal bovine serum (FBS). 6 M-CSF, GM-CSF, and IL3 were purchased from PeproTech and were cultured at a final concentration of 10 Cytokines were added to the cultures every 2 or 3 days.

[0039] RNA interference Human primary monocytes were transfected using the Human Monocyte Nucleofector Kit (Lonza) according to the manufacturer's instructions. And 5×10 6Monocytes were resuspended in 100 μl of nucleofector solution containing 0.5 nmol of either Hom-1 siRNA (forward: 5'-UUCAGAAUCGCCGCAUGAAACACAAACGG-3' (SEQ ID NO: 7); reverse: 5'-CCGUUUGUGUUUCAUGCGGCGAUUCUGAA-3' (SEQ ID NO: 8)) or ineffective GFP siRNA (forward: 5'-UGACCACCCUGACCUACGGCGUGCAGUGC-3' (SEQ ID NO: 9); 5'-reverse: GCACUGCACGCCGUAGGUCAGGGUGGUCA-3' (SEQ ID NO: 10)) and then electroporated with a nucleofector II Device (Lonza). The cells were then immediately removed from the device and electroporated with a nucleofector II Device (Lonza). The cells were then incubated overnight with 1 ml of pre-warmed Human Monocyte Nucleofector Medium containing 1 mM RPMI, 2 mM glutamine and 10% FBS. The cells were then resuspended in complete RPMI medium and treated with the appropriate cytokines to induce differentiation into macrophages. Similarly, monocyte-derived macrophages were generated using the Human Macrophage Nucleofector Kit (Lonza) according to the manufacturer's instructions. Transfection was carried out according to the manufacturer's instructions.

[0040] FACS analysis Monocyte / macrophage phenotypic analysis was performed using flow cytometry after immunolabeling of cells with fluorescent dye-conjugated antibodies. The following antibodies were used: PE-conjugated anti-CD71, CD11b, CD11c, CD16, CD64, CD80, CD86, HLA-DR, CD14, TLR4, IL1-β and TNF-α, and FITC-conjugated anti-CD40, CD36 (eBioscience); FITC-conjugated anti-mannose Receptor (MR), as well as unconjugated mouse anti-MCSFR (R&D Systems). Control labeling was performed in parallel. Antibodies were diluted as recommended by the supplier. A PE-conjugated rabbit anti-mouse IgG antibody was used for secondary M-CSFR staining. Labeled cells were analyzed using a FACScan flow cytometer (BD Bioscience ) and analyzed using CellQuest software. Results are expressed as the percentage of positive cells and / or mean fluorescence intensity (MFI) values ​​subtracted from the MFI obtained with an isotype control antibody.

[0041] RT-PCR Total RNA was isolated using TRIzol reagent, and equal amounts of RNA were analyzed using SuperScript For first-strand cDNA synthesis, the AccuPrime™ Taq DNA polymerase system (Invitrogen) was used according to the manufacturer's protocol. To amplify Hom-1 cDNA by conventional PCR, the AccuPrime™ Taq DNA polymerase system (Invitrogen) was used according to the manufacturer's instructions. PCR products were separated on 2% agarose gels and stained with ethidium bromide. GAPDH was used as an internal control. Quantitative measurement of Hom-1 and cytokine cDNA using SYBR Green was performed on a LightCycler™ (480 Real-Time PCR System; Roche).

[0042] Cytokine measurements Macrophages treated with E. coli LPS (Sigma) and IFN-γ (PeproTech) The levels of IL-1β, TNF-α and IL12p70 in the supernatants of U937 cells treated with phages or LPS were quantified using ELISA kits obtained from eBiosciences. The assays were performed according to the manufacturer's instructions.

[0043] Detection of reactive oxygen species (ROS) and nitric oxide (NO) ROS levels in activated macrophages were measured using Image-iT™ LIVE Green Reactive Oxygen Species Detection The NO levels were detected using a Grifollicle-derived Immunoglobulin (GRI) kit (Invitrogen) essentially according to the manufacturer's instructions, except that the results were analyzed by both fluorescence microscopy and flow cytometry. Nitrite determination was performed using the ess Reagent Kit for Nitrite Determination (Invitrogen) according to the protocol provided by the manufacturer.

[0044] Cell staining (Cytostaining) For Wright-Giemsa staining, a staining kit from Sigma was used according to the manufacturer's instructions. Used.

[0045] statistical analysis Data were analyzed using Student's paired t-test (two-tailed) and Wilcoxon rank sum tests. Differences with p values ​​less than 0.05 were considered statistically significant.

[0046] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0047] From the above detailed description, those skilled in the art can easily identify the essential characteristics of the described embodiments, and can make various changes and modifications to the above embodiments to suit various usages and conditions without departing from the spirit and scope thereof. Thus, other embodiments are within the scope of the claims.

Claims

1. A composition comprising modified macrophages, monocytes or dendritic cells for use in a method for treating an inflammatory disorder in a subject, wherein the cells have been treated with a Hom-1 inhibitor or contain an expression construct for expressing a Hom-1 inhibitor, and the cells express lower levels of Hom-1 compared to control levels.

2. The composition described in claim 1, wherein the Hom-1 inhibitor is an RNAi agent or an antisense oligonucleotide.

3. The composition described in claim 1, wherein the Hom-1 inhibitor is a morpholino oligonucleotide.

4. The composition described in claim 1, wherein the Hom-1 inhibitor inhibits the expression of Hom-1.

5. The composition described in claim 3, wherein the morpholino oligonucleotide comprises the sequence of SEQ ID NO: 3, 4, 5 or 6.

6. The composition described in claim 1, wherein an inflamed tissue sample obtained from the subject expresses higher levels of Hom-1 compared to control levels.

7. The composition described in claim 1, wherein, prior to administering the cells to the subject, an inflamed tissue sample obtained from the subject is determined to have a higher expression level of Hom-1 compared to a control level.

8. The composition described in claim 7, wherein the control level corresponds to the expression level of Hom-1 in a non-inflamed tissue sample.

9. A composition described in any one of claims 1 to 8, wherein the inflammatory disorder is inflammatory bowel disease.

10. The composition of any one of claims 1 to 8, wherein the inflammatory disorder is selected from the group consisting of inflammatory skin diseases, acute respiratory distress syndrome, fulminant hepatitis, pancreatitis, hypersensitivity pulmonary disease, asthma, COPD, allergic rhinitis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, myasthenia gravis, juvenile-onset diabetes mellitus, glomerulonephritis, autoimmune thyroiditis, ankylosing spondylitis, systemic sclerosis, multiple sclerosis, primary lateral sclerosis, amyotrophic lateral sclerosis, anaphylaxis, systemic anaphylaxis, hypersensitivity response, systemic inflammatory state, drug allergy, insect sting allergy, allograft rejection, graft-versus-host disease, Sjogren's syndrome, human immunodeficiency, viral infection, atherosclerosis, hypertension, diabetes mellitus, chronic kidney disease, ocular inflammatory disease, uveitis, conjunctivitis, or neuritis.

11. The composition of any one of claims 1 to 8, wherein the inflammatory disorder is selected from the group consisting of dermatitis, psoriasis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, necrotizing vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, eosinophilic myositis, polymyositis, dermatomyositis, or eosinophilic fasciitis, Crohn's disease and ulcerative colitis, acute respiratory distress syndrome, fulminant hepatitis, pancreatitis, hypersensitivity pneumonitis, eosinophilic pneumonia, delayed hypersensitivity, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, and ILD associated with rheumatoid arthritis.