Neutralization Of IL-22 Research Articles

Abstract 1405: Development of a humanized anti-IL-22 antibody for cancer and inflammation therapy

Abstract Interleukin-22 (IL-22) is an inflammatory cytokine involved in the pathology of autoimmune diseases such as psoriasis, atopic dermatitis, and ulcerative colitis. IL-22 has also been shown to promote epithelial cell proliferation, stemness and tumorigenesis in cancer, and is associated with a more aggressive phenotype in a variety of cancers including inflammatory colon cancer models. IL-22 plays a central role in this pathogenic process by driving activation of STAT3 and other signaling cascades. Recent studies demonstrated that neutralization of IL-22 reduced dysplasia and tumor development in preclinical models. However, there has not been a clear application for anti-IL-22 therapy for cancer and autoimmune diseases, likely due to identifying the right disease indication and their low potency and efficacy. Thus, there remains an opportunity to explore the potential of more potent anti-IL-22 therapeutics. We have developed humanized anti-IL-22 antibodies with high affinities to human, cynomolgus and mouse IL-22 using BioAtla’s proprietary antibody discovery and engineering platforms. In vitro data demonstrated that our anti-IL-22 antibodies inhibited IL-22-induced p-STAT3 activities. In addition, our antibodies showed a 10-fold increased binding activity to human IL-22 compared to Fezakinumab, which was previously developed by Pfizer for the treatment of autoimmune diseases. To test the functions of our antibodies in vivo, we established an inflammation-driven sporadic colitis-associated colorectal (CAC) cancer mouse model. Our humanized anti-IL-22 antibody, a potent, species cross-reactive anti-IL-22 blocking antibody, significantly reduced tumor progression in CAC model. These data suggest that targeting IL-22 reduces tumor occurrence by reducing inflammation-induced tumorigenesis. In addition, we evaluated our anti-IL22 antibodies in an imiquimod-induced psoriasiform skin inflammation mouse model. The mice treated with anti-IL22 antibodies had significantly reduced skin lesions compared to isotype antibody-treated mice. Q-PCR analysis of the mouse skin demonstrated that treatment with our anti-IL-22 antibody led to significantly decreased RNA levels of the imiquimod-induced inflammatory marker CXCL3. In conclusion, the development of a potent, species cross-reactive anti-IL-22 antibody using BioAtla’s antibody discovery and engineering platforms addresses the past challenges of anti-IL-22 therapeutics and allows for translational studies in relevant animal efficacy and safety models. Citation Format: Jian Chen, Cathy Chang, Gerhard Frey, Haizhen Liu, Jing Wang, William J. Boyle, Jay M. Short. Development of a humanized anti-IL-22 antibody for cancer and inflammation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1405.

IL-22 promotes liver regeneration after portal vein ligation

BackgroundInsufficient remnant liver volume (RLV) after the resection of hepatic malignancy could lead to liver failure and mortality. Portal vein ligation (PVL) prior to hepatectomy is subsequently introduced to increase the remnant liver volume and improve the outcome of hepatic malignancy. IL-22 has previously been reported to promote liver regeneration, while facilitating tumor development in the liver via Steap4 upregulation. Here we performed PVL in mouse models to study the role of IL-22 in liver regeneration post-PVL. MethodsLiver weight and volume was measured via magnetic resonance imaging (MRI). Immunohistochemistry for Ki67 and hepatocyte growth factor (HGF) was performed. IL-22 was analyzed by flow cytometry and quantitative polymerase chain reaction (qPCR) was used for acquisition of Il-33, Steap4, Fga, Fgb and Cebpd. To analyze signaling pathways, mice with deletion of STAT3 and a neutralizing antibody for IL-22 were used. ResultsThe remnant liver weight and volume increased over time after PVL. Additionally, we found that liver regenerative molecules, including Ki67 and HGF, were significantly increased in remnant liver at day 3 post-PVL, as well as IL-22. Administration of IL-22 neutralizing antibody could reduce Ki67 expression after PVL. The upregulation of IL-22 after PVL was mainly derived from innate cells. IL-22 blockade resulted in lower levels of IL-33 and Steap4 in the remnant liver, which was also the case in mice with deletion of STAT3, the main downstream signaling molecule of IL-22, in hepatocytes. ConclusionIL-22 promotes liver regeneration after PVL. Thus, a combination of IL-22 supplementation and Steap4 blockade could potentially be applied as a novel therapeutic approach to boost liver regeneration without facilitating tumor progression after PVL.

CD4+ T cell-derived IL-22 enhances liver metastasis by promoting angiogenesis

ABSTRACT Metastasis is a cancer-related systemic disease and is responsible for the greatest mortality rate among cancer patients. Interestingly, the interaction between the immune system and cancer cells seems to play a key role in metastasis formation in the target organ. However, this complex network is only partially understood. We previously found that IL-22 produced by tissue resident iNKT17 cells promotes cancer cell extravasation, the early step of metastasis. Based on these data, we aimed here to decipher the role of IL-22 in the last step of metastasis formation. We found that IL-22 levels were increased in established metastatic sites in both human and mouse. We also found that Th22 cells were the key source of IL-22 in established metastasis sites, and that deletion of IL-22 in CD4+ T cells was protective in liver metastasis formation. Accordingly, the administration of a murine IL-22 neutralizing antibody in the establishment of metastasis formation significantly reduced the metastatic burden in a mouse model. Mechanistically, IL-22-producing Th22 cells promoted angiogenesis in established metastasis sites. In conclusion, our findings highlight that IL-22 is equally as important in contributing to metastasis formation at late metastatic stages, and thus, identify it as a novel therapeutic target in established metastasis.

Abstract 6335: Developing anti-IL-22 therapeutics for inflammation and cancer

Abstract Chronic inflammation facilitates the development of cancer, age-related, inflammatory autoimmune and other diseases. Proinflammatory cytokines play a central role in this pathogenic process by driving activation of NF-ƙB, STAT3 and other signaling cascades. IL-22 is an inflammatory cytokine involved in the pathology of autoimmune diseases such as psoriasis, atopic dermatitis, and ulcerative colitis. IL-22 has also been shown to promote epithelial cell proliferation, stemness and tumorigenesis in cancer. Recent studies demonstrated that neutralization of IL-22 reduced dysplasia and tumor development in preclinical models. To date, there has not been a clear application for anti-IL-22 therapy for autoimmune diseases and cancer, likely due to identifying the right disease indication and to their low potency and low efficacy. Thus, there remains an opportunity to explore the potential of more potent anti-IL-22 therapeutics. Using BioAtla’s proprietary antibody discovery and engineering platforms, we have generated humanized anti-IL-22 antibodies with high affinities to human, cynomolgus and mouse IL-22. In vitro data demonstrated that our anti-IL-22 antibodies inhibited IL-22-induced p-STAT3, CXCL-1 and IL-10 activities. In addition, our antibodies showed a 10-fold increased binding activity to human IL-22 compared to Fezakinumab, which was previously developed by Pfizer for the treatment of autoimmune diseases. BioAtla’s high affinity anti-IL-22 antibodies were shown to be more potent relative to Fezakinumab using multiple in vitro assays. To test the functions of our antibodies in vivo, we also established mouse models to determine anti-IL22 efficacy. More specifically, to study whether targeting IL-22 in an inflammation-induced tumor microenvironment reduces tumor occurrence, we developed an inflammation-driven sporadic colitis-associated colorectal cancer mouse model for ongoing cancer studies that will be discussed. In addition, we evaluated our anti-IL22 antibodies in an imiquimod-induced psoriasiform skin inflammation mouse model. The mice treated with potent anti-IL-22 antibodies had significantly reduced skin lesions, including erythema, scales, and skin thickness compared to isotype antibody-treated mice. Q-PCR analysis of the mouse skin demonstrated that treatment with our anti-IL-22 antibody led to significantly decreased RNA levels of the imiquimod-induced inflammatory marker CXCL3. Thus, the enhanced anti-IL-22 therapy is a promising strategy for targeting anti-inflammation in autoimmune diseases, such as psoriasis and atopic dermatitis, and inflammatory cancers, including colorectal cancer. In conclusion, the development of a potent, species cross reactive anti-IL-22 antibody using BioAtla’s antibody discovery and engineering platforms addresses the challenges of anti-IL-22 therapeutics and allows for translational studies in relevant animal efficacy and safety models. Citation Format: Christina Wheeler, Jian Chen, Cathy Chang, Gerhard Frey, Haizhen Liu, Jing Wang, Kathryn Woodard, Solmarie Joyner, Wei Zhou, William J. Boyle, Jay M. Short. Developing anti-IL-22 therapeutics for inflammation and cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6335.

Local immune cell contributions to fracture healing in aged individuals – A novel role for interleukin 22

With increasing age, the risk of bone fractures increases while regenerative capacity decreases. This variation in healing potential appears to be linked to adaptive immunity, but the underlying mechanism is still unknown. This study sheds light on immunoaging/inflammaging, which impacts regenerative processes in aging individuals. In an aged preclinical model system, different levels of immunoaging were analyzed to identify key factors that connect immunoaged/inflammaged conditions with bone formation after long bone fracture. Immunological facets, progenitor cells, the microbiome, and confounders were monitored locally at the injury site and systemically in relation to healing outcomes in 12-month-old mice with distinct individual levels of immunoaging. Bone tissue formation during healing was delayed in the immunoaged group and could be associated with significant changes in cytokine levels. A prolonged and amplified pro-inflammatory reaction was caused by upregulated immune cell activation markers, increased chemokine receptor availability and a lack of inhibitory signaling. In immunoaged mice, interleukin-22 was identified as a core cell signaling protein that played a central role in delayed healing. Therapeutic neutralization of IL-22 reversed this specific immunoaging-related disturbed healing. Immunoaging was found to be an influencing factor of decreased regenerative capacity in aged individuals. Furthermore, a novel therapeutic strategy of neutralizing IL-22 may successfully rejuvenate healing in individuals with advanced immune experiences.

IL-36α Enhances Host Defense against Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas.

The IL-36 cytokines are known to play various roles in mediating the immune response to infection in a tissue- and pathogen-dependent manner. The present study seeks to investigate the role of IL-36R signaling in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. IL-36α-/-, IL-36γ-/-, and IL-36R-/- mice had significantly more severe keratitis than wild-type mice. At six hours postinfection, IL-36α pretreatment augmented P. aeruginosa-induced expression of IL-1Ra, IL-36γ, LCN2, and S100A8/A9. At one day postinfection, exogenous IL-36α suppressed, whereas IL-36α deficiency promoted, the expression of IL-1β. At three days postinfection, exogenous IL-36α suppressed Th1 but promoted Th2 immune response. IL-36α stimulated the infiltration of IL-22-expressing immune cells, and IL-22 neutralization resulted in more severe keratitis. IL-36α alone stimulated dendritic cell infiltration in B6 mouse corneas. Taken together, our study suggests that IL-36R signaling plays a protective role in the pathogenesis of P. aeruginosa keratitis by promoting the innate immune defense, Th2, and/or Th22/IL-22 immune responses. Exogenous IL-36α might be a potential therapy for improving the outcome of P. aeruginosa keratitis.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

Amphiregulin alleviated concanavalin A-induced acute liver injury via IL-22

Objectives Amphiregulin (Areg), a glycoprotein from the epidermal growth factor receptor (EGFR) ligand family, has a well-documented protective role against tissue injury; however, its effects on immune-mediated liver injury are still unclear. Here, we used a concanavalin A (ConA)-induced acute liver hepatitis model to explore the effects of Areg on immune-mediated acute liver injury. Materials and methods Some C57BL/6 mice were administered ConA at a dose of 20 mg/kg (model mice), and some received 5 µg of Areg (treated mice). Then, their survival rates over 36 h were analyzed. After 5 h of treatment, liver function, hepatic histology, and apoptosis in liver tissue were investigated, and cytokine expression and neutrophil infiltration and activity in the liver were detected. Moreover, the protective effects of Areg were also evaluated without IL-22 in vivo. Results Our results showed that Areg administration increased acute liver failure (ALF) mouse survival, restored liver function, and alleviated liver damage. Interestingly, Areg administration increased IL-22 production in hepatic T cells and upregulated IL-22 concentrations in the serum and liver, whereas IL-22 neutralization completely abolished the therapeutic effect of Areg. Meanwhile, Areg administration was concomitant with increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, which are important in the hepatoprotective mechanism of IL-22. Conclusions Areg showed direct protective effects against ConA-induced acute liver injury, which suggests the potential therapeutic application of Areg in immune-mediated ALF.

Angelica sinensis polysaccharide attenuates CCl4-induced liver fibrosis via the IL-22/STAT3 pathway

Angelica sinensis polysaccharide (ASP) has hepatoprotective effects in liver injury models. However, its role and mechanism in chronic liver fibrosis have not been fully elucidated. In this study, a carbon tetrachloride (CCl4)-induced chronic liver fibrosis mouse model was established. The results showed that ASP treatment reduced serum alanine aminotransferase by approximately 50% and liver fibrosis areas by approximately 70%. Hepatic stellate cell (HSC) activation was inhibited in ASP-treated mice. Furthermore, the mechanism was studied in-depth, focusing on the interleukin 22/signal transducer and activator of transcription 3 (IL-22/STAT3) axis. Concentrations of 50 μg/ml and 100 μg/ml ASP induced the secretion of IL-22 in vitro, which further increased at a concentration of 200 μg/ml. Moreover, in vivo data showed that ASP significantly promoted IL-22 production in splenocytes and liver tissues. The antifibrotic effects of ASP were abolished after IL-22 neutralization. In addition, ASP activated the STAT3 pathway in the liver, as demonstrated by a 2-fold increase compared to that of the CCl4 group, which was abrogated by the IL-22 antibody. Subsequently, we showed that the antifibrotic effects of ASP were abrogated by blocking STAT3 with S3I-201. In conclusion, ASP effectively alleviates chronic liver fibrosis by inhibiting HSC activation through the IL-22/STAT3 pathway.

A46 THE INFLAMMATORY CASPASES COORDINATE MUCOSAL RESTRICTION OF SALMONELLA THROUGH THE EPITHELIAL-INTRINSIC INFLAMMASOME AND IL-22 DRIVEN MUCIN SECRETION

Abstract Background Intestinal epithelial cells (IECs) are located at the interface between the gut lumen and the underlying mucosal defense system. Here, they play a central role in the coordination of intestinal homeostasis, tempering pro-inflammatory responses but remaining rapidly responsive to noxious stimuli such as enteric pathogens. One early response mechanism by which IECs engage in immune defense is through the activation of the inflammasome which mobilizes the inflammatory caspases; caspase-1 and -11. Aims Here, we investigated the role of the inflammasome in overall mucosal defense against the enteric pathogen Salmonella enterica serovar Typhimurium. Methods Streptomycin-pretreated C57BL/6 (wildtype), Casp1/11 deficient (−/−), Casp1−/− and Casp11−/− mice were orally infected with Salmonella and burdens determined in intestinal tissues at 18h post infection (p.i.). Results Increased pathogen loads were observed for all caspase-deficient mice compared to wildtype, which correlated with increased IEC intracellular Salmonella burdens. Interestingly, despite increased bacterial loads, pathology scores for all inflammatory caspase mice were decreased, especially with regard to ‘IEC damage’ and ‘goblet cell loss’. To determine if the increased burdens were due to the loss of IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. This revealed significantly increased intracellular burdens in caspase-deficient monolayers as compared to wildtype, in concert with a marked decrease in IEC shedding and cell death. Peak inflammatory caspase activity was displayed in shedding wildtype IECs, suggesting the IEC-intrinsic inflammasome restricts Salmonella infection through infected IEC expulsion. The role of inflammasome signaling in acute mucosal defense was also examined. Wildtype tissue demonstrated a dramatic increase in mucus thickness (as evaluated by Muc2 immunostaining) and antimicrobial Reg3γ and β lectin transcript levels compared to caspase-deficient mice. Mucin release and Reg3 induction has been previously linked to IL-22, therefore we measured IL-22 expression and observed increased secretion in infected wildtype mice compared to Casp1/11−/−. This correlated with increased cecal infiltration of IL-22 producing ILC3 and NK T-cells. When infected mice were treated with IL-22 neutralizing antibody, this increased Salmonella burdens and decreased infection-induced mucus secretion, while no differences were observed in Casp1/11−/− treated with neutralizing antibody or isotype control. Conclusions Therefore the intestinal epithelium utilizes inflammasome signaling to coordinate multiple layers of innate defense at the gut mucosal surface to ultimately restrict enteric pathogen infections and their systemic spread. Funding Agencies CCC, CIHR, NRCUBC

Progress in mechanisms and immunological treatment of chronic mucocutaneous candidiasis associat-ed with congenital IL-17 pathway deficiency

Chronic mucocutaneous candidiasis (CMC) is a rare, persistent and recurrent infection affecting skin, nails, and oral and genital mucosae. It is mainly caused by Candida albicans and hard to be cured with routine antifungal therapy. Usually, CMC is a primary immunodeficiency disease and can be divided into two categories. The most common one is CMC disease (CMCD), which defined as Candida infection confined to the surface of the skin and mucous membranes and not complicated by systemic Candida albicans infection or other clinical symptoms. The other category is systemic CMC (SCMC) complicated by infections caused by other pathogens, systemic invasive fungal infections, or other clinical symptoms apart from the symptoms of CMCD. It is currently believed that both CMCD and SCMC are related to immunodeficiency caused by gene mutations related to IL-17 signal pathway. The inhibited Th17 proliferation, decreased secretion of IL-17 or IL-22 cytokine, or increased IL-17 or IL-22 neutralizing antibody induced by the mutations promoted the susceptibility to Candida or other pathogens. In the treatment of CMC, in addition to the traditional antifungal drugs such as azoles, polyenes and echinocandins, biological agents and target gene therapy offer potential new therapeutic strategies. This article reviewed the association between congenital immunodeficiency in the IL-17 signaling pathway and CMC, and the possible immunological therapeutic approaches and new therapeutic targets. Key words: Chronic mucocutaneous candidiasis; Candidia albicans; IL-17

Indole-3-carbinol prevents colitis and associated microbial dysbiosis in an IL-22-dependent manner.

Colitis, an inflammatory bowel disease, is caused by a variety of factors, but luminal microbiota are thought to play crucial roles in disease development and progression. Indole is produced by gut microbiota and is believed to protect the colon from inflammatory damage. In the current study, we investigated whether indole-3-carbinol (I3C), a naturally occurring plant product found in numerous cruciferous vegetables, can prevent colitis-associated microbial dysbiosis and attempted to identify the mechanisms. Treatment with I3C led to repressed colonic inflammation and prevention of microbial dysbiosis caused by colitis, increasing a subset of gram-positive bacteria known to produce butyrate. I3C was shown to increase production of butyrate, and when mice with colitis were treated with butyrate, there was reduced colonic inflammation accompanied by suppression of Th17 and induction of Tregs, protection of the mucus layer, and upregulation in Pparg expression. Additionally, IL-22 was increased only after I3C but not butyrate administration, and neutralization of IL-22 prevented the beneficial effects of I3C against colitis, as well as blocked I3C-mediated dysbiosis and butyrate induction. This study suggests that I3C attenuates colitis primarily through induction of IL-22, which leads to modulation of gut microbiota that promote antiinflammatory butyrate.

Interleukin-22 regulates interferon lambda expression in a mice model of pseudomonas aeruginosa pneumonia

BackgroundInterleukin (IL)-22 is a cytokine involved in tissue protection and repair following lung pathologies. Interferon (IFN)-λ cytokines displayed similar properties during viral infection and a synergy of action between these two players has been documented in the intestine. We hypothesize that during Pseudomonas aeruginosa challenge, IL-22 up-regulates IFN-λ and that IFN-λ exhibits protective functions during Pseudomonas aeruginosa acute pneumonia model in mice. MethodsUsing an in vitro human alveolar epithelial cell line A549, we assessed the ability of IL-22 to enhance IFN-λ expression during infection. IFN-λ protective function was evaluated in an acute mouse pneumonia model. ResultsWe first demonstrated in murine lungs that only type-II alveolar cells express IL-22 receptor and that IL-22 treatment of A549 cell line up-regulates IFN-λ expression. In a murine acute pneumonia model, IL-22 administration maintained significant IFN-λ levels in the broncho-alveolar fluids whereas IL-22 neutralization abolished IFN-λ up-regulation. In vivo administration of IFN-λ during Pseudomonas aeruginosa pneumonia improves mice outcome by dampening neutrophil recruitment and decreasing epithelium damages. DiscussionWe show here that IL-22 regulates IFN-λ levels during Pseudomonas aeruginosa pneumonia.

IL-22 Confers EGFR-TKI Resistance in NSCLC via the AKT and ERK Signaling Pathways

Background: The efficacy of an EGFR-targeted treatment strategy for non-small cell lung cancer (NSCLC) is reduced by drug resistance. IL-22 enhances tumor growth and induces chemotherapy resistance in human lung cancer cells. The present study elucidated the IL-22-induced mechanism underlying EGFR-tyrosine kinase inhibitor (TKI) resistance in NSCLC.Methods: The plasma and tissues of patients who received EGFR-TKIs were utilized to determine the association between IL-22 expression and gefitinib efficacy. The IL-22 effect on the EGFR/ERK/AKT pathways in NSCLC HCC827 and PC-9 cells was determined using the CCK-8 assay, western blot, and flow cytometric analysis. A PC-9 xenograft model of IL-22 exposure was established. Gefitinib was administered to mice in combination with IL-22 or vehicle.Results: We showed that IL-22 expression was higher in the EGFR-TKI-resistant group compared to EGFR-TKI-sensitive group. IL-22 expression was associated with EGFR-TKI efficacy in plasma. Additional treatment of IL-22 induced gefitinib resistance and reduced apoptosis in PC-9 and HCC827 cell lines. Furthermore, we showed that the effects of IL-22 attributed to p-ERK, p-EGFR, and p-AKT up-regulation. IL-22 neutralizing antibody completely abrogated the effects of IL-22 on apoptosis and AKT/EGFR/ERK signaling. Finally, we showed that IL-22 enhanced tumor growth and induced gefitinib resistance in the PC-9 xenograft model. Moreover, compared with gefitinib alone, the combination of IL-22 and gefitinib led to an increase in Ki67-positive staining and a reduction in TUNEL staining.Conclusions: Our findings indicate that IL-22 plays a role in tumor progression and EGFR-TKI resistance in NSCLC. Thus, IL-22 might serve as a novel biomarker to overcome resistance of EGFR-TKI.

IL-22-Independent Protection from Colitis in the Absence of Nkx2.3 Transcription Factor in Mice.

The transcription factor Nkx2.3 regulates the vascular specification of Peyer patches in mice through determining endothelial addressin preference and may function as a susceptibility factor in inflammatory bowel diseases in humans. We wished to analyze the role of Nkx2.3 in colonic solitary intestinal lymphoid tissue composition and in colitis pathogenesis. We studied the colonic solitary intestinal lymphoid tissue of Nkx2.3-deficient mice with immunofluorescence and flow cytometry. Colitis was induced in mice using 2.5% dextran sodium sulfate, and severity was assessed with histology, flow cytometry, and quantitative PCR. We found that the lack of Nkx2.3 impairs maturation of isolated lymphoid follicles and attenuates dextran sodium sulfate-induced colitis independent of endothelial absence of mucosal addressin cell-adhesion molecule-1 (MAdCAM-1), which was also coupled with enhanced colonic epithelial regeneration. Although we observed increased numbers of group 3 innate lymphoid cells and Th17 cells and enhanced transcription of IL-22, Ab-mediated neutralization of IL-22 did not abolish the protection from colitis in Nkx2.3-deficient mice. Nkx2.3-/- hematopoietic cells could not rescue wild-type mice from colitis. Using LacZ-Nkx2.3 reporter mice, we found that Nkx2.3 expression was restricted to VAP-1+ myofibroblast-like pericryptal cells. These results hint at a previously unknown stromal role of Nkx2.3 as driver of colitis and indicate that Nkx2.3+ stromal cells play a role in epithelial cell homeostasis.

IL-22 Preserves Gut Epithelial Integrity and Promotes Disease Remission during Chronic Salmonella Infection.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic Salmonella gastroenteritis model, Ab-mediated neutralization of IL-22 impaired intestinal epithelial barrier integrity and, consequently, exaggerated expression of proinflammatory cytokines. As disease normally resolved, neutralization of IL-22 caused luminal narrowing of the cecum-a feature reminiscent of fibrotic strictures seen in Crohn disease patients. Corresponding to the exaggerated immunopathology caused by IL-22 suppression, Salmonella burdens in the gut were reduced. This enhanced inflammation and pathogen clearance was associated with alterations in gut microbiome composition, including the overgrowth of Bacteroides acidifaciens Our findings thus indicate that IL-22 plays a protective role by limiting infection-induced gut immunopathology but can also lead to persistent pathogen colonization.

Th22 Cells Promote Osteoclast Differentiation via Production of IL-22 in Rheumatoid Arthritis.

T helper (Th) cells can differentiate into functionally distinct subsets and play a pivotal role in inflammatory and autoimmune diseases such as rheumatoid arthritis (RA). Th22 cells have been identified as a new subset secreting interleukin (IL)-22. Although elevated levels of IL-22 in the synovial fluids of RA patients were reported, its pathological roles remain unclear. Here, we demonstrated that IL-22 was characteristically produced from CD3+CD4+CC-chemokine receptor (CCR)4+CCR6+CCR10+ cells and their ability of the production of IL-22 markedly exceeded that of other Th subsets and the subset, thereby, designated Th22 cells. Th22 cells were efficiently induced by the stimulation with tumor necrosis factor-α, IL-6, and IL-1β. Th22 cells were markedly infiltrated in synovial tissue in patients with active RA, but not in patients with osteoarthritis (OA). CCL17, CCL20, and CCL28, which are chemokine ligands of CCR4, CCR6, and CCR10, respectively, were abundantly expressed in RA synovial tissue compared to OA. By in vitro Trans-well migration assay, Th22 cells efficiently migrated toward CCL28. Co-culture of Th22 cells, which were sorted from peripheral blood, with monocytes in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor (NF)-κB ligand induced osteoclasts formation more efficiently than that of either Th1 cells or Th17 cells. Furthermore, IL-22 markedly augmented osteoclast differentiation by promoting nuclear factor of activated T cells c1 expression in CD14+ monocytes. Contrarily, the addition of IFN-γ to the culture significantly decreased osteoclasts number, whereas IL-17 had marginal effects. IL-22 neutralizing antibody inhibited osteoclast formation in the co-culture of Th22 cells with CD14+ monocytes. Collectively, the results indicated that Th22 cells, which co-express chemokine receptors CCR4, CCR6, and CCR10, possess strong potency of tissue migration and accumulate into inflamed synovial tissues where the ligands such as CCL28 are highly expressed. Thus, Th22 cells have the capacity to promote osteoclast differentiation through production of IL-22 and thus play a pivotal role in bone destruction in patients with RA.

Limited Presence of IL-22 Binding Protein, a Natural IL-22 Inhibitor, Strengthens Psoriatic Skin Inflammation.

Psoriasis is a chronic inflammatory disease resulting from dysregulated immune activation associated with a large local secretion of cytokines. Among them, IL-22 largely contributes to epithelial remodeling and inflammation through inhibiting the terminal differentiation of keratinocytes and inducing antimicrobial peptides and selected chemokines. The activity of IL-22 is regulated by IL-22 binding protein (IL-22BP); however, the expression and role of IL-22BP in psoriatic skin has remained unknown so far. Here we showed that nonaffected skin of psoriasis patients displayed lower expression of IL-22BP than skin of healthy controls. Furthermore, the strong IL-22 increase in lesional psoriatic skin was accompanied by a moderate induction of IL-22BP. To investigate the role of IL-22BP in controlling IL-22 during skin inflammation, we used imiquimod-induced skin disease in rodents and showed that rats with genetic IL-22BP deficiency (Il22ra2-/-) displayed exacerbated disease that associated with enhanced expression of IL-22-inducible antimicrobial peptides. We further recapitulated these findings in mice injected with an anti-IL-22BP neutralizing Ab. Hypothesizing that the IL-22/IL-22BP expression ratio reflects the level of bioactive IL-22 in psoriasis skin, we found positive correlations with the expression of IL-22-inducible molecules (IL-20, IL-24, IL-36γ, CXCL1, and BD2) in keratinocytes. Finally, we observed that serum IL-22/IL-22BP protein ratio strongly correlated with psoriasis severity. In conclusion, we propose that although IL-22BP can control deleterious actions of IL-22 in the skin, its limited production prevents a sufficient neutralization of IL-22 and contributes to the development and maintenance of epidermal alterations in psoriasis.

IL-1R and IL-1RA differentially contribute to immunopathogenesis during fungal asthma

Abstract Severe asthma with fungal sensitization defines a subset of asthmatics with allergy to multiple fungal species, including Aspergillus fumigatus. We have previously shown genetic deficiency in or neutralization of IL-22 improves fungal asthma severity, which correlated with lower levels of IL-1α and IL-1β. Here, we show that bronchoalveolar lavage fluid (BALF) and induced sputum from human asthmatics sensitized to fungi demonstrate increased levels of IL-1α, IL-1β and IL-1 Receptor Antagonist (IL-1RA) compared to fungal negative asthmatics, suggesting that members of the IL-1 cytokine family may contribute to fungal asthma severity. To this end, we examined fungal asthma severity in mice deficient in IL-1R and IL-1RA with the hypothesis being that severity would be decreased and increased in these animals respectively. Surprisingly, the absence of IL-1R signaling had no impact on biomarkers of asthma severity (i.e. levels of proallergic CCL17, CCL22, and IL-33), yet Il1r−/−mice demonstrated better lung function compared to WT mice. Interestingly, the absence of IL-1R signaling resulted in a profound defect in the production of IL-22, suggesting a mechanism of improved lung function in Il1r−/− mice. In direct contrast, Il1rn−/− mice exhibited elevated proallergic biomarker levels, but no changes in the levels of IL-22. These preliminary results identify differential roles for IL-1R signaling (IL-22 mediated immunopathogenesis) and IL-1RA signaling (type 2 mediated immunopathogenesis) in fungal asthma severity.

Increased levels of the T-helper 22-associated cytokine (interleukin-22) and transcription factor (aryl hydrocarbon receptor) in patients with periodontitis are associated with osteoclast resorptive activity and severity of the disease.

Two new T-helper (Th) phenotypes have been recently described and named Th9 and Th22 lymphocytes; however, their role in the pathogenesis of periodontitis remains unclear. This study was aimed to assess whether Th9 and Th22 lymphocytes, through interleukin (IL)-9 and IL-22 production, respectively, are associated with the severity of periodontitis and bone resorption. Gingival crevicular fluid samples and biopsies were obtained from patients with moderate-to-advanced chronic periodontitis and gingivitis, and healthy controls. The levels for the Th9 and Th22-associated cytokines and master-switch transcription factors Spi-B and aryl hydrocarbon receptor (AhR) were quantified by enzyme-linked immunosorbent assay, real-time reverse-transcription quantitative polymerase chain reaction and flow cytometry. In addition, the osteoclast activity in response to tissue homogenates from periodontitis and healthy samples was analyzed quantifying the number of TRAP-positive cells and areas of bone resorption pits produced, in the presence or absence of recombinant human IL-22 and anti-IL-22 neutralization antibody. Higher levels of IL-22 and AhR were detected in patients with periodontitis compared with gingivitis and healthy individuals. In addition, higher levels of IL-9 and Spi-B were detected in gingivitis patients compared with periodontitis and healthy individuals. In patients with periodontitis, a significant positive correlation was detected between secreted levels of IL-22 and clinical attachment level of the sampled periodontal pockets. When osteoclasts were exposed to tissue homogenates obtained from patients with periodontitis, higher levels of resorptive activity were observed as compared with the same cells exposed to tissue homogenates obtained from healthy individuals, and this increment was dependent on the presence and neutralization of IL-22. Increased levels of IL-22 produced by Th22 lymphocytes are associated with the pathogenesis of periodontitis, in particular, with osteoclast resorptive activity and severity of disease.