Discovery of orally efficacious RORγt inverse agonists. Part 2: Design, synthesis, and biological evaluation of novel tetrahydroisoquinoline derivatives.

A series of tetrahydroisoquinoline derivatives were designed, synthesized, and evaluated for their potential as novel orally efficacious retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of Th17-driven autoimmune diseases. We carried out cyclization of the phenylglycinamide core by structure-based drug design and successfully identified a tetrahydroisoquinoline carboxylic acid derivative 14 with good biochemical binding and cellular reporter activity. Interestingly, the combination of a carboxylic acid tether and a central fused bicyclic ring was crucial for optimizing PK properties, and the compound 14 showed significantly improved PK profile. Successive optimization of the carboxylate tether led to the discovery of compound 15 with increased inverse agonistic activity and an excellent PK profile. Oral treatment of mice with compound 15 robustly and dose-dependently inhibited IL-17A production in an IL23-induced gene expression assay.

TLR9-dependent IL-23/IL-17 is required for the generation of Stachybotrys chartarum-induced hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease that develops after repeated exposure to inhaled particulate Ag. Stachybotrys chartarum is a dimorphic fungus that has been implicated in a number of respiratory illnesses, including HP. In this study, we have developed a murine model of S. chartarum-induced HP that reproduces pathology observed in human HP, and we have hypothesized that TLR9-mediated IL-23 and IL-17 responses are required for the generation of granulomatous inflammation induced by inhaled S. chartarum. Mice that undergo i.p. sensitization and intratracheal challenge with 10(6) S. chartarum spores developed granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of T cells. S. chartarum sensitization and challenge resulted in robust pulmonary expression of IL-17 and IL-23. S. chartarum-mediated granulomatous inflammation required intact IL-23 or IL-17 responses and required TLR9, because TLR9(-/-) mice displayed reduced IL-17 and IL-23 expression in whole lung associated with decreased accumulation of IL-17 expressing CD4(+) and γδ T cells. Compared with S. chartarum-sensitized dendritic cells (DC) isolated from WT mice, DCs isolated from TLR9(-/-) mice had a reduced ability to produce IL-23 in responses to S. chartarum. Moreover, shRNA knockdown of IL-23 in DCs abolished IL-17 production from splenocytes in response to Ag challenge. Finally, the intratracheal reconstitution of IL-23 in TLR9(-/-) mice recapitulated the immunopathology observed in WT mice. In conclusion, our studies suggest that TLR9 is critical for the development of Th17-mediated granulomatous inflammation in the lung in response to S. chartarum.

Membrane-bound IL-12 and IL-23 serve as potent mucosal adjuvants when co-presented on whole inactivated influenza vaccines.

BACKGROUND: Potent and safe adjuvants are needed to improve the efficacy of parenteral and mucosal vaccines. Cytokines, chemokines and growth factors have all proven to be effective immunomodulatory adjuvants when administered with a variety of antigens. We have previously evaluated the efficacy of membrane-anchored interleukins (IL) such as IL-2 and IL-4 co-presented as Cytokine-bearing Influenza Vaccines (CYT-IVACs) using a mouse model of influenza challenge. FINDINGS: Here, we describe studies evaluating the parenteral and mucosal adjuvanticity of membrane-bound IL-12 and IL-23 CYT-IVACs in young adult mice. Mucosal immunization using IL-12 and IL-23 bearing whole influenza virus vaccine (WIV) was more effective at eliciting virus-specific nasal IgA and reducing viral lung burden following challenge compared to control WIV vaccinated animals. Both IL-12 and IL-23 bearing WIV elicited the highest anti-viral IgA levels in serum and nasal washes. CONCLUSIONS: This study highlights for the first time the mucosal adjuvant potential of IL-12 and IL-23 CYT-IVAC formulations in eliciting mucosal immune responses and reducing viral lung burden. The co-presentation of immunomodulators in direct context with viral antigen in whole inactivated viral vaccines may provide a means to significantly lower the dose of vaccine required for protection.

IL-23-mediated psoriasis-like epidermal hyperplasia is dependent on IL-17A.

IL-23 and Th17 cells producing IL-17A and IL-22 are found in excess in skin affected by psoriasis. Previous studies showed that IL-22, but not IL-17A, mediates psoriasis-like epidermal hyperplasia following recombinant murine (rm)IL-23 injections into skin. To further investigate the role of IL-17A, ears of mice were injected with rmIL-23. Investigators blinded to treatment conditions and mouse genotypes measured ear swelling, epidermal thickness, and cytokine expression. In wild-type (WT) mice, rmIL-23 induced ear swelling (p < 0.001, all p values versus saline), epidermal hyperplasia by histology (p < 0.001) and confocal microscopy (p < 0.004), and expression of both IL-17A and IL-22. As expected, rmIL-23 injections into IL-22(-/-) mice resulted in relatively little ear swelling (p < 0.09) and epidermal hyperplasia (p < 0.51 by histology and p < 0.75 by confocal microscopy). Notably, rmIL-23 injections into IL-17A(-/-) mice produced little ear swelling (p < 0.001, versus IL-23-injected WT mice) and epidermal hyperplasia (p < 0.001 by histology and p < 0.005 by confocal microscopy), even though IL-22 was readily induced in these mice. Furthermore, systemic delivery of blocking Abs directed against either IL-22 or IL-17A completely inhibited IL-23-induced epidermal hyperplasia in WT mice. These results demonstrate that IL-17A, like IL-22, is a downstream mediator for IL-23-induced changes in murine skin and that both of these Th17 cytokines are necessary to produce IL-23-mediated skin pathology. IL-17A may represent an attractive therapeutic target in individuals with psoriasis by blocking downstream effects of IL-23.

Epidermal CCR6+ γδ T cells are major producers of IL-22 and IL-17 in a murine model of psoriasiform dermatitis.

Cytokine components of Th17 pathway play vital roles in human psoriasis. Although much is known about TCR αß T cells in psoriasis, the role of unconventional T cells, including γδ T cells, is unclear. In this study, using an IL-23 skin injection model of psoriasiform dermatitis in mice, we demonstrate that IL-22, IL-17A, and the IL-23R were highly enriched in a population of CCR6(+), TCR γδ-low expressing (GDL) T cells that accumulated in the epidermis after IL-23 injections. GDL cells were distinct from resident TCR γδ-high, Vγ3(+),CCR6(-) T cells in the epidermis that did not change appreciably in numbers following IL-23 injection. Large numbers of CCR6(+) cells were detected at or above the level of the epidermal basement membrane by confocal microscopy 5 d after repeated IL-23 injections at the same time GDL cells increased in numbers in the epidermis. TCR δ-deficient mice (lacking γδ T cells) exhibited decreased ear swelling and downregulated expression of IL-22 and IL-17A in the epidermis following IL-23 injection. Our data suggest that a subset of γδ T cells play a critical role in IL-23-mediated psoriasiform dermatitis.

Regulation of the development of acute hepatitis by IL-23 through IL-22 and IL-17 production.

IL-23 plays a critical role in the expansion of highly proinflammatory Th17 cells secreting IL-17 and IL-22. Recently, we demonstrated that Notch signaling drives IL-22 secretion through the aryl hydrocarbon receptor (AHR) and plays a protective role in Con A-induced hepatitis. In this study, we investigated the role of IL-23 in hepatitis using IL-23p19- and IL-17-deficient mice. In WT mice, the injection of Con A induced the upregulation of various cytokines, which included IL-23, IL-22, IL-17, IFN-γ and TNF-α. In IL-23p19-deficient mice, exacerbated hepatitis was observed and serum IL-22 and IL-17 levels were greatly reduced, whereas in IL-17-deficient mice, ameliorated hepatitis was observed. The injection of exogenous IL-22 protected p19-deficient mice from hepatitis, whereas the injection of exogenous IL-23 significantly increased the serum levels of not only IL-22 but also IL-17, and less effectively protected against hepatitis in IL-17-dependent and -independent manners. Finally, it was revealed that STAT3, STAT4 and Notch contributed to the production of both the cytokines, and that the AHR was important only for IL-22 production in response to Con A and IL-23 in liver mononuclear cells. These results suggest that IL-23 plays a protective role in hepatitis through IL-22 production and also a pathological role via IL-17-dependent and -independent mechanisms.

Th17 cells in type 1 diabetes.

T1D is an autoimmune disorder, which involves the CD4(+) as well as CD8(+) T-cell-mediated destruction of ß cells. Recently, another population of T cells (Th17) is found to be involved in T1D pathology. This review will discuss the characteristics of Th17 cells and the mechanism of Th17-mediated T1D development. Th17 cell expansion is unstrained under T1D condition. Certain Treg cells are defective in T1D and lose the control of Th17 expansion. In addition, the altered function of APCs and a subset of monocytes which spontaneously secrete IL-1ß and IL-6 in T1D determine the abnormal expansion of Th17 as well. The pathogenic Th17 cells can cause the imbalance between Teff and Treg cells. Conversion from Th17 to Th1 phenotype and Th17 stimulated CTL responses may play an accessory role in T1D as well. Due to the effects of Th17 on T1D, therapeutic strategies designed to inhibit these cells are applicable and the positive effects are obvious. Taken together, Th17 may exert essential effects on the development of T1D. Identification of the underlying mechanism may inspire new viewpoints for the therapy of this disease.

Administration of IL-23 engages innate and adaptive immune mechanisms during fungal infection.

IL-23 is a key cytokine in promotion of chronic inflammation. Here, we address if its pro-inflammatory potential can be harnessed to protect against chronic cryptococcosis. Mice were infected with Cryptococcus neoformans and treated with recombinant IL-23. Administration of IL-23 led to prolonged survival and reduced fungal burden but was inferior to IL-12 treatment. Independent of endogenous IL-23/IL-12, IL-23 treatment induced an altered cytokine profile accompanied by marked changes in composition of the inflammatory infiltrate characterized by T cell and dendritic cell recruitment. Although IL-23 induced hallmarks of the T(h)17 pathway, also non-T cells produced IL-17A and IL-22. IL-23 treatment of T-cell-deficient mice resulted in increased IL-17A and IL-22 production and modulation of the cellular response at the site of infection with elevated expression of CD86 on macrophages. Our data show that IL-23 treatment induces innate and adaptive tissue inflammation with limited impact on resistance to chronic cryptococcosis.

FUT8 Remodeling of EGFR Regulates Epidermal Keratinocyte Proliferation during Psoriasis Development.

α-(1,6)-fucosyltransferase 8 (FUT8) is implicated in the pathogenesis of several malignancies, but its role in psoriasis is poorly understood. In this study, we show that FUT8 remodeling of EGFR plays a critical role in the development of psoriasis phenotypes. Notably, elevated FUT8 expression was associated with disease severity in the lesional epidermis of a patient with psoriasis. FUT8 gain of function promoted HaCaT cell proliferation, whereas short hairpin FUT8 reduced cell proliferation and induced a longer S phase with downregulation of cyclin A1 expression. Furthermore, cell proliferation, which is controlled by the activation of EGFR, was shown to be regulated by FUT8 core fucosylation of EGFR. Short hairpin FUT8 significantly reduced EGFR/protein kinase B signaling and slowed EGF‒EGFR complex trafficking to the perinuclear region. Moreover, short hairpin FUT8 reduced ligand-induced EGFR dimerization. Overactivated EGFR was observed in the lesional epidermis of both human patient and psoriasis-like mouse model, whereas conditional knockout of FUT8 in an IL-23 psoriasis-like mouse model ameliorated disease phenotypes and reduced EGFR activation in the epidermis. These findings implied that elevated FUT8 expression in the lesional epidermis is implicated in the development of psoriasis phenotypes, being required for EGFR overactivation and leading to keratinocyte hyperproliferation.

Galectin-8 Is Upregulated in Keratinocytes by IL-17A and Promotes Proliferation by Regulating Mitosis in Psoriasis.

Psoriasis is a chronic inflammatory skin disease that develops under the influence of the IL-23/T helper 17 cell axis and is characterized by intense inflammation and prominent epidermal hyperplasia. In this study, we demonstrate that galectin-8, a ß-galactoside‒binding lectin, is upregulated in the epidermis of human psoriatic skin lesions as well as in a mouse model of psoriasis induced by intradermal IL-23 injections and in IL-17A‒treated keratinocytes. We show that keratinocyte proliferation is less prominent in galectin-8‒knockout mice after intradermal IL-23 treatment than in wild-type mice. In addition, we show that galectin-8 levels in keratinocytes are positively correlated with the ability of the cells to proliferate and that transitioning from mitosis into G1 phase is delayed in galectin-8‒knockout HaCaT cells after cell-cycle synchronization and release. We demonstrate by immunofluorescence staining and immunoblotting the presence of galectin-8 within the mitotic apparatus. We reveal by coimmunoprecipitation and mass spectrometry analysis that α-tubulin interacts with galectin-8 during mitosis. Finally, we show that in the absence of galectin-8, pericentrin compactness is lessened and mitotic microtubule length is shortened, as demonstrated by immunofluorescence staining. We conclude that galectin-8 is upregulated in psoriasis and contributes to the hyperproliferation of keratinocytes by maintaining centrosome integrity during mitosis through interacting with α-tubulin.

ACT1 Is Required for Murine IL-23-Induced Psoriasiform Inflammation Potentially Independent of E3 Ligase Activity.

Psoriasis is a debilitating skin disease characterized by epidermal thickening, abnormal keratinocyte differentiation, and proinflammatory immune cell infiltrate into the affected skin. IL-17A plays a critical role in the etiology of psoriasis. ACT1, an intracellular adaptor protein and a putative ubiquitin E3 ligase, is essential for signal transduction downstream of the IL-17A receptor. Thus, IL-17A signaling in general, and ACT1 specifically, represent attractive targets for the treatment of psoriasis. We generated Act1 knockout and Act1 L286G knockin (ligase domain) mice to investigate the potential therapeutic effects of targeting ACT1 and its U-box domain, respectively. Act1 knockout, but not Act1 L286G knockin, mice were resistant to increases in CXCL1 plasma levels induced by subcutaneous injection of recombinant IL-17A. Moreover, in a mouse model of psoriasiform dermatitis induced by intradermal IL-23 injection, Act1 knockout, but not Act1 L286G knockin, was protective against increases in ear thickness, keratinocyte hyperproliferation, expression of genes for antimicrobial peptides and chemokines, and infiltration of monocytes and macrophages. Our studies highlight the critical contribution of ACT1 to proinflammatory skin changes mediated by the IL-23/IL-17 signaling axis and illustrate the need for further insight into ACT1 E3 ligase activity.

Suppression of IL-23-mediated psoriasis-like inflammation by regulatory B cells.

Psoriasis is an inflammatory cutaneous disease mediated by T-cell dependent immune responses; however, B cells are also considered to play an important role its development. Regulatory B cells (Bregs) regulate immune responses negatively through interleukin-10 (IL-10) production. This study aimed to investigate the role of Bregs in IL-23-mediated psoriasis-like inflammation in mice. Psoriasis-like inflammation was induced in B cell-specific phosphatase and tensin homolog (PTEN)-deficient mice, in which Bregs were significantly expanded, and in their controls, by intradermal injection of 20 µL phosphate-buffered saline (PBS) containing 0.5 µg rmIL-23 into one ear, every other day for 16 days. IL-23-mediated psoriasis-like inflammation was suppressed in B cell-specific PTEN-deficient mice along with decreased ear thickness and epidermal thickness on day 15. Moreover, adoptive transfer of B1 B cells suppressed IL-23-mediated psoriasis-like inflammation. rmIL-23-injected B cell-specific PTEN-deficient mice showed expanded regulatory T cells (Tregs) in the spleen and draining lymph nodes along with increased Bregs. Further, T helper (Th) 17 differentiation in the rmIL-23-injected ear was suppressed in B cell-specific PTEN-deficient mice. Overall, these results indicate that increased Bregs suppress IL-23-mediated psoriasis-like inflammation through Treg expansion and inhibition of Th17 differentiation. Thus, targeting Bregs may be a feasible treatment strategy for psoriasis.

Prostaglandin E2 synergistically with interleukin-23 favors human Th17 expansion.

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.

Aryl Hydrocarbon Receptor in Cutaneous Vascular Endothelial Cells Restricts Psoriasis Development by Negatively Regulating Neutrophil Recruitment.

Vascular endothelial cells (VECs) that line the interiors of blood vessels participate in physiological and inflammatory processes. All skin cell types express the aryl hydrocarbon receptor (AhR), which is involved in the pathogenesis of psoriasis. However, the role of the cutaneous VEC AhR in the pathogenesis of psoriasis remains elusive. In the present study, we found that AhR protein expression and activation were downregulated in psoriatic VECs. Furthermore, cutaneous VEC-specific AhR-knockout (AhRcVECs-KO) mice were established. Using imiquimod and IL-23-induced psoriasis models, we found that skin inflammation was exacerbated with excessive neutrophil recruitment in AhRcVECs-KO mice. Furthermore, neutrophil neutralization alleviates exacerbated inflammation in imiquimod-treated AhRcVECs-KO mice. In addition, cutaneous VECs in AhRcVECs-KO mice exhibited increased dilation and activation compared with those in control mice. Finally, AhR-deficient microvascular endothelial cells stimulated by proinflammatory cytokines showed increased ICAM-1 expression in vivo and in vitro, which may have facilitated neutrophil recruitment. In summary, our study demonstrates that AhR in dermal VECs restricts psoriasis development by negatively regulating neutrophil recruitment, thereby providing insight into the pathogenesis of psoriasis.

GPR15 is not critically involved in the regulation of murine psoriasiform dermatitis.

BACKGROUND: GPR15 has been implicated in the pathogenesis of T cell-driven inflammation of the skin and the gut. Expression levels of the GPR15 ligand GPR15 L are increased in psoriatic skin and considered as potential biomarker for the treatment response to anti-IL-17 antibody therapies. However, the significance of the GPR15 L/GPR15 for the pathogenesis of psoriasis and the mechanisms regulating GPR15 L expression are still elusive. OBJECTIVE: To determine the significance of GPR15 signaling in mouse models of psoriasis. METHODS: We addressed the role of the GPR15 L/GPR15 in the Aldara™-induced psoriasiform dermatitis (AIPD) and the IL-23-induced dermatitis model. In both models, we charted the expression levels of GPR15 L in the skin and assessed the significance of GPR15 L/GPR15 by examining Gpr15-/- mice. RESULTS: GPR15 L levels were increased in the AIPD, but not in the IL-23-induced dermatitis model. Deficiency in Gpr15 did not alter the course of disease neither in the AIPD, nor in the IL-23-induced dermatitis model. In neither model, deficiency in Gpr15 modulated disease on the histopathological or the molecular level. Despite the induction of GPR15 L in the AIPD model, GPR15+ cells did not accumulate in the skin. CONCLUSION: GPR15 L expression is induced in psoriasiform dermatitis, but the activation of the IL-23/IL-17 axis alone is not sufficient for its induction. This restricts the potential use of GPR15 L levels as biomarker for the treatment response to anti-IL-17 antibody therapy. Our results leave a significant role of GPR15 in the pathogenesis of psoriasiform dermatitis rather unlikely. Hence, GPR15 L probably modulates psoriasiform dermatitis via GPR15-independent pathways.

Anti-interleukin-12 and anti-interleukin-23 agents in Crohn's disease.

INTRODUCTION: Blockers of IL-12/23, as well as specific blockers of IL-23, have been investigated as options for medical therapy in inflammatory bowel disease. These biological agents include ustekinumab - the first agent of this pharmacological class which has shown clinical efficacy in psoriasis, psoriatic arthritis, and moderate-to-severe Crohn's disease (CD) - and other monoclonal antibodies under investigation, including brazikumab, risankizumab, mirikizumab, and guselkumab. AREAS COVERED: This review will focus on the rationale of the blockade of IL-12/23 axis in CD, efficacy and safety data of ustekinumab derived from randomized controlled trials and real-life observational studies, and the preliminary data of the highly promising selective IL-23 inhibitors. EXPERT OPINION: Data from literature have demonstrated that ustekinumab holds the potential to deserve a relevant role in the management of patients with CD thanks to several properties, including the fast onset of action, the long duration of efficacy, the favorable safety profile, the systemic anti-inflammatory effect, and the peculiar way of administration. Nonetheless, additional research is warranted to determine the real value of ustekinumab, as current data are not able to answer all the questions about its effectiveness in real-life practice, and the external validity of the available results is not absolute.

IL-23 induces regulatory T cell plasticity with implications for inflammatory skin diseases.

Foxp3+ regulatory T cells (Tregs) represent a major fraction of skin resident T cells. Although normally protective, Tregs have been shown to produce pro-inflammatory cytokines in human diseases, including psoriasis. A significant hurdle in the Treg field has been the identification, or development, of model systems to study this Treg plasticity. To overcome this gap, we analyzed skin resident Tregs in a mouse model of IL-23 mediated psoriasiform dermatitis. Our results demonstrate that IL-23 drove the accumulation of Tregs; including a subpopulation that co-expressed RORγt and produced IL-17A. Genesis of this population was attenuated by a RORγt inverse agonist compound and clinically relevant therapeutics. In vitro, IL-23 drove the generation of CD4+Foxp3+RORγt+IL-17A+ cells from Treg cells. Collectively, our data shows that IL-23 drives Treg plasticity by inducing a population of CD4+Foxp3+RORγt+IL-17A+ cells that could play a role in the disease pathogenesis. Through this work, we define an in vitro system and a pre-clinical in vivo mouse model that can be used to further study Treg homeostasis and plasticity in the context of psoriasis.

Neutralization of IL-17C Reduces Skin Inflammation in Mouse Models of Psoriasis and Atopic Dermatitis.

IL-17C is a functionally distinct member of the IL-17 family that was believed to play a role in the pathogenesis of psoriasis. Here we confirmed that IL-17C is involved in psoriasis and explored potential roles for IL-17C in atopic dermatitis (AD). An anti-IL-17C antibody, MOR106, was generated that potently and selectively binds to human and mouse IL-17C, thereby inhibiting the binding of IL-17C to its IL-17RE receptor. The antibody inhibited cutaneous inflammation in an IL-23-induced psoriatic-like skin inflammation model. In lesional skin of patients with AD, IL-17C expression levels were increased and localized to keratinocytes and infiltrating immune cells. To determine the contribution of IL-17C to AD pathogenesis, MOR106 was tested in two distinct in vivo models. In the calcipotriol-induced AD model, ear skin inflammation, TSLP, and IL-33 protein production in ears was suppressed by MOR106. Consistently, in the flaky tail strain mouse model, spontaneous development of AD-like skin inflammation was reduced by MOR106. Moreover, serum IgE levels, number of mast cells in skin and T helper type 2-related cytokines IL-4 and CCL17 in serum were all reduced. Overall, our results indicate that IL-17C is a central mediator of skin inflammation beyond psoriasis and is relevant in particular in AD.

Continuous IL-23 stimulation drives ILC3 depletion in the upper GI tract and, in combination with TNFα, induces robust activation and a phenotypic switch of ILC3.

Mutations in the Interleukin (IL)-23/IL-23 receptor loci are associated with increased inflammatory bowel disease (IBD) susceptibility, and IL-23 neutralization has shown efficacy in early clinical trials. To better understand how an excess of IL-23 affects the gastrointestinal tract, we investigated chronic systemic IL-23 exposure in healthy wildtype mice. As expected, IL-23 exposure resulted in early activation of intestinal type 3 innate lymphoid cells (ILC3), followed by infiltration of activated RORγt+ T helper cells. Surprisingly, however, sustained IL-23 stimulus also dramatically reduced classical ILC3 populations within the proximal small intestine, and a phenotypically distinct T-bet expressing ILC3 population emerged. TNFα neutralization, a widely used IBD therapy, reduced several aspects of the IL-23 driven ILC3 response, suggesting a synergy between IL-23 and TNFα in ILC3 activation. In vitro studies supported these findings, revealing previously unappreciated effects of IL-23 and TNFα within the intestine.