Treg specialization and functions beyond immune suppression.

The actions of the immune system are finely tuned, involving complex communication and coordination between diverse immune and non-immune cells across the tissues of the body. A healthy immune system requires a precise balance between immunity and tolerance. Regulatory T cells (Tregs) have long been appreciated as one of the master regulators of this balance; their importance is underscored by the autoimmunity that develops in mice and humans when Tregs are missing or dysfunctional. In addition to the immunoregulatory roles of Tregs in suppressing autoimmunity and inflammation via control of adaptive and innate immune responses, several non-immune modulatory functions of Tregs have been identified in recent years. In this review, we have highlighted the growing literature on the action of Tregs in metabolism, stem cell maintenance, tissue repair, and angiogenesis. Alongside Tregs' immune suppressive role, these non-suppressive activities comprise a key function of Tregs in regulating health and disease. As Tregs receive increasing attention as therapeutic targets, understanding their non-canonical functions may become an important feature of Treg-directed interventions.

RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota.

Lymphoid cells that express the nuclear hormone receptor RORγt are involved in containment of the large intestinal microbiota and defense against pathogens through the production of interleukin 17 (IL-17) and IL-22. They include adaptive IL-17-producing helper T cells (T(H)17 cells), as well as innate lymphoid cells (ILCs) such as lymphoid tissue-inducer (LTi) cells and IL-22-producing NKp46+ cells. Here we show that in contrast to T(H)17 cells, both types of RORγt+ ILCs constitutively produced most of the intestinal IL-22 and that the symbiotic microbiota repressed this function through epithelial expression of IL-25. This function was greater in the absence of adaptive immunity and was fully restored and required after epithelial damage, which demonstrates a central role for RORγt+ ILCs in intestinal homeostasis. Our data identify a finely tuned equilibrium among intestinal symbionts, adaptive immunity and RORγt+ ILCs.

Identification of quinazoline based inhibitors of IRAK4 for the treatment of inflammation.

Interleukin-1 receptor associated kinase 4 (IRAK4) has been implicated in IL-1R and TLR based signaling. Therefore selective inhibition of the kinase activity of this protein represents an attractive target for the treatment of inflammatory diseases. Medicinal chemistry optimization of high throughput screening (HTS) hits with the help of structure based drug design led to the identification of orally-bioavailable quinazoline based IRAK4 inhibitors with excellent pharmacokinetic profile and kinase selectivity. These highly selective IRAK4 compounds show activity in vivo via oral dosing in a TLR7 driven model of inflammation.

IL25 elicits a multipotent progenitor cell population that promotes T(H)2 cytokine responses.

CD4(+) T helper 2 (T(H)2) cells secrete interleukin (IL)4, IL5 and IL13, and are required for immunity to gastrointestinal helminth infections. However, T(H)2 cells also promote chronic inflammation associated with asthma and allergic disorders. The non-haematopoietic-cell-derived cytokines thymic stromal lymphopoietin, IL33 and IL25 (also known as IL17E) have been implicated in inducing T(H)2 cell-dependent inflammation at mucosal sites, but how these cytokines influence innate immune responses remains poorly defined. Here we show that IL25, a member of the IL17 cytokine family, promotes the accumulation of a lineage-negative (Lin(-)) multipotent progenitor (MPP) cell population in the gut-associated lymphoid tissue that promotes T(H)2 cytokine responses. The IL25-elicited cell population, termed MPP(type2) cells, was defined by the expression of Sca-1 (also known as Ly6a) and intermediate expression of c-Kit (c-Kit(int)), and exhibited multipotent capacity, giving rise to cells of monocyte/macrophage and granulocyte lineages both in vitro and in vivo. Progeny of MPP(type2) cells were competent antigen presenting cells, and adoptive transfer of MPP(type2) cells could promote T(H)2 cytokine responses and confer protective immunity to helminth infection in normally susceptible Il25(-/-) mice. The ability of IL25 to induce the emergence of an MPP(type2) cell population identifies a link between the IL17 cytokine family and extramedullary haematopoiesis, and suggests a previously unrecognized innate immune pathway that promotes T(H)2 cytokine responses at mucosal sites.

IL-25 exhibits disparate roles during Th2-cell differentiation versus effector function.

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL-25 has been shown to enhance Th2-type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL-25(-/-) C57BL/6 mice with G4 IL-4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL-4-independent Th2-cell differentiation. These assays were used to determine whether IL-25 was critical for the formation of Th2 cells. We found there was no physiological role for IL-25 in either the differentiation of Th2 cells or their development to effector or memory Th2-cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL-25 and its proposed role in promoting Th2-cell responses.

Identification of N-(1H-pyrazol-4-yl)carboxamide inhibitors of interleukin-1 receptor associated kinase 4: Bicyclic core modifications.

IRAK4 plays a critical role in the IL-1R and TLR signalling, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of permeable N-(1H-pyrazol-4-yl)carboxamides was developed by introducing lipophilic bicyclic cores in place of the polar pyrazolopyrimidine core of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides. Replacement of the pyrazolo[1,5-a]pyrimidine core with the pyrrolo[2,1-f][1,2,4]triazine, the pyrrolo[1,2-b]pyridazine, and thieno[2,3-b]pyrazine cores guided by cLogD led to the identification of highly permeable IRAK4 inhibitors with excellent potency and kinase selectivity.

IL-25 regulates Th17 function in autoimmune inflammation.

Interleukin (IL)-25 is a member of the IL-17 family of cytokines. However, unlike the other members of this family, IL-25 promotes T helper (Th) 2 responses. We now show that IL-25 also regulates the development of autoimmune inflammation mediated by IL-17-producing T cells. We have generated IL-25-deficient (il25-/-) mice and found that they are highly susceptible to experimental autoimmune encephalomyelitis (EAE). The accelerated disease in the il25-/- mice is associated with an increase of IL-23 in the periphery and a subsequent increase in the number of inflammatory IL-17-, IFNgamma-, and TNF-producing T cells that invade the central nervous system. Neutralization of IL-17 but not IFNgamma in il25-/- mice prevented EAE, suggesting that IL-17 is a major disease-promoting factor. IL-25 treatment at several time points during a relapse-remitting model or chronic model of EAE completely suppressed disease. IL-25 treatment induced elevated production of IL-13, which is required for suppression of Th17 responses by direct inhibition of IL-23, IL-1beta, and IL-6 expression in activated dendritic cells. Thus, IL-25 and IL-17, being members of the same cytokine family, play opposing roles in the pathogenesis of organ-specific autoimmunity.

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.

Development of Gut-Selective Pan-Janus Kinase Inhibitor TD-1473 for Ulcerative Colitis: A Translational Medicine Programme.

BACKGROUND AND AIMS: Oral systemic pan-Janus kinase [JAK] inhibition is effective for ulcerative colitis [UC] but is limited by toxicities. We describe preclinical to clinical translation of TD-1473-an oral gut-selective pan-JAK inhibitor-from in vitro characterization through a Phase 1b study in patients with UC. METHODS: TD-1473 JAK inhibition potency was evaluated in vitro; plasma pharmacokinetics, safety and efficacy were assessed in mice. In a first-time-in-human study, plasma pharmacokinetics and safety were assessed after single and multiple [14 days] ascending doses administered orally to healthy subjects. The Phase 1b study randomized patients with moderately to severely active UC to receive once-daily oral TD-1473 20, 80 or 270 mg, or placebo for 28 days. Plasma and colonic tissue concentrations were measured; safety was assessed; and efficacy was evaluated by UC clinical parameters, disease-surrogate biomarkers, endoscopy, histology and colonic tissue JAK signalling. RESULTS: TD-1473 exhibited potent pan-JAK inhibitory activity in vitro. Oral TD-1473 administration to mice achieved high, biologically active colonic tissue concentrations with low plasma exposure and decreased oxazolone-induced colitis activity without reducing blood cell counts vs placebo. TD-1473 administration in healthy human subjects and patients with UC yielded low plasma exposure and was generally well tolerated; treatment in patients with UC resulted in biologically active colonic tissue concentrations and descriptive trends toward reduced clinical, endoscopic and histological disease activity vs placebo. CONCLUSION: Gut-selective pan-JAK inhibition with TD-1473 administration resulted in high intestinal vs plasma drug exposure, local target engagement, and trends toward reduced UC disease activity. [Clinicaltrials.gov NCT02657122, NCT02818686].

IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6.

Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10-deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases.

The NLRP3 inflammasome mediates DSS-induced intestinal inflammation in Nod2 knockout mice.

Crohn's disease (CD) is a chronic disorder of the gastrointestinal tract characterized by inflammation and intestinal epithelial injury. Loss of function mutations in the intracellular bacterial sensor NOD2 are major risk factors for the development of CD. In the absence of robust bacterial recognition by NOD2 an inflammatory cascade is initiated through alternative PRRs leading to CD. In the present study, MCC950, a specific small molecule inhibitor of NLR pyrin domain-containing protein 3 (NLRP3), abrogated dextran sodium sulfate (DSS)-induced intestinal inflammation in Nod2-/- mice. NLRP3 inflammasome formation was observed at a higher rate in NOD2-deficient small intestinal lamina propria cells after insult by DSS. NLRP3 complex formation led to an increase in IL-1ß secretion in both the small intestine and colon of Nod2ko mice. This increase in IL-1ß secretion in the intestine was attenuated by MCC950 leading to decreased disease severity in Nod2ko mice. Our work suggests that NLRP3 inflammasome activation may be a key driver of intestinal inflammation in the absence of functional NOD2. NLRP3 pathway inhibition can prevent intestinal inflammation in the absence of robust NOD2 signaling.

Intestinally-restricted Janus Kinase inhibition: a potential approach to maximize the therapeutic index in inflammatory bowel disease therapy.

BACKGROUND: An unmet need remains for safe and effective treatments to induce and maintain remission in inflammatory bowel disease (IBD) patients. The Janus kinase (JAK) inhibitor, tofacitinib, has demonstrated robust efficacy in ulcerative colitis patients although, like other systemic immunosuppressants, there may be safety concerns associated with its use. This preclinical study evaluated whether modulating intestinal inflammation via local JAK inhibition can provide efficacy without systemic immunosuppression. METHODS: The influence of tofacitinib, dosed orally or intracecally, on oxazolone-induced colitis, oxazolone or interferon-γ (IFNγ)-induced elevation of colonic phosphorylated signal transducer and activator of transcription1 (pSTAT1) levels, and basal splenic natural killer (NK) cell counts was investigated in mice. RESULTS: Tofacitinib, dosed orally or intracecally, inhibited, with similar efficacy, oxazolone-induced colitis, represented by improvements in the disease activity index and its sub-scores (body weight, stool consistency and blood content). Intracecal dosing of tofacitinib resulted in a higher colon:plasma drug exposure ratio compared to oral dosing. At equieffective oral and intracecal doses, colonic levels of tofacitinib were similar, while the plasma levels for the latter were markedly lower, consistent with a lack of effect on splenic NK cell counts. Tofacitinib, dosed orally, intracecally, or applied to the colonic lumen in vitro, produced dose-dependent, and maximal inhibition of oxazolone or IFNγ-induced STAT1 phosphorylation in the colon. CONCLUSIONS: Localized colonic JAK inhibition, by intracecal delivery of tofacitinib, provides colonic target engagement and efficacy in a mouse colitis model at doses which do not impact splenic NK cell counts. Intestinal targeting of JAK may permit separation of local anti-inflammatory activity from systemic immunosuppression, and thus provide a larger therapeutic index compared to systemic JAK inhibitors.

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system.

While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the 'immune fingerprint' of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.

Discovery of 5-Amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide Inhibitors of IRAK4.

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing.

IL-10 elicits IFNγ-dependent tumor immune surveillance.

Tumor immune surveillance and cancer immunotherapies are thought to depend on the intratumoral infiltration of activated CD8(+) T cells. Intratumoral CD8(+) T cells are rare and lack activity. IL-10 is thought to contribute to the underlying immune suppressive microenvironment. Defying those expectations we demonstrate that IL-10 induces several essential mechanisms for effective antitumor immune surveillance: infiltration and activation of intratumoral tumor-specific cytotoxic CD8(+) T cells, expression of the Th1 cytokine interferon-γ (IFNγ) and granzymes in CD8(+) T cells, and intratumoral antigen presentation molecules. Consequently, tumor immune surveillance is weakened in mice deficient for IL-10 whereas transgenic overexpression of IL-10 protects mice from carcinogenesis. Treatment with pegylated IL-10 restores tumor-specific intratumoral CD8(+) T cell function and controls tumor growth.

Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation.

The C-type lectin-like receptor CD161, which has recently been described to promote T cell expansion, is expressed on a discrete subset of human CD4 T cells. The function of such cells, however, has remained elusive. We now demonstrate that CD161(+) CD4 T cells comprise a circulating and gut-resident T helper 17 (Th17) cell population. During Crohn's disease (CD), these CD161(+) cells display an activated Th17 phenotype, as indicated by increased expression of interleukin (IL)-17, IL-22, and IL-23 receptor. CD161(+) CD4 T cells from CD patients readily produce IL-17 and interferon gamma upon stimulation with IL-23, whereas, in healthy subjects, priming by additional inflammatory stimuli such as IL-1beta was required to enable IL-23-induced cytokine release. Circulating CD161(+) Th17 cells are imprinted for gut homing, as indicated by high levels of CC chemokine receptor 6 and integrin beta7 expression. Supporting their colitogenic phenotype, CD161(+) Th17 cells were found in increased numbers in the inflammatory infiltrate of CD lesions and induced expression of inflammatory mediators by intestinal cells. Our data identify CD161(+) CD4 T cells as a resting Th17 pool that can be activated by IL-23 and mediate destructive tissue inflammation.

IL-23 enhances the inflammatory cell response in Cryptococcus neoformans infection and induces a cytokine pattern distinct from IL-12.

IL-23, a heterodimeric cytokine composed of the p40 subunit of IL-12 and a novel p19 subunit, has been shown to be a key player in models of autoimmune chronic inflammation. To investigate the role of IL-23 in host resistance during chronic fungal infection, wild-type, IL-12- (IL-12p35-/-), IL-23- (IL-23p19-/-), and IL-12/IL-23- (p40-deficient) deficient mice on a C57BL/6 background were infected with Cryptococcus neoformans. Following infection, p40-deficient mice demonstrated higher mortality than IL-12p35-/- mice. Reconstitution of p40-deficient mice with rIL-23 prolonged their survival to levels similar to IL-12p35-/- mice. IL-23p19-/- mice showed a moderately reduced survival time and delayed fungal clearance in the liver. Although IFN-gamma production was similar in wild-type and IL-23p19-/- mice, production of IL-17 was strongly impaired in the latter. IL-23p19-/- mice produced fewer hepatic granulomata relative to organ burden and showed defective recruitment of mononuclear cells to the brain. Moreover, activation of microglia cells and expression of IL-1beta, IL-6, and MCP-1 in the brain was impaired. These results show that IL-23 complements the more dominant role of IL-12 in protection against a chronic fungal infection by an enhanced inflammatory cell response and distinct cytokine regulation.