Dynamic chromatin accessibility licenses STAT5- and STAT6-dependent innate-like function of TH9 cells to promote allergic inflammation.

Allergic diseases are a major global health issue. Interleukin (IL)-9-producing helper T (TH9) cells promote allergic inflammation, yet TH9 cell effector functions are incompletely understood because their lineage instability makes them challenging to study. Here we found that resting TH9 cells produced IL-9 independently of T cell receptor (TCR) restimulation, due to STAT5- and STAT6-dependent bystander activation. This mechanism was seen in circulating cells from allergic patients and was restricted to recently activated cells. STAT5-dependent Il9/IL9 regulatory elements underwent remodeling over time, inactivating the locus. A broader 'allergic TH9' transcriptomic and epigenomic program was also unstable. In vivo, TH9 cells induced airway inflammation via TCR-independent, STAT-dependent mechanisms. In allergic patients, TH9 cell expansion was associated with responsiveness to JAK inhibitors. These findings suggest that TH9 cell instability is a negative checkpoint on bystander activation that breaks down in allergy and that JAK inhibitors should be considered for allergic patients with TH9 cell expansion.

IL-9 receptor signaling in memory B cells regulates humoral recall responses.

Memory B cells (Bmem cells) are the basis of long-lasting humoral immunity. They respond to re-encountered antigens by rapidly producing specific antibodies and forming germinal centers (GCs), a recall response that has been known for decades but remains poorly understood. We found that the receptor for the cytokine IL-9 (IL-9R) was induced selectively on Bmem cells after primary immunization and that IL-9R-deficient mice exhibited a normal primary antibody response but impaired recall antibody responses, with attenuated population expansion and plasma-cell differentiation of Bmem cells. In contrast, there was augmented GC formation, possibly due to defective downregulation of the ligand for the co-stimulatory receptor ICOS on Bmem cells. A fraction of Bmem cells produced IL-9. These findings indicate that IL-9R signaling in Bmem cells regulates humoral recall responses.

Germinal-center development of memory B cells driven by IL-9 from follicular helper T cells.

Germinal centers (GCs) support high-affinity, long-lived humoral immunity. How memory B cells develop in GCs is not clear. Through the use of a cell-cycle-reporting system, we identified GC-derived memory precursor cells (GC-MP cells) that had quit cycling and reached G0 phase while in the GC, exhibited memory-associated phenotypes with signs of affinity maturation and localized toward the GC border. After being transferred into adoptive hosts, GC-MP cells reconstituted a secondary response like genuine memory B cells. GC-MP cells expressed the interleukin 9 (IL-9) receptor and responded to IL-9. Acute treatment with IL-9 or antibody to IL-9 accelerated or retarded the positioning of GC-MP cells toward the GC edge and exit from the GC, and enhanced or inhibited the development of memory B cells, which required B cell-intrinsic responsiveness to IL-9. Follicular helper T cells (TFH cells) produced IL-9, and deletion of IL-9 from T cells or, more specifically, from GC TFH cells led to impaired memory formation of B cells. Therefore, the GC development of memory B cells is promoted by TFH cell-derived IL-9.

Retinoic Acid Receptor Alpha Represses a Th9 Transcriptional and Epigenomic Program to Reduce Allergic Pathology.

CD4+ T helper (Th) differentiation is regulated by diverse inputs, including the vitamin A metabolite retinoic acid (RA). RA acts through its receptor RARα to repress transcription of inflammatory cytokines, but is also essential for Th-mediated immunity, indicating complex effects of RA on Th specification and the outcome of the immune response. We examined the impact of RA on the genome-wide transcriptional response during Th differentiation to multiple subsets. RA effects were subset-selective and were most significant in Th9 cells. RA globally antagonized Th9-promoting transcription factors and inhibited Th9 differentiation. RA directly targeted the extended Il9 locus and broadly modified the Th9 epigenome through RARα. RA-RARα activity limited murine Th9-associated pulmonary inflammation, and human allergic inflammation was associated with reduced expression of RA target genes. Thus, repression of the Th9 program is a major function of RA-RARα signaling in Th differentiation, arguing for a role for RA in interleukin 9 (IL-9) related diseases.

The DNA-binding inhibitor Id3 regulates IL-9 production in CD4(+) T cells.

The molecular mechanisms by which signaling via transforming growth factor-ß (TGF-ß) and interleukin 4 (IL-4) control the differentiation of CD4(+) IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4(+) T cells. Mechanistically, TGF-ß1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4(+) T cells in vitro. Thus, our study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 differentiation.

The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells.

The TH9 subset of helper T cells was initially shown to contribute to the induction of autoimmune and allergic diseases, but subsequent evidence has suggested that these cells also exert antitumor activities. However, the molecular events that account for their effector properties are elusive. Here we found that the transcription factor IRF1 enhanced the effector function of TH9 cells and dictated their anticancer properties. Under TH9-skewing conditions, interleukin 1ß (IL-1ß) induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to the promoters of Il9 and Il21 and enhanced secretion of the cytokines IL-9 and IL-21 from TH9 cells. Furthermore, IL-1ß-induced TH9 cells exerted potent anticancer functions in an IRF1- and IL-21-dependent manner. Our findings thus identify IRF1 as a target for controlling the function of TH9 cells.

TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells.

The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely target for the treatment of chronic intestinal inflammation.

More Is Less: IL-9 in the Resolution of Inflammation.

In a recent study in Nature Medicine, Rauber et al. (2017) identify interleukin-9 (IL-9) derived from group 2 innate lymphoid cells as crucial regulators inducing resolution of chronic inflammation in rheumatoid arthritis. Their findings provide insight into the varied functions of IL-9 and open the door to novel therapeutic interventions.

Histone Deacetylase SIRT1 Negatively Regulates the Differentiation of Interleukin-9-Producing CD4(+) T Cells.

Distinct metabolic programs support the differentiation of CD4(+) T cells into separate functional subsets. In this study, we investigated metabolic mechanisms underlying the differentiation of IL-9-producing CD4(+) T cells (Th9) in allergic airway inflammation and cancerous tumors. We found that histone deacetylase SIRT1 negatively regulated Th9 cell differentiation. A deficiency of SIRT1 induced by either conditional deletion in mouse CD4(+) T cells or the use of small interfering RNA (siRNA) in mouse or human T cells increased IL-9 production, whereas ectopic SIRT1 expression inhibited it. Notably, SIRT1 inhibited Th9 cell differentiation that regulated anti-tumor immunity and allergic pulmonary inflammation. Glycolytic activation through the mTOR-hypoxia-inducible factor-1α (HIF1α) was required for the differentiation of Th9 cells that conferred protection against tumors and is involved in allergic airway inflammation. Our results define the essential features of SIRT1-mTOR-HIF1α signaling-coupled glycolytic pathway in inducing Th9 cell differentiation, with implications for metabolic reprogramming as an immunotherapeutic approach.

The World according to IL-9.

Among the cytokines regulating immune cells, IL-9 has gained considerable attention for its ability to act on multiple cell types as a regulator of beneficial and pathologic immune responses. Yet, it is still not clearly defined how IL-9 impacts immune responses. IL-9 demonstrates a remarkable degree of tissue-specific functionality and has cellular sources that vary by tissue site and the context of the inflammatory milieu. Here, we provide perspective to summarize the biological activities of IL-9 and highlight cell type-specific roles in the immune pathogenesis of diseases. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial and where it has the potential to complicate clinical outcomes.

Upregulated Expression of the IL-9 Receptor on TRAF3-Deficient B Lymphocytes Confers Ig Isotype Switching Responsiveness to IL-9 in the Presence of Antigen Receptor Engagement and IL-4.

The pleiotropic cytokine IL-9 signals to target cells by binding to a heterodimeric receptor consisting of the unique subunit IL-9R and the common subunit γ-chain shared by multiple cytokines of the γ-chain family. In the current study, we found that the expression of IL-9R was strikingly upregulated in mouse naive follicular B cells genetically deficient in TNFR-associated factor 3 (TRAF3), a critical regulator of B cell survival and function. The highly upregulated IL-9R on Traf3-/- follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3-/- B cells, which was not observed in littermate control B cells. We further demonstrated that blocking the JAK-STAT3 signaling pathway abrogated the enhancing effect of IL-9 on class switch recombination to IgG1 induced by BCR crosslinking plus IL-4 in Traf3-/- B cells. Our study thus revealed, to our knowledge, a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Taken together, our findings provide (to our knowledge) new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation such as autoimmune disorders.

ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Harnessing TH9 cells in cancer immunotherapy.

CD4 T cell effector subsets not only profoundly affect cancer progression, but recent evidence also underscores their critical contribution to the anticancer efficacy of immune checkpoint inhibitors. In 2012, the two seminal studies suggested the superior antimelanoma activity of TH9 cells over other T cell subsets upon adoptive T cell transfer. While these findings provided great impetus to investigate further the unique functions of TH9 cells and explore their relevance in cancer immunotherapy, the following questions still remain outstanding: are TH9 cell anticancer functions restricted to melanoma? What are the factors favouring TH9 cell effector functions? What is the contribution of TH9 cells to cancer immunotherapy treatments? Can TH9 cells be identified in humans and, if so, what is their clinical relevance? By reviewing the studies addressing these questions, we will discuss how TH9 cells could be therapeutically harnessed for cancer immunotherapy strategies.

Staphylococcus aureus-derived factors promote human Th9 cell polarization and enhance a transcriptional program associated with allergic inflammation.

T helper (Th) 9 cells, characterized by robust secretion of IL-9, have been increasingly associated with allergic diseases. However, whether and how Th9 cells are modulated by environmental stimuli remains poorly understood. In this study, we show that in vitro exposure of human PBMCs or isolated CD4 T-cells to Staphylococcus (S.) aureus-derived factors, including its toxins, potently enhances Th9 cell frequency and IL-9 secretion. Furthermore, as revealed by RNA sequencing analysis, S. aureus increases the expression of Th9-promoting factors at the transcriptional level, such as FOXO1, miR-155, and TNFRSF4. The addition of retinoic acid (RA) dampens the Th9 responses promoted by S. aureus and substantially changes the transcriptional program induced by this bacterium, while also altering the expression of genes associated with allergic inflammation. Together, our results demonstrate a strong influence of microbial and dietary factors on Th9 cell polarization, which may be important in the context of allergy development and treatment.

Interleukin-9 protects from microglia- and TNF-mediated synaptotoxicity in experimental multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. METHODS: Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated the effects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. RESULTS: We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability, reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking its synaptotoxic effects. CONCLUSIONS: The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable of interfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting the neurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of the central nervous system.

The many lives of IL-9: a question of survival?

Although the cytokine interleukin 9 (IL-9) was discovered decades ago, it remains one of the most enigmatic cytokines identified so far, in particular because its functional activities remain far from clear. Breakthroughs made through the use of IL-9 reporter mice have allowed the identification of cell types that produce IL-9 in vivo and, contrary to expectations based on previous results obtained in vitro, it is not T cells but instead a previously unknown type of innate lymphoid cell, called the 'ILC2 cell', that is the main cell type that expresses IL-9 in vivo. In this perspective, we put forward a hypothesis about the potential biological functions of IL-9 in the immune system and beyond.

OX40 signaling favors the induction of T(H)9 cells and airway inflammation.

The mechanisms that regulate the T(H)9 subset of helper T cells and diseases mediated by T(H)9 cells remain poorly defined. Here we found that the costimulatory receptor OX40 was a powerful inducer of T(H)9 cells in vitro and T(H)9 cell-dependent airway inflammation in vivo. In polarizing conditions based on transforming growth factor-ß (TGF-ß), ligation of OX40 inhibited the production of induced regulatory T cells and the T(H)17 subset of helper T cells and diverted CD4(+)Foxp3(-) T cells to a T(H)9 phenotype. Mechanistically, OX40 activated the ubiquitin ligase TRAF6, which triggered induction of the kinase NIK in CD4(+) T cells and the noncanonical transcription factor NF-κB pathway; this subsequently led to the generation of T(H)9 cells. Thus, our study identifies a previously unknown mechanism for the induction of T(H)9 cells and may have important clinical implications in allergic inflammation.

Oxytocin-induced increases in cytokines and clinical effect on the core social features of autism: Analyses of RCT datasets.

Although oxytocin may provide a novel therapeutics for the core features of autism spectrum disorder (ASD), previous results regarding the efficacy of repeated or higher dose oxytocin are controversial, and the underlying mechanisms remain unclear. The current study is aimed to clarify whether repeated oxytocin alter plasma cytokine levels in relation to clinical changes of autism social core feature. Here we analyzed cytokine concentrations using comprehensive proteomics of plasmas of 207 adult males with high-functioning ASD collected from two independent multi-center large-scale randomized controlled trials (RCTs): Testing effects of 4-week intranasal administrations of TTA-121 (A novel oxytocin spray with enhanced bioavailability: 3U, 6U, 10U, or 20U/day) and placebo in the crossover discovery RCT; 48U/day Syntocinon or placebo in the parallel-group verification RCT. Among the successfully quantified 17 cytokines, 4 weeks TTA-121 6U (the peak dose for clinical effects) significantly elevated IL-7 (9.74, 95 % confidence interval [CI] 3.59 to 15.90, False discovery rate corrected P (PFDR) < 0.001), IL-9 (56.64, 20.46 to 92.82, PFDR < 0.001) and MIP-1b (18.27, 4.96 to 31.57, PFDR < 0.001) compared with placebo. Inverted U-shape dose-response relationships peaking at TTA-121 6U were consistently observed for all these cytokines (IL-7: P < 0.001; IL-9: P < 0.001; MIP-1b: P = 0.002). Increased IL-7 and IL-9 in participants with ASD after 4 weeks TTA-121 6U administration compared with placebo was verified in the confirmatory analyses in the dataset before crossover (PFDR < 0.001). Furthermore, the changes in all these cytokines during 4 weeks of TTA-121 10U administration revealed associations with changes in reciprocity score, the original primary outcome, observed during the same period (IL-7: Coefficient = -0.05, -0.10 to 0.003, P = 0.067; IL-9: -0.01, -0.02 to -0.003, P = 0.005; MIP-1b: -0.02, -0.04 to -0.007, P = 0.005). These findings provide the first evidence for a role of interaction between oxytocin and neuroinflammation in the change of ASD core social features, and support the potential role of this interaction as a novel therapeutic seed. Trial registration: UMIN000015264, NCT03466671/UMIN000031412.

Licensed to Ill: IL-9 Generation in Immature Mast Cells Permits Food-Elicited Anaphylaxis.

Food-specific IgE is central to the pathobiology of food allergy, but not sufficient to induce disease. Chen et al. (2015) demonstrate that food-elicited reactions require an immature mast cell that generates IL-9 to induce its own maturation.