Bacterial sepsis remains a devastating clinical problem. Here, we describe a protective role for the recently discovered acid-sensitive, proton-activated chloride channel, PACC1 (PAC/ASOR/TMEM206), during sepsis. Initially, we found PACC1 was enriched in healthy human and mouse mononuclear phagocytes, particularly macrophages, and differentially regulated by inflammatory stimuli, suggesting PACC1 involvement in innate immunity. To further investigate, we generated de novo Pacc1 knockout (-/-) mice, which presented without major immunologic abnormalities at baseline. Compared to wild-type (WT), Pacc1-/- myeloid cells showed normal phagocytic uptake of acid-insensitive Escherichia coli BioParticles, but impaired development of the acidifying phagolysosome using acid-sensitive E. coli BioParticles. Transcriptomic profiling of Pacc1-/- macrophages revealed dysregulated phagolysosomal and cytokine networks (e.g., interferons). Because phagolysosomal bacterial clearance is essential to resolve infection, we challenged Pacc1-/- mice with intraperitoneal gram-negative E. coli sepsis. Pacc1-/- mice displayed increased bacterial burden, immune cell infiltration, inflammation, and lethality. In contrast, phagocytosis-independent E. coli lipopolysaccharide (LPS)-induced endotoxemia yielded comparable WT and Pacc1-/- survival, as well as similar inflammatory responses. Finally, we engineered Pacc1-floxed (fl/fl) mice crossed with a myeloid lineage Cre-deleter strain to interrogate myeloid cell-intrinsic PACC1 in vivo. Consistent with a predominate role for PACC1 during phagocytosis and bacterial clearance in these cells, LysM-Cre/Pacc1fl/fl mice exhibited impaired E. coli sepsis survival but indifferent endotoxemia phenotypes. In conclusion, PACC1 links sterilizing phagolysosomal activity with immune networks in sepsis pathobiology.

IFN-β, a type I interferon, has been used as a first-line therapy for patients with multiple sclerosis (MS) for more than 30 years; however, the cellular and molecular basis of its therapeutic efficacy remains unclear. Here, we first used experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, to show that the therapeutic effects of IFN-β were associated with a down-regulation of microRNA-21 (miR-21) and pathogenic TH17 (pTH17) cells. In vitro experiments demonstrated that genetic knockout of miR-21 directly inhibited pathogenic TH17 cell differentiation. Further mechanistic investigations revealed that miR-21 promoted pathogenic TH17 differentiation by inhibiting the transcription factor Forkhead box protein O1 (Foxo1). Accordingly, miR-21 loss abrogated pathogenic TH17 differentiation and conferred resistance to EAE. Treatment of T cell monocultures with IFN-β showed that IFN-β did not directly limit miR-21 expression. Instead, IFN-β treatment inhibited the secretion of miR-21-inducing cytokines from myeloid cells, reduced miR-21 induction within cocultured T cells, and inhibited pathogenic TH17 development. In patient samples, immunophenotypic and targeted transcriptomic analyses revealed that compared with IFN-β treatment responders, nonresponders expressed elevated miR-21-inducing cytokines within myeloid cells, alongside increased miR-21 and pathogenic TH17 cytokines within CD4+ T cells. Direct miR-21 inhibition reduced pathogenic TH17 differentiation in nonresponder CD4+ T cells. These results suggest that type I IFN signaling limits central nervous system autoimmunity by inhibiting miR-21-mediated pathogenic TH17 development. miR-21 inhibition may be of potential therapeutic value specifically for the IFN-β nonresponder cohort.

In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided.

Transforming growth factor β (TGF-β) is critical to the maintenance of intestinal immune homeostasis. Here, we present techniques for analyzing Smad molecules downstream of TGF-β receptor signaling in dextran-sulfate-sodium-induced colitic mice. We describe colitis induction, cell isolation, and flow cytometric cell sorting of dendritic cells and T cells. We then detail intracellular staining of phosphorylated Smad2/3 and western blotting analysis of Smad7. This protocol can be performed on a limited number of cells from many sources. For complete details on the use and execution of this protocol, please refer to Garo et al.1.

A disequilibrium between immunosuppressive Tregs and inflammatory IL-17–producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, the molecular mechanisms underlying the Treg and Th17 imbalance in CNS autoimmunity remain largely unclear. Identifying the factors that drive this imbalance is of high clinical interest. Here, we report a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. miR-92a was elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuated EAE. Mechanistically, miR-92a mediated EAE susceptibility in a T cell–intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses, by directly repressing the transcription factor Foxo1. Although miR-92a did not directly alter Th1 differentiation, it appeared to indirectly promote Th1 cells by inhibiting Treg responses. Correspondingly, miR-92a inhibitor therapy ameliorated EAE by concomitantly boosting Treg responses and dampening inflammatory T cell responses. Analogous to our findings in mice, miR-92a was elevated in CD4+ T cells from patients with MS, and miR-92a silencing in patients’ T cells promoted Treg development but limited Th17 differentiation. Together, our results demonstrate that miR-92a drives CNS autoimmunity by sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.

A disequilibrium between immunosuppressive regulatory T cells (Tregs) and inflammatory interleukin (IL)-17-producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, molecular mechanisms underlying Treg and Th17 imbalance in the central nervous system (CNS) autoimmunity remain largely unclear and thus identifying factors which drive this imbalance is of high clinical interest. Recently, we found a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. MiR-92a is elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuates EAE. Mechanistically, miR-92a mediates EAE susceptibility in a T cell-intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses by directly repressing the transcription factor, Foxo1. Correspondingly, miR-92a inhibitor therapy ameliorates EAE by modulating the balance between Tregs and Th17 cells. Analogous to mice, miR-92a is elevated in MS patient CD4+ T cells, and miR-92a silencing in patient T cells promotes Treg development while limiting Th17 differentiation. Together, our results identify a previously unknown function by which miR-92a drives CNS autoimmunity via sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS. Supported by NIH R01 R01AI127853 NMSS RG-1507-05164

IL-27 is a heterodimeric IL-12 family cytokine formed by noncovalent association of the promiscuous EBI3 subunit and selective p28 subunit. IL-27 is produced by mononuclear phagocytes and unfolds pleiotropic immune-modulatory functions through ligation to IL-27 receptor α (IL-27RA). Although IL-27 is known to contribute to immunity and to limit inflammation after various infections, its relevance for host defense against multicellular parasites is still poorly defined. Here, we investigated the role of IL-27 during infection with the soil-transmitted hookworm, Nippostrongylus brasiliensis, in its early host intrapulmonary life cycle. IL-27(p28) was detectable in bronchoalveolar lavage fluid of C57BL/6J wild-type mice on day 1 after s.c. inoculation. IL-27RA expression was most abundant on lung-invading γδ T cells. Il27ra-/- mice showed increased lung parasite burden together with aggravated pulmonary hemorrhage and higher alveolar total protein leakage as a surrogate for epithelial-vascular barrier disruption. Conversely, injections of recombinant mouse (rm)IL-27 into wild-type mice reduced lung injury and parasite burden. In multiplex screens, higher airway accumulations of IL-6, TNF-α, and MCP-3 (CCL7) were observed in Il27ra-/- mice, whereas rmIL-27 treatment showed a reciprocal effect. Importantly, γδ T cell numbers in airways were enhanced by endogenous or administered IL-27. Further analysis revealed a direct antihelminthic function of IL-27 on γδ T cells as adoptive intratracheal transfer of rmIL-27-treated γδ T cells during primary N. brasiliensis lung infection conferred protection in mice. In summary, this report demonstrates protective functions of IL-27 to control the early lung larval stage of hookworm infection.

Interleukin-17 (IL-17) is a major inflammatory cytokine implicated in colorectal cancer (CRC) development. However, the mechanisms that control tumorigenic IL-17 signaling are poorly understood. Recently, expression changes and polymorphisms in the small non-coding RNA, microRNA-146a (miR-146a), have been associated with clinical outcomes in inflammatory bowel disease and CRC patients. Here, we identified a novel role for miR-146a as a major negative regulator of colonic inflammation and tumorigenesis via modulation of IL-17 responses. MiR-146a-deficient mice are susceptible to both colitis-associated and sporadic CRC, and present with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, an intermediate in NOD2 signaling, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17 levels. Accordingly, myeloid cell-specific deletion of miR-146a leads to CRC susceptibility. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an intermediate in IL-17R signaling, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, an enzyme for PGE2 synthesis. IEC-specific deletion of miR-146a confers marked CRC susceptibility. Importantly, preclinical administration of miR-146a mimic or direct inhibition of miR-146a targets, TRAF6/RIPK2 can ameliorate CRC. In conclusion, miR-146a prevents CRC by two interlinked mechanisms: 1) by limiting myeloid cell-mediated IL-17 production; and 2) by inhibiting tumorigenic IL-17R signaling in IECs. Overexpression of miR-146a may be a promising therapeutic approach for CRC to limit multiple pathways converging on tumorigenic IL-17 signaling.

Chronic inflammation can drive tumor development. Here, we have identified microRNA-146a (miR-146a) as a major negative regulator of colonic inflammation and associated tumorigenesis by modulating IL-17 responses. MiR-146a-deficient mice are susceptible to both colitis-associated and sporadic colorectal cancer (CRC), presenting with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, a NOD2 signaling intermediate, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17. Accordingly, myeloid-specific miR-146a deletion promotes CRC. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an IL-17R signaling intermediate, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, a PGE2 synthesis enzyme. IEC-specific miR-146a deletion therefore promotes CRC. Importantly, preclinical administration of miR-146a mimic, or small molecule inhibition of the miR-146a targets, TRAF6 and RIPK2, ameliorates colonic inflammation and CRC. MiR-146a overexpression or miR-146a target inhibition represent therapeutic approaches that limit pathways converging on tumorigenic IL-17 signaling in CRC.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are mediated by dysregulated autoreactive T cell responses in the central nervous system (CNS). This dysregulation consists of an imbalance between inflammatory T helper cells, such as Th17 cells, and Foxp3+ T regulatory cells (Tregs). MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to play a key role in immune function, and to be dysregulated in EAE and MS. However, identifying specific miRNA pathways that directly connect clinical activity with immune mechanisms in CNS autoimmunity has been a challenge. Previous work has identified miR-92a as one of the most significantly elevated miRNAs in the sera of MS patients, which positively correlates with neurological symptoms and brain atrophy. We now report a major functional role for miR-92a in CNS autoimmunity. MiR-92a is increased during EAE, and its loss strikingly attenuates clinical disease. This attenuation is accompanied by reduced Th17 and increased Treg cells in the CNS. Mechanistically, we found that T cell-intrinsic miR-92a inhibits the development and function of Treg cells while promoting those of Th17 cells by targeting Foxo1, a key transcription factor in T helper cell biology. Preclinical administration of miR-92a inhibitor phenocopies miR-92a loss and ameliorates EAE. Analogous to mice, miR-92a is significantly elevated in MS patient T cells, and reciprocally regulates human Treg and Th17 differentiation. These findings suggest miR-92a skews the Treg/Th17 balance to promote CNS autoimmunity, and that miR-92a silencing may be of therapeutic benefit for MS patients.

Programmed death 1 (PD1) has emerged as a major inhibitor of antitumor T cells, and anti-PD1 therapies have demonstrated clinical efficacy in multiple cancers. However, the impact of PD1 on other immune cells had remained unclear. A recent study by Strauss et al. describes how myeloid cell-intrinsic PD1 signaling limits myelopoiesis in cancer pertinent to anti-PD1 therapies.

Interleukin-17 (IL-17) is a major inflammatory cytokine implicated in colorectal cancer (CRC) development. However, the mechanisms that control tumorigenic IL-17 signaling remain unclear. Recently, expression changes and polymorphisms in the small non-coding RNA, microRNA-146a (miR-146a), have been associated with clinical outcomes in inflammatory bowel disease and CRC patients. Here, we identified a novel role for miR-146a as a major negative regulator of colonic inflammation and tumorigenesis via modulation of IL-17 responses. MiR-146a-deficient mice are highly susceptible to both colitis-associated and sporadic CRC, and present with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, an intermediate in NOD2 signaling, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17 levels. Accordingly, myeloid cell-specific deletion of miR-146a leads to CRC susceptibility. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an intermediate in IL-17R signaling, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, an enzyme for PGE2 synthesis. IEC-specific deletion of miR-146a therefore confers marked CRC susceptibility. Importantly, preclinical administration of miR-146a mimic can ameliorate colonic inflammation and CRC. In conclusion, miR-146a prevents CRC by two interlinked mechanisms: 1) by limiting myeloid cell-mediated inflammatory IL-17 production; and 2) by inhibiting tumorigenic IL-17R signaling in IECs. Overexpression of miR-146a may be a promising therapeutic approach for CRC to limit multiple pathways converging on tumorigenic IL-17 signaling.

Inflammation in the central nervous system (CNS) has been linked to demyelination and remyelination. Using zebrafish and mouse models of demyelination and remyelination, Cunha et al. now describe a novel role for myeloid differentiation factor 88 (MyD88) signaling in supporting remyelination by promoting myeloid cell-mediated inflammatory responses via TNF-α, which are essential for phagocytic myelin debris clearance and for oligodendrogenesis.

Smad7, a negative regulator of TGF-β signaling, has been implicated in the pathogenesis and treatment of inflammatory bowel diseases (IBDs), including Crohn's disease (CD) and ulcerative colitis (UC). Here, we found that Smad7 mediates intestinal inflammation by limiting the PDL2/1-PD1 axis in dendritic cells (DCs) and CD4+T cells. Smad7 deficiency in DCs promotes TGF-β responsiveness and the co-inhibitory molecules PDL2/1 on DCs, and it further imprints T cell-PD1 signaling to promote Treg differentiation. DC-specific Smad7 deletion mitigates DSS-induced colitis by inducing CD103+PDL2/1+DCs and Tregs. In addition, Smad7 deficiency in CD4+T cells promotes PD1 and PD1-induced Tregs in vitro. The transfer of Smad7-deficient CD4+T cells enhances Tregs in vivo and protects against T cell-mediated colitis. Furthermore, Smad7 antisense ameliorates DSS-induced UC, increasing TGF-β and PDL2/1-PD1 signaling. Enhancing PD1 signaling directly via Fc-fused PDL2/1 is also beneficial. Our results identify how Smad7 mediates intestinal inflammation and leverages these pathways therapeutically, providing additional strategies for IBD intervention.

MicroRNAs (miRNAs) are a family of small noncoding RNAs that regulate gene expression at the posttranscriptional level. miRNAs have diverse expression patterns and regulate various physiological processes, including immune system development and function. Altered expression of specific miRNAs in immune cells has been associated with several inflammatory disorders and autoimmunity. Emerging evidence indicates that miRNAs can modulate epigenetic pathways, and that the production of miRNAs can in turn be modulated by epigenetic mechanisms. This chapter discusses the association between miRNAs and epigenetic pathways in the regulation of T helper cell differentiation and function.

IL-9-producing Th9 cells are a novel subset of T helper cells that develop independently of other T helper subsets. Th9 cells have been implicated in the pathogenesis of allergic asthma and autoimmunity, while also serving as critical effector T cells in mediating antitumor immune responses. Concomitant presence of TGF-β and IL-4 lead to the differentiation of naïve CD4+ T cells towards the Th9 phenotype. In addition, several cytokines, including IL-1β, IL-2, IL-25, and IL-33, further amplify Th9 responses. Negative regulators of Th9 cells include other cytokines such as IFN-γ, IL-23, and IL-27. Here, we describe a detailed protocol for the analysis of STAT molecules involved in the differentiation of Th9 cells and Th9 inhibition by IL-27.

Anti-LAP antibody induces antitumor immunity by affecting both adaptive and innate immune mechanisms.