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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

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., Activation of interleukin-17 (IL-17) receptor signaling within intestinal epithelial cells (IECs) promotes colorectal cancer development. Here, the authors show that miR-146a limits inflammation-induced colorectal carcinogenesis by inhibiting both IL-17 induction from myeloid cells and inhibiting IL-17R signaling within IECs.

Summary Myeloid suppressor cells promote tumor growth by a variety of mechanisms which are not fully characterized. We identified myeloid cells (MCs) expressing the latency-associated peptide (LAP) of TGF-β on their surface and LAPHi MCs that stimulate Foxp3+ Tregs while inhibiting effector T cell proliferation and function. Blocking TGF-β inhibits the tolerogenic ability of LAPHi MCs. Furthermore, adoptive transfer of LAPHi MCs promotes Treg accumulation and tumor growth in vivo. Conversely, anti-LAP antibody, which reduces LAPHi MCs, slows cancer progression. Single-cell RNA-Seq analysis on tumor-derived immune cells revealed LAPHi dominated cell subsets with distinct immunosuppressive signatures, including those with high levels of MHCII and PD-L1 genes. Analogous to mice, LAP is expressed on myeloid suppressor cells in humans, and these cells are increased in glioma patients. Thus, our results identify a previously unknown function by which LAPHi MCs promote tumor growth and offer therapeutic intervention to target these cells in cancer., Graphical abstract, Highlights • Several myeloid cell subsets express surface LAP • Myeloid cells that express surface LAP possess immunosuppressive properties • LAP expressing myeloid cells induce Tregs and inhibit effector T cell function • LAP expressing myeloid cells promote tumor growth, Immunology; Cancer

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.

MicroRNAs (miRNAs) are short, non-coding, single-stranded RNA molecules that play a critical role in regulating gene expression. miRNAs govern numerous immunological processes by regulating immune cell development, maturation, proliferation, activation, differentiation, and other functions that include cytokine and antibody secretion, and antigen presentation. Such regulation has been implicated in a variety of immune cells, not limited to T cells, B cells, dendritic cells and macrophages that are discussed in this chapter. Consequently, miRNAs have been identified as critical regulators of immune homeostasis and pathology. Growing evidence suggests the dysregulation of miRNAs within various immune cell subsets and related tissues is a crucial driver of autoimmunity. This chapter summarizes key studies investigating miRNAs in transgenic mice and preclinical autoimmune models, as well as in human samples from patients with autoimmune diseases. These studies have advanced our understanding of the role of miRNAs in regulating various immune cell subsets in specific autoimmune conditions, including multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Such compelling studies highlight the potential utility of miRNAs in the diagnosis and treatment of autoimmune diseases.

The role of TLR signaling in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is unclear. This role is especially controversial in models of adoptive transfer EAE in which no adjuvant and no TLR ligands are administered. We recently reported that a microbiome-derived TLR2 ligand, Lipid 654 (L654), is present in healthy human serum but significantly decreased in the serum of MS patients. This suggested that microbiome products that gain access to the systemic circulation, rather than being proinflammatory, may normally play an immune-regulatory role by maintaining a state of relative TLR tolerance. Therefore, a loss of microbiome-mediated TLR tolerance, as suggested by lower serum levels of L654, may play a role in the pathogenesis of MS. As proof of concept we asked whether administering low-level TLR2 ligands in adoptive transfer EAE induces TLR2 tolerance and attenuates disease. We administered low-level Pam2CSK4 or L654 to mice receiving encephalitogenic cells and in doing so induced both TLR2 tolerance and attenuation of EAE. Disease attenuation was accompanied in the CNS by a decrease in macrophage activation, a decrease in a specific proinflammatory macrophage population, and a decrease in Th17 cells. In addition, disease attenuation was associated with an increase in splenic type 1 regulatory T cells. Kinetic tolerance induction studies revealed a critical period for TLR2 involvement in adoptive transfer EAE. Overall, these results suggest that inducing TLR tolerance may offer a new approach to treating CNS autoimmune diseases such as MS.

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.

Summary The roles of the microbiome and innate immunity in the pathogenesis of multiple sclerosis (MS) remain unclear. We have previously documented abnormally low levels of a microbiome-derived Toll-like receptor (TLR)2-stimulating bacterial lipid in the blood of MS patients and postulated that this is indicative of a deficiency in the innate immune regulating function of the microbiome in MS. We postulated further that the resulting enhanced TLR2 responsiveness plays a critical role in the pathogenesis of MS. As proof-of-concept, we reported that decreasing systemic TLR2 responsiveness by administering very low-dose TLR2 ligands attenuated significantly the mouse model of MS, experimental autoimmune encephalomyelitis. Studies of Toll-like receptor responses in patients with MS have been conflicting. Importantly, most of these investigations have focused on the response to TLR4 ligation and few have characterized TLR2 responses in MS. In the present study, our goal was to characterize TLR2 responses of MS patients using multiple approaches. Studying a total of 26 MS patients and 32 healthy controls, we now document for the first time that a large fraction of MS patients (50%) demonstrate enhanced responsiveness to TLR2 stimulation. Interestingly, the enhanced TLR2 responders include a significant fraction of those with progressive forms of MS, a subset of patients considered unresponsive to adaptive immune system-targeting therapies. Our results suggest the presence of a pathologically relevant TLR2 related innate immune abnormality in patients with both relapsing–remitting and progressive MS. These findings may have significant implications for understanding the role of innate immunity in the pathogenesis of MS.

Vesicular stomatitis virus (VSV) has been extensively studied as a vaccine vector and oncolytic agent. Nevertheless, safety concerns have limited its widespread use in humans. The type III lambda interferon (IFN-λ) family of cytokines shares common signaling pathways with the IFN-α/β family and thus evokes similar antiviral activities. However, IFN-λ signals through a distinct receptor complex that is expressed in a cell type-specific manner, which restricts its activity to epithelial barriers, particularly those corresponding to the respiratory and gastrointestinal tracts. In this study, we determined how IFN-λ expression from recombinant VSV would influence vector replication, spread, and immunogenicity. We demonstrate that IFN-λ expression severely attenuates VSV in cell culture. In vivo , IFN-λ limits VSV replication in the mouse lung after intranasal administration and reduces virus spread to other organs. Despite this attenuation, however, the vector retains its capacity to induce protective CD8 T cell and antibody responses after a single immunization. These findings demonstrate a novel method of viral vector attenuation that could be used in both vaccine and oncolytic virus applications. IMPORTANCE Viruses such as VSV that are used as vaccine vectors can induce protective T cell and antibody responses after a single dose. Additionally, IFN-λ is a potent antiviral agent that has certain advantages for clinical use compared to IFN-α/β, such as fewer patient side effects. Here, we demonstrate that IFN-λ attenuates VSV replication and spread following intranasal virus delivery but does not reduce the ability of VSV to induce potent protective immune responses. These findings demonstrate that the type III IFN family may have widespread applicability for improving the safety and efficacy of viral vaccine and oncolytic vectors.

The total cellular lipids of Porphyromas gingivalis , a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis , called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro , both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2 −/− ) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2 −/− mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

The variable response to therapy in multiple sclerosis (MS) suggests a need for personalized approaches based on individual genetic differences. GWAS have linked CBLB gene polymorphisms with MS and recent evidence demonstrated that these polymorphisms can be associated with abnormalities in T cell function and response to interferon-β therapy. Cbl-b is an E3 ubiquitin ligase that regulates T cell activation and Cbl-b-deficient (Cbl-b(-/-)) mice show T cell abnormalities described in MS patients. We now show that Cbl-b(-/-) T cells demonstrate significant lymph node trafficking abnormalities. We thus asked whether the MS-approved drug, FTY720, postulated to trap T cells in lymphoid tissues, is less effective in the context of Cbl-b dysfunction. We now report that FTY720 significantly inhibits EAE in Cbl-b(-/-) mice. Our results newly document a role for Cbl-b in T cell trafficking but suggest nevertheless that MS patients with Cbl-b abnormalities may still be excellent candidates for FTY720 treatment.

TLR2 expression is enhanced in MS and EAE, but its role in these diseases is controversial. In adoptive transfer EAE, exogenous TLR ligands are not administered yet TLR2-deficient mice have been reported to develop attenuated disease. Despite this evidence for a disease-promoting function of TLR2, others have shown the opposite, i.e. that administration of TLR2 ligands can inhibit EAE in WT mice. To understand this paradox we tested the postulate that although TLR2 signaling may be EAE-promoting, repeated TLR2 ligand administration can lead to diminished TLR2 responsiveness (tolerance) and attenuated disease. To establish our approach, we administered Pam2Cys to SJL/J mice for 5 days and induced TLR2 tolerance that persisted for 4–5 days, reflected in decreased serum TNFα in response to a second TLR2 ligand (p In sum, we have used TLR tolerance as a novel probe to identify a narrow kinetic window in which TLR2 is required for EAE. Additionally, we have demonstrated the potential use of TLR tolerance as a new therapeutic approach in EAE and MS. Ongoing studies will characterize the relevant TLR2-expressing cells in EAE and the potential role of the microbiome in inducing homeostatic TLR tolerance that regulates autoimmunity.

The PD-L1/PD-1 pathway is a critical regulator of T cell responses that has become a significant focus in cancer immunotherapy. Cbl-b is an E3 ubiquitin ligase that regulates many aspects of T cell activation. In humans, polymorphisms in the CBLB gene are associated with autoimmunity. Cbl-b deficient (Cbl-b−/−) mice demonstrate spontaneous autoimmunity and enhanced anti-tumor T cell responses. Thus, there is now great interest in manipulating Cbl-b to enhance anti-tumor T cell responses. We now report that, in contrast to WT T cells, in vitro TCR-stimulated proliferative responses of Cbl-b−/− CD4+ and CD8+ T cells are not suppressed by a recombinant PD-L1 fusion protein (PD-L1 Ig) (% suppression in CD4+: WT 40%, Cbl-b 0%, p In sum, we report for the first time that Cbl-b−/− T cells are resistant in vitro and in vivo to suppression by PD-L1/PD-1. Our finding of Cbl-b−/− T cell resistance to PD-L1/PD-1-mediated inhibition broadens our understanding of Cbl-b’s role in autoimmunity and suggests a new mechanism by which manipulation of Cbl-b function may lead to enhanced anti-tumor T cell responses.

The role of the microbiome in multiple sclerosis (MS) remains unknown. We have identified a novel commensal bacteria-produced lipodipeptide, Lipid 654 (L654), which functions as a toll-like receptor 2 agonist. We reported that L654 can be recovered in healthy human serum and that levels are significantly lower in patients with MS. The purpose of this study was to understand the functional relevance of the decreased serum L654 level in MS by determining: 1) if L654 attenuates clinical disease in a transfer model of EAE, and 2) the mechanism by which L654 mediates this attenuation. L654 administration significantly attenuated clinical disease severity in an adoptive transfer model of EAE in SJL/J mice (p < 0.0001). Cellular analysis of mononuclear cells in the spinal cord at day 18 showed a significant decrease in total cell number (p = 0.02) as well as a decrease in percentage of IL-17 producing, IFNγ producing and IL-17/IFNγ double-producing CD4+ T cells in mice administered L654. At day 11, we found a decrease in total mononuclear cell number and an increase in percentage of CD39+ Tregs and IL-10 producing CD11b+ cells in mice administered L654. These results suggest that L654, normally present in human serum but at significantly lower levels in MS serum, has the ability to mediate an immunoregulatory and disease inhibitory effect on CNS autoimmunity. L654 may thus have a role both in the abnormal immunoregulation in MS and as a potential therapeutic in this disease.

FTY720 is a sphingosine-1-phosphate receptor 1 (S1P1) modulator used to treat Multiple Sclerosis (MS). While FTY720 is postulated to mediate its effect by enhancing T cell S1P1 internalization and degradation, the mechanisms underlying the therapeutic effect of FTY720 in MS are not fully understood. To further clarify the functional effects of FTY720 we utilized Cbl-b-deficient (Cbl-b-/-) mice. Cbl-b is an E3 ubiquitin ligase that regulates the PI3K-Akt pathway in T cells and its absence leads to resistance to regulatory T cells (Tregs) and multi-organ autoimmunity in mice. Given that MS has been associated with both SNPs in the CBLB gene and resistance to Tregs, Cbl-b-/- mice represent an important model for studying MS. We now report that Cbl-b-/- T cells demonstrate multiple functional abnormalities in S1P1. These include significantly less lymphopenia induced by the S1P lyase inhibitor, THI, (Cbl-b-/- blood CD4+ T cells decreased 19% vs 59% in wild type (WT) at 48 hrs), suggesting abnormal regulation of Cbl-b-/- T cell S1P1 in response to the endogenous ligand S1P. Paradoxically, Cbl-b-/- T cells demonstrate enhanced lymphopenia induced by the therapeutic agent FTY720 (Cbl-b-/- blood CD4+ T cells decreased 51% vs 26% in WT at 120 hrs). These results for the first time identify a complex role for Cbl-b in regulating expression and turnover of T cell S1P1 and suggest that SNPs in the CBLB gene may associate with a differential response to FTY720 in MS patients.

Multiple sclerosis (MS) is an autoimmune disease of unknown etiology. Infectious agents have been suggested to have a role as environmental factors in MS, but this concept remains controversial. Recently, gastrointestinal commensal bacteria have been implicated in the pathogenesis of autoimmune diseases, but mechanisms underlying the relationship of human systemic autoimmunity with the commensal microbiome have yet to be identified. Consistent with the lack of understanding of pathogenic mechanisms and relevant environmental factors in MS, no blood biomarkers have been identified that distinguish MS patients from healthy individuals. We recently identified a unique gastrointestinal and oral bacteria-derived lipodipeptide, Lipid 654, which is produced by commensal bacteria and functions as a human and mouse Toll-like receptor 2 ligand. Using multiple-reaction-monitoring mass spectrometry, a critical approach in targeted lipidomics, we now report that Lipid 654 can be recovered in the serum of healthy individuals. Most interestingly, we find that Lipid 654 is expressed at significantly lower levels in the serum of patients with MS compared with both healthy individuals and patients with Alzheimer's disease. These results thus identify for the first time a potential mechanism relating the gastrointestinal and oral commensal microbiome to a human systemic autoimmune disease. In addition, these results also identify a potential etiologic environmental factor and novel clinically relevant serum biomarker for MS.

Introduction Vaccines based on replicating viral vectors that express foreign antigens can generate superior durable antibody and memory CD8 T cell responses compared to inactivated virus or recombinant protein, with the added benefit of potential needle-free intranasal administration. Despite these significant advantages, safety concerns related to viral vectors as vaccine platforms can be an issue with this approach. Replicating viral vectors are perceived as less safe alternatives compared to other immunization modalities, especially in the context of intranasal administration, where the potential exists for systemic virus spread from the lung. The type III interferons (IL-29, IL-28A, IL-28B; also known as IFN-lambda1, 2, and 3) play a critical role in protecting epithelial cells in the lung and gut from virus infection, and can also stimulate adaptive immunity. We therefore examined the ability of this cytokine family to improve the safety and efficacy of a viral vector by limiting its systemic spread and vector-associated pathological effects after intranasal delivery. Methods Vesicular stomatitis virus (VSV) based vectors are promising candidates for use as vaccines and oncolytic agents. We generated recombinant VSV vectors expressing mouse IL-28A either from the fifth position or the more highly expressing first position of the viral genome, and measured virus replication in cell culture and mice. Results We found that IL-28A is properly expressed, secreted, and active when produced by VSV-infected cells. In cell culture, expression of IL-28A from VSV attenuated virus replication in type III IFN-sensitive cells, but not in type III IFN-resistant cells. Following intranasal inoculation in mice, spread of the VSV.IL28A vector from the lung to the lymph nodes, spleen, blood, liver, and brain was significantly reduced compared to recombinant wild-type VSV. Despite this attenuation, the VSV.mIL28A vector generated virus-specific CD8 T cell and antibody responses in mice that were comparable to wild-type VSV. Conclusion These studies demonstrate the ability of the type III interferon cytokine family to improve the safety and efficacy of a clinically promising viral vaccine vector. These results may potentially also inform the enhancement of VSV oncolytic activity by increasing selectivity of the virus for type III IFN-resistant tumor cells.