The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis.

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Regulation and functions of the IL-10 family of cytokines in inflammation and disease.

The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29. Evolutionarily, IL-10 family cytokines emerged before the adaptive immune response. These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation. Finally, IL-10 itself can repress proinflammatory responses and limit unnecessary tissue disruptions caused by inflammation. Thus, IL-10 family cytokines have indispensable functions in many infectious and inflammatory diseases.

IL-10 Signaling Remodels Adipose Chromatin Architecture to Limit Thermogenesis and Energy Expenditure.

Signaling pathways that promote adipose tissue thermogenesis are well characterized, but the limiters of energy expenditure are largely unknown. Here, we show that ablation of the anti-inflammatory cytokine IL-10 improves insulin sensitivity, protects against diet-induced obesity, and elicits the browning of white adipose tissue. Mechanistic studies define bone marrow cells as the source of the IL-10 signal and adipocytes as the target cell type mediating these effects. IL-10 receptor alpha is highly enriched in mature adipocytes and is induced in response to differentiation, obesity, and aging. Assay for transposase-accessible chromatin sequencing (ATAC-seq), ChIP-seq, and RNA-seq reveal that IL-10 represses the transcription of thermogenic genes in adipocytes by altering chromatin accessibility and inhibiting ATF and C/EBPß recruitment to key enhancer regions. These findings expand our understanding of the relationship between inflammatory signaling pathways and adipose tissue function and provide insight into the physiological control of thermogenesis that could inform future therapy.

Hard CiDRE revs up macrophage infectivity in COVID-19.

IL-10 has been linked to COVID-19 severity, but whether it is causal is unclear. In this issue of Immunity, Mitsui et al. find that a specific single-nucleotide polymorphism (SNP) results in expression of an IL10RB-IFNAR2 fusion protein that intensifies IL-10-mediated induction of ACE2 expression and viral infectivity of macrophages.

Expression of the readthrough transcript CiDRE in alveolar macrophages boosts SARS-CoV-2 susceptibility and promotes COVID-19 severity.

Lung infection during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin-I-converting enzyme 2 (ACE2) receptor induces a cytokine storm. However, the precise mechanisms involved in severe COVID-19 pneumonia are unknown. Here, we showed that interleukin-10 (IL-10) induced the expression of ACE2 in normal alveolar macrophages, causing them to become vectors for SARS-CoV-2. The inhibition of this system in hamster models attenuated SARS-CoV-2 pathogenicity. Genome-wide association and quantitative trait locus analyses identified a IFNAR2-IL10RB readthrough transcript, COVID-19 infectivity-enhancing dual receptor (CiDRE), which was highly expressed in patients harboring COVID-19 risk variants at the IFNAR2 locus. We showed that CiDRE exerted synergistic effects via the IL-10-ACE2 axis in alveolar macrophages and functioned as a decoy receptor for type I interferons. Collectively, our data show that high IL-10 and CiDRE expression are potential risk factors for severe COVID-19. Thus, IL-10R and CiDRE inhibitors might be useful COVID-19 therapies.

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation.

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.

Regulatory Innate Lymphoid Cells Control Innate Intestinal Inflammation.

An emerging family of innate lymphoid cells (termed ILCs) has an essential role in the initiation and regulation of inflammation. However, it is still unclear how ILCs are regulated in the duration of intestinal inflammation. Here, we identify a regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and harbors a unique gene identity that is distinct from that of ILCs or regulatory T cells (Tregs). During inflammatory stimulation, ILCregs can be induced in the intestine and suppress the activation of ILC1s and ILC3s via secretion of IL-10, leading to protection against innate intestinal inflammation. Moreover, TGF-ß1 is induced by ILCregs during the innate intestinal inflammation, and autocrine TGF-ß1 sustains the maintenance and expansion of ILCregs. Therefore, ILCregs play an inhibitory role in the innate immune response, favoring the resolution of intestinal inflammation.

IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

Interleukin-10 Prevents Pathological Microglia Hyperactivation following Peripheral Endotoxin Challenge.

Microglia, the resident macrophages of the brain parenchyma, are key players in central nervous system (CNS) development, homeostasis, and disorders. Distinct brain pathologies seem associated with discrete microglia activation modules. How microglia regain quiescence following challenges remains less understood. Here, we explored the role of the interleukin-10 (IL-10) axis in restoring murine microglia homeostasis following a peripheral endotoxin challenge. Specifically, we show that lipopolysaccharide (LPS)-challenged mice harboring IL-10 receptor-deficient microglia displayed neuronal impairment and succumbed to fatal sickness. Addition of a microglial tumor necrosis factor (TNF) deficiency rescued these animals, suggesting a microglia-based circuit driving pathology. Single cell transcriptome analysis revealed various IL-10 producing immune cells in the CNS, including most prominently Ly49D+ NK cells and neutrophils, but not microglia. Collectively, we define kinetics of the microglia response to peripheral endotoxin challenge, including their activation and robust silencing, and highlight the critical role of non-microglial IL-10 in preventing deleterious microglia hyperactivation.

Apoptosis in response to microbial infection induces autoreactive TH17 cells.

Microbial infections often precede the onset of autoimmunity. How infections trigger autoimmunity remains poorly understood. We investigated the possibility that infection might create conditions that allow the stimulatory presentation of self peptides themselves and that this might suffice to elicit autoreactive T cell responses that lead to autoimmunity. Self-reactive CD4(+) T cells are major drivers of autoimmune disease, but their activation is normally prevented through regulatory mechanisms that limit the immunostimulatory presentation of self antigens. Here we found that the apoptosis of infected host cells enabled the presentation of self antigens by major histocompatibility complex class II molecules in an inflammatory context. This was sufficient for the generation of an autoreactive TH17 subset of helper T cells, prominently associated with autoimmune disease. Once induced, the self-reactive TH17 cells promoted auto-inflammation and autoantibody generation. Our findings have implications for how infections precipitate autoimmunity.

IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation.

Cytokines are among the most important effector and messenger molecules in the immune system. They profoundly participate in immune responses during infection and inflammation, protecting against or contributing to diseases such as allergy, autoimmunity, and cancer. Manipulating cytokine pathways, therefore, is one of the most effective strategies to treat various diseases. IL-10 family cytokines exert essential functions to maintain tissue homeostasis during infection and inflammation through restriction of excessive inflammatory responses, upregulation of innate immunity, and promotion of tissue repairing mechanisms. Their important functions in diseases are supported by data from many preclinical models, human genetic studies, and clinical interventions. Despite significant efforts, however, there is still no clinically approved therapy through manipulating IL-10 family cytokines. Here, we summarize the recent progress in understanding the biology of this family of cytokines, suggesting more specific strategies to maneuver these cytokines for the effective treatment of inflammatory diseases and cancers.

IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Adipocyte autophagy limits gut inflammation by controlling oxylipin and IL-10.

Lipids play a major role in inflammatory diseases by altering inflammatory cell functions, either through their function as energy substrates or as lipid mediators such as oxylipins. Autophagy, a lysosomal degradation pathway that limits inflammation, is known to impact on lipid availability, however, whether this controls inflammation remains unexplored. We found that upon intestinal inflammation visceral adipocytes upregulate autophagy and that adipocyte-specific loss of the autophagy gene Atg7 exacerbates inflammation. While autophagy decreased lipolytic release of free fatty acids, loss of the major lipolytic enzyme Pnpla2/Atgl in adipocytes did not alter intestinal inflammation, ruling out free fatty acids as anti-inflammatory energy substrates. Instead, Atg7-deficient adipose tissues exhibited an oxylipin imbalance, driven through an NRF2-mediated upregulation of Ephx1. This shift reduced secretion of IL-10 from adipose tissues, which was dependent on the cytochrome P450-EPHX pathway, and lowered circulating levels of IL-10 to exacerbate intestinal inflammation. These results suggest an underappreciated fat-gut crosstalk through an autophagy-dependent regulation of anti-inflammatory oxylipins via the cytochrome P450-EPHX pathway, indicating a protective effect of adipose tissues for distant inflammation.

Production of IL-10 by CD4(+) regulatory T cells during the resolution of infection promotes the maturation of memory CD8(+) T cells.

Memory CD8(+) T cells are critical for host defense upon reexposure to intracellular pathogens. We found that interleukin 10 (IL-10) derived from CD4(+) regulatory T cells (Treg cells) was necessary for the maturation of memory CD8(+) T cells following acute infection with lymphocytic choriomeningitis virus (LCMV). Treg cell-derived IL-10 was most important during the resolution phase, calming inflammation and the activation state of dendritic cells. Adoptive transfer of IL-10-sufficient Treg cells during the resolution phase 'restored' the maturation of memory CD8(+) T cells in IL-10-deficient mice. Our data indicate that Treg cell-derived IL-10 is needed to insulate CD8(+) T cells from inflammatory signals, and reveal that the resolution phase of infection is a critical period that influences the quality and function of developing memory CD8(+) T cells.

Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue.

Invariant natural killer T cells (iNKT cells) are lipid-sensing innate T cells that are restricted by the antigen-presenting molecule CD1d and express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, as well as their targets in adipose tissue, are unknown. Here we found that iNKT cells in adipose tissue had a unique transcriptional program and produced interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lacked PLZF but expressed the transcription factor E4BP4, which controlled their IL-10 production. The adipose iNKT cells were a tissue-resident population that induced an anti-inflammatory phenotype in macrophages and, through the production of IL-2, controlled the number, proliferation and suppressor function of regulatory T cells (Treg cells) in adipose tissue. Thus, iNKT cells in adipose tissue are unique regulators of immunological homeostasis in this tissue.

LAG-3 Inhibitory Receptor Expression Identifies Immunosuppressive Natural Regulatory Plasma Cells.

B lymphocytes can suppress immunity through interleukin (IL)-10 production in infectious, autoimmune, and malignant diseases. Here, we have identified a natural plasma cell subset that distinctively expresses the inhibitory receptor LAG-3 and mediates this function in vivo. These plasma cells also express the inhibitory receptors CD200, PD-L1, and PD-L2. They develop from various B cell subsets in a B cell receptor (BCR)-dependent manner independently of microbiota in naive mice. After challenge they upregulate IL-10 expression via a Toll-like receptor-driven mechanism within hours and without proliferating. This function is associated with a unique transcriptome and epigenome, including the lowest amount of DNA methylation at the Il10 locus compared to other B cell subsets. Their augmented accumulation in naive mutant mice with increased BCR signaling correlates with the inhibition of memory T cell formation and vaccine efficacy after challenge. These natural regulatory plasma cells may be of broad relevance for disease intervention.

Regulatory T Cells Promote Macrophage Efferocytosis during Inflammation Resolution.

Regulatory T (Treg) cell responses and apoptotic cell clearance (efferocytosis) represent critical arms of the inflammation resolution response. We sought to determine whether these processes might be linked through Treg-cell-mediated enhancement of efferocytosis. In zymosan-induced peritonitis and lipopolysaccharide-induced lung injury, Treg cells increased early in resolution, and Treg cell depletion decreased efferocytosis. In advanced atherosclerosis, where defective efferocytosis drives disease progression, Treg cell expansion improved efferocytosis. Mechanistic studies revealed the following sequence: (1) Treg cells secreted interleukin-13 (IL-13), which stimulated IL-10 production in macrophages; (2) autocrine-paracrine signaling by IL-10 induced Vav1 in macrophages; and (3) Vav1 activated Rac1 to promote apoptotic cell engulfment. In summary, Treg cells promote macrophage efferocytosis during inflammation resolution via a transcellular signaling pathway that enhances apoptotic cell internalization. These findings suggest an expanded role of Treg cells in inflammation resolution and provide a mechanistic basis for Treg-cell-enhancement strategies for non-resolving inflammatory diseases.

CAR directs T cell adaptation to bile acids in the small intestine.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

ALS iPSC-derived microglia and motor neurons respond to astrocyte-targeted IL-10 and CCL2 modulation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons (MNs). The loss of MNs in ALS leads to muscle weakness and wasting, respiratory failure, and death often within two years of diagnosis. Glial cells in ALS show aberrant expression of pro-inflammatory and neurotoxic proteins associated with activation and have been proposed as ideal therapeutic targets. In this study, we examined astrocyte-targeted treatments to reduce glial activation and neuron pathology using cells differentiated from ALS patient-derived iPSC carrying SOD1 and C9ORF72 mutations. Specifically, we tested the ability of increasing interleukin 10 (IL-10) and reducing C-C motif chemokine ligand 2 (CCL2/MCP-1) signaling targeted to astrocytes to reduce activation phenotypes in both astrocytes and microglia. Overall, we found IL10/CCL2NAb treated astrocytes to support anti-inflammatory phenotypes and reduce neurotoxicity, through different mechanisms in SOD1 and C9ORF72 cultures. We also found altered responses of microglia and motor neurons to astrocytic influences when cells were cultured together rather than in isolation. Together these data support IL-10 and CCL2 as non-mutation-specific therapeutic targets for ALS and highlight the role of glial-mediated pathology in this disease.

The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4⁺ T cells.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.