Effector TH17 Cells Give Rise to Long-Lived TRM Cells that Are Essential for an Immediate Response against Bacterial Infection.

Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (TRM) cells. However, the cellular origin of CD4 TRM cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 TRM cells derive from IL-17A-producing effector (TH17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exTH17 TRM cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exTH17 TRM cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 TRM cells during bacterial infection, offering novel strategies for targeted vaccine design.

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.

TFH cells progressively differentiate to regulate the germinal center response.

Germinal center (GC) B cells undergo affinity selection, which depends on interactions with CD4(+) follicular helper T cells (TFH cells). We found that TFH cells progressed through transcriptionally and functionally distinct stages and provided differential signals for GC regulation. They initially localized proximally to mutating B cells, secreted interleukin 21 (IL-21), induced expression of the transcription factor Bcl-6 and selected high-affinity B cell clones. As the GC response evolved, TFH cells extinguished IL-21 production and switched to IL-4 production, showed robust expression of the co-stimulatory molecule CD40L, and promoted the development of antibody-secreting B cells via upregulation of the transcription factor Blimp-1. Thus, TFH cells in the B cell follicle progressively differentiate through stages of localization, cytokine production and surface ligand expression to 'fine tune' the GC reaction.

Oct-1 regulates IL-17 expression by directing interchromosomal associations in conjunction with CTCF in T cells.

Interchromosomal associations can regulate gene expression, but little is known about the molecular basis of such associations. In response to antigen stimulation, naive T cells can differentiate into Th1, Th2, and Th17 cells expressing IFN-γ, IL-4, and IL-17, respectively. We previously reported that in naive T cells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sites cooperatively, and CTCF deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF.

Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation.

Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-ß signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.

Gatekeeper role of brain antigen-presenting CD11c+ cells in neuroinflammation.

Multiple sclerosis is the most frequent chronic inflammatory disease of the CNS. The entry and survival of pathogenic T cells in the CNS are crucial for the initiation and persistence of autoimmune neuroinflammation. In this respect, contradictory evidence exists on the role of the most potent type of antigen-presenting cells, dendritic cells. Applying intravital two-photon microscopy, we demonstrate the gatekeeper function of CNS professional antigen-presenting CD11c(+) cells, which preferentially interact with Th17 cells. IL-17 expression correlates with expression of GM-CSF by T cells and with accumulation of CNS CD11c(+) cells. These CD11c(+) cells are organized in perivascular clusters, targeted by T cells, and strongly express the inflammatory chemokines Ccl5, Cxcl9, and Cxcl10. Our findings demonstrate a fundamental role of CNS CD11c(+) cells in the attraction of pathogenic T cells into and their survival within the CNS. Depletion of CD11c(+) cells markedly reduced disease severity due to impaired enrichment of pathogenic T cells within the CNS.

Th17 cells express interleukin-10 receptor and are controlled by Foxp3⁻ and Foxp3+ regulatory CD4+ T cells in an interleukin-10-dependent manner.

T helper 17 (Th17) cells are important for host defense against extracellular microorganisms. However, they are also implicated in autoimmune and chronic inflammatory diseases, and as such need to be tightly regulated. The mechanisms that directly control committed pathogenic Th17 cells in vivo remain unclear. We showed here that IL-17A-producing CD4+ T cells expressed interleukin-10 receptor α (IL-10Rα) in vivo. Importantly, T cell-specific blockade of IL-10 signaling led to a selective increase of IL-17A+IFN-γ⁻ (Th17) and IL-17A+IFN-γ+ (Th17+Th1) CD4+ T cells during intestinal inflammation in the small intestine. CD4+Foxp3⁻ IL-10-producing (Tr1) cells and CD4+Foxp3+ regulatory (Treg) cells were able to control Th17 and Th17+Th1 cells in an IL-10-dependent manner in vivo. Lastly, IL-10 treatment of mice with established colitis decreased Th17 and Th17+Th1 cell frequencies via direct signaling in T cells. Thus, IL-10 signaling directly suppresses Th17 and Th17+Th1 cells.

Intestinal type 1 regulatory T cells migrate to periphery to suppress diabetogenic T cells and prevent diabetes development.

Growing insight into the pathogenesis of autoimmune diseases and numerous studies in preclinical models highlights the potential of regulatory T cells to restore tolerance. By using non-obese diabetic (NOD) BDC2.5 TCR-transgenic (Tg), and IL-10 and Foxp3 double-reporter mice, we demonstrate that alteration of gut microbiota during cohousing experiments or treatment with anti-CD3 mAb significantly increase intestinal IL-10-producing type 1 regulatory T (Tr1) cells and decrease diabetes incidence. These intestinal antigen-specific Tr1 cells have the ability to migrate to the periphery via a variety of chemokine receptors such as CCR4, CCR5, and CCR7 and to suppress proliferation of Th1 cells in the pancreas. The ability of Tr1 cells to cure diabetes in NOD mice required IL-10 signaling, as Tr1 cells could not suppress CD4+ T cells with a dominant-negative IL-10R. Taken together, our data show a key role of intestinal Tr1 cells in the control of effector T cells and development of diabetes. Therefore, modulating gut-associated lymphoid tissue to boost Tr1 cells may be important in type 1 diabetes management.

IL-10 Receptor Signaling Is Essential for TR1 Cell Function In Vivo.

IL-10 is essential to maintain intestinal homeostasis. CD4+ T regulatory type 1 (TR1) cells produce large amounts of this cytokine and are therefore currently being examined in clinical trials as T cell therapy in patients with inflammatory bowel disease. However, factors and molecular signals sustaining TR1 cell regulatory activity still need to be identified to optimize the efficiency and ensure the safety of these trials. We investigated the role of IL-10 signaling in mature TR1 cells in vivo. Double IL-10eGFP Foxp3mRFP reporter mice and transgenic mice with impairment in IL-10 receptor signaling were used to test the activity of TR1 cells in a murine inflammatory bowel disease model, a model that resembles the trials performed in humans. The molecular signaling was elucidated in vitro. Finally, we used human TR1 cells, currently employed for cell therapy, to confirm our results. We found that murine TR1 cells expressed functional IL-10Rα. TR1 cells with impaired IL-10 receptor signaling lost their regulatory activity in vivo. TR1 cells required IL-10 receptor signaling to activate p38 MAPK, thereby sustaining IL-10 production, which ultimately mediated their suppressive activity. Finally, we confirmed these data using human TR1 cells. In conclusion, TR1 cell regulatory activity is dependent on IL-10 receptor signaling. These data suggest that to optimize TR1 cell-based therapy, IL-10 receptor expression has to be taken into consideration.

Control of TH17 cells occurs in the small intestine.

Interleukin (IL)-17-producing T helper cells (T(H)17) are a recently identified CD4(+) T cell subset distinct from T helper type 1 (T(H)1) and T helper type 2 (T(H)2) cells. T(H)17 cells can drive antigen-specific autoimmune diseases and are considered the main population of pathogenic T cells driving experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis. The factors that are needed for the generation of T(H)17 cells have been well characterized. However, where and how the immune system controls T(H)17 cells in vivo remains unclear. Here, by using a model of tolerance induced by CD3-specific antibody, a model of sepsis and influenza A viral infection (H1N1), we show that pro-inflammatory T(H)17 cells can be redirected to and controlled in the small intestine. T(H)17-specific IL-17A secretion induced expression of the chemokine CCL20 in the small intestine, facilitating the migration of these cells specifically to the small intestine via the CCR6/CCL20 axis. Moreover, we found that T(H)17 cells are controlled by two different mechanisms in the small intestine: first, they are eliminated via the intestinal lumen; second, pro-inflammatory T(H)17 cells simultaneously acquire a regulatory phenotype with in vitro and in vivo immune-suppressive properties (rT(H)17). These results identify mechanisms limiting T(H)17 cell pathogenicity and implicate the gastrointestinal tract as a site for control of T(H)17 cells.

Enhanced anti-serpin antibody activity inhibits autoimmune inflammation in type 1 diabetes.

Intracellular (clade B) OVA-serpin protease inhibitors play an important role in tissue homeostasis by protecting cells from death in response to hypo-osmotic stress, heat shock, and other stimuli. It is not known whether these serpins influence immunological tolerance and the risk for autoimmune diseases. We found that a fraction of young autoimmune diabetes-prone NOD mice had elevated levels of autoantibodies against a member of clade B family known as serpinB13. High levels of anti-serpinB13 Abs were accompanied by low levels of anti-insulin autoantibodies, reduced numbers of islet-associated T cells, and delayed onset of diabetes. Exposure to anti-serpinB13 mAb alone also decreased islet inflammation, and coadministration of this reagent and a suboptimal dose of anti-CD3 mAb accelerated recovery from diabetes. In a fashion similar to that discovered in the NOD model, a deficiency in humoral activity against serpinB13 was associated with early onset of human type 1 diabetes. These findings suggest that, in addition to limiting exposure to proteases within the cell, clade B serpins help to maintain homeostasis by inducing protective humoral immunity.

miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling.

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.

Fatty acid binding protein 5 suppression attenuates obesity-induced hepatocellular carcinoma by promoting ferroptosis and intratumoral immune rewiring.

Due to the rise in overnutrition, the incidence of obesity-induced hepatocellular carcinoma (HCC) will continue to escalate; however, our understanding of the obesity to HCC developmental axis is limited. We constructed a single-cell atlas to interrogate the dynamic transcriptomic changes during hepatocarcinogenesis in mice. Here we identify fatty acid binding protein 5 (FABP5) as a driver of obesity-induced HCC. Analysis of transformed cells reveals that FABP5 inhibition and silencing predispose cancer cells to lipid peroxidation and ferroptosis-induced cell death. Pharmacological inhibition and genetic ablation of FABP5 ameliorates the HCC burden in male mice, corresponding to enhanced ferroptosis in the tumour. Moreover, FABP5 inhibition induces a pro-inflammatory tumour microenvironment characterized by tumour-associated macrophages with increased expression of the co-stimulatory molecules CD80 and CD86 and increased CD8+ T cell activation. Our work unravels the dual functional role of FABP5 in diet-induced HCC, inducing the transformation of hepatocytes and an immunosuppressive phenotype of tumour-associated macrophages and illustrates FABP5 inhibition as a potential therapeutic approach.

Dynamic metabolism of endothelial triglycerides protects against atherosclerosis in mice.

Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelial-dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.

Development of autoimmune diabetes in the absence of detectable IL-17A in a CD8-driven virally induced model.

Recent studies have shown that IL-17 can contribute beneficially to pathogen defense but also that excessive IL-17 levels are associated with chronic inflammation and autoimmune disorders. To date, the role of IL-17 in viral infections and type 1 diabetes is ambiguous. In this study, we used IL-17A enhanced green fluorescent protein bicistronic reporter mouse strains to analyze in situ production of IL-17A. Upon Klebsiella pneumoniae bacterial infection, CD4(+) and γδ T cells produce IL-17A. In contrast, CD4(+) or CD8(+) T cells do not produce IL-17A in response to acute or protracted viral infection with lymphocytic choriomeningitis virus or during autoimmune diabetes development in the CD8-driven lymphocytic choriomeningitis virus-induced model of type 1 diabetes. We conclude that viral elimination and type 1 diabetes can occur in the absence of detectable IL-17A production, suggesting IL-17A is not essential in these settings.

SARS-CoV-2-encoded small RNAs are able to repress the host expression of SERINC5 to facilitate viral replication.

Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro. Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection.

Enhanced antitumor immunity in mice deficient in CD69.

We investigated the in vivo role of CD69 by analyzing the susceptibility of CD69-/- mice to tumors. CD69-/- mice challenged with MHC class I- tumors (RMA-S and RM-1) showed greatly reduced tumor growth and prolonged survival compared with wild-type (WT) mice. The enhanced anti-tumor response was NK cell and T lymphocyte-mediated, and was due, at least in part, to an increase in local lymphocytes. Resistance of CD69-/- mice to MHC class I- tumor growth was also associated with increased production of the chemokine MCP-1, diminished TGF-beta production, and decreased lymphocyte apoptosis. Moreover, the in vivo blockade of TGF-beta in WT mice resulted in enhanced anti-tumor response. In addition, CD69 engagement induced NK and T cell production of TGF-beta, directly linking CD69 signaling to TGF-beta regulation. Furthermore, anti-CD69 antibody treatment in WT mice induced a specific down-regulation in CD69 expression that resulted in augmented anti-tumor response. These data unmask a novel role for CD69 as a negative regulator of anti-tumor responses and show the possibility of a novel approach for the therapy of tumors.

CD69 limits early inflammatory diseases associated with immune response to Listeria monocytogenes infection.

Mouse infection with intracellular bacteria induces a potent inflammatory response that requires protective mechanisms to avoid infection-induced immune pathology. CD69 is expressed in all leukocytes during activation after infection with a wide range of microbial pathogens. This study explores the way in which CD69 affects cell activation after Listeria monocytogenes (Lm) infection and its effects on host protection. We show that infectivity and bacterial clearance capability are unaltered in CD69(-/-) peritoneal macrophages, bone marrow-derived macrophages and dendritic cells. We found no major altered cell populations in splenocytes of Lm-infected CD69(-/-) mice. However, an increase in the expression of Th1 cytokines was observed after infection, with increased production of type I and II interferon (IFN). In addition, CD69(-/-) splenocytes showed increased apoptosis, consistent with IFN enhancement of lymphocyte apoptosis in response to Lm infection. CD69(-/-) mice showed liver and spleen damage, and greatly increased susceptibility to Lm infection, compared with wild-type controls. Lm-specific T cells were decreased in CD69(-/-) mice even if T-cell cross-presentation and T-cell intrinsic priming response were not compromised. As listeriosis was increased as early as day 1 post-infection but CD69(-/-)RAG2(-/-) mice were more efficient at controlling Listeria, we propose that CD69 controls the cross-talk between innate components and lymphocytes. These results highlight a role for CD69 in preventing infection-induced immunopathology.

The induction and function of the anti-inflammatory fate of TH17 cells.

TH17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of TH17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-ß signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in TH17 cells. Our data thus indicate a key function of TH17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of TH17 cells with regard to environmental changes.

Microbiota-driven interleukin-17-producing cells and eosinophils synergize to accelerate multiple myeloma progression.

The gut microbiota has been causally linked to cancer, yet how intestinal microbes influence progression of extramucosal tumors is poorly understood. Here we provide evidence implying that Prevotella heparinolytica promotes the differentiation of Th17 cells colonizing the gut and migrating to the bone marrow (BM) of transgenic Vk*MYC mice, where they favor progression of multiple myeloma (MM). Lack of IL-17 in Vk*MYC mice, or disturbance of their microbiome delayed MM appearance. Similarly, in smoldering MM patients, higher levels of BM IL-17 predicted faster disease progression. IL-17 induced STAT3 phosphorylation in murine plasma cells, and activated eosinophils. Treatment of Vk*MYC mice with antibodies blocking IL-17, IL-17RA, and IL-5 reduced BM accumulation of Th17 cells and eosinophils and delayed disease progression. Thus, in Vk*MYC mice, commensal bacteria appear to unleash a paracrine signaling network between adaptive and innate immunity that accelerates progression to MM, and can be targeted by already available therapies.