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The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.

The murine bone marrow has a central role in immune function and health as the primary source of leukocytes in adult mice. Laboratory mice provide a human-homologous, genetically manipulable and reproducible model that has enabled an immeasurable volume of high-quality immunological research. However, recent research has questioned the translatability of laboratory mouse research into humans and proposed that the exposure of mice to their wild and natural environment may hold the key to further immunological breakthroughs. To date, there have been no studies providing an in-depth cellular analysis of the wild mouse bone marrow. This study utilized wild mice from an isolated island population (Isle of May, Scotland, UK) and performed flow cytometric and histological analysis to characterize the myeloid, lymphoid, hematopoietic progenitor, and adipocyte compartments within the wild mouse bone marrow. We find that, compared to laboratory mouse bone marrow, the wild mouse bone marrow differs in every cell type assessed. Some of the major distinctions include; a smaller B cell compartment with an enriched presence of plasma cells, increased proportions of KLRG1+ CD8+ T cells, diminished CD11b expression in the myeloid lineage and a five-fold enlargement of the eosinophil compartment. We conclude that the wild mouse bone marrow is dramatically distinct from its laboratory counterparts, with multiple phenotypes that to our knowledge have never been observed in laboratory models. Further research into these unique features may uncover novel immunological mechanisms and grant a greater understanding of the role of the immune system in a natural setting.

The gut microbiota is important for host health and immune system function. Moreover autoimmune diseases, such as rheumatoid arthritis, are associated with significant gut microbiota dysbiosis, although the causes and consequences of this are not fully understood. It has become clear that the composition and metabolic outputs of the microbiome exhibit robust 24 h oscillations, a result of daily variation in timing of food intake as well as rhythmic circadian clock function in the gut. Here, we report that experimental inflammatory arthritis leads to a re-organization of circadian rhythmicity in both the gut and associated microbiome. Mice with collagen induced arthritis exhibited extensive changes in rhythmic gene expression in the colon, and reduced barrier integrity. Re-modeling of the host gut circadian transcriptome was accompanied by significant alteration of the microbiota, including widespread loss of rhythmicity in symbiont species of Lactobacillus, and alteration in circulating microbial derived factors, such as tryptophan metabolites, which are associated with maintenance of barrier function and immune cell populations within the gut. These findings highlight that altered circadian rhythmicity during inflammatory disease contributes to dysregulation of gut integrity and microbiome function.

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in the C57BL/6 strain. Here, we provide a comprehensive analysis of immune cells in the pleural cavity using both C57BL/6 and BALB/c mice. Unlike C57BL/6 mice, naïve tissue-resident Large Cavity Macrophages (LCM) of BALB/c mice failed to fully implement the tissue residency program. Following infection with a pleural-dwelling nematode these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6 but not BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte to macrophage conversion required both T cells and IL-4Rα signalling. Host genetics are therefore a key influence on tissue resident macrophage biology, and during nematode infection Th2 cells control the differentiation pathway of tissue resident macrophages.Graphical Abstract

The cytokine TGFβ1 induces epidermal Langerhans cell (LC) differentiation from human precursors, an effect mediated through BMPR1a/ALK3 signaling, as revealed from ectopic expression and receptor inhibition studies. Whether TGFβ1‒BMPR1a signaling is required for LC differentiation in vivo remained incompletely understood. We found that TGFβ1-deficient mice show defective perinatal expansion and differentiation of LCs. LCs can be identified within the normal healthy human epidermis by anti-BMPR1a immunohistology staining. Deletion of BMPR1a in all (vav

Circulating microRNAs (miRNAs) are exceptional mechanism-based correlates of disease, yet their potential remains largely untapped in COVID-19. Here, we determined circulating miRNA and cytokine and chemokine (CC) profiles in 171 blood plasma samples from 58 hospitalised COVID-19 patients. Thirty-two miRNAs were differentially expressed in severe cases when compared to moderate and mild cases. These miRNAs and their predicted targets reflected key COVID-19 features including cell death and hypoxia. Compared to mild cases, moderate and severe cases were characterised by a global decrease in circulating miRNA levels. Partial least squares regression using miRNA and CC measurements allowed for discrimination of severe cases with greater accuracy (87%) than using miRNA or CC levels alone. Correlation analysis revealed severity group-specific associations between CC and miRNA levels. Importantly, the miRNAs that correlated with IL6 and CXCL10, two cardinal COVID-19-associated cytokines, were distinct between severity groups, providing a novel qualitative way to stratify patients with similar levels of proinflammatory cytokines but different disease severity. Integration of miRNA and CC levels with clinical parameters revealed severity-specific signatures associated with clinical hallmarks of COVID-19. Our study highlights the existence of severity-specific circulating CC/miRNA networks, providing insight into COVID-19 pathogenesis and a novel approach for monitoring COVID-19 progression.

Background Emerging studies indicate that some COVID-19 patients suffer from persistent symptoms including breathlessness and chronic fatigue; however the long-term immune response in these patients presently remains ill-defined. Methods Here we describe the phenotypic and functional characteristics of B and T cells in hospitalised COVID-19 patients during acute disease and at 3-6 months of convalescence. Findings We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed continued alterations with persistence of a cytotoxic programme evident in CD8+ T cells as well as elevated production of type-1 cytokines and IL-17. Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/IL-10 cytokine imbalance in response to toll-like receptor activation, skewed towards a pro-inflammatory phenotype. Whereas the frequency of IL-6+ B cells was restored in convalescent patients irrespective of clinical outcome, recovery of IL-10+ B cells was associated with resolution of lung pathology. Conclusions Our data detail lymphocyte alterations in previously hospitalized COVID-19 patients up to 6 months following hospital discharge and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes, with one subgroup associated with poorer clinical outcome. We propose that alterations in B and T cell function following hospitalisation with COVID-19 could impact longer term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. Funding Provided by UKRI, Lister Institute of Preventative Medicine, The Wellcome Trust, The Kennedy Trust for Rheumatology Research and 3M Global Giving., Graphical Abstract, Shuwa et al examine lymphocyte characteristics in acute and convalescent COVID-19 patients, detailing persistent alterations in lymphocyte phenotype up to 6 months following hospital discharge. In this report they identify 3 subgroups of convalescent patients based on distinct lymphocyte signatures, with one subgroup associated with poorer clinical outcome.

Krishnan et al. outline a novel pathway of myeloid cell development in healthy gingiva. They identify HSPCs in noninflamed gingiva tissue, which support the generation of a proportion of the innate immune cells that police this barrier site., Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier., Graphical Abstract

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalised COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g. IL6, CCL20) or clinical hallmarks of COVID-19 (e.g. neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Critically, analysis of an independent cohort of 108 patients from a different centre (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19. Funding: The study was funded by the UKRI MRC/NIHR award to the UK Coronavirus Immunology Consortium (UK-CIC, MR/V028448/1). Sample collection at Manchester NHS Trusts was supported by the NIHR Manchester Biomedical Research Centre (TH and AS) and 3M Global Giving award (JRG and TH). Sample collection at York and Scarborough NHS Trust was supported by the Wellcome Trust (ISSF grant WT204829 through the Centre for Future Health at the University of York) and the Hull York Medical School. JRG is supported by a senior fellowship by The Kennedy Trust for Rheumatology Research. This report contains independent research supported by the North West Lung Centre Charity and the NIHR Manchester Clinical Research Facility at Wythenshawe Hospital. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: For the cohort 1, ethics approval was obtained from the North West-Haydock Research Ethics Committee for ManARTS (reference 15/NW/0409) and from the Wales Research Ethics Committee 4 for NCARC (reference 18/WA/0368). For the cohort 2, ethics approval was obtained from Yorkshire & The Humber - Leeds West Research Ethics Committee (REC reference 19/YH/0394 with approved 26 SA002 amendment of IRAS project 269597).

The role of the human microbiome in health and disease is becoming increasingly apparent. Emerging evidence suggests that the microbiome is affected by solid organ transplantation. Kidney transplantation is the gold standard treatment for End-Stage Renal Disease (ESRD), the advanced stage of Chronic Kidney Disease (CKD). The question of how ESRD and transplantation affect the microbiome and vice versa includes how the microbiome is affected by increased concentrations of toxins such as urea and creatinine (which are elevated in ESRD), whether restoration of renal function following transplantation alters the composition of the microbiome, and the impact of lifelong administration of immunosuppressive drugs on the microbiome. Changes in microbiome composition and activity have been reported in ESRD and in therapeutic immunosuppression, but the effect on the outcome of transplantation is not well-understood. Here, we consider the current evidence that changes in kidney function and immunosuppression following transplantation influence the oral, gut, and urinary microbiomes in kidney transplant patients. The potential for changes in these microbiomes to lead to disease, systemic inflammation, or rejection of the organ itself is discussed, along with the possibility that restoration of kidney function might re-establish orthobiosis.

Helminth parasites are effective in biasing Th2 immunity and inducing regulatory pathways that minimize excessive inflammation within their hosts, thus allowing chronic infection to occur whilst also suppressing bystander atopic or autoimmune diseases. Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory lesions within the central nervous system; there are very limited therapeutic options for the progressive forms of the disease and none are curative. Here, we used the experimental autoimmune encephalomyelitis (EAE) model to examine if the intestinal helminth Heligmosomoides polygyrus and its excretory/secretory products (HES) are able to suppress inflammatory disease. Mice infected with H. polygyrus at the time of immunization with the peptide used to induce EAE (myelin-oligodendrocyte glycoprotein, pMOG), showed a delay in the onset and peak severity of EAE disease, however, treatment with HES only showed a marginal delay in disease onset. Mice that received H. polygyrus 4 weeks prior to EAE induction were also not significantly protected. H. polygyrus secretes a known TGF-β mimic (Hp-TGM) and simultaneous H. polygyrus infection with pMOG immunization led to a significant expansion of Tregs; however, administering the recombinant Hp-TGM to EAE mice failed to replicate the EAE protection seen during infection, indicating that this may not be central to the disease protecting mechanism. Mice infected with H. polygyrus also showed a systemic Th2 biasing, and restimulating splenocytes with pMOG showed release of pMOG-specific IL-4 as well as suppression of inflammatory IL-17A. Notably, a Th2-skewed response was found only in mice infected with H. polygyrus at the time of EAE induction and not those with a chronic infection. Furthermore, H. polygyrus failed to protect against disease in IL-4Rα−/− mice. Together these results indicate that the EAE disease protective mechanism of H. polygyrus is likely to be predominantly Th2 deviation, and further highlights Th2-biasing as a future therapeutic strategy for MS.

Emerging studies indicate that some COVID-19 patients suffer from persistent symptoms including breathlessness and chronic fatigue; however the long-term immune response in these patients presently remains ill-defined. Here we describe the phenotypic and functional characteristics of B and T cells in healthy individuals and individuals with acute or convalescent COVID-19. We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed long-term alterations with persistence of a cytotoxic programme evident in CD8+ T cells as well as elevated production of type-1 cytokines and IL-17. Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/ IL-10 cytokine imbalance in response to toll-like receptor activation, skewed towards a pro-inflammatory phenotype. Whereas the frequency of IL-10+ B cells was restored in a subset of convalescent patients, IL-6 production remained elevated. Our data are the first to define long-term alterations in the lymphocyte compartment of previously hospitalized COVID-19 patients, at up to 19 weeks of convalescence, and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes. We propose that alterations in B and T cell function following hospitalisation with COVID-19 could impact long-term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. Funding: This study was funded by the BBSRC (BB/M025977/1 to JEK, BB/S01103X/1 to TNS), by the Lister Institute (Prize Fellowship to JEK), by PTDF (SHS/1327/18 to HAS), The Wellcome Trust (202865/Z/16/Z to TH), The Kennedy Trust for Rheumatology Research (Rapid Response Award to JRG) and Versus Arthritis (FAM and JEK studentship). This report is independent research supported by the North West Lung Centre Charity and the NIHR Manchester Clinical Research Facility at Wythenshawe Hospital. We acknowledge the Manchester Allergy, Respiratory and Thoracic Surgery Biobank for supporting this project and thank the study participants for their contribution. Ethical Approval: Ethical approval obtained from the National Research Ethics Service (REC reference 15/NW/0409 for ManARTS and 18/WA/0368 for NCARC). Informed consent was obtained from each patient, clinical information was extracted from written/electronic medical records including demographic data, presenting symptoms, comorbidities, radiographic findings, vital signs, and laboratory data. Declaration of Interest: None to declare.

D-mannose, a C-2 epimer of glucose, exists naturally in many plants and fruits, and is found in human blood at concentrations less than one-fiftieth of that of glucose. However, although the roles of glucose in T cell metabolism, diabetes and obesity are well characterized, the function of D-mannose in T cell immune responses remains unknown. Here we show that supraphysiological levels of D-mannose safely achievable by drinking-water supplementation suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3+ regulatory T cells (Treg cells) in mice. In vitro, D-mannose stimulated Treg cell differentiation in human and mouse cells by promoting TGF-β activation, which in turn was mediated by upregulation of integrin αvβ8 and reactive oxygen species generated by increased fatty acid oxidation. This previously unrecognized immunoregulatory function of D-mannose may have clinical applications for immunopathology.

MLL4 is an essential subunit of the histone H3 Lys4 (H3K4)-methylation complexes. We found that MLL4 deficiency compromised the development of regulatory T cells (Treg cells) and resulted in a substantial decrease in monomethylated H3K4 (H3K4me1) and chromatin interaction at putative gene enhancers, a considerable portion of which were not direct targets of MLL4 but were enhancers that interacted with MLL4-bound sites. The decrease in H3K4me1 and chromatin interaction at the enhancers not bound by MLL4 correlated with MLL4 binding at distant interacting regions. Deletion of an upstream MLL4-binding site diminished the abundance of H3K4me1 at the regulatory elements of the gene encoding the transcription factor Foxp3 that were looped to the MLL4-binding site and compromised both the thymic differentiation and the inducible differentiation of Treg cells. We found that MLL4 catalyzed methylation of H3K4 at distant unbound enhancers via chromatin looping, which identifies a previously unknown mechanism for regulating the T cell enhancer landscape and affecting Treg cell differentiation.

Immuno-surveillance networks operating at barrier sites are tuned by local tissue cues to ensure effective immunity. Site-specific commensal bacteria provide key signals ensuring host defense in the skin and gut. However, how the oral microbiome and tissue-specific signals balance immunity and regulation at the gingiva, a key oral barrier, remains minimally explored. In contrast to the skin and gut, we demonstrate that gingiva-resident T helper 17 (Th17) cells developed via a commensal colonization-independent mechanism. Accumulation of Th17 cells at the gingiva was driven in response to the physiological barrier damage that occurs during mastication. Physiological mechanical damage, via induction of interleukin 6 (IL-6) from epithelial cells, tailored effector T cell function, promoting increases in gingival Th17 cell numbers. These data highlight that diverse tissue-specific mechanisms govern education of Th17 cell responses and demonstrate that mechanical damage helps define the immune tone of this important oral barrier.

Intestinal macrophages represent the last tissue macrophages thought to entirely adhere to van Furth's decades-old continuous monocyte replenishment model. In this study, Shaw et al. identify a population of intestinal macrophages that are long lived and maintained independently of monocyte replenishment over long periods of time., A defining feature of resident gut macrophages is their high replenishment rate from blood monocytes attributed to tonic commensal stimulation of this site. In contrast, almost all other tissues contain locally maintained macrophage populations, which coexist with monocyte-replenished cells at homeostasis. In this study, we identified three transcriptionally distinct mouse gut macrophage subsets that segregate based on expression of Tim-4 and CD4. Challenging current understanding, Tim-4+CD4+ gut macrophages were found to be locally maintained, while Tim-4–CD4+ macrophages had a slow turnover from blood monocytes; indeed, Tim-4–CD4– macrophages were the only subset with the high monocyte-replenishment rate currently attributed to gut macrophages. Moreover, all macrophage subpopulations required live microbiota to sustain their numbers, not only those derived from blood monocytes. These findings oppose the prevailing paradigm that all macrophages in the adult mouse gut rapidly turn over from monocytes in a microbiome-dependent manner; instead, these findings supplant it with a model of ontogenetic diversity where locally maintained subsets coexist with rapidly replaced monocyte-derived populations., Graphical Abstract

The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.

CD4+ regulatory T cells expressing the transcription factor Foxp3 can be generated in the thymus (tTreg cells), but the cellular and molecular pathways driving their development remain incompletely understood. Transforming growth factor-beta (TGF-β) is essential for the generation of Foxp3+ Treg cells converted from peripheral naive CD4+ T cells (pTreg cells), yet a role for TGF-β in tTreg-cell development was initially refuted. Nevertheless, recent studies have unmasked a requirement for TGF-β in the generation of tTreg cells. Experimental evidence reveals that TGF-β in the context of TCR stimulation induces Foxp3 gene transcription in thymic Treg precursors, CD4+CD8−CD25− semi-mature and mature single-positive (SP) thymocytes. Intriguingly, thymic apoptosis was found to be intrinsically linked to the generation of tTreg cells, as apoptosis induced expression of TGF-β intra-thymically. In this short review, we will highlight key data, discuss the experimental evidence and propose a modified model of tTreg-cell development involving TGF-β. We will also outline the remaining unresolved questions concerning generation of thymic Foxp3+ Treg cells and provide our personal perspectives on the mechanisms controlling tTreg-cell development.

Our recent work highlights unique requirements for the induction of Th17 cellimmunity at the oral and gingival mucosal barrier. Unlike other barrier sites, such as the skin and gastrointestinal tract, we found that Th17 cells can develop at thegingiva independently of commensal microbiota colonization. Instead, we identifiedthat damage, which occurs physiologically due to mastication, promotes induction of Th17 cells and therefore tones homeostatic immunity at the gingiva.

Regulatory T cells (Treg cells) perform suppressive functions in disparate tissue environments and against many inflammatory insults, yet the tissue-enriched factor(s) that influence Treg cell phenotype and function remain largely unknown. We have shown a vital role for transforming growth factor-β (TGF-β) signals in safe-guarding specific Treg cell functions. TGF-β signals were dispensable for steady-state Treg cell homeostasis and for Treg cell suppression of T cell proliferation and T helper-1 (Th1) cell differentiation. However, Treg cells require TGF-β signals to appropriately dampen Th17 cells and regulate responses in the gastrointestinal tract. TGF-β signaling maintains CD103 expression, promotes expression of the colon-specific trafficking molecule GPR15, and inhibits expression of GPR174, a receptor for lysophosphatidylserine, on Treg cells, collectively supporting the accumulation and retention of Treg cells in the colon and control of colitogenic responses. Thus, we reveal an unrecognized function for TGF-β signaling as an upstream factor controlling Treg cell activity in specific tissue environments.

Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells (non-HSCs) that can support the function of HSCs in bone marrow (BM). MSCs have been shown to possess regenerative properties and unique immunoregulatory functions that make them an attractive option for cellular therapy in patients with autoimmune diseases and chronic inflammation (1). We have previously shown that allogeneic BM- and umbilical cord (UC)–derived MSC transplantation is a safe and effective treatment of active systemic lupus erythematosus (SLE) (2,3) and other autoimmune diseases, such as systemic sclerosis (4), Sjogren's syndrome (5), and myositis (6). Conversely, autologous MSCs from lupus patients cannot offer therapeutic benefits due to intrinsic abnormal functions (7–9). However, the mechanisms by which allogeneic MSC transplantation ameliorates SLE remain largely unknown. It is now clear that MSCs exert immunoregulatory properties on various immune cells. This includes suppression of T cell proliferation, regulation of dendritic cell (DC) maturation and function, modulation of B cell proliferation and terminal differentiation, and regulation of natural killer cells and macrophage function (10–12). Many factors are involved in MSC immunomodulation, including but not limited to, production of transforming growth factor β (TGFβ), hepatocyte growth factor (HGF), prostaglandin E2 (PGE2), interleukin-10 (IL-10), indolamine 2,3-dioxygenase (IDO), nitric oxide (NO), heme oxygenase 1 (HO-1), and HLA–G (13–16). IDO, which is mainly produced by DCs and macrophages, is an enzyme that degrades the essential amino acid tryptophan and participates in immune tolerance (17,18). In 2004, a study demonstrated that human MSCs could secrete IDO in vitro in the presence of mixed lymphocyte reaction. The IDO that was secreted by MSCs mediated inhibition of normal T cell proliferation (19). However, other studies have demonstrated that IDO plays a dispensable role in human MSC suppression of T cell proliferation and have instead suggested that HLA–G and IL-10 have a cell-contact–dependent role (20). In animal studies, it has been suggested that NO rather than IDO is involved in immunomodulation by MSCs (21). Importantly, the precise mechanisms responsible for the regulatory effects of MSCs in lupus patients remain unknown. In this study, we determined that high levels of interferon-γ (IFNγ), produced predominantly by CD8+ T cells in lupus patients, are a key factor involved in the stimulation of allogeneic UC-MSCs to produce IDO, which can then inhibit the proliferation of T cells from lupus patients. Thus, we uncovered a previously unrecognized CD8+ T cell/IFNγ/IDO axis that mediates the therapeutic benefit of allogeneic MSCs in lupus.

The membrane-anchored serine protease, matriptase, is consistently dysregulated in a range of human carcinomas, and high matriptase activity correlates with poor prognosis. Furthermore, matriptase is unique among tumor-associated proteases in that epithelial stem cell expression of the protease suffices to induce malignant transformation. Here, we use genetic epistasis analysis to identify proteinase-activated receptor (PAR)-2-dependent inflammatory signaling as an essential component of matriptase-mediated oncogenesis. In cell-based assays, matriptase was a potent activator of PAR-2, and PAR-2 activation by matriptase caused robust induction of nuclear factor (NF)κB through Gαi. Importantly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced pre-malignant progression, including inflammatory cytokine production, inflammatory cell recruitment, epidermal hyperplasia and dermal fibrosis. Selective ablation of PAR-2 from bone marrow-derived cells did not prevent matriptase-driven pre-malignant progression, indicating that matriptase activates keratinocyte stem cell PAR-2 to elicit its pro-inflammatory and pro-tumorigenic effects. When combined with previous studies, our data suggest that dual induction of PAR-2-NFκB inflammatory signaling and PI3K-Akt-mTor survival/proliferative signaling underlies the transforming potential of matriptase and may contribute to pro-tumorigenic signaling in human epithelial carcinogenesis.

Three homologues of TGF-β exist in mammals as follows: TGF-β1, TGF-β2, and TGF-β3. All three proteins share high homology in their amino acid sequence, yet each TGF-β isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-β proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-β knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-β1 ligand with a sequence from TGF-β3 using targeted recombination to create chimeric TGF-β1/3 knock-in mice (TGF-β1Lβ3/Lβ3). In the TGF-β1Lβ3/Lβ3 mouse, localization and activation still occur through the TGF-β1 latent associated peptide, but cell signaling is triggered through the TGF-β3 ligand that binds to TGF-β receptors. Unlike TGF-β1−/− mice, the TGF-β1Lβ3/Lβ3 mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-β3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-β1 deficiency. However, the TGF-β1Lβ3/Lβ3 mice have a shortened life span and display tooth and bone defects, indicating that the TGF-β homologues are not completely interchangeable. Remarkably, the TGF-β1Lβ3/Lβ3 mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-β isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-β pathway in human disease. Background: The mammalian TGF-β homologues have highly conserved heterologous motifs that are believed to impart isoform-specific functions. Results: Exchange of TGF-β1 ligand for TGF-β3 in a mouse reveals functional redundancies/differences in cell signaling. Conclusion: TGF-β1 and TGF-β3 are only partially interchangeable. Significance: These findings have relevance for therapeutic approaches aimed at targeting the TGF-β pathway in disease conditions.

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell-specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4⁻CD8⁻TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.

Human γδ T-cell lymphoma is a rare clinicopathologic entity with aggressive course and poor prognosis. The etiology and pathogenesis of γδ T-cell lymphoma is unknown. We show here that mice with deficiency in inhibitory helix-loop-helix protein Id3 (Id3−/−) developed γδ T-cell lymphoma that resembled human γδ T-cell lymphoma. The Id3−/− mice with lymphoma showed splenomegaly, hepatomegaly, and lymphadenopathy with involvement of bone marrow, thymus, kidney, and lungs between 6 and 15 months of age. Phenotypic analysis revealed that lymphomatous cells were cluster of differentiation (CD)3+, γδ T-cell receptor (TCR)+, and αβ TCR−, and expressed CD8+CD4−, CD4+CD8−, or a mixture of the two. Id3−/− γδ T-cell lymphoma used predominantly Vγ1.1, some Vγ3, yet no Vγ2 TCR, and some showed increased levels of the oncogene c-Myc. Strikingly, adoptive transfer of the γδ T-cell lymphoma into syngeneic Rag1−/− mice resulted in aggressive γδ T-cell lymphoma, identical to the Id3−/− donor. Thus, our data demonstrate that Id3 regulates the development of γδ T-cell lymphoma in mice, raising a possibility of Id3 gene mutation in human γδ T-cell lymphoma. Our model will provide a tool for studying the molecular mechanisms and development of human γδ T-cell lymphoma.

Summary The ultimate outcome of T-cell recognition of peptide–major histocompatibility complex (MHC) complexes is determined by the molecular context in which antigen presentation is provided. The paradigm is that, after exposure to peptides presented by steady-state dendritic cells (DCs), inhibitory signals dominate, leading to the deletion and/or functional inactivation of antigen-reactive T cells. This has been utilized in a variety of models providing peptide antigen in soluble form in the absence of adjuvant. A co-inhibitory molecule of considerable current interest is PD-1. Here we show that there is the opportunity for the PD-1/PD-L1 interaction to function in inhibiting the T-cell response during tolerance induction. Using traceable CD4+ T-cell receptor (TCR) transgenic cells, together with a blocking antibody to disrupt PD-1 signalling, we explored the roles of PD-1 in the induction of tolerance versus a productive immune response. Intact PD-1 signalling played a role in limiting the extent of CD4+ T-cell accumulation in response to an immunogenic stimulus. However, PD-1 signalling was not required for either the induction, or the maintenance, of peptide-induced tolerance; a conclusion underlined by successful tolerance induction in TCR transgenic cells genetically deficient for PD-1. These observations contrast with the reported requirement for PD-1 signals in CD8+ T-cell tolerance.

In naïve T cells transforming growth factor-beta (TGF-beta) induces Foxp3, a transcription factor essential for programming and developing T regulatory cells (Treg cells). This finding reveals a physiological factor which can turn on the Foxp3 gene and establishes an experimental approach to induce antigen-specific Treg cells as a potential therapy for human diseases. While this role for TGF-beta is well confirmed, several critical questions remain largely unanswered and await further investigation. In this regard, it is imperative to understand the molecular pathways by which TGF-beta signaling initiates and regulates Foxp3 expression. It is also important to elucidate which factors and/or cytokines influence the TGF-beta-mediated conversion of naïve T cells and how to create an immunologically regulatory milieu to facilitate Treg cell generation in vivo. In this short article, we will highlight the key findings and recent progress in the field, discuss the molecular mechanisms underlying the TGF-beta-mediated induction of Foxp3, and attempt to outline the challenges ahead.

The oral mucosa is a barrier site constantly exposed to rich and diverse commensal microbial communities, yet little is known of the immune cell network maintaining immune homeostasis at this interface. We have performed a detailed characterization of the immune cell subsets of the oral cavity in a large cohort of healthy subjects. We focused our characterization on the gingival interface, a particularly vulnerable mucosal site, with thin epithelial lining and constant exposure to the tooth adherent biofilm. In health, we find a predominance of T cells, minimal B cells, a large presence of granulocytes/neutrophils, a sophisticated network of professional antigen-presenting cells (APCs), and a small population of innate lymphoid cells (ILCs) policing the gingival barrier. We further characterize cellular subtypes in health and interrogate shifts in immune cell populations in the common oral inflammatory disease periodontitis. In disease, we document an increase in neutrophils and an upregulation of interleukin-17 (IL-17) responses. We identify the main source of IL-17 in health and Periodontitis within the CD4(+) T-cell compartment. Collectively, our studies provide a first view of the landscape of physiologic oral immunity and serve as a baseline for the characterization of local immunopathology.

Psoriasis is an inflammatory skin disease affecting ∼2% of the world's population, but the aetiology remains incompletely understood. Recently, microbiota have been shown to differentially regulate the development of autoimmune diseases, but their influence on psoriasis is incompletely understood. We show here that adult mice treated with antibiotics that target Gram-negative and Gram-positive bacteria develop ameliorated psoriasiform dermatitis induced by imiquimod, with decreased pro-inflammatory IL-17- and IL-22-producing T cells. Surprisingly, mice treated neonatally with these antibiotics develop exacerbated psoriasis induced by imiquimod or recombinant IL-23 injection when challenged as adults, with increased IL-22-producing γδ+ T cells. 16S rRNA gene compositional analysis reveals that neonatal antibiotic-treatment dysregulates gut and skin microbiota in adults, which is associated with increased susceptibility to experimental psoriasis. This link between neonatal antibiotic-mediated imbalance in microbiota and development of experimental psoriasis provides precedence for further investigation of its specific aetiology as it relates to human psoriasis., Commensal microbes are necessary for proper development of the immune system. Here Zanvit et al. show that neonatal antibiotics treatment causes long-term changes in the gut and skin microbiomes, and exacerbates immune-mediated skin pathology at adult age in mouse experimental models of psoriasis.

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation. DOI: http://dx.doi.org/10.7554/eLife.03416.001, eLife digest The immune system protects the body from dangerous microbes and removes damaged cells. However, in some cases, the immune system can malfunction and attack healthy tissues, which can lead to type-1 diabetes, multiple sclerosis, and other autoimmune diseases. Many of the current treatments for these disorders suppress the immune system, which can make the individuals more susceptible to infections. It may be possible to treat autoimmune diseases using small pieces of protein—called peptides—that are based on proteins found on the cells that the immune system attacks by mistake. This strategy would target the specific immune cells that are malfunctioning, but allow the rest of the immune system to continue to work as normal. Peptide-based therapies for autoimmune diseases are currently being tested in clinical trials, and although the results look promising, it is not known precisely how they work. McPherson et al. used mice that develop a disease similar to multiple sclerosis because some of their immune cells, known as effector T cells, attack a protein found in the mouse brain called MBP. The mice were treated with a peptide based on part of MBP, which prevented them from developing the autoimmune disease. The success of the peptide therapy depended on the T cells producing large amounts of a protein called PD-1. This protein stops the T-cells from activating immune responses when they detect the MBP protein. The gene that makes PD-1 can have a methyl-tag—a chemical modification to DNA—which alters how much PD-1 is made in the T cells. When the gene has this methyl-tag, it can only be switched on for a short time to make a small amount of PD-1, which helps to control the immune responses activated by the T cell. However, when the methyl-tag was removed as a result of the peptide therapy the gene could be switched on for much longer, so that much more PD-1 was produced. This work helps us to understand how peptide therapy works and should improve the chances of using this therapy to successfully treat patients with autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.03416.002

The export of secretory cargo from the endoplasmic reticulum is mediated by the COPII complex. In common with other aspects of intracellular transport, this step is regulated by protein kinase signalling. Recruitment of the COPII complex to the membrane is known to require ATP and to be blocked by the protein kinase inhibitor H-89. The identity of the specific protein kinase or kinases involved remains equivocal. Here we show that the Sec23p subunit of COPII interacts with PCTAIRE protein kinases. This interaction is shown using two-hybrid screening, direct binding and immunoprecipitation. Inhibition of PCTAIRE kinase activity by expression of a kinase-dead mutant, or specific depletion of PCTAIRE using RNAi, leads to defects in early secretory pathway function including cargo transport, as well as vesicular-tubular transport carrier (VTC) and Golgi localization. These data show a role for PCTAIRE protein kinase function in membrane traffic through the early secretory pathway.

At barrier sites, resident immune cell populations help to maintain tissue homeostasis and function. These cells receive and integrate key signals from the local environment including stromal/epithelial cells and the commensal microbiome. Studies of the skin and gastrointestinal tract have revealed the importance of these signals for the development of host immune response. However, which commensal or tissue-specific cues are important for the immune system at the oral barrier remains minimally explored. Th17 cells have been described as key mediators of immunity at the oral barrier but also essential for periodontitis, a highly prevalent inflammatory pathology that affects the gingiva. In this study we focused in the identification of the mechanisms controlling the induction and regulation of Th17 cells in the gingiva. Our data show that IL-17-producing CD4+ T cells increase with age and their accumulation at the oral barrier occurs independently of commensal colonization. Moreover, we demonstrate that IL-6 elicited by physiological mechanical damage during mastication shapes the function of T cells at the oral mucosa, promoting Th17 differentiation. Finally, we observe that long-term mechanical damage through mastication induces IL-17 mediated bone loss at the gingival barrier. Our data highlight the notion that a variety of signals may be essential to shape the immune responses at different barrier sites, and particularly at the oral cavity, unique mechanisms modulates homeostatic and also pathogenic Th17 responses.

SummaryRegulatory T (Treg) cells are essential for maintenance of immune homeostasis. Here we found that hydrogen sulfide (H2S) was required for Foxp3+ Treg cell differentiation and function and that H2S deficiency led to systemic autoimmune disease. H2S maintained expression of methylcytosine dioxygenases Tet1 and Tet2 by sulfhydrating nuclear transcription factor Y subunit beta (NFYB) to facilitate its binding to Tet1 and Tet2 promoters. Transforming growth factor-β (TGF-β)-activated Smad3 and interleukin-2 (IL-2)-activated Stat5 facilitated Tet1 and Tet2 binding to Foxp3. Tet1 and Tet2 catalyzed conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in Foxp3 to establish a Treg-cell-specific hypomethylation pattern and stable Foxp3 expression. Consequently, Tet1 and Tet2 deletion led to Foxp3 hypermethylation, impaired Treg cell differentiation and function, and autoimmune disease. Thus, H2S promotes Tet1 and Tet2 expression, which are recruited to Foxp3 by TGF-β and IL-2 signaling to maintain Foxp3 demethylation and Treg-cell-associated immune homeostasis.

Harnessing regulatory T (Treg) cells is a promising approach for treating autoimmune disease. However, inducing antigen-specific Treg cells that target inflammatory immune cells without compromising beneficial immune responses has remained an unmet challenge. We developed a pathway to generate autoantigen-specific Treg cells in vivo, which showed therapeutic effects on experimental autoimmune encephalomyelitis and nonobese diabetes in mice. Specifically, we induced apoptosis of immune cells by systemic sublethal irradiation or depleted B and CD8(+) T cells with specific antibodies and then administered autoantigenic peptides in mice with established autoimmune diseases. We demonstrated mechanistically that apoptotic cells triggered professional phagocytes to produce transforming growth factor-β, under which the autoantigenic peptides directed naive CD4(+) T cells to differentiate into Foxp3(+) Treg cells instead of into T effector cells in vivo. These antigen-specific Treg cells specifically ameliorated autoimmunity without compromising immune responses to bacterial antigen. We have thus successfully generated antigen-specific Treg cells with therapeutic activity toward autoimmunity. The findings may lead to the development of antigen-specific Treg cell-mediated immunotherapy for multiple sclerosis and type 1 diabetes and also other autoimmune diseases.

Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of β₂ integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)-deficient mice--which exhibit the LAD-I periodontal phenotype--was associated with excessive production of predominantly T cell-derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1-deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1-deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17-driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17-targeted therapy for periodontitis in LAD-I patients.

Maintenance of immune tolerance critically depends upon regulatory T cells that express the transcription factor forkhead box P3 (Foxp3). These CD4(+) T cells can be generated in the thymus, termed thymus-derived regulatory T cells (tTregs), but their developmental pathway remains incompletely understood. tTreg development has been shown to be delayed compared with that of CD4(+) single positive (SP) thymocytes, with tTregs being detected only in neonatal thymi by day 3 after birth. Here, we outline the reasons for this delayed emergence of Foxp3(+) tTregs and demonstrate that thymocyte apoptosis is intrinsically tied to tTreg development. We show that thymic apoptosis leads to the production of TGFβ intrathymically from thymic macrophages, dendritic cells, and epithelial cells. This TGFβ then induces foxp3 expression and drives tTreg generation. Thymocyte apoptosis has previously been shown to accelerate after birth, which drives increases in TGFβ in the neonatal thymus. We highlight a paucity of TGFβ in the neonatal thymus, accounting for the delayed development of tTregs compared with CD4(+) SP thymocytes. Importantly, we show that enhanced levels of apoptosis in the thymus result in an augmented tTreg population and, moreover, that decreasing thymic apoptosis results in reduced tTregs. In addition to this, we also show that T-cell receptor (TCR) signals of different affinity were all capable of driving tTreg development; however, to achieve this TGFβ signals must also be received concomitant with the TCR signal. Collectively, our results indicate that thymic apoptosis is a key event in tTreg generation and reveal a previously unrecognized apoptosis-TGFβ-Foxp3 axis that mediates the development of tTregs.

Transforming growth factor-β (TGF-β) receptors (TβRs) are essential components for TGF-β signal transduction in T cells, yet the mechanisms by which the receptors are regulated remain poorly understood. We show here that Poly(ADP-ribose) polymerase-1 (PARP-1) regulates TGF-β receptor I (TβRI) and II (TβRII) expression in CD4(+) T cells and subsequently affects Smad2/3-mediated TGF-β signal transduction. Inhibition of PARP-1 led to the upregulation of both TβRI and TβRII, yet the underlying molecular mechanisms were distinct. PARP-1 selectively bound to the promoter of TβRII, whereas the enzymatic activity of PARP-1 was responsible for the inhibition of TβRI expression. Importantly, inhibition of PARP-1 also enhanced expression of TβRs in human CD4(+) T cells. Thus, PARP-1 regulates TβR expression and TGF-β signaling in T cells.

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme and transcription factor that is involved in inflammatory response, but its role in T cell response remains largely unknown. We show here that PARP-1 regulates the suppressive function of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Specifically, Tregs in mice with a null mutation of the PARP-1 gene (PARP-1(-/-)) showed significantly stronger suppressive activity than did wild-type Tregs in culture. We elucidate that this enhanced suppressive function is attributed to sustained higher expression of Foxp3 and CD25 in PARP-1(-/-) Tregs. Furthermore, in PARP-1(-/-) Tregs, Foxp3 protein shows substantially higher levels of binding to the conserved non-coding DNA sequence 2 (CNS2) at the foxp3 gene, a region important in maintaining Foxp3 gene expression in Tregs. Thus, our data reveal a role for PARP-1 in controlling the function of Tregs through modulation of the stable expression of Foxp3.