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Introduction: Stroke is characterized by deleterious oxidative stress. Selenoprotein enzymes are essential endogenous antioxidants, and detailed insight into their role after stroke could define new therapeutic treatments. This systematic review aimed to elucidate how blood selenoprotein concentration and activity change in the acute phase of stroke. Methods: We searched PubMed, EMBASE, and Medline databases for studies measuring serial blood selenoprotein concentration or activity in acute stroke patients or in stroke patients compared to non-stroke controls. Meta-analyses of studies stratified by the type of stroke, blood compartment, and type of selenoprotein measurement were conducted. Results: Eighteen studies and data from 941 stroke patients and 708 non-stroke controls were included in this review. Glutathione peroxidase (GPx) was the only identified selenoprotein, and its activity was most frequently measured. Results from 12 studies and 693 patients showed that compared to non-stroke controls in acute ischaemic stroke patients, the GPx activity increased in haemolysate (standardized mean difference [SMD]: 0.27, 95% CI: 0.07–0.47) but decreased in plasma (mean difference [MD]: −1.08 U/L, 95% CI: −1.94 to −0.22) and serum (SMD: −0.54, 95% CI: −0.91 to −0.17). From 4 identified studies in 106 acute haemorrhagic stroke patients, the GPx activity decreased in haemolysate (SMD: −0.40, 95% CI: −0.68 to −0.13) and remained unchanged in plasma (MD: −0.10 U/L, 95% CI: −0.81 to 0.61) and serum (MD: −5.00 U/mL, 95% CI: −36.17 to 26.17) compared to non-stroke controls. Results from studies assessing the GPx activity in the haemolysate compartment were inconsistent and characterized by high heterogeneity. Conclusions: Our results suggest a reduction of the blood GPx activity in acute ischaemic stroke patients, a lack of evidence regarding a role for GPx in haemorrhagic stroke patients, and insufficient evidence for other selenoproteins.

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.

Eosinophils are key contributors to allergic pathology, however, increasingly eosinophils are described to have important roles in organ health and immunoregulation. Factors that impact these diverse functions of eosinophils are not understood. Here we show in allergic-type lung inflammation, metabolically distinct populations of eosinophils can be identified based on expression of Siglec-F (Siglec-Fhi and Siglec-Fint). Notably, the lung Siglec-Fhi population was responsive to the commensal microbiome, expressing the short-chain fatty acid receptor GPR109A. Animals deficient in GPR109A displayed augmented eosinophilia during allergy. Moreover, transferred GPR109A-deficient eosinophils released more eosinophil peroxidase than controls. Treatment with butyrate or vitamin B3, both GPR109A ligands, reduced Siglec-Fhi eosinophil frequency and activation, which was associated with apoptosis of Siglec-Fhi eosinophils. These findings identify GPR109A as an unappreciated regulator of glycolytic Siglec-Fhi eosinophils, raising the possibility of depleting pathological eosinophil populations in disease states while sparing those with homeostatic functions.

Acute ischaemic and haemorrhagic stroke account for significant disability and morbidity burdens worldwide. The myeloid arm of the peripheral innate immune system is critical in the immunological response to acute ischaemic and haemorrhagic stroke. Neutrophils, monocytes, and dendritic cells (DC) contribute to the evolution of pathogenic local and systemic inflammation, whilst maintaining a critical role in ongoing immunity protecting against secondary infections. This review aims to summarise the key alterations to myeloid immunity in acute ischaemic stroke, intracerebral haemorrhage (ICH), and subarachnoid haemorrhage (SAH). By integrating clinical and preclinical research, we discover how myeloid immunity is affected across multiple organ systems including the brain, blood, bone marrow, spleen, and lung, and evaluate how these perturbations associate with real-world outcomes including infection. These findings are placed in the context of the rapidly developing field of human immunology, which offers a wealth of opportunity for further research.

Stroke affects millions of people across the globe and infections following a stroke often lead to death or disability in patients. Looking rapidly after a stroke in patients, we identify early alterations to the innate and adaptive cells in circulation. Our findings highlight novel changes to dendritic cells, monocytes, haematopoietic stem and progenitor cells, B and T cells to be further investigated as they could have implications the development of post‐stroke infection and poorer patient outcomes., Summary Systemic immune changes following ischaemic stroke are associated with increased susceptibility to infection and poor patient outcome due to their role in exacerbating the ischaemic injury and long‐term disability. Alterations to the abundance or function of almost all components of the immune system post‐stroke have been identified, including lymphocytes, monocytes and granulocytes. However, subsequent infections have often confounded the identification of stroke‐specific effects. Global understanding of very early changes to systemic immunity is critical to identify immune targets to improve clinical outcome. To this end, we performed a small, prospective, observational study in stroke patients with immunophenotyping at a hyperacute time point (

The emergence of SARS-CoV-2 triggering the COVID-19 pandemic ranks as arguably the greatest medical emergency of the last century. COVID-19 has highlighted health disparities both within and between countries and will leave a lasting impact on global society. Nonetheless, substantial investment in life sciences over recent decades has facilitated a rapid scientific response with innovations in viral characterization, testing, and sequencing. Perhaps most remarkably, this permitted the development of highly effective vaccines, which are being distributed globally at unprecedented speed. In contrast, drug treatments for the established disease have delivered limited benefits so far. Innovative and rapid approaches in the design and execution of large-scale clinical trials and repurposing of existing drugs have saved many lives; however, many more remain at risk. In this review we describe challenges and unmet needs, discuss existing therapeutics, and address future opportunities. Consideration is given to factors that have hindered drug development in order to support planning for the next pandemic challenge and to allow rapid and cost-effective development of new therapeutics with equitable delivery.

Post-stroke infection is a common complication of stroke that is associated with increased mortality and morbidity. We previously found that experimental stroke induces an ablation of multiple sub-populations of B cells and reduced levels of IgM antibody that coincide with the development of spontaneous bacterial pneumonia. Reduced circulating IgM concentrations were also observed in acute stroke patients. The loss of IgM antibody after stroke could be an important determinant of infection susceptibility and highlights this pathway as an important target for intervention.We treated mice with a low (replacement), dose of IgM-enriched intravenous immunoglobulin (IgM-IVIg) prior to and 24 h after experimental stroke induced by middle cerebral artery occlusion (MCAO) or sham surgery, then recovered mice for 2 d or 5 d. The effect of treatment on lung bacterial burden, lung pathology, brain infarct volume, antibody levels and both lung and systemic cellular and cytokine immune profiles was determined. Treatment with IgM-IVIg enhanced bacterial clearance from the lung after MCAO and improved pathology but did not impact infarct volume. IgM-IVIg treatment induced immunomodulatory effects systemically including rescue of splenic plasma B cell numbers and endogenous mouse IgM and IgA circulating immunoglobulin concentrations that were reduced by MCAO, and treatment also reduced concentrations of pro-inflammatory cytokines in the lung. The effects of MCAO and IgM-IVIg treatment on the immune system were tissue specific as no impact on B cells or mouse immunoglobulins were found within the lung. However, the presence of human immunoglobulins from the IgM-IVIg treatment led to increased total lung immunoglobulin concentration. IgM-IVIg treatment did not increase the number of lung mononuclear phagocytes or directly modulate macrophage phagocytic capacity but enhanced their capability to phagocytose Staphylococcus aureus bioparticles in vitro by increasing opsonisation.Low dose IgM-IVIg contributes to increased clearance of spontaneous lung bacteria after MCAO likely via increasing availability of antibody in the lung to enhance phagocytic activity. Immunomodulatory effects of IgM-IVIg treatment, including reduced pro-inflammatory cytokine production, may also contribute to reduced levels of damage in the lung after MCAO. IgM-IVIg shows promise as an antibacterial and immunomodulatory agent to use in the treatment of post-stroke infection.GRAPHICAL ABSTRACT

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.

Chronic inflammation is a hallmark of impaired healing in a plethora of tissues, including skin, and is associated with aging and diseases such as diabetes. Diabetic chronic skin wounds are characterized by excessive myeloid cells that display an aberrant phenotype, partially mediated by stable intrinsic changes induced during hematopoietic development.However, the relative contribution of myeloid cell-intrinsic factors to chronic inflammation versus aberrant signals from the local environmental was unknown. Moreover, identificationof myeloid cell-intrinsic factors that contribute to chronic inflammation in diabetic wounds remained elusive. Here we show that Gr-1+CD11b+ myeloid cells are retained specifically within the presumptive granulation tissue region of the wound at a higher density in diabetic mice and associate with endothelial cells at the site of injury with a higher frequency than innon-diabetic mice. Adoptive transfer of myeloid cells demonstrated that aberrant wound retention is due to myeloid cell-intrinsic factors and not the local environment. RNAsequencing of bone marrow and wound-derived myeloid cells identified Selplg as a myeloid cell-intrinsic factor that is deregulated in chronic wounds. In vivo blockade of this protein significantly accelerated wound healing in diabetic mice and may be a potential therapeutictarget in chronic wounds and other chronic inflammatory diseases.

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

Background/Aims Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may develop acute respiratory inflammation, due to an exaggerated immune response and some develop chronic complications. Neutrophils play a major role in the pathology of inflammatory diseases and have been shown to contribute to lung and vascular damage in COVID-19. Our aim was to establish a relationship between neutrophil phenotype and disease severity and to determine whether neutrophil abnormalities persist in convalescent patients. Methods Peripheral blood samples were obtained from acute COVID-19 patients (n = 74), follow-up (FU) patients discharged following inpatient admission (n = 56), a median of 87 days after discharge, and healthy controls (HCs, n = 22). Patients were stratified by disease severity based on inspired oxygen (FiO2) and admission to intensive care (ICU). Neutrophils were isolated from whole blood by negative selection for phenotyping and functional analysis. PBMC Isolation Tubes were used to quantify and phenotype low density neutrophils (LDNs) within the PBMC fraction. For quantification of reactive oxygen species (ROS) production, isolated neutrophils were incubated with a ROS reactive dye, DHR-123 and stimulated with PMA. All samples were stained and fixed prior to analysis by flow cytometry. Results There was a marked increase in neutrophils expressing the activation and degranulation markers, CD64 (P < 0.0001) and CD63 (P < 0.0001) and a reduction in neutrophils expressing the maturity markers, CD10 (P < 0.0005) and CD101 (P < 0.0005) in patients with acute COVID-19 compared to HCs. Increased frequency of neutrophils expressing CD64 (P < 0.005), CD63 (P < 0.01) and expressing decreased CD101 (P < 0.0001) were also detected in FU patients compared to HCs. Notably, 42.3 ± 4.4% of neutrophils were CD101lo in FU patients, compared to 29.0 ± 3.7% in acute patients and 9.6 ± 4.1% in HCs. These changes were most apparent in FU patients recovering from severe COVID-19 compared to mild or moderate disease. The frequency of LDNs in PBMCs from acute patients was significantly higher than HCs (P < 0.0001), and correlated with disease severity. Similarly, the frequency of LDNs in FU patients was significantly higher than in HCs (P < 0.0005). We found a trend towards higher basal ROS production in acute and FU patients, but a blunted response to PMA stimulated ROS production in neutrophils from acute patients versus HCs (P < 0.0001). Impaired ROS production persisted in FU patients compared to HCs (P < 0.01). Conclusion Circulating neutrophils in acute COVID-19 have an altered phenotype and comprise immature and activated cells. This altered phenotype persisted in convalescence and may contribute to the persistence of symptoms and an increased susceptibility to subsequent infections. Future work will aim to investigate the functional implications of these findings. Disclosure T.O. Williams: None. V. Kästele: None. E.R. Mann: None. S.B. Knight: None. M. Menon: None. C. Jagger: None. S. Khan: None. J.E. Konkel: None. T.N. Shaw: None. M. Rattray: Consultancies; M.R. has a paid consultancy with AstraZeneca. L. Pearmain: None. A. Horsley: None. A. Ustianowski: None. I. Prise: None. N.D. Bakerly: None. P.M. Dark: None. G.M. Lord: Corporate appointments; G.M.L. is cofounder and scientific advisory board member of Gritstone Oncology Inc., which is a public company that develops therapeutic vaccines (primarily for the treatment of cancer). A. Simpson: None. T. Felton: None. L. Ho: None. M. Feldmann: None. I. Bruce: None. J.R. Grainger: None. T. Hussell: None.

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).

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.

The cornerstone of life-saving therapy in immune-mediated thrombotic thrombocytopenic purpura (iTTP) has been plasma exchange (PEX) combined with immunomodulatory strategies. Caplacizumab, a novel anti–von Willebrand factor nanobody trialed in 2 multicenter randomized controlled trials (RCTs) leading to European Union and US Food and Drug Administration approval, has been available in the United Kingdom (UK) through a patient access scheme. Data were collected retrospectively from 2018 to 2020 for 85 patients (4 children) receiving caplacizumab from 22 UK hospitals. Patient characteristics and outcomes in the real-world clinical setting were compared with caplacizumab trial end points and historical outcomes in the precaplacizumab era. Eighty-four of 85 patients received steroid and rituximab alongside PEX; 26% required intubation. Median time to platelet count normalization (3 days), duration of PEX (7 days), and hospital stay (12 days) were comparable with RCT data. Median duration of PEX and time from PEX initiation to platelet count normalization were favorable compared with historical outcomes (P < .05). Thrombotic thrombocytopenic purpura (TTP) recurred in 5 of 85 patients; all had persistent ADAMTS13 activity < 5 IU/dL. Of 31 adverse events in 26 patients, 17 of 31 (55%) were bleeding episodes, and 5 of 31 (16%) were thrombotic events (2 unrelated to caplacizumab); mortality was 6% (5/85), with no deaths attributed to caplacizumab. In 4 of 5 deaths, caplacizumab was introduced >48 hours after PEX initiation (3-21 days). This real-world evidence represents the first and largest series of TTP patients, including pediatric patients, receiving caplacizumab outside of clinical trials. Representative of true clinical practice, the findings provide valuable information for clinicians treating TTP globally.

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.

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

Recent research has shed light on the plethora of mechanisms by which the gastrointestinal commensal microbiome can influence the local immune response in the gut (in particular, the impact of the immune system on epithelial barrier homeostasis and ensuring microbial diversity). However, an area that is much less well explored but of tremendous therapeutic interest is the impact the gut microbiome has on systemic cell-mediated immune responses. In this commentary, we highlight some key studies that are beginning to broadly examine the different mechanisms by which the gastrointestinal microbiome can impact the systemic immune compartment. Specifically, we discuss the effects of the gut microbiome on lymphocyte polarisation and trafficking, tailoring of resident immune cells in the liver, and output of circulating immune cells from the bone marrow. Finally, we explore contexts in which this new understanding of long-range effects of the gut microbiome can have implications, including cancer therapies and vaccination.

SummaryShifts in commensal microbiota composition are emerging as a hallmark of gastrointestinal inflammation. In particular, outgrowth of γ-proteobacteria has been linked to the etiology of inflammatory bowel disease and the pathologic consequences of infections. Here we show that following acute Toxoplasma gondii gastrointestinal infection of mice, control of commensal outgrowth is a highly coordinated process involving both the host response and microbial signals. Notably, neutrophil emigration to the intestinal lumen results in the generation of organized intraluminal structures that encapsulate commensals and limit their contact with the epithelium. Formation of these luminal casts depends on the high-affinity N-formyl peptide receptor, Fpr1. Consequently, after infection, mice deficient in Fpr1 display increased microbial translocation, poor commensal containment, and increased mortality. Altogether, our study describes a mechanism by which the host rapidly contains commensal pathobiont outgrowth during infection. Further, these results reveal Fpr1 as a major mediator of host commensal interaction during dysbiosis.

The commensal flora can promote both immunity to pathogens and mucosal inflammation. How commensal-driven inflammation is regulated in the context of infection remains poorly understood. Here, we show that during acute mucosal infection of mice with Toxoplasma gondii, inflammatory monocytes acquire a tissue-specific regulatory phenotype associated with production of the lipid mediator prostaglandin E2 (PGE2). Notably, in response to commensals, inflammatory monocytes can directly inhibit neutrophil activation in a PGE2-dependent manner. Further, in the absence of inflammatory monocytes, mice develop severe neutrophil-mediated pathology in response to pathogen challenge that can be controlled by PGE2 analog treatment. Complementing these findings, inhibition of PGE2 led to enhanced neutrophil activation and host mortality after infection. These data demonstrate a previously unappreciated dual action of inflammatory monocytes in controlling pathogen expansion while limiting commensal-mediated damage to the gut. Collectively, our results place inflammatory monocyte-derived PGE2 at the center of a commensal-driven regulatory loop required to control host-commensal dialog during pathogen-induced inflammation.

Retinoic acid is required to maintain pre-DC–derived CD11b+CD8α−Esamhigh dendritic cells (DCs) in the spleen and CD11b+CD103+ DCs in the gut., Dendritic cells (DCs) comprise distinct populations with specialized immune-regulatory functions. However, the environmental factors that determine the differentiation of these subsets remain poorly defined. Here, we report that retinoic acid (RA), a vitamin A derivative, controls the homeostasis of pre-DC (precursor of DC)–derived splenic CD11b+CD8α−Esamhigh DCs and the developmentally related CD11b+CD103+ subset within the gut. Whereas mice deprived of RA signaling significantly lost both of these populations, neither pre-DC–derived CD11b−CD8α+ and CD11b−CD103+ nor monocyte-derived CD11b+CD8α−Esamlow or CD11b+CD103− DC populations were deficient. In fate-tracking experiments, transfer of pre-DCs into RA-supplemented hosts resulted in near complete conversion of these cells into the CD11b+CD8α− subset, whereas transfer into vitamin A–deficient (VAD) hosts caused diversion to the CD11b−CD8α+ lineage. As vitamin A is an essential nutrient, we evaluated retinoid levels in mice and humans after radiation-induced mucosal injury and found this conditioning led to an acute VAD state. Consequently, radiation led to a selective loss of both RA-dependent DC subsets and impaired class II–restricted auto and antitumor immunity that could be rescued by supplemental RA. These findings establish a critical role for RA in regulating the homeostasis of pre-DC–derived DC subsets and have implications for the management of patients with immune deficiencies resulting from malnutrition and irradiation.

SummaryIt is thought that monocytes rapidly differentiate to macrophages or dendritic cells (DCs) upon leaving blood. Here we have shown that Ly-6C+ monocytes constitutively trafficked into skin, lung, and lymph nodes (LNs). Entry was unaffected in gnotobiotic mice. Monocytes in resting lung and LN had similar gene expression profiles to blood monocytes but elevated transcripts of a limited number of genes including cyclo-oxygenase-2 (COX-2) and major histocompatibility complex class II (MHCII), induced by monocyte interaction with endothelium. Parabiosis, bromodoxyuridine (BrdU) pulse-chase analysis, and intranasal instillation of tracers indicated that instead of contributing to resident macrophages in the lung, recruited endogenous monocytes acquired antigen for carriage to draining LNs, a function redundant with DCs though differentiation to DCs did not occur. Thus, monocytes can enter steady-state nonlymphoid organs and recirculate to LNs without differentiation to macrophages or DCs, revising a long-held view that monocytes become tissue-resident macrophages by default.

The microbiota contributes to the induction of both effector and regulatory responses in the gastrointestinal (GI) tract. However, the mechanisms controlling these distinct properties remain poorly understood. We previously showed that commensal DNA promotes intestinal immunity. Here, we find that the capacity of bacterial DNA to stimulate immune responses is species specific and correlated with the frequency of motifs known to exert immunosuppressive function. In particular, we show that the DNA of Lactobacillus species, including various probiotics, is enriched in suppressive motifs able to inhibit lamina propria dendritic cell activation. In addition, immunosuppressive oligonucleotides sustain T(reg) cell conversion during inflammation and limit pathogen-induced immunopathology and colitis. Altogether, our findings identify DNA-suppressive motifs as a molecular ligand expressed by commensals and support the idea that a balance between stimulatory and regulatory DNA motifs contributes to the induction of controlled immune responses in the GI tract and gut immune homeostasis. Further, our findings suggest that the endogenous regulatory capacity of DNA motifs enriched in some commensal bacteria could be exploited for therapeutic purposes.

SummaryT helper (Th) cells are critical for defenses against infection and recognize peptides bound to class II major histocompatibility complex (MHC II) molecules. Although transcription factors have been identified that direct Th cells into specific effector fates, whether a “master” regulator controls the developmental program common to all Th cells remains unclear. Here, we showed that the two transcription factors Thpok and LRF share this function. Although disruption of both factors did not prevent the generation of MHC II-specific T cells, these cells failed to express Th cell genes or undergo Th cell differentiation in vivo. In contrast, T cells lacking Thpok, which only displayed LRF-dependent functions, contributed to multiple effector responses, both in vitro and in vivo, with the notable exception of Th2 cell responses that control extracellular parasites. These findings identify the Thpok-LRF pair as a core node of Th cell differentiation and function.

The intestinal nematode parasite Heligmosomoides polygyrus bakeri exerts widespread immunomodulatory effects on both the innate and adaptive immune system of the host. Infected mice adopt an immunoregulated phenotype, with abated allergic and autoimmune reactions. At the cellular level, infection is accompanied by expanded regulatory T cell populations, skewed dendritic cell and macrophage phenotypes, B cell hyperstimulation and multiple localised changes within the intestinal environment. In most mouse strains, these act to block protective Th2 immunity. The molecular basis of parasite interactions with the host immune system centres upon secreted products termed HES (H. polygyrus excretory-secretory antigen), which include a TGF-β-like ligand that induces de novo regulatory T cells, factors that modify innate inflammatory responses, and molecules that block allergy in vivo. Proteomic and transcriptomic definition of parasite proteins, combined with biochemical identification of immunogenic molecules in resistant mice, will provide new candidate immunomodulators and vaccine antigens for future research.

Vitamin A elicits a broad array of immune responses through its metabolite, retinoic acid (RA). Recent evidence indicates that loss of RA leads to impaired immunity, whereas excess RA can potentially promote inflammatory disorders. In this review, we discuss recent advances showcasing the crucial contributions of RA to both immunological tolerance and the elicitation of adaptive immune responses. Further, we provide a comprehensive overview of the cell types and factors that control the production of RA and discuss how host perturbations may affect the ability of this metabolite to control tolerance and immunity or to instigate pathology.

SummaryVitamin A and its metabolite, retinoic acid (RA) are implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we showed RA was also required to elicit proinflammatory CD4+ helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) was the critical mediator of these effects. Antagonism of RAR signaling and deficiency in RARα (Rara−/−) resulted in a cell-autonomous CD4+ T cell activation defect, which impaired intermediate signaling events, including calcium mobilization. Altogether, these findings reveal a fundamental role for the RA-RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses.

Interleukin 2 (IL-2), a cytokine linked to human autoimmune disease, limits IL-17 production. Here we found that deletion of the gene encoding the transcription factor STAT3 in T cells abrogated IL-17 production and attenuated autoimmunity associated with IL-2 deficiency. Whereas STAT3 induced IL-17 and the transcription factor RORγt and inhibited the transcription factor Foxp3, IL-2 inhibited IL-17 independently of Foxp3 and RORγt. STAT3 and STAT5 bound to multiple common sites across the locus encoding IL-17. The induction of STAT5 binding by IL-2 was associated with less binding of STAT3 at these sites and the inhibition of associated active epigenetic marks. 'Titration' of the relative activation of STAT3 and STAT5 modulated the specification of cells to the IL-17-producing helper T cell (T(H)17 cell) subset. Thus, the balance rather than the absolute magnitude of these signals determined the propensity of cells to make a key inflammatory cytokine.

Neutrophil function is perhaps best studied in bacterial infection, during which they are directly involved in pathogen killing. After helminth invasion, however, neutrophils acquire an alternative transcriptional profile that allows them to 'train' macrophages to acquire long-term protective features.

Each microenvironment is controlled by a specific set of regulatory elements that have to be finely and constantly tuned to maintain local homeostasis. These environments could be site specific, such as the gut environment, or induced by chronic exposure to microbes. Various populations of dendritic cells are central to the orchestration of this control. In this review, we discuss some new findings associating dendritic cells from defined compartments with the induction and control of regulatory T cells in the context of exposure to both commensal and pathogenic microbes.

Helminths are masterful immunoregulators. A characteristic feature of helminth infection is a Th2-dominated immune response, but stimulation of immunoregulatory cell populations, such as regulatory T cells and alternatively activated macrophages, is equally common. Typically, Th1/17 immunity is blocked and productive effector responses are muted, allowing survival of the parasite in a “modified Th2” environment. Drug treatment to clear the worms reverses the immunoregulatory effects, indicating that a state of active suppression is maintained by the parasite. Hence, research has focussed on “excretory–secretory” products released by live parasites, which can interfere with every aspect of host immunity from initial recognition to end-stage effector mechanisms. In this review, we survey our knowledge of helminth secreted molecules, and summarise current understanding of the growing number of individual helminth mediators that have been shown to target key receptors or pathways in the mammalian immune system.

SUMMARYThe transforming growth factor-β (TGF-β) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-β homologues from 2 laboratory model nematodes (Heligmosomoides polygyrusandNippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortusandTeladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as inC. elegansDAF-7,Brugia malayiTGH-2 and mammalian TGF-β. Surprisingly, only theH. contortushomologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that inH. contortusandN. brasiliensisexpression was maximal in the infective larval stage, and very low in adult worms. In contrast, inH. polygyrusandT. circumcincta, tgh-2transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-β homologues while conserving the structure of the active domain.

The immune system of the gastrointestinal tract must be tightly regulated to limit pathologic responses towards innocuous antigens while simultaneously allowing for rapid development of effector responses against invading pathogens. Highly specialized antigen-presenting cell (APC) subsets present in the gut play a dominant role in balancing these seemingly disparate functions. In this review, we discuss new findings associated with the function of gut APCs and particularly the contextual role of these cells in both establishing tolerance to orally acquired antigens in the steady state and regulating acute inflammation during infection.

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.

Background: The TPO receptor agonist romiplostim is approved for use in adults with chronic ITP. The use of romiplostim in children with ITP has been evaluated in phase 1/2 and 3 studies. In this study, children with ITP will receive open-label SC romiplostim for up to 3 years (y). Methods: Eligible children, recruited in 16 countries worldwide, had ITP for ≥6 months, ≥1 prior ITP therapy, and platelet (plt) count ≤30×109/L. Weekly SC dosing started at 1 μg/kg and was titrated in 1 μg/kg increments up to 10 μg/kg to target plt counts of 50-200×109/L. In patients in Europe, bone marrow aspirates and biopsies were obtained at baseline and after 1 or 2 y of exposure. The 1º endpoint was the % of time in the first 6 months with a plt response, with response defined as a plt count ≥50×109/L without rescue medication use in the past 4 weeks (wk). Results: As of 15 Mar 2016, 147 patients enrolled; 145 received ≥1 romiplostim dose. At baseline, median (min-max) age was 10 (2-17) y; 51% were female; 4% had prior splenectomy. Median (min-max) ITP duration was 1.9 (0.5-12.3) y and plt count was 13 (2-168)×109/L. The median (Q1, Q3) % of time with a plt response in the first 6 months patients were on study was 50% (0%, 83.3%); that of months 7-12 was 92% (33%, 100%). Over the course of the study, 80% (114/143) of patients had a plt response. The median (Q1, Q3) % of time with an increase in plt counts ≥20×109/L above baseline was 60% (25%, 84%). The median dose increased to 10 μg/kg by wk 32 (Figure). Median (min-max) treatment duration as of data cutoff was 25 (1-67) wk for a total exposure to date of 79 patient-years; 67 (46%) patients (or caregivers) self-administered romiplostim. Median (min-max) average weekly romiplostim dose was 6.1 (0.4-9.0) µg/kg. Thirty-two patients (22%) discontinued treatment for lack of efficacy (n = 17), required other therapy (n = 5), patient request (n = 4), noncompliance (n = 2), adverse event (AE) (n = 2) (interstitial lung disease unrelated to treatment in a 15 y old boy and abdominal pain, vomiting, and headache related to treatment per investigator in a 9 y old girl), administrative decision (n = 1), and investigator decision (n = 1). After wk 12, median plt counts remained ≥50×109/L (Figure). Thirty-four (23%) patients received rescue medications. The most frequently reported AEs were headache (27.6%), epistaxis (22.8%), and nasopharyngitis (23%); 15 (10.3%) patients had serious AEs (SAEs) including epistaxis (n = 4), petechiae (n = 2), decreased plt count (n = 2), and thrombocytopenia (n = 2). A case of abdominal pain was the only SAE deemed treatment-related by the investigator. Bleeding over the course of the study was seen in 52% of patients. CTCAE grade 3 bleeding was seen in 8 patients (6%) and included epistaxis (n = 5), ecchymosis (n = 2), petechiae (n = 2), and 1 case each of hematemesis, hematoma, SC hemorrhage, injection site hemorrhage, and mouth hemorrhage. No grade 4 or 5 bleeding was observed. One event of grade 2 phlebitis/thrombophlebitis in deep veins in the left arm lasting 14 days was reported in a 13 y old girl; plt counts were 40-70×109/L and the dose was 7-8 µg/kg during this time. Per investigator, this event was not serious or related to romiplostim. She was treated with antibiotics (oral amoxicillin/clavulanic acid and transdermal muciprocin) and continued romiplostim treatment as before. No neutralizing antibodies against romiplostim or TPO were identified. Of 30 patients with baseline bone marrow biopsies [50% female, median (min-max) age 10.5 (6-12) y, ITP duration 3.1 (0.6-11) y], all had modified Bauermeister scores of grade 0 (no reticulin) or 1 (fine fibers) and bone marrow typical for ITP. Of these 30 patients, 21 had evaluable on-study biopsies, with no increases in 2 or more grades, findings of collagen, or bone marrow abnormalities. Conclusion: In this year 1 datacut of an ongoing open-label study of romiplostim in children with ITP, the % of time in the first 6 months with a platelet response was 50%, with 80% of children having a platelet response at some point on study. The median romiplostim dose reached 10 μg/kg and there were no new safety signals. No effects of romiplostim were observed in the subset of patients with bone marrow biopsies. Future datacuts for year 2 and 3 in this study, the largest of romiplostim in children with ITP with 79 patient-years of exposure to date, will provide more information on platelet response, dose requirements, and safety. Disclosures Grainger: Baxter: Honoraria, Research Funding; Amgen Inc.: Honoraria; Novartis: Honoraria; GlaxoSmithKline: Honoraria. Bussel:Shionogi: Membership on an entity's Board of Directors or advisory committees; Symphogen: Membership on an entity's Board of Directors or advisory committees; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Research Funding; BiologicTx: Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prophylix Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Protalex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Physicians Education Resource: Speakers Bureau; Genzyme: Research Funding; UpToDate: Patents & Royalties; Momenta Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Immunomedics: Research Funding; Cangene: Research Funding; Sysmex: Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cooper:UK ITP support association: Research Funding; National Organization for Rare Disorders: Research Funding; Imperial College BRC: Research Funding; Eisai: Consultancy, Honoraria, Other: Received honoraria for speaking at educational meetings; GlaxoSmithKline: Consultancy, Honoraria, Other: Received honoraria for speaking at educational meetings ; Amgen Inc.: Consultancy, Honoraria, Other: Received honoraria for speaking at educational meetings . Tarantino:Pfizer: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Grifols: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Biogen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees. Blanchette:Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Data safety monitoring boards , Research Funding; Bayer Healthcare: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Octapharma: Other: Data safety monitoring boards . Carpenter:Amgen Inc.: Employment, Equity Ownership. Eisen:Amgen Inc.: Employment, Equity Ownership. Mehta:Amgen Inc.: Employment, Equity Ownership.

Heligmosomoides polygyrus is a widely used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary Ab response as a subset of the secreted products in H. polygyrus excretory–secretory (HES) Ag. An immunodominant epitope is an O-linked glycan (named glycan A) carried on three highly expressed HES glycoproteins (venom allergen Ancylostoma-secreted protein-like [VAL]-1, -2, and -5), which stimulates only IgM Abs, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (glycan B), present on both a non-protein high molecular mass component and a 65-kDa molecule, is widely distributed in adult somatic tissues. Whereas the high molecular mass component and 65-kDa molecules bear phosphorylcholine, the glycan B epitope itself is not phosphorylcholine. Class-switched IgG1 Abs are found to glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary Ab responses include the same specificities while also recognizing VAL-7. Although vaccination with HES conferred complete protection against challenge H. polygyrus infection, mAbs raised against each of the glycan epitopes and against VAL-1, VAL-2, and VAL-4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective Abs against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity.

The intestinal helminth parasite, Heligmosomoides polygyrus bakeri offers a tractable experimental model for human hookworm infections such as Ancylostoma duodenale and veterinary parasites such as Haemonchus contortus. Parasite excretory-secretory (ES) products represent the major focus for immunological and biochemical analyses, and contain immunomodulatory molecules responsible for nematode immune evasion. In a proteomic analysis of adult H. polygyrus secretions (termed HES) matched to an extensive transcriptomic dataset, we identified 374 HES proteins by LC-MS/MS, which were distinct from those in somatic extract HEx, comprising 446 identified proteins, confirming selective export of ES proteins. The predominant secreted protein families were proteases (astacins and other metalloproteases, aspartic, cysteine and serine-type proteases), lysozymes, apyrases and acetylcholinesterases. The most abundant products were members of the highly divergent venom allergen-like (VAL) family, related to Ancylostoma secreted protein (ASP); 25 homologues were identified, with VAL-1 and -2 also shown to be associated with the parasite surface. The dominance of VAL proteins is similar to profiles reported for Ancylostoma and Haemonchus ES products. Overall, this study shows that the secretions of H. polygyrus closely parallel those of clinically important GI nematodes, confirming the value of this parasite as a model of helminth infection.

Tregs not only keep immune responses to autoantigens in check, but also restrain those directed toward pathogens and the commensal microbiota. Control of peripheral immune homeostasis by Tregs relies on their capacity to accumulate at inflamed sites and appropriately adapt to their local environment. To date, the factors involved in the control of these aspects of Treg physiology remain poorly understood. Here, we show that the canonical Th2 transcription factor GATA3 is selectively expressed in Tregs residing in barrier sites including the gastrointestinal tract and the skin. GATA3 expression in both murine and human Tregs was induced upon TCR and IL-2 stimulation. Although GATA3 was not required to sustain Treg homeostasis and function at steady state, GATA3 played a cardinal role in Treg physiology during inflammation. Indeed, the intrinsic expression of GATA3 by Tregs was required for their ability to accumulate at inflamed sites and to maintain high levels of Foxp3 expression in various polarized or inflammatory settings. Furthermore, our data indicate that GATA3 limits Treg polarization toward an effector T cell phenotype and acquisition of effector cytokines in inflamed tissues. Overall, our work reveals what we believe to be a new facet in the complex role of GATA3 in T cells and highlights what may be a fundamental role in controlling Treg physiology during inflammation.

CD4(+) T-helper cells that selectively produce interleukin (IL)-17 (T(H)17), are critical for host defence and autoimmunity. Although crucial for T(H)17 cells in vivo, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been proposed to be the factors responsible for initiating specification. Here we show that T(H)17 differentiation can occur in the absence of TGF-β signalling. Neither IL-6 nor IL-23 alone efficiently generated T(H)17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naive precursors, independently of TGF-β. Epigenetic modification of the Il17a, Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed RORγt (encoded by Rorc) and T-bet. T-bet(+)RORγt(+) T(H)17 cells are generated in vivo during experimental allergic encephalomyelitis, and adoptively transferred T(H)17 cells generated with IL-23 without TGF-β1 were pathogenic in this disease model. These data indicate an alternative mode for T(H)17 differentiation. Consistent with genetic data linking IL23R with autoimmunity, our findings re-emphasize the importance of IL-23 and therefore may have therapeutic implications.

The gastrointestinal tract is home to the highest density of commensal bacteria in the human body and is a primary site of pathogen exposure. Understanding how the immune system recognizes and responds to friend or foe in the gut is central to developing treatments for allergic and inflammatory diseases. Both specialized cells and structural compartmentalization of the gut are key controllers of immune responses. In particular, a mucus layer covers the entire gastrointestinal tract, physically separating the microbiota from host tissue and preventing pathogen invasion ( 1 ). On page 447 of this issue, Shan et al. ( 2 ) reveal that mucus does more than act as a shield—it also influences the function of intestinal antigen-presenting cells and epithelial cells to sustain the ability of the host to maintain tolerance toward food and commensal antigens.

SummaryTissue-infiltrating Ly6Chi monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DCs) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals after tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function.