Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Ferritin regulates organismal energy balance and thermogenesis.

OBJECTIVE: The ferritin heavy/heart chain (FTH) gene encodes the ferroxidase component of the iron (Fe) sequestering ferritin complex, which plays a central role in the regulation of cellular Fe metabolism. Here we tested the hypothesis that ferritin regulates organismal Fe metabolism in a manner that impacts energy balance and thermal homeostasis. METHODS: We developed a mouse strain, referred herein as FthR26 fl/fl, expressing a tamoxifen-inducible Cre recombinase under the control of the Rosa26 (R26) promoter and carrying two LoxP (fl) sites: one at the 5'end of the Fth promoter and another the 3' end of the first Fth exon. Tamoxifen administration induces global deletion of Fth in adult FthR26Δ/Δ mice, testing whether FTH is required for maintenance of organismal homeostasis. RESULTS: Under standard nutritional Fe supply, Fth deletion in adult FthR26Δ/Δ mice led to a profound deregulation of organismal Fe metabolism, oxidative stress, inflammation, and multi-organ damage, culminating in death. Unexpectedly, Fth deletion was also associated with a profound atrophy of white and brown adipose tissue as well as with collapse of energy expenditure and thermogenesis. This was attributed mechanistically to mitochondrial dysfunction, as assessed in the liver and in adipose tissue. CONCLUSION: The FTH component of ferritin acts as a master regulator of organismal Fe homeostasis, coupling nutritional Fe supply to organismal redox homeostasis, energy expenditure and thermoregulation.

Renal control of disease tolerance to malaria.

Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Disease Tolerance as an Inherent Component of Immunity.

Pathogenic organisms exert a negative impact on host health, revealed by the clinical signs of infectious diseases. Immunity limits the severity of infectious diseases through resistance mechanisms that sense and target pathogens for containment, killing, or expulsion. These resistance mechanisms are viewed as the prevailing function of immunity. Under pathophysiologic conditions, however, immunity arises in response to infections that carry health and fitness costs to the host. Therefore, additional defense mechanisms are required to limit these costs, before immunity becomes operational as well as thereafter to avoid immunopathology. These are tissue damage control mechanisms that adjust the metabolic output of host tissues to different forms of stress and damage associated with infection. Disease tolerance is the term used to define this defense strategy, which does not exert a direct impact on pathogens but is essential to limit the health and fitness costs of infection. Under this argument, we propose that disease tolerance is an inherent component of immunity.

Targeting of Rac1 prevents bronchoconstriction and airway hyperresponsiveness.

BACKGROUND: The molecular mechanisms responsible for airway smooth muscle cells' (aSMCs) contraction and proliferation in airway hyperresponsiveness (AHR) associated with asthma are still largely unknown. The small GTPases of the Rho family (RhoA, Rac1, and Cdc42) play a central role in SMC functions including migration, proliferation, and contraction. OBJECTIVE: The objective of this study was to identify the role of Rac1 in aSMC contraction and to investigate its involvement in AHR associated with allergic asthma. METHODS: To define the role of Rac1 in aSMC, ex and in vitro analyses of bronchial reactivity were performed on bronchi from smooth muscle (SM)-specific Rac1 knockout mice and human individuals. In addition, this murine model was exposed to allergens (ovalbumin or house dust mite extract) to decipher in vivo the implication of Rac1 in AHR. RESULTS: The specific SMC deletion or pharmacological inhibition of Rac1 in mice prevented the bronchoconstrictor response to methacholine. In human bronchi, a similar role of Rac1 was observed during bronchoconstriction. We further demonstrated that Rac1 activation is responsible for bronchoconstrictor-induced increase in intracellular Ca2+ concentration and contraction both in murine and in human bronchial aSMCs, through its association with phospholipase C ß2 and the stimulation of inositol 1,4,5-trisphosphate production. In vivo, Rac1 deletion in SMCs or pharmacological Rac1 inhibition by nebulization of NSC23766 prevented AHR in murine models of allergic asthma. Moreover, nebulization of NSC23766 decreased eosinophil and neutrophil populations in bronchoalveolar lavages from mice with asthma. CONCLUSIONS: Our data reveal an unexpected and essential role of Rac1 in the regulation of intracellular Ca2+ and contraction of aSMCs, and the development of AHR. Rac1 thus appears as an attractive therapeutic target in asthma, with a combined beneficial action on both bronchoconstriction and pulmonary inflammation.

Role of TLRs and DAMPs in allograft inflammation and transplant outcomes.

Graft inflammation impairs the induction of solid organ transplant tolerance and enhances acute and chronic rejection. Elucidating the mechanisms by which inflammation is induced after organ transplantation could lead to novel therapeutics to improve transplant outcomes. In this Review we describe endogenous substances--damage-associated molecular patterns (DAMPs)--that are released after allograft reperfusion and induce inflammation. We also describe innate immune signalling pathways that are activated after solid organ transplantation, with a focus on Toll-like receptors (TLRs) and their signal adaptor, MYD88. Experimental and clinical studies have yielded a large body of evidence that TLRs and MYD88 are instrumental in initiating allograft inflammation and promoting the development of acute and chronic rejection. Ongoing clinical studies are testing TLR inhibition strategies in solid organ transplantation, although avoiding compromising host defence to pathogens is a key challenge. Further elucidation of the mechanisms by which sterile inflammation is induced, maintained and amplified within the allograft has the potential to lead to novel anti-inflammatory treatments that could improve outcomes for solid organ transplant recipients.

Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.

Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti-inflammatory therapy in allergic asthma. However, the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)-induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f-sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and decreased ex vivo carbachol-induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To evaluate in vivo MSC survival, MSCs were labeled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digested to obtain single-cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26(+) lung cells expressed specific macrophage markers in control and Der f mice, respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26(+) macrophages expressed M2 phenotype, while the innate PKH26(-) macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26(+) MSCs expressed 10- to 100-fold more COX-2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. Stem Cells 2016;34:1836-1845.

Deciphering the role of TRIB1 in regulatory T-cells.

The role of regulatory T-cells (Tregs) is crucial to maintain immune homoeostasis by controlling peripheral tolerance. A better understanding in the molecular mechanisms involved in the biology of these Tregs could improve their expansion and selection to treat immune-related diseases, achieve immunosuppression-free organ transplantation and to specifically target them in cancer. We reported on the overexpression of tribbles-1 (TRIB1) in Tregs compared with their counterpart naive T-cells and that TRIB1 interacts with the master molecule of Tregs, forkhead box P3 (FOXP3), a transcription factor essential for Treg suppressive activity. We demonstrated that these two molecules interact together in the nucleus of Tregs and TRIB1 overexpression is associated with a decrease in their proliferative capacities. Since TRIB1 was reported to be overexpressed in the blood of renal transplanted patients with chronic antibody-mediated rejection (CAMR), altogether, these results suggest TRIB1 could be linked to the decrease proportion of Tregs in patients exhibiting CAMR and a key player in Tregs through its FOXP3 interaction. In addition, yeast two-hybrid screening experiments highlighted that TRIB1 potentially interacts with molecules playing roles in intracellular events following T-cell activation and particularly cluster of differentiation (CD)4(+) T-cells. This suggests still non explored potential links between TRIB1 in Tregs. Our goal is thus to decipher the role of TRIB1 in the Treg biology, notably in pathways known to involved its partner and main transcriptional factor of Tregs, FOXP3 and to determine the role of TRIB1 in immune pathologies.

Phenotype and functions of B cells in patients with acute brain injuries.

BACKGROUND: Brain injuries (BI) induce a state of systemic immunosuppression, leading to a high risk of pneumonia. In this pilot study, we investigated the status of B cell compartment in BI patients. METHODS: A prospective observational study was performed in 2 intensive care units in a university hospital. Blood samples were collected in 14 patients at day 1 and day 7 after acute BI. The phenotype and the ability of B cells to secrete IL-10 were compared to 11 healthy volunteers (HV). RESULTS: Among the circulating lymphocytes, the frequency of B cells was significantly higher in BI patients compared to HV (p<0.001). B cells from BI patients displayed an activated profil on day 7 after BI, reflected by a significantly higher proportion of CD27(+) memory (p=0.01) and CD27(+) IgD(-) switched memory B cells (p=0.02), as well as a significantly higher blood level of IgA (p=0.001) and IgM (p<0.001) as compared to day 1. The frequency of IL-10 secreting B cells (IL-10(+) B cells) on day 1 and day 7 was significantly lower in BI patients compared to HV (p<0.05). Interestingly, we observed that all BI patients with high frequency of IL-10(+) B cells on day 1 displayed an episode of pneumonia, and had a longer duration of mechanical ventilation and ICU stay compared to BI patients with low proportion of IL-10(+) B cells. CONCLUSION: This study provides an extensive description of the phenotype and function of B cells in BI patients. Our results suggest that IL-10(+) B cells could play a major role in immunosuppression after BI.

Tolerant Kidney Transplant Patients Produce B Cells with Regulatory Properties.

Whereas a B cell-transcriptional profile has been recorded for operationally tolerant kidney graft patients, the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine production by effector CD4(+)CD25(-) T cells were measured after anti-CD3/anti-CD28 stimulation with or without autologous B cells. We report that B cells inhibit CD4(+)CD25(-) effector T cell response in a dose-dependent manner. This effect required B cells to interact with T-cell targets and was achieved through a granzyme B (GzmB)-dependent pathway. Tolerant recipients harbored a higher number of B cells expressing GzmB and displaying a plasma cell phenotype. Finally, GzmB(+) B-cell number was dependent on IL-21 production, and B cells from tolerant recipients but not from other patients positively regulated both the number of IL-21(+) T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases excessive B cell activation and allows regulation to take place. These data provide insights into the characterization of B cell-mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on effector T cells in blood from patients with operationally tolerant kidney grafts.

Central Role of CD45RA- Foxp3hi Memory Regulatory T Cells in Clinical Kidney Transplantation Tolerance.

The role of Foxp3(+) regulatory T cells (Tregs) in operational tolerance remains elusive, as initial results revealed an increased frequency of this subset in tolerant patients but no functional differences compared with immunosuppressed recipients. In addition, recent studies of regulatory B cells strongly suggest that Tregs may not have a central role in kidney transplantation tolerance. However, recent investigations of the crucial role of Foxp3 demethylation in Treg function and the possibility of identifying distinct Foxp3 T cell subsets prompted us to more thoroughly characterize Tregs in operationally tolerant patients. Thus, we studied the level of demethylation of the Foxp3 Treg-specific demethylated region (TSDR) in circulating CD4(+) T cells and analyzed Treg subset frequency in tolerant patients, healthy volunteers, patients with stable graft function under immunosuppression, and chronically rejecting recipients. We observed a higher proportion of CD4(+) T cells with demethylated Foxp3 and a specific expansion of CD4(+) CD45RA(-) Foxp3(hi) memory Tregs exclusively in tolerant patients. The memory Tregs of tolerant recipients exhibited increased Foxp3 TSDR demethylation, expressed higher levels of CD39 and glucocorticoid-induced TNF-related receptor, and harbored greater suppressive properties than memory Tregs from patients with stable graft function. Taken together, our data demonstrate that operationally tolerant patients mobilize an array of potentially suppressive cells, including not only regulatory B cells but also Tregs. Our results also indicate that tolerant patients have potent CD4(+)CD45RA(-) Foxp3(hi) memory Tregs with a specific Foxp3 TSDR demethylation pattern, which may contribute to the maintenance of graft tolerance.

Food allergy enhances allergic asthma in mice.

BACKGROUND: Atopic march refers to the typical transition from a food allergy in early childhood to allergic asthma in older children and adults. However the precise interplay of events involving gut, skin and pulmonary inflammation in this process is not completely understood. OBJECTIVES: To develop a mouse model of mixed food and respiratory allergy mimicking the atopic march and better understand the impact of food allergies on asthma. METHODS: Food allergy to ovalbumin (OVA) was induced through intra-peritoneal sensitization and intra-gastric challenge, and/or a respiratory allergy to house dust mite (HDM) was obtained through percutaneous sensitization and intra-nasal challenges with dermatophagoides farinae (Der f) extract. Digestive, respiratory and systemic parameters were analyzed. RESULTS: OVA-mediated gut allergy was associated with an increase in jejunum permeability, and a worsening of Der f-induced asthma with stronger airway hyperresponsiveness and pulmonary cell infiltration, notably eosinophils. There was overproduction of the pro-eosinophil chemokine RANTES in broncho-alveolar lavages associated with an enhanced Th2 cytokine secretion and increased total and Der f-specific IgE when the two allergies were present. Both AHR and lung inflammation increased after a second pulmonary challenge. CONCLUSION: Gut sensitization to OVA amplifies Der f-induced asthma in mice.

IL-17 in severe asthma. Where do we stand?

Asthma is a major chronic disease ranging from mild to severe refractory disease and is classified into various clinical phenotypes. Severe asthma is difficult to treat and frequently requires high doses of systemic steroids. In some cases, severe asthma even responds poorly to steroids. Several studies have suggested a central role of IL-17 (also called IL-17A) in severe asthma. Indeed, high levels of IL-17 are found in induced sputum and bronchial biopsies obtained from patients with severe asthma. The recent identification of a steroid-insensitive pathogenic Th17 pathway is therefore of major interest. In addition, IL-17A has been described in multiple aspects of asthma pathogenesis, including structural alterations of epithelial cells and smooth muscle contraction. In this perspective article, we frame the topic of IL-17A effects in severe asthma by reviewing updated information from human studies. We summarize and discuss the implications of IL-17 in the induction of neutrophilic airway inflammation, steroid insensitivity, the epithelial cell profile, and airway remodeling.

MicroRNAs, Major Players in B Cells Homeostasis and Function.

As a main actor in humoral immunity, B cells participate in various antibody-related disorders. However, a deeper understanding of B-cell differentiation and function is needed in order to decipher their immune-modulatory roles, notably with the recent highlighting of regulatory B cells. microRNAs (miRNAs), key factors in various biological and pathological processes, have been shown to be essential for B-cell homeostasis, and therefore understanding their participation in B-cell biology could help identify biomarkers and contribute toward curing B-cell-related immune disorders. This review aims to report studies casting light on the roles played by miRNAs in B-cell lineage and function and B-cell-related immune pathologies.

Block copolymer/DNA vaccination induces a strong allergen-specific local response in a mouse model of house dust mite asthma.

BACKGROUND: Allergic asthma is caused by abnormal immunoreactivity against allergens such as house dust mites among which Dermatophagoides farinae (Der f) is a common species. Currently, immunotherapy is based on allergen administration, which has variable effect from patient to patient and may cause serious side effects, principally the sustained risk of anaphylaxis. DNA vaccination is a promising approach by triggering a specific immune response with reduced allergenicity. OBJECTIVE: The aim of the study is to evaluate the effects of DNA immunization with Der f1 allergen specific DNA on allergic sensitization, inflammation and respiratory function in mice. METHODS: Mice were vaccinated 28 and 7 days before allergen exposure with a Der f1-encoding plasmid formulated with a block copolymer. Asthma was induced by skin sensitization followed by intra-nasal challenges with Der f extract. Total lung, broncho-alveolar lavage (BAL) and spleen cells were analyzed by flow cytometry for their surface antigen and cytokine expression. Splenocytes and lung cell IFN-γ production by CD8+ cells in response to Der f CMH1-restricted peptides was assessed by ELISPOT. IgE, IgG1 and IgG2a were measured in serum by ELISA. Specific bronchial hyperresponsiveness was assessed by direct resistance measurements. RESULTS: Compared to animals vaccinated with an irrelevant plasmid, pVAX-Der f1 vaccination induced an increase of B cells in BAL, and an elevation of IL-10 and IFN-γ but also of IL-4, IL-13 and IL-17 producing CD4+ lymphocytes in lungs and of IL-4 and IL-5 in spleen. In response to CD8-restricted peptides an increase of IFN-γ was observed among lung cells. IgG2a levels non-specifically increased following block copolymer/DNA vaccination although IgE, IgG1 levels and airways resistances were not impacted. CONCLUSIONS & CLINICAL RELEVANCE: DNA vaccination using a plasmid coding for Der f1 formulated with the block copolymer 704 induces a specific immune response in the model of asthma used herein.

Reconsidering the bio-detection of tolerance in renal transplantation.

Operational tolerance in kidney transplantation tolerance is rare phenomenon. It concerns recipients who keep a good function of their graft without immunosuppressors for more than one year. A critical need in the field of transplantation tolerance is the identification of biomarkers able to detect precociously tolerance phenotype in stable recipient in order to adapt treatment and progressively stop immunosuppressive therapy. But many limitations in these studies slow the application in clinics of such tolerance signature. In this addendum article we talk about these limitations and potential new directions to improve our approach in the quest of tolerance biomarkers.

The role of B lymphocytes in the progression from autoimmunity to autoimmune disease.

Autoimmunity, defined as the presence of autoreactive T and/or B lymphocytes in the periphery, is a frequent and probably even physiological condition. It is mainly caused by the fact that the central tolerance mechanisms, which are responsible for counter-selection of autoreactive lymphocytes, are not perfect and thus a limited number of these autoreactive cells can mature and enter the periphery. Nonetheless, autoreactive cells do not lead automatically to autoimmune disease as evidenced by a multitude of experimental and human data sets. Interestingly, the progression from autoimmunity to autoimmune disease is not only determined by the degree of central tolerance leakage and thus the amount of autoreactive lymphocytes in the periphery, but also by peripheral mechanism of activation and control of the autoreactive cells. In this review, we discuss the contribution of peripheral B lymphocytes in this process, ranging from activation of T cells and epitope spreading to control of the autoimmune process by regulatory mechanisms. We also discuss the parallels with the role of B cells in the induction and control of alloimmunity in the context of organ transplantation, as more precise knowledge of the pathogenic antigens and time of initiation of the immune response in allo- versus auto-immunity allows better dissection of the exact role of B cells. Since peripheral mechanisms may be easier to modulate than central tolerance, a more thorough understanding of the role of peripheral B cells in the progression from autoimmunity to autoimmune disease may open new avenues for treatment and prevention of autoimmune disorders.

Gene expression signature in transplantation tolerance.

Human kidney transplantation tolerance exists, but the definition of true tolerance as defined by Billingham and colleagues suffers several key elements that cannot be demonstrated in humans. Indeed, human tolerance in transplantation is defined by different functional and clinical parameters, this is why in clinical transplantation we preferentially talk about operational tolerance. These patients are very rare and defined in the literature as immunocompetent patients with stable graft function without any immunosuppressive treatments. These patients are characterized by a stable graft function in the absence of histologic information since the biopsy is often lacking. In kidney transplantation, this state of operational tolerance is observed in two situations. It is sometimes detected by chance in patients who stop their immunosuppressive treatments due to incompliance or because of secondary effects (cancer, opportunistic infections). The principal goal in kidney transplantation and one of the reasons why so many people are interested in cases of operational tolerance in humans, is to be able to identify patients who are developing spontaneous tolerance to their transplants while under classical immunosuppression. Consequently, there is an increasing need to develop an assay to identify and differentiate such patients with specific and noninvasive methods. In this review we will discuss the various studies that attempt to identify these new biomarkers of tolerance thanks to gene expression profiling using microarrays or quantitative PCR that have become a benchmark for research in novel and informative transplant monitoring assays.