The gut microbiota posttranslationally modifies IgA1 in autoimmune glomerulonephritis.

Mechanisms underlying the disruption of self-tolerance in acquired autoimmunity remain unclear. Immunoglobulin A (IgA) nephropathy is an acquired autoimmune disease where deglycosylated IgA1 (IgA subclass 1) auto-antigens are recognized by IgG auto-antibodies, forming immune complexes that are deposited in the kidneys, leading to glomerulonephritis. In the intestinal microbiota of patients with IgA nephropathy, there was increased relative abundance of mucin-degrading bacteria, including Akkermansia muciniphila. IgA1 was deglycosylated by A. muciniphila both in vitro and in the intestinal lumen of mice. This generated neo-epitopes that were recognized by autoreactive IgG from the sera of patients with IgA nephropathy. Mice expressing human IgA1 and the human Fc α receptor I (α1KI-CD89tg) that underwent intestinal colonization by A. muciniphila developed an aggravated IgA nephropathy phenotype. After deglycosylation of IgA1 by A. muciniphila in the mouse gut lumen, IgA1 crossed the intestinal epithelium into the circulation by retrotranscytosis and became deposited in the glomeruli of mouse kidneys. Human α-defensins-a risk locus for IgA nephropathy-inhibited growth of A. muciniphila in vitro. A negative correlation observed between stool concentration of α-defensin 6 and quantity of A. muciniphila in the guts of control participants was lost in patients with IgA nephropathy. This study demonstrates that gut microbiota dysbiosis contributes to generation of auto-antigens in patients with IgA nephropathy and in a mouse model of this disease.

Cholesterol and Ceramide Facilitate Membrane Fusion Mediated by the Fusion Peptide of the SARS-CoV-2 Spike Protein.

SARS-CoV-2 entry into host cells is mediated by the Spike (S) protein of the viral envelope. The S protein is composed of two subunits: S1 that induces binding to the host cell via its interaction with the ACE2 receptor of the cell surface and S2 that triggers fusion between viral and cellular membranes. Fusion by S2 depends on its heptad repeat domains that bring membranes close together and its fusion peptide (FP) that interacts with and perturbs the membrane structure to trigger fusion. Recent studies have suggested that cholesterol and ceramide lipids from the cell surface may facilitate SARS-CoV-2 entry into host cells, but their exact mode of action remains unknown. We have used a combination of in vitro liposome-liposome and in situ cell-cell fusion assays to study the lipid determinants of S-mediated membrane fusion. Our findings reveal that both cholesterol and ceramide lipids facilitate fusion, suggesting that targeting these lipids could be effective against SARS-CoV-2. As a proof of concept, we examined the effect of chlorpromazine (CPZ), an antipsychotic drug known to perturb membrane structure. Our results show that CPZ effectively inhibits S-mediated membrane fusion, thereby potentially impeding SARS-CoV-2 entry into the host cell.

Basophils and IgE contribute to mixed connective tissue disease development.

BACKGROUND: Mixed connective tissue disease (MCTD) is a rare and complex autoimmune disease that presents mixed features with other connective tissue diseases, such as systemic lupus erythematosus, systemic sclerosis, and myositis. It is characterized by high levels of anti-U1 small nuclear ribonucleoprotein 70k autoantibodies and a high incidence of life-threatening pulmonary involvement. The pathophysiology of MCTD is not well understood, and no specific treatment is yet available for the patients. Basophils and IgE play a role in the development of systemic lupus erythematosus and thus represent new therapeutic targets for systemic lupus erythematosus and other diseases involving basophils and IgE in their pathogenesis. OBJECTIVE: We sought to investigate the role of basophils and IgE in the pathophysiology of MCTD. METHODS: Basophil activation status and the presence of autoreactive IgE were assessed in peripheral blood of a cohort of patients with MCTD and in an MCTD-like mouse model. Basophil depletion and IgE-deficient animals were used to investigate the contribution of basophils and IgE in the lung pathology development of this mouse model. RESULTS: Patients with MCTD have a peripheral basopenia and activated blood basophils overexpressing C-C chemokine receptor 3. Autoreactive IgE raised against the main MCTD autoantigen U1 small nuclear ribonucleoprotein 70k were found in nearly 80% of the patients from the cohort. Basophil activation and IgE anti-U1 small nuclear ribonucleoprotein 70k were also observed in the MCTD-like mouse model along with basophil accumulation in lymph nodes and lungs. Basophil depletion dampened lung pathology, and IgE deficiency prevented its development. CONCLUSIONS: Basophils and IgE contribute to MCTD pathophysiology and represent new candidate therapeutic targets for patients with MCTD.

Acyl-CoA-Binding Protein Is a Lipogenic Factor that Triggers Food Intake and Obesity.

Autophagy facilitates the adaptation to nutritional stress. Here, we show that short-term starvation of cultured cells or mice caused the autophagy-dependent cellular release of acyl-CoA-binding protein (ACBP, also known as diazepam-binding inhibitor, DBI) and consequent ACBP-mediated feedback inhibition of autophagy. Importantly, ACBP levels were elevated in obese patients and reduced in anorexia nervosa. In mice, systemic injection of ACBP protein inhibited autophagy, induced lipogenesis, reduced glycemia, and stimulated appetite as well as weight gain. We designed three approaches to neutralize ACBP, namely, inducible whole-body knockout, systemic administration of neutralizing antibodies, and induction of antiACBP autoantibodies in mice. ACBP neutralization enhanced autophagy, stimulated fatty acid oxidation, inhibited appetite, reduced weight gain in the context of a high-fat diet or leptin deficiency, and accelerated weight loss in response to dietary changes. In conclusion, neutralization of ACBP might constitute a strategy for treating obesity and its co-morbidities.

CD89 Is a Potent Innate Receptor for Bacteria and Mediates Host Protection from Sepsis.

Direct bacterial recognition by innate receptors is crucial for bacterial clearance. Here, we show that the IgA receptor CD89 is a major innate receptor that directly binds bacteria independently of its cognate ligands IgA and c-reactive protein (CRP). This binding is only partially inhibited by serum IgA and induces bacterial phagocytosis by CD11c+ dendritic cells and monocytes and/or macrophages, suggesting a physiological role in innate host defense. Blood phagocytes from common variable immunodeficiency patients bind, internalize, and kill bacteria in a CD89-dependent manner, confirming the IgA independence of this mechanism. In vivo, CD89 transgenic mice are protected in two different models of sepsis: a model of pneumonia and the cecal ligation and puncture (CLP) polymicrobial model of infection. These data identify CD89 as a first-line innate receptor for bacterial clearance before adaptive responses can be mounted. Fc receptors may emerge as a class of innate receptors for various bacteria with pleiotropic roles.

Modulation of the microbiota by oral antibiotics treats immunoglobulin A nephropathy in humanized mice.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. IgA is mainly produced by the gut-associated lymphoid tissue (GALT). Both experimental and clinical data suggest a role of the gut microbiota in this disease. We aimed to determine if an intervention targeting the gut microbiota could impact the development of disease in a humanized mouse model of IgAN, the α1KI-CD89Tg mice. METHODS: Four- and 12-week old mice were divided into two groups to receive either antibiotics or vehicle control. Faecal bacterial load and proteinuria were quantified both at the beginning and at the end of the experiment, when blood, kidneys and intestinal tissue were collected. Serum mouse immunoglobulin G (mIgG) and human immunoglobulin A1 (hIgA1)-containing complexes were quantified. Renal and intestinal tissue were analysed by optical microscopy after haematoxylin and eosin colouration and immunohistochemistry with anti-hIgA and anti-mouse CD11b antibodies. RESULTS: Antibiotic treatment efficiently depleted the faecal microbiota, impaired GALT architecture and impacted mouse IgA production. However, while hIgA1 and mIgG serum levels were unchanged, the antibiotic treatment markedly prevented hIgA1 mesangial deposition, glomerular inflammation and the development of proteinuria. This was associated with a significant decrease in circulating hIgA1-mIgG complexes. Notably, final faecal bacterial load strongly correlated with critical clinical and pathophysiological features of IgAN such as proteinuria and hIgA1-mIgG complexes. In addition, treatment with broad-spectrum antibiotics reverted established disease. CONCLUSIONS: These data support an essential role of the gut microbiota in the generation of mucosa-derived nephrotoxic IgA1 and in IgAN development, opening new avenues for therapeutic approaches in this disease.

Autoantibodies against podocytic UCHL1 are associated with idiopathic nephrotic syndrome relapses and induce proteinuria in mice.

Idiopathic steroid sensitive nephrotic syndrome (INS), the most frequent childhood nephropathy, is thought to be mediated by a circulating soluble factor that reversibly affects the renal protein sieving. The efficiency of rituximab therapy recently highlighted the involvement of B cells. Here we studied the involvement of a specific immunoglobulin G (IgG) in the disease. After plasma fractionation by size exclusion chromatography, a detachment of cultured podocyte was observed with one IgG-containing fraction from 47% patients in relapse, 9% of patients in remission and 0% of controls. Podocyte protein lysates were immunoprecipitated by IgG from those plasma fractions identifying a list of 41 podocyte proteins after proteomic analysis. Five podocyte targets were selected on statistical and biological criteria. Specific antibodies were tested and only anti-Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) IgG led to podocyte detachment. UCHL1 was mainly found inside the podocyte but also weakly expressed on podocyte cell surface. Incubation of either anti-UCHL1 IgG or plasma fractions with recombinant UCHL1 prevented podocyte detachment. Plasma levels of anti-UCHL1 IgG were significantly increased in relapsing INS patients compared to patients in remission and controls. Proteinuria correlated with anti-UCHL1 IgG level at various stages of the disease. Purified patient anti-UCHL1 antibodies induced proteinuria and podocyte foot effacement in mice. Altogether, these results identified UCHL1 as a target podocyte protein of autoantibodies in a set of relapsing patients and support a causative role of anti-UCHL1 autoantibodies in the development of INS.

IgA1 Protease Treatment Reverses Mesangial Deposits and Hematuria in a Model of IgA Nephropathy.

IgA nephropathy (IgAN), characterized by mesangial IgA1 deposits, is a leading cause of renal failure worldwide. IgAN pathogenesis involves circulating hypogalactosylated IgA1 complexed with soluble IgA Fc receptor I (sCD89) and/or anti-hypogalactosylated-IgA1 autoantibodies, but no specific treatment is available for IgAN. The absence of IgA1 and CD89 homologs in the mouse has precluded in vivo proof-of-concept studies of specific therapies targeting IgA1. However, the α1KI­CD89Tg mouse model of IgAN, which expresses human IgA1 and human CD89, allows in vivo testing of recombinant IgA1 protease (IgA1­P), a bacterial protein that selectively cleaves human IgA1. Mice injected with IgA1­P (1-10 mg/kg) had Fc fragments of IgA1 in both serum and urine, associated with a decrease in IgA1-sCD89 complexes. Levels of mesangial IgA1 deposits and the binding partners of these deposits (sCD89, transferrin receptor, and transglutaminase 2) decreased markedly 1 week after treatment, as did the levels of C3 deposition, CD11b(+) infiltrating cells, and fibronectin. Antiprotease antibodies did not significantly alter IgA1­P activity. Moreover, hematuria consistently decreased after treatment. In conclusion, IgA1­P strongly diminishes human IgA1 mesangial deposits and reduces inflammation, fibrosis, and hematuria in a mouse IgAN model, and therefore may be a plausible treatment for patients with IgAN.

Transcriptional and Linkage Analyses Identify Loci that Mediate the Differential Macrophage Response to Inflammatory Stimuli and Infection.

Macrophages display flexible activation states that range between pro-inflammatory (classical activation) and anti-inflammatory (alternative activation). These macrophage polarization states contribute to a variety of organismal phenotypes such as tissue remodeling and susceptibility to infectious and inflammatory diseases. Several macrophage- or immune-related genes have been shown to modulate infectious and inflammatory disease pathogenesis. However, the potential role that differences in macrophage activation phenotypes play in modulating differences in susceptibility to infectious and inflammatory disease is just emerging. We integrated transcriptional profiling and linkage analyses to determine the genetic basis for the differential murine macrophage response to inflammatory stimuli and to infection with the obligate intracellular parasite Toxoplasma gondii. We show that specific transcriptional programs, defined by distinct genomic loci, modulate macrophage activation phenotypes. In addition, we show that the difference between AJ and C57BL/6J macrophages in controlling Toxoplasma growth after stimulation with interferon gamma and tumor necrosis factor alpha mapped to chromosome 3, proximal to the Guanylate binding protein (Gbp) locus that is known to modulate the murine macrophage response to Toxoplasma. Using an shRNA-knockdown strategy, we show that the transcript levels of an RNA helicase, Ddx1, regulates strain differences in the amount of nitric oxide produced by macrophage after stimulation with interferon gamma and tumor necrosis factor. Our results provide a template for discovering candidate genes that modulate macrophage-mediated complex traits.

Shifting FcγRIIA-ITAM from activation to inhibitory configuration ameliorates arthritis.

Rheumatoid arthritis-associated (RA-associated) inflammation is mediated through the interaction between RA IgG immune complexes and IgG Fc receptors on immune cells. Polymorphisms within the gene encoding the human IgG Fc receptor IIA (hFcγRIIA) are associated with an increased risk of developing RA. Within the hFcγRIIA intracytoplasmic domain, there are 2 conserved tyrosine residues arranged in a noncanonical immunoreceptor tyrosine-based activation motif (ITAM). Here, we reveal that inhibitory engagement of the hFcγRIIA ITAM either with anti-hFcγRII F(ab')2 fragments or intravenous hIgG (IVIg) ameliorates RA-associated inflammation, and this effect was characteristic of previously described inhibitory ITAM (ITAMi) signaling for hFcαRI and hFcγRIIIA, but only involves a single tyrosine. In hFcγRIIA-expressing mice, arthritis induction was inhibited following hFcγRIIA engagement. Moreover, hFcγRIIA ITAMi-signaling reduced ROS and inflammatory cytokine production through inhibition of guanine nucleotide exchange factor VAV-1 and IL-1 receptor-associated kinase 1 (IRAK-1), respectively. ITAMi signaling was mediated by tyrosine 304 (Y304) within the hFcγRIIA ITAM, which was required for recruitment of tyrosine kinase SYK and tyrosine phosphatase SHP-1. Anti-hFcγRII F(ab')2 treatment of inflammatory synovial cells from RA patients inhibited ROS production through induction of ITAMi signaling. These data suggest that shifting constitutive hFcγRIIA-mediated activation to ITAMi signaling could ameliorate RA-associated inflammation.

Usefulness in clinical practice of a point-of-care rapid test for simultaneous detection of nontreponemal and Treponema pallidum-specific antibodies in patients suffering from documented syphilis.

The usefulness of a point-of-care immunochromatographic dual test for the simultaneous detection of both nontreponemal and Treponema pallidum-specific antibodies (Chembio Diagnostics Systems Inc., Medford, NY, USA) was assessed in various situations related to syphilis, by reference to conventional syphilis serology. Thawed sera were obtained from 100 adults including 36 primary syphilis, 6 secondary syphilis, 6 re-infection, 9 recently-treated syphilis, and 43 old syphilis. Doubtful reactivities for the treponemal line were considered positive; doubtful reactivities for the nontreponemal line were considered positive only when the treponemal line was present. The sensitivity, the specificity, and its concordance to gold standard serology of treponemal line were high, around 90%. The sensitivity of nontreponemal line was 96.3%, its specificity 76.7%, and its concordance 83.4%. In conclusion, the dual rapid test from Chembio Diagnostics Systems Inc. is useful for rapid point-of-care diagnosis in the various situations encountered with patients suffering from syphilis.

Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation.

European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection.