Development of Urinary Diagnostic Biomarker for IgA Nephropathy by Lectin Microarray

<b><i>Introduction:</i></b> The pathogenic roles of aberrantly glycosylated IgA1 have been reported. However, it is unexplored whether the profiling of urinary glycans contributes to the diagnosis of IgAN. <b><i>Methods:</i></b> We conducted a retrospective study enrolling 493 patients who underwent renal biopsy at Okayama University Hospital between December 2010 and September 2017. We performed lectin microarray in urine samples and investigated whether c-statistics of the reference standard diagnosis model employing hematuria, proteinuria, and serum IgA were improved by adding the urinary glycan intensity. <b><i>Results:</i></b> Among 45 lectins, 3 lectins showed a significant improvement of the models: <i>Amaranthus caudatus</i> lectin (ACA) with the difference of c-statistics 0.038 (95% CI: 0.019–0.058, <i>p</i> &#x3c; 0.001), <i>Agaricus bisporus</i> lectin (ABA) 0.035 (95% CI: 0.015–0.055, <i>p</i> &#x3c; 0.001), and <i>Maackia amurensis</i> lectin (MAH) 0.035 (95% CI: 0.015–0.054, <i>p</i> &#x3c; 0.001). In 3 lectins, each signal plus reference standard showed good reclassification (category-free NRI and relative IDI) and good model fitting associated with the improvement of AIC and BIC. Stratified by eGFR, the discriminatory ability of ACA plus reference standard was maintained, suggesting the robust renal function-independent diagnostic performance of ACA. By decision curve analysis, there was a 3.45% net benefit by adding urinary glycan intensity of ACA to the reference standard at the predefined threshold probability of 40%. <b><i>Conclusions:</i></b> The reduction of Gal(β1-3)GalNAc (T-antigen), Sia(α2-3)Gal(β1-3)GalNAc (Sialyl T), and Sia(α2-3)Gal(β1-3)Sia(α2-6)GalNAc (disialyl-T) was suggested by binding specificities of 3 lectins. C1GALT1 and COSMC were responsible for the biosynthesis of these glycans, and they were known to be downregulated in IgAN. The urinary glycan analysis by ACA is a useful and robust noninvasive strategy for the diagnosis of IgAN.

Celiac Disease After Administration of Immune Checkpoint Inhibitors: A Case Report

Immune checkpoint inhibitors (ICI) reinvigorate the immune system to recognize and destroy tumor cells. Because of this biological mechanism, patients might develop autoimmune toxicities, notably in the digestive tract (most frequently, hepatitis or colitis). A 70-year-old man with relapsed mesothelioma was treated with nivolumab in 3rd line. He was hospitalized for watery and foul-smelling diarrhea. He underwent gastrointestinal endoscopy, showing duodenitis and villous atrophy and measurement of serum IgA antibodies to tissue transglutaminase (tTG-IgA+), leading to the diagnosis of ICI-induced celiac disease. He was treated with steroids, proton pump inhibitors, and a gluten-free diet. If ICI-induced celiac disease is rare in the literature, increasing reports suggest that celiac disease might represent an underestimated ICI toxicity. This case highlights the necessity of complementary investigation (including tTG-IgA and endoscopic biopsies) in patients with atypical digestive symptoms during immunotherapy.

Monomeric IgA Antagonizes IgG-Mediated Enhancement of DENV Infection

Dengue virus (DENV) is a prevalent human pathogen, infecting approximately 400 million individuals per year and causing symptomatic disease in approximately 100 million. A distinct feature of dengue is the increased risk for severe disease in some individuals with preexisting DENV-specific immunity. One proposed mechanism for this phenomenon is antibody-dependent enhancement (ADE), in which poorly-neutralizing IgG antibodies from a prior infection opsonize DENV to increase infection of Fc gamma receptor-bearing cells. While IgM and IgG are the most commonly studied DENV-reactive antibody isotypes, our group and others have described the induction of DENV-specific serum IgA responses during dengue. We hypothesized that monomeric IgA would be able to neutralize DENV without the possibility of ADE. To test this, we synthesized IgG and IgA versions of two different DENV-reactive monoclonal antibodies. We demonstrate that isotype-switching does not affect the antigen binding and neutralization properties of the two mAbs. We show that DENV-reactive IgG, but not IgA, mediates ADE in Fc gamma receptor-positive K562 cells. Furthermore, we show that IgA potently antagonizes the ADE activity of IgG. These results suggest that levels of DENV-reactive IgA induced by DENV infection might regulate the overall IgG mediated ADE activity of DENV-immune plasma in vivo, and may serve as a predictor of disease risk.

Elevated Myeloperoxidase-DNA Complex Levels in Sera of Patients with IgA Vasculitis

<b><i>Introduction:</i></b> IgA vasculitis is a systemic disease that results from the entrapment of circulating IgA-containing immune complexes in small-vessel walls in the skin, kidneys, and gastrointestinal tract. An excessive formation of neutrophil extracellular traps (NETs) is involved in the pathogenesis of vasculitis, especially in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. This study aimed to clarify whether NETs are implicated in IgA vasculitis. <b><i>Methods:</i></b> Twenty-two patients with IgA vasculitis and 4 healthy volunteers were enrolled in this study. Serum levels of myeloperoxidase (MPO)-DNA complex, a fragment derived from NETs, were determined by enzyme-linked immunosorbent assay (ELISA), and the association between MPO-DNA complex levels and clinical parameters was examined. The presence of the ANCA was also assessed by ELISA specific for MPO and proteinase 3 (PR3) and indirect immunofluorescence (IIF), followed by assessing the differences in clinical parameters with and without the ANCA. <b><i>Results:</i></b> Serum MPO-DNA complex levels were significantly higher in patients with IgA vasculitis than those in healthy controls. A significant positive correlation between the serum MPO-DNA complex and IgA levels was noted. Interestingly, 63.6% of IgA vasculitis patients were ANCA-positive in IIF with an atypical pattern, whereas neither MPO-ANCA nor PR3-ANCA was detected by ELISA. These findings indicated that some IgA vasculitis patients possessed the so called minor ANCA. Serum IgA and MPO-DNA complex levels and the frequency of hematuria in the minor ANCA-positive group were significantly higher than in the minor ANCA-negative group. <b><i>Conclusion:</i></b> The collective findings suggested that NETs are certainly involved in the pathogenesis of IgA vasculitis.

GWAS defines pathogenic signaling pathways and prioritizes drug targets for IgA nephropathy

IgA nephropathy (IgAN) is a progressive form of kidney disease defined by glomerular deposition of IgA. We performed a genome-wide association study involving 10,146 kidney biopsy-diagnosed IgAN cases and 28,751 matched controls across 17 international cohorts. We defined 30 independent genome-wide significant risk loci jointly explaining 11% of disease risk. A total of 16 loci were novel, including TNFSF4, REL, CD28, CXCL8/PF4V1, LY86, LYN, ANXA3, TNFSF8/15, REEP3, ZMIZ1, RELA, ETS1, IGH, IRF8, TNFRSF13B and FCAR. The SNP-based heritability of IgAN was estimated at 23%. We observed a positive genetic correlation between IgAN and total serum IgA levels, allergy, tonsillectomy, and several infections, and a negative correlation with inflammatory bowel disease. All significant non-HLA loci shared with serum IgA levels had a concordant effect on the risk of IgAN. Moreover, IgAN loci were globally enriched in gene orthologs causing abnormal IgA levels when genetically manipulated in mice. The explained heritability was enriched in the regulatory elements of cells from the immune and hematopoietic systems and intestinal mucosa, providing support for the pathogenic role of extra-renal tissues. The polygenic risk of IgAN was associated with early disease onset, increased lifetime risk of kidney failure, as well as hematuria and several other traits in a phenome-wide association study of 590,515 individuals. In the comprehensive functional annotation analysis of candidate causal genes across genome-wide significant loci, we observed the convergence of biological candidates on a common set of inflammatory signaling pathways and cytokine ligand-receptor pairs, prioritizing potential new drug targets.

Genetic regulation of serum IgA levels and susceptibility to common immune, infectious, kidney, and cardio-metabolic traits

Immunoglobulin A (IgA) mediates mucosal responses to food antigens and the intestinal microbiome and has a known role in susceptibility to mucosal pathogens, celiac disease, inflammatory bowel disease, and IgA nephropathy. We performed genetic analyses of serum IgA levels in 41,263 individuals of diverse ancestries. We observed unexpected variability in IgA levels across major ancestral populations, with African ancestry being reproducibly associated with higher serum IgA levels compared to other ancestries. The trans-ethnic GWAS analysis identified 20 genome-wide significant loci associated with serum IgA levels, including nine known and 11 novel loci. Systematic co-localization analysis with blood and primary immune cell expression QTLs prioritized candidate genes for 14 of 20 loci. Most GWAS loci encoded genes that produce immune defects and IgA abnormalities when genetically manipulated in mice. We uncovered positive genetic correlations of serum IgA levels with IgA nephropathy, type 2 diabetes and body mass index, as well as negative genetic correlations with celiac disease, inflammatory bowel disease, several infections, and intestinal microbiome diversity. Our findings provide novel insights into the genetic regulation of IgA production and its potential role in human disease.

Prevention and partial reversion of the lupus phenotype in ABIN1[D485N] mice by an IRAK4 inhibitor

ObjectiveWe have reported previously that the IRAK4 inhibitor PF06426779 given to ubiquitin-binding-defective ABIN1[D485N] mice at 6 weeks of age prevents the major facets of lupus that develop 10 weeks later. The present study was undertaken to investigate whether PF06426779 could reverse the lupus phenotype when administered to 13-week-old ABIN1[D485N] mice that had already developed symptoms of lupus.MethodsSplenomegaly, the number of splenic neutrophils, TFH and Germinal Centre B (GCB) cells, serum levels of immunoglobulins, the extent of kidney, liver and lung pathology, and glomerular IgA and IgM were measured after feeding 13-week-old ABIN1[D485N] and wild-type mice for another 10 weeks with R&M3 diet with and without PF06426779 (4 g/kg).ResultsFollowing drug treatment, spleen size and weight, splenic neutrophil numbers, and serum IgA and glomerular IgA levels of ABIN1[D485N] mice returned to those seen in wild-type mice. The rise in splenic TFH and GCB numbers, the increase in kidney and liver pathology, and the concentrations of serum IgG1, IgG2A and IgE between 13 and 23 weeks were suppressed. There was no reduction in the level of anti-self double-stranded DNA, anti-self nuclear antigens or IgM during the drug treatment.ConclusionsThe results demonstrate the therapeutic potential of IRAK4 inhibitors for the treatment of lupus and raise the possibility of monitoring efficacy by measuring decreases in the serum levels of IgA. Our results support the view that there may be a closer connection between lupus and IgA nephropathy than realised previously.

Broad-Spectrum and Gram-Negative-Targeting Antibiotics Differentially Regulate Antibody Isotype Responses to Injected Vaccines

Antibiotics are extensively used worldwide for the treatment of common infections by agents such as E. coli and Salmonella. They also represent the most common cause of alteration of the microbiota in people. We addressed whether broad-spectrum and Gram-negative-targeting antibiotics differentially regulate systemic and mucosal immune responses to vaccines. Antibiotics treatment enhances serum IgG1 responses in mice immunized systemically with a model polyvalent vaccine. This increase was not seen for other IgG subclasses and was dependent on the immunogenicity of vaccine antigens. The broad-spectrum antibiotic cocktail also enhanced serum IgA responses. Interestingly, both the broad spectrum and the antibiotic targeting Gram-negative bacteria enhanced the number of IgA antibody secreting cells in the intestinal lamina propria. This effect was unlikely to be due to an increase in cells expressing gut-homing receptors (i.e., CCR9 and α4β7) in peripheral tissues. On the other hand, the microbiome in mice treated with antibiotics was characterized by an overall reduction of the number of firmicutes. Furthermore, Bacteroidetes were increased by either treatment, and Proteobacteria were increased by the broad-spectrum antibiotics cocktail. Thus, immunoglobulin isotype and subclass responses are differentially regulated by oral antibiotics treatment and the gut microbiota shapes mucosal antibody responses after systemic immunization.

Human genital antibody-mediated inhibition of Chlamydia trachomatis infection and evidence for ompA genotype-specific neutralization

The endocervix, the primary site of Chlamydia trachomatis (Ct) infection in women, has a unique repertoire of locally synthesized IgG and secretory IgA (SIgA) with contributions from serum IgG. Here, we assessed the ability of genital and serum-derived IgG and IgA from women with a recent positive Ct test to neutralize Ct elementary bodies (EBs) and inhibit inclusion formation in vitro in human endocervical epithelial cells. We also determined if neutralization was influenced by the major outer membrane protein (MOMP) of the infecting strain, as indicated by ompA gene sequencing and genotyping. At equivalent low concentrations of Ct EB (D/UW-3/Cx + E/UW-5/Cx)-specific antibody, genital-derived IgG and IgA and serum IgA, but not serum IgG, significantly inhibited inclusion formation, with genital IgA being most effective, followed by genital IgG, then serum IgA. The well-characterized Ct genotype D strain, D/UW-3/Cx, was neutralized by serum-derived IgG from patients infected with genotype D strains, genital IgG from patients infected with genotype D or E strains, and by genital IgA from patients infected with genotype D, E, or F strains. Additionally, inhibition of D/UW-3/Cx infection by whole serum, rather than purified immunoglobulin, was associated with levels of serum EB-specific IgG rather than the genotype of infecting strain. In contrast, a Ct genotype Ia clinical isolate, Ia/LSU-56/Cx, was neutralized by whole serum in a genotype and genogroup-specific manner, and inhibition also correlated with EB-specific IgG concentrations in serum. Taken together, these data suggest that (i) genital IgA most effectively inhibits Ct infection in vitro, (ii) human antibody-mediated inhibition of Ct infection is significantly influenced by the ompA genotype of the infecting strain, (iii) the genital antibody repertoire develops or matures differently compared to systemic antibody, and (iv) ompA genotype-specificity of inhibition of infection by whole serum can be overcome by high concentrations of Ct-specific IgG.

Induced antigen-binding polyspecificity in human serum IgA

Recent studies have shown that polyspecific antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyspecificity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgE and IgG antibodies to acquire polyspecificity following contact with protein-modifying agents has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to pro-oxidative ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using Western blotting and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyspecificity.