Immunoglobulin A Nephropathy Is Characterized By Anticommensal Humoral Immune Responses

IgA nephropathy (IgAN) is a leading cause of kidney failure, yet little is known about the immunopathogenesis of this disease. IgAN is characterized by the deposition of IgA in the kidney glomeruli, but the source and stimulus for IgA production are not known. Clinical and experimental data suggest a role for aberrant immune responses to mucosal microbiota in IgAN, and in some countries with high disease prevalence, tonsillectomy is regarded as standard-of-care therapy. To evaluate the relationship between microbiota and mucosal immune responses, we characterized the tonsil microbiota in patients with IgAN versus nonrelated household-matched control group participants and identified increased carriage of the genus Neisseria and elevated Neisseria-targeted serum IgA in IgAN patients. We reverse-translated these findings in experimental IgAN driven by BAFF overexpression in BAFF-transgenic mice rendered susceptible to Neisseria infection by the introduction of a humanized CEACAM-1 transgene (B × hC-Tg). Colonization of B × hC-Tg mice with Neisseria yielded augmented levels of systemic Neisseria-specific IgA. Using a custom ELISPOT assay, we discovered anti–Neisseria–specific IgA-secreting cells within the kidneys of these mice. These findings suggest a role for cytokine-driven aberrant mucosal immune responses to oropharyngeal pathobionts, such as Neisseria, in the immunopathogenesis of IgAN. Furthermore, in the presence of excess BAFF, pathobiont-specific IgA can be produced in situ within the kidney.

NEW HERBAL FOR IGA NEPHROPATHY BY THE CAUSE OF KIDNEY FAILURE

Introduction 

The mechanisms underlying the pathogenesis of IgA nephropathy (IgAN) are poorly understood, yet it is one of the most common causes of kidney failure. The widely accepted "multi-hit hypothesis" for the development of IgAN focuses on the production of pathogenic galactose-deficient IgA1-containing immune complexes in the circulation and deposited within kidney glomeruli of patients with IgAN (1). The source (i.e., mucosal-associated lymphoid tissues and/or bone marrow) and stimuli for immune complex production are not known. We have recently demonstrated that IgA-producing plasma cells from the gut can migrate to non-immune system tissues, such as the inflamed brain in the experimental encephalitis model of multiple sclerosis, radically shifting prevailing views on the properties of plasma cells (2). It is therefore tempting to speculate that mucosal-derived IgA-secreting cells could also be recruited to other organs, such as the kidney. 

Clinical and experimental evidence suggest a link between IgAN and mucosal microbial exposures. Patients with IgAN experience macroscopic hematuria concurrently with pharyngitis (3), and tonsillectomy is considered standard-of-care for patients with IgAN in Japan (4–6). Our previous studies of a murine IgAN model suggest that commensal microbial colonization is essential for the development of IgAN in mice overexpressing the TNF-family member BAFF (7, 8). This model shares many features with human IgAN, including underglycosylated IgA; clinical and histological measures of kidney injury, such as proteinuria; and IgA-dominant glomerulonephritis (7, 8). The TNFSF13 (APRIL) locus was subsequently linked to IgAN in genome-wide association studies (9). Since APRIL and BAFF share similar receptors, the BAFF/APRIL axis has been implicated in IgAN disease pathogenesis.

We therefore hypothesized that in the setting of high levels of BAFF/APRIL, mucosal-derived IgA-secreting cells are recruited to the kidney and contribute to the development of IgAN. To test this hypothesis, we characterized the tonsil and stool microbiome profiles of a large cohort of patients with IgAN compared with household-matched nonrelated control group participants. We identified increased tonsil Neisseria carriage and enhanced production of anti-Neisseria-targeted IgA in the blood of our patient cohort. We reverse-translated these observations into an experimental IgAN model and found that elevated levels of BAFF provoked an enhanced IgA-biased systemic immune response to mucosal Neisseria exposure. We noted increased expression of RNA encoding the secreted-spliced variant of IgA in the kidneys of our experimental mice, suggesting local IgA transcription. Using a custom ELISPOT, we discovered that anti-Neisseria–-specific IgA-secreting cells were detectable in the kidneys of these mice. Our findings showed that a maladaptive host response to a commensal organism was associated with IgAN disease and recruitment of commensal-specific IgA-secreting cells to the kidney

Results 

16s rRNA analysis reveals alterations in the tonsillar microbiome of patients with IgAN compared with healthy controls. 

Given the association between pharyngitis and hematuria in IgAN (4), we compared the tonsillar and stool microbiota of a cohort of patients with IgAN (n = 93) with that of healthy nonrelated household-matched individuals (n = 58) using 16S rRNA V4 sequencing. Clinical characteristics are provided in Table 1. We did not observe any statistically significant differences in Shannon diversity or taxonomic richness between cases and healthy control tonsil samples (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.141289DS1). The dominant tonsillar genus differed between cases and controls (Figure 1A), with a trend toward differences in Neisseria as the dominant genus in IgAN tonsil samples (χ2 test, P = 0.15). We therefore evaluated the difference in relative abundance (RA) of Neisseria (Figure 1B) and confirmed the presence of a significantly greater abundance of tonsil Neisseria in IgAN compared with controls (2-tailed unadjusted t-test P = 0.002). No differences were noted in the diversity or taxonomic richness of stool microbiota in IgAN compared with controls (Supplemental Figure 2).

Humoral immune responses to Neisseria are biased toward IgA versus IgG in patients with IgAN. Based on the observed differences in tonsillar microbial abundance, we hypothesized that patients may exhibit altered titers of anticommensal antibodies in their circulation. Because Neisseria represented the most abundant genus in patients, we measured anti-Neisseria humoral responses, evaluating the ratio of anti-Neisseria IgA to anti-Neisseria IgG in serum. This ratio is reported as an average IgA/IgG response against a panel of 4 commensal Neisseria species (anti–N. lactamica, N. sicca, N. cinerea, and N. flavescens) and 4 pathogenic N. mengingivitis (Nme) strains (90/18311, H4476, 208, and 860800). As illustrated in Figure 2, patients with IgAN exhibited an increase in the ratio of plasma IgA/IgG anti-Neisseria antibody titers. Increased IgA titers against Neisseria were observed in response to both pathogenic Nme and commensal strains (Supplemental Figure 3). Therefore, patients with IgAN exhibited a bias toward generating an enhanced IgA response to a variety of commensal Neisseria species and Nme strains compared with healthy controls.

Effect of BAFF overexpression on the immune response to Nme in BAFF-transgenic mice. We previously reported that the cytokine APRIL (TNFSF13) was elevated in the serum of patients with IgAN compared with controls in 2 independent cohorts (8). We confirmed that APRIL was elevated in patients with IgAN in the current cohort (median 1.98 vs. 1.55; IQR 1.75, 0.38; P < 0.01). Moreover, we observed a positive correlation between serum APRIL levels and proteinuria (Spearman's ρ = 0.28, P = 0.01). As in our previous study, we did not observe differences in serum BAFF levels.

APRIL binds to TACI, a TNF receptor that promotes a class switch to IgA (10). TACI is also stimulated by higher-order multimers of BAFF, and overexpression of BAFF in BAFF-transgenic (BAFF-Tg) mice is sufficient to stimulate TACI, thus mimicking an APRIL/TACI signal (11). Importantly, as they age, BAFFTg mice exhibit an IgAN-like disease characterized by underglycosylated IgA in the serum, IgA deposition in the kidney, elevated proteinuria, and kidney pathology (8). We previously showed that the microbiota is an essential cofactor promoting the deposition of IgA immune complexes in the kidneys of BAFF overpressing BAFF-Tg mice (8). This implies that a host-microbiome interaction may play a role in the etiology of IgAN, at least in mice. However, in our previous studies, we did not assess whether specific microbial candidates could accelerate the pathogenesis of IgAN-like disease in BAFF-Tg mice.

Therefore, we next explored the possibility that Neisseira-targeted, IgA-biased immune responses may be observed in BAFF-overexpressing mice. Name infects epithelium via the CEACAM-1 receptor. Because Name will bind to humans but not mouse CEACAM-1 we crossed BAFF-Tg mice with mice that express the human form of CEACAM-1 (hC-Tg) to generate double transgenic mice (B × hC-Tg). Transgenic mice and their littermate controls were nasally infected with Nme, and serum and nasal lavages were subsequently collected for analysis. As illustrated in Figure 3A, although hC-Tgneg (WT) mice were not appreciably colionized by Nme, hC-Tg, and B × hC-Tg mice exhibited similar rates of Nme colonization (80% vs. 100%, P = NS) and similar bacterial burdens (192.9 vs. 116.7, P = NS) at 5 days after infection. These data indicate that B × hC-Tg mice did not have altered rates of Nme colonization in comparison to WT mice.

We have previously shown that 2 nasal infections are required to induce immunity against a subsequent third nasal infection in hC-Tg mice (12). To determine whether B × hC-Tg mice exhibited improved clearance of Nme compared with hC-Tg controls, we nasally infected mice twice, on days 1 and 14. At 24 hours after secondary infection, the rate of infection of hC-Tg mice and B × hC-Tg mice was similar (85% vs. 80%), demonstrating a lack of neutralizing immunity induced by 1 previous exposure of Nme (Figure 3B). This implies that overexpression of BAFF did not eliminate the need for 2 infections to induce protection against colonization in our mouse model.

Given that there was no obvious impact of BAFF overexpression on nasal susceptibility to Nme, we looked at the mouse antibody response to Nme colonization. First, we assessed the local IgA and IgG response to Nme. Nasal Nme-specific IgG, as evaluated in nasopharyngeal lavage fluid, was not detected in any mice, and nasal anti-Nme IgA titers were similar for both B × hC-Tg and hC-Tg mice (Figure 3C). We are next measuring the systemic antibody response to Nme infection. After the second nasal infection, both hC-Tg and B × hC-Tg mice exhibited an Nme-specific IgG response in serum above baseline, with hC-Tg mice exhibiting a mean anti-Nme IgG titer 2-fold greater than that in B × hC-Tg mice (164 ng/mL vs. 54.6 ng/mL) (Figure 3D). In contrast, B × hC-Tg mice exhibited a mean anti-Nme IgA titer in serum that was 10-fold higher than in hC-Tg controls (321.7 ng/mL vs. 33.09 ng/mL) (Figure 3E). Taken together, our findings demonstrated that whereas the local IgA response to Nme was comparable between hC-Tg and B × hC-Tg mice, the systemic humoral immune response to nasal Nme exposure was IgA-biased in B × hC-Tg mice (Figure 3F).

Effect of Nme colonization on IgA in the kidneys of BAFF-Tg mice. Given that systemic Nme-specific IgA levels were significantly elevated in BAFF-Tg mice, we hypothesized that a combination of the BAFF transgene and Nme infection may accelerate kidney pathology compared with uninfected BAFF-Tg mice. We therefore assessed glomerular mesangial expansion by staining kidney sections with periodic acid–Schiff (PAS). Using a semiquantitative score of mesangial expansion, we observed a nonsignificant trend toward increased mesangial expansion in the B × hC-Tg mice (Figure 4, A and B).

We next examined the impact of Nme infection on IgA deposition in the kidney, the diagnostic hallmark of IgAN. Using immunohistochemistry, deposition of mesangial IgG was not detected in any mice (data not shown). Moreover, we did not observe mesangial IgA in the kidneys of WT or hC-Tg mice. In contrast, B × hC-Tg and BAFF-Tg mice exhibited IgA deposition in areas of PAS+ mesangial expansion (representative section in Figure 4C). For a more quantitative analysis, the expression of mRNA encoding the secreted splice form of IgA was evaluated by quantitative PCR in the kidneys of all groups of mice (Figure 4D). There was a significant difference in the expression of the secreted splice form of IgA across groups (Kruskal-Wallis 1-way ANOVA, P = 0.01), with double transgenic mice (B × hC-Tg) demonstrating significantly higher levels of IgA expression compared with other experimental groups (Dunn's multiple-comparison test adjusted P < 0.05 for all comparisons). These results showed that colonization of BAFF-Tg mice with Nme resulted in augmented IgA expression within the kidney itself.

Having recently discovered that mucosal-derived IgA-secreting cells migrate to brain tissue in mice with experimental encephalitis (2), we hypothesized that the intrarenal expression of IgA mRNA may reflect the presence of anticommensal IgA-producing cells that have migrated from the site of initial pathobiont exposure. We therefore designed a custom ELISPOT assay to detect Nme-specific anti-body-secreting cells. High-affinity ELISPOT plates were coated with heat-killed Nme and incubated with a single-cell suspension of kidney-derived immune cells from our experimental mice; HRP-conjugated IgA detection antibodies were subsequently added. We discovered that Nme-specific IgA-secreting cells were identified in the kidney parenchyma of Nme-infected mice, predominantly in the setting of increased BAFF levels (B × hC-Tg mice, Figure 5).

Figure 1. Tonsil microbiota. (A) The most abundant genus (>2× the relative abundance of the next most abundant genus) in tonsil swabs in patients with IgAN was Neisseria. Each color represents an individual genus, and the y-axis indicates the proportion of samples with that genus as the most abundant organism (χ2, P = 0.18). (B) The relative abundance of the Neisseria genus was significantly higher in IgAN compared with the nonrelated household-matched healthy control individuals (2-tailed unadjusted t-test P = 0.002).