Lupus nephritis is linked to disease-activity associated expansions and immunity to a gut commensal

Abstract

Background/purpose: To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity.

Methods: In a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts.

Results: Compared with controls, the microbiome in patients with SLE showed decreased species richness diversity, with reductions in taxonomic complexity most pronounced in those with high SLE disease activity index (SLEDAI). Notably, patients with SLE had an overall 5-fold greater representation of Ruminococcus gnavus (RG) of the Lachnospiraceae family, and individual communities also displayed reciprocal contractions of a species with putative protective properties. Gut RG abundance correlated with serum antibodies to only 1/8 RG strains tested. Anti-RG antibodies correlated directly with SLEDAI score and antinative DNA levels, but inversely with C3 and C4. These antibodies were primarily against antigen(s) in an RG strain-restricted pool of cell wall lipoglycans. Novel structural features of these purified lipoglycans were characterised by mass spectrometry and NMR. Highest levels of serum anti-RG strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts.

Conclusion: These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.

Keywords: autoantibodies; commensal; gut barrier; microbiome; pathobiont.

Figure 1

Patients with SLE have altered faecal communities of commensal taxa, elevated faecal immunoglobulin levels and biomarker evidence of altered gut barrier function. (A) Chao1 estimates of alpha diversity represent the total expected number of OTUs that represent quasi-species identified from 16S rRNA amplicon sequencing surveys, with larger values representing higher diversity. Patients with SLE (n=61) have less diverse faecal microbiota than healthy controls (n=17), indicating that SLE commonly have intestinal dysbioses. At far right, the distribution of Chao1 values in patients with SLE with high activity (ie, SLEDAI≥8) was significantly contracted compared with healthy adult controls (HC), with a trend towards more limited diversity in SLE with high disease activity when compared with SLE with low disease activity. We used a cut-point that was associated with greatest statistically significant differences. The SLE low disease activity group had a range of 0–7 SLEDAI scores (n=47), and SLE high disease activity group had scores of 8–18 (n=14). p values were based on the Mann-Whitney test. (B) PcoA showed that the beta diversity within bacterial communities in the faecal microbiomes in healthy controls was less different than the communities from patients with SLE low disease activity (PERMANOVA, p=0.02). Furthermore, healthy subjects were more like one another than were patients with SLE. (C) Results indicate average within group beta diversity differences in the three different groups, with comparisons for Control-SLEDAI^hi-SLEDAI^low, p=0.002. The binary comparison, control vs SLE had p=0.02. Both comparisons were done using PERMANOVA test. Examinations of faecal extract demonstrated patients with SLE commonly display: (D) elevated faecal sIgA levels, (E) elevated faecal IgM levels, (F) elevated faecal IgG levels, and (G) elevated faecal calprotectin. Patients with SLE also displayed: (H) elevated serum sCD14 levels and (I) elevated serum α1-acid glycoprotein levels, as measured by commercial assay. Results are for the NYU cohort of adult female patients with SLE, were compared to adult female controls without inflammatory or autoimmune disease (CTL). (D–F) and (H–I) using unpaired two-tailed t test with Welch’s correction, (G) used Mann-Whitney test. Significance for p<0.05. OTUs, operational taxonomic units; PCoA, principal coordinates analysis; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index.

Figure 2

Patients with LN have serum IgG that recognise RG2 strain-restricted non-protein antigen(s) and that levels correlate with scores forlupus disease activity. Immunoblot results for IgG from (A) patient with LN (S-134), (B) patients with LN (S-047), (C) patient with non-active lupus (S-096) and (D) healthy control (CTL23). Bacterial extracts were prepared with nuclease and lysozyme treatment; lanes 1–8 represent R. gnavus RG1-RG8 strains (see online supplementary table 4), and lanes 9 and 10 contain extracts from gut commensal species, Bacteroides thetaiotaomicron and Prevotella copri. After electrophoretic transfer, replicate membranes were incubated with different sera at 1:100 dilution, with detection for human IgG deposition. Only patients with highly active lupus had serum IgG that recognised the oligomeric antigenic band in RG2 (red arrow) that was not detected in other RG strains. (E) Immunoblot of human gut isolated strains of R. gnavus, RG1-RG8. For each strain, molecular species were electrophoretically separated side-by-side, after nuclease and lysozyme treatment (left lane) and after additional thorough proteinase K treatment (right lane). Reactivity for serum IgG from patient S-134 with active LN is shown, with the serum tested at 1:100. Inset shows magnified view of non-protein oligomeric bands in RG2 extract, which are also seen in panels A and B. (F) In patients with SLE, RG faecal abundance by 16S rRNA analysis correlates with the levels of serum IgG anti-RG2 antibody. (G) Levels of serum IgG anti-RG2 in individual patients with lupus in the NYU cohort were compared with unaffected adults (see Methods section). Patients with SLE with high disease activity (SLEDAI ≥8) had higher levels of anti-RG2 IgG antibodies than healthy controls and than SLE with low disease activity. (H) Levels of serum IgG antinative DNA directly correlate with IgG anti-RG2 antibodies. Results from multiplex assay using extracts treated with lysozyme and a broad endonuclease, which was separately coupled to a set of paramagnetic beads (Luminex) in parallel with other antigens. (I) Levels of IgG anti-RG2 directly correlate with IgG to an extract of human glomeruli. Results from multiplex assay. (J) Levels of serum C3 inversely correlated with levels of serum lupus IgG anti-RG2 antibodies in patients with SLE. (K) Levels of serum C4 inversely correlated with levels of serum lupus IgG anti-RG2 antibodies in patients with SLE. (L) IgG anti-RG2 levels strongly correlate with serum IL-6 levels. (M) Levels of IgG anti-dsDNA, determined by commercial ELISA (INOVA), have only a modest correlation with serum IL-6 levels. (N) IgG anti-RG2 levels correlate with IFNα2 levels, determined by commercial bead-based assay (Luminex). (O) Levels of IgG anti-dsDNA antibodies do not correlate with serum IFNα2 levels. IgG anti-DNA and RG2 levels were measured by in-house custom bead-based assays. C3 and C4 were measured with commercial ELISA, while cytokines were measured by commercial bead-based assays. Significance was based on Mann-Whitney test or Spearman correlations. LN, lupus nephritis; SLE, systemic lupus erythematosus; SLEDAI, SLE disease activity index.

Figure 3

The LG3 pool contains two major series of lipoglycan molecules. (A) The ¹H NMR spectrum of native LG3, recorded in D₂O at 300 K, shows signals typical for lipoglycans, for example, for methyl groups of N-acetyl sugars, other carbohydrates and aliphatic chains of the lipid anchor. (B) The charge-deconvoluted MS spectrum of negative ion mode analysis of native LG3. (C) The charge-deconvoluted MS spectrum of negative ion mode analysis of de-O-acylated LG3. (Alternate version of this spectrum with additional assigned peaks is shown in online supplementary figure 1L.) (C) The charge-deconvoluted MS spectrum of negative ion mode analysis of de-O-acylated LG3. LG3 contains two major series of lipoglycans (termed series A [red] and B [dark blue]), starting from molecules with molecular weights of 2499.091/2513.107/2527.122 Da (series A) and 3380.395/3394.410/3408.426 Da (series B), respectively. These molecules are present in a series of further hexose-substituted (△m =+162.05 Da) molecular species. All peaks are present with variations in fatty acid composition (△m=14.02 Da is equivalent to CH₂) (B). The complexity of the mass spectrum of de-O-acyl LG3 (C) is significantly decreased because of the removal of fatty acids. The overall signal pattern of the two series of lipoglycan species is well preserved. For series B, subspecies with a further alanine-substitution are apparent (light blue). Sodium adducts are represented as △m=21.98 Da, labelled in italic style. Abundances were normalised to the respective base peak of the depicted spectral region.

Figure 4

Lipoglycans from RG2 represent immunodominant antigens for patients with SLE. Immunoblots of purified LG2 and LG3 pools, and RG2 and RG1 extracts, were performed with sera from (A) SLE-07, (B) SLE-127, (C) SLE-080 that react with the same protease-resistant bands in LG2, LG3 and RG2 (red box), whereas (D) a healthy control reacts with distinct immunodominant antigens also recognised by patients with active SLE that are generally lower MW (blue box). (E) Immune recognition by serum antibodies in SLE-134 with oligomeric bands in LG2 and LG3, and in RG2, (F) are entirely inhibited by preincubation with LG2 at 2 µg/mL. (G) Periodate treatment before blotting destroys antigens in LG2 and LG3. Loading per lane is indicated above at µg/lane. Immunoblots were performed with serum at 1:200, with detection of IgG reactivity. (H) Serum levels of antibodies to LG3 pool and RG2 extract were highly correlated. Levels were determined by bead-based assay side-by-side in sera from 40 patients with SLE with diverse disease activity scores. SLE, systemic lupus erythematosus.

Figure 5

Active lupus nephritis is associated with elevated IgG anti-RG2 antibodies. (A) Results from individuals in NYU cohort are shown, with comparisons to unaffected controls. (B) Results from individuals in the Temple University cohort with 16 patients with lupus without evidence of renal (ie, non-renal) are compared with 12 with active LN. (C) Results from 17 patients with biopsy-proven active LN from Ohio State University are compared with 6 patients with biopsy-proven idiopathic primary mesnagial glomerulonephritis (MGN) and 20 with IgA nephropathy (IgAN). The cut-off MFI value of 1900 for IgG anti-RG2 (dotted line) was determined by mean for 57 + 2 SD from unaffected 23 healthy controls with gut microbiome data, 15 other healthy individuals, 13 with psoriatic arthritis, and 12 with osteoarthritis). Renal disease was determined by clinical criteria and/or biopsy. Anti-RG2 values were determined by bead-based assay, with significance assigned by Mann-Whitney test.