While a transmissible agent has long been suspected in lupus pathogenesis, only recently has suitable technology become available that enable in-depth consideration of the potential roles of the immense dynamic communities of commensal microorganisms that coevolved with our species. The largest microbiome community resides within our gut, where these microbes play essential roles, including for the early priming of our immune systems 13 and subsequent immune regulation. 14 Mounting evidence has implicated imbalances within these gut microbial communities, also termed dysbioses, in the autoimmune pathogenesis of several diseases: inflammatory bowel disease (IBD), type 1 diabetes, multiple sclerosis and rheumatoid arthritis. 15 Yet, there have only been a few reports on the human lupus microbiome, in small cohorts that have included only a few active patients. 16–18

Serum autoantibodies to native DNA are a specific diagnostic criterion for SLE, 2 and a prognostic factor for the development of lupus nephritis (LN) that affects 30%–60% of patients. 3 However, the earliest reports of antibody responses to nucleic acids/nucleoproteins were documented in association with clinically apparent bacterial infections. 4–6 Yet two decades later autoantibodies to nuclear antigens were recognised to be a common feature of SLE. 7–9 Indeed, some DNA-reactive autoantibodies are directly nephritogenic in animal models. 10 Conversely, only ~20% of the IgG eluted from lupus kidneys is DNA-reactive, 11 suggesting that other antibody reactivities may also contribute to the pathogenesis of LN. 12

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with hallmarks of B-cell abnormalities, circulating autoantibodies to nuclear antigens and immune-complex formation. 1 The heterogeneity of disease presentation and organ involvement in different individuals, and the variability of disease activity from remission to exacerbations and progression, all contribute to clinical challenges for diagnosis and effective management. Indeed, such heterogeneity suggests that SLE may not represent a single disease but rather several.

Patients were consecutively recruited from the NYU Langone Medical Center and Bellevue Hospital. All patients fulfilled the American College of Rheumatology Criteria for the diagnosis of SLE. 2 Further details on trial-specific inclusion and exclusion criteria, clinical evaluations, sampling and 16S rRNA amplicon analyses are described in the supplementary data . Sections on immunoassays, as well as culture of bacterial strains and purification of R. gnavus lipoglycans (LGs) are also provided (see supplementary materials ).

The study was conducted according to the Declaration of Helsinki. Before study inclusion, written informed consent, approved by the NYU IRB, was obtained from all subjects for research use and publication of their data.

Results

Patients with lupus have distinctive patterns of dysbiosis that parallel disease activity In the discovery phase of our studies, we analysed the faecal microbiota of 61 female patients with lupus in a cross-sectional urban cohort and 17 female healthy controls (online supplementary table 1). Our patients displayed great heterogeneity in their organ involvement, and in disease activity from clinical remission to highly active, that was scored using the composite SLE disease activity index (SLEDAI).19 Supplemental material [annrheumdis-2018-214856supp002.xlsx] To define the richness of alpha diversity in gut communities, Chao1 estimates of the total number of operational taxonomic units20 demonstrated that the overall biodiversity of the intestinal microbiota in patients with lupus was significantly restricted compared with unaffected female adult controls (Mann-Whitney, p=0.038) (figure 1A). Among patients with lupus, there was a numerical trend towards an inverse correlation of Chao1 estimates of alpha diversity with SLEDAI score (Mann-Whitney, p=0.08) (data not shown). Moreover, after empirically considering all possible cut-off values to dichotomise patients based on SLEDAI score, those with high disease activity (defined as SLEDAI ≥8) displayed significantly restricted microbiota diversity compared with controls (Mann-Whitney, p=0.003) (figure 1A). 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-SLEDAIhi-SLEDAIlow, 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. The human gut microbiome is dominated by four bacterial phyla—Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria—with thousands of different species and countless strains in complex dynamic relationships within individual gut communities.21 22 To visualise community variation, we performed principal coordinates analyses that confirmed that taxonomic distributions within patients with SLE were significantly different than in controls (PERMANOVA, p=0.02) (figure 1B). When the patients with SLE were stratified by high or low disease activity, the beta diversity differences were even more pronounced (PERMANOVA, p=0.002, data not shown). Furthermore, the variability in beta diversity of healthy subjects was lower than in patients with lupus (figure 1B, C). The within group variability between communities in patients with lupus with low disease activity was also lower than that in patients with high disease activity (figure 1C). However, the severity of the detected disease-associated dysbiosis did not correlate with disease duration (online supplementary figure 1A). Supplemental material [annrheumdis-2018-214856supp003.pdf] With 16S rRNA amplicon sequence analysis, the taxa in each community with the most highly abundant (>1%) representation at all phylogenetic levels were identified by univariate Kruskal Wallis ANOVA analysis (Table 1 and online supplementary table 2). At the species level, patients with SLE displayed a mean 5-fold overabundance of an anaerobic Gram-positive taxon in the Firmicutes phylum and Lachnospiraceae family, identified as Ruminococcus gnavus 23 (RG) (range 0.00%–10.79%, mean±SD 1.35%±2.01%) compared with controls (0.00%–1.27%, 0.25%±0.39%, Mann-Whitney, p=0.01). Strikingly, RG relative abundance correlated with lupus disease activity, as even patients with SLE with low disease activity had a mean 4-fold RG overrepresentation, while those with high disease activity had >8 fold greater RG abundance (Mann-Whitney, p=0.01) (table 1, complete data in online supplementary table 2). Compared with those without a history of renal disease, patients with a history of renal involvement displayed an even greater abundance of a RG specific amplicon sequence variant (ASV) (Mann-Whitney, p=0.04), as well as several other ASVs that included two assigned to the Veillonella genus (online supplementary table 3). In an individual community these Veillonella species were highly correlated with each other (CLR-transformed, r=0.89, p<10–16) although less strongly with RG (39% V. parvula, 43% V. dispar, p<0.005). Hence, the microbiota in patients with lupus commonly displayed concurrent expansions of both RG and Veillonella species. Supplemental material [annrheumdis-2018-214856supp004.docx] Supplemental material [annrheumdis-2018-214856supp005.xlsx] Table 1 Shifts in taxonomic abundance in SLE by disease activity As an alternative approach, DADA224 was used to assign ASVs (ie, quasi-species): patients with a history of renal disease displayed increased representation of ASV31 (Mann-Whitney, p<0.05) (online supplementary tables 2-4). In fact, this ASV identifies RG and has the predictive effect size of AUC=0.65 for renal involvement (see Ref. 25 for description of predictive effect size analysis protocol), although this study was not adequately powered for multiple comparison correction. Nonetheless, these findings collectively indicate that patients with LN commonly have intestinal expansions of the RG species.

The lupus microbiome is associated with altered dynamic relationships between species Within microbiome communities, we also looked for evidence of coordinated shifts in the representation of species within individual lupus communities, and outgrowths of RG were found to be commonly associated with reciprocal reductions of the Bacteroides uniformis species (r=−0.43, Pearson correlation for CLR-transformed abundances, p<0.001). Indeed, lupus patients with the highest disease activity scores also had the lowest abundance of B. uniformis (Mann-Whitney, p=0.01) (table 1), suggesting that these species may be in dynamic reciprocal relationships. Notably, as B. uniformis conveys anti-inflammatory properties that can ameliorate metabolic abnormalities in diabetes-prone mice,26 a paucity in active lupus patients could also be relevant to understanding the drivers of autoimmune pathogenesis.

Evidence of in vivo gut recognition of RG and altered gut barrier function To assess in vivo local recognition of specific gut taxa, we evaluated the representation of secretory IgA (sIgA)-coated faecal bacteria recovered from 15 unaffected controls and 23 patients with SLE (online supplementary figure 1B). Overall, RG bacteria were prominent among sIgA-coated subcommunities by 16S rRNA analysis, and lupus samples displayed a 2-fold greater abundance of RG among sIgA-coated faecal bacteria than healthy controls, However, in these limited surveys, the numerical differences did not attain significance (Wilcoxon rank sum, p=0.14, not significant) (online supplementary figure 1C). In healthy individuals, an intact intestinal barrier prevents leakage of luminal contents out of, and also systemic IgG from entering into, the gut lumen. In these faecal samples, patients with lupus had 2.6-fold overall sIgA elevations vs controls (Welch test, p=0.002) (figure 1D). Furthermore, the elevations of faecal IgM in patients with lupus, as well as the increases in faecal IgG, provide additional circumstantial evidence of an impairment of the epithelial barrier in these patients (figure 1E, F). To further search for evidence of impaired gut barriers, we measured faecal levels of calprotectin, an accepted biomarker for intestinal barrier defect.27 In these pilot studies, we foud that 12/61 patients with lupus exceeded the level in any of the tested control subjects (Mann-Whitney, p=0.03) (figure 1G). Furthermore, patients with lupus also displayed raised serum soluble CD14 (Welch test, p=0.0002) and raised α1-acid glycoprotein levels (Welch test, p<0.0001) (figure 1H, I), which in other conditions have been attributed to gut bacterial translocation.28 29 Taken together, these findings suggest that patients with lupus may at times experience impaired gut barrier function, which potentially may enable commensals, or their components, to escape the intestinal lumen.