Eggerthella lenta augments preclinical autoantibody production and metabolic shift mimicking senescence in arthritis

Baskar Balakrishnan; David Luckey; Kerry Wright; John M Davis; Jun Chen; Veena Taneja

doi:10.1126/sciadv.adg1129

. 2023 Sep 1;9(35):eadg1129. doi: 10.1126/sciadv.adg1129

Eggerthella lenta augments preclinical autoantibody production and metabolic shift mimicking senescence in arthritis

Baskar Balakrishnan ^1,^†, David Luckey ¹, Kerry Wright ², John M Davis ², Jun Chen ³, Veena Taneja ^1,^*

PMCID: PMC10854426 PMID: 37656793

Abstract

Although the etiology of rheumatoid arthritis (RA) is unknown, a strong genetic predisposition and the presence of preclinical antibodies before the onset of symptoms is documented. An expansion of Eggerthella lenta is associated with severe disease in RA. Here, using a humanized mouse model of collagen-induced arthritis, we determined the impact of E. lenta abundance on RA severity. Naïve mice gavaged with E. lenta produce preclinical rheumatoid factor and, when induced for arthritis, develop severe disease. The augmented antibody response was much higher in female mice, and among patients with RA, women had higher average load of E. lenta. Expansion of E. lenta increased CXCL5 and CD4 T cells, and both interleukin-17– and interferon-γ–producing B cells. Further, E. lenta gavage caused gut dysbiosis and decline in amino acids and nicotinamide adenine dinucleotide with an increase in microbe-dependent bile acids and succinyl carnitine causing systemic senescent-like inflammation.

Expansion of gut bacterium Eggerthella lenta initiates preclinical autoreactive response increasing the risk of severe arthritis.

INTRODUCTION

Rheumatoid arthritis (RA) is a chronic inflammatory disease with a strong genetic predisposition leading to an aberrant immune response. Among all the genetic factors, human leukocyte antigen (HLA) class II molecules contribute most substantially to RA susceptibility. This association is further strengthened by the observations of the presence of preclinical autoantibodies in genetically predisposed individuals. However, the low concordance rate of RA in monozygotic twins and the fact that not all individuals with susceptibility-associated HLA-DR/DQ molecules develop RA implicate environmental factors. An infectious episode before the onset of RA has been considered (1), however in the absence of a specific pathogen, the observations do not provide any conclusive evidence. On the contrary, a population-based study showed that gastrointestinal infection diminished the risk for RA as compared to lung or respiratory tract infections (2). With the recent knowledge that the gut microbiome can affect mucosal immunity with consequent effects on systemic immunity, there is a growing realization about the role of gut microbes in RA pathogenesis.

Recent studies have established that RA is characterized by gut dysbiosis. Alterations in the gut microbiota with an abundance of certain taxa in patients with RA implicate a link between gut microbiota and pathogenesis or progression of arthritis (3–6). Because the gut microbiome is affected by a host of factors, including genetic, environment (e.g., diet, geographic location, and infections), or behaviors (e.g., smoking and exercise), a causal role of the gut bacteria in disease requires a model organism. The question arises as to whether certain endogenous gut microbes in concert with the RA-associated genetic factors predispose to production of preclinical autoantibodies. An event that would lead to dysbiosis in a genetically susceptible host may augment the autoimmune response and onset of RA.

To determine whether genetic factors dictate colonization of gut microbiota, we generated humanized mice that express RA-associated HLA class II molecules, DQ8, in the absence of endogenous class II molecules. These mice express HLA molecules similar to humans and generate HLA-restricted immune response to antigens observed in humans (7). When immunized with type II collagen (CII), DQ8 mice develop cellular and humoral response to CII and develop arthritis that mimics human RA in histopathology and autoantibody profile (8). Using naïve humanized mice, we showed that HLA molecules play a role in microbial diversity of gut microbiota (9).

Recently two studies, including ours, showed an expansion of Eggerthella lenta, belonging to phylum Actinobacteria, in patients with RA (3, 6). However, both studies did not determine whether dysbiosis is a cause or an effect of the disease. In this study, we investigated pathogenic ability of E. lenta in arthritis using a humanized mouse model of RA. We show a plausible role of gut bacteria like E. lenta in the production of preclinical autoantibodies and augmentation of disease severity. Our observations suggest that the gut E. lenta expansion causes microbial alterations and a metabolic shift instigating reduced short-chain fatty acid production (SCFA) and amino acids, with alterations in tryptophan (Trp) catabolites and bile acid (BA) metabolites. These changes are associated with senescence-like alterations in the immune system, increases in both antibody-producing B cells and T helper 17 (T_H17)–producing immune cells.

RESULTS

E. lenta enhances arthritis severity by augmenting cellular and humoral response to CII

On the basis of our published data showing association between RA and E. lenta (3), we determined whether E. lenta abundance can cause onset or progression of arthritis in DQ8.AEo mice. Increasing abundance of E. lenta by gavage did not cause arthritis, suggesting that microbial abundance alone may not precipitate disease onset (Fig. 1A). However, E. lenta enhanced arthritis severity in DQ8.AEo mice when mice were gavaged with E. lenta for 5 weeks starting 1 week before the induction of arthritis (Fig. 1B; P < 0.05). Also, E. lenta gavage led to increased incidence and earlier onset of arthritis as compared to control media-gavaged mice (Fig. 1A; P < 0.05). Arthritic mice gavaged with E. lenta did not show an increase in collagen II–specific antibodies though there was a significant increase in IgG rheumatoid factor (assessed in two separate experiments) (Fig. 1C and fig. S1; P = 0.01). The increase in humoral response was associated with an increase in cellular response. CD4⁺ T cells from CII-immunized mice cultured in vitro with dendritic cells (DCs) primed with CII or CII and E. lenta mounted significantly higher response when cultured with DCs primed with CII and E. lenta (Fig. 1D; P = 0.0003). To further delineate whether the augmented response in E. lenta–gavaged mice was due to the immunizing antigen or E. lenta proteins, we analyzed the T cell response to CII and lysate of E. lenta in combination and separately in naïve and CII-immunized mice (Fig. 1E). Naïve mice generated response to E. lenta lysate as well as CII and lysate combination but not to CII alone. Also, CII-immunized mice generated higher response in the presence of E. lenta lysate compared to CII alone, raising the speculation of molecular mimicry. A data search revealed sequence similarity between CII-peptide amino acids 754 to 773 and an unknown E. lenta protein (fig. S2). In vitro proliferation of lymph node cells from arthritic mice gavaged with E. lenta generated a higher response to CII-derived peptides (amino acids 184 to 203 and 754 to 773) compared to control mice not gavaged with E. lenta (Fig. 1F; P = 0.005, P = 0.03 respectively), suggesting that molecular mimicry may also play a role in augmenting T cell response.

Fig. 1. — DQ8 mice pregavaged with *E. lenta* (EL) for 7 days and then immunized with CII considered week 0 and continued EL gavage for four more weeks. Mice were divided and evaluated in three independent experiments, Females three to five, males three to five per group per experiment; a total of 75 mice were used, including four groups (Naïve, Naïve/EL, CIA, and CIA/EL). (A) Incidence and onset of arthritis, compared between groups, CIA/EL (CII immunized + *E. lenta* gavaged), CIA (CII immunized only) and Naïve/EL (*E. lenta* gavage only). P < 0.05, n = 15 per group (Female 7–8 and Male 7–8 per group, and (B) arthritis severity, compared between group CIA versus CIA/EL, *P < 0.05 (n = 15 per group) were used as controls. (C) Anti-CII antibodies and rheumatoid factor (RF) were analyzed by ELISA in sera collected from mice 5 weeks after CII immunization (OD.-optical density (D) Dendritic cells primed with *E. lenta* or not were cultured in vitro with CII and CD4⁺ T cells from CII-immunized mice. The higher T cell response was observed when DCs were pre-cultured with *E. lenta* compared to CII alone. Naïve DQ8 mice generate a response to the lysate of *E. lenta*/ (E) In vitro response of lymph node cells isolated from Naïve and CII-immunized mice in the presence of CII and *E. lenta* lysate alone or in combination, n = 4 to 5 per group. (F) Lymph node cell response to CII-derived peptide amino acids 184 to 203 and amino acids 754 to 773 in mice immunized with CII and gavaged *E. lenta* as compared to CII-immunized only (n = 3 to 5 per group). All experiments were conducted in replicates in at least two independent experiments. The data shown are a compilation of all experiments. The difference in the incidence of arthritis between groups was analyzed using chi-square test and arthritis onset was analyzed using analysis of variance (ANOVA). Antibody levels, mean scores for arthritic mice, and proliferation responses were compared using an unpaired t test.

Augmented immune response in E. lenta–gavaged mice was accompanied with higher levels of interleukin-1β (IL-1β), IL-23, and interferon-γ (IFN-γ) in response to CII challenge in vitro (fig. S3A; P = 0.0001) (10). Sera of mice in the in vivo protocol demonstrated that CII-immunized mice gavaged with E. lenta produced higher levels of proinflammatory cytokines, IL-9, IL-17, IL-23, IFN-γ, and tumor necrosis factor–α (TNF-α) (Fig. 2) than mice without E. lenta gavage although no difference was observed in IL-10 levels (fig. S3B). In addition, eotaxin (CXCL5), a chemokine with angiogenic properties, was increased in E. lenta–gavaged mice (P = 0.01).

Fig. 2. — Serum cytokines in arthritic DQ8 mice gavaged with *E. lenta* (CIA/EL) and control (CIA). Unpaired Student’s t test was used for significance (n = 5 to 9 per group, females 2 to 5 and males 2 to 4 per group).

Changes in few cytokines were observed within 3 weeks of E. lenta gavage (fig. S3C), suggesting that dysbiosis was associated with inflammatory adaptive immune response within 3 weeks of E. lenta treatment (Fig. 1 and fig. S3C). To define whether E. lenta is required or the metabolites produced by E. lenta can also change immune response, we cultured Caco-2 cells in the presence of E. lenta or supernatants of E. lenta cultures (fig. S4). The observations suggest that E. lenta interaction with host is required for modulation of immunity as cytokines, including IL-6, IL23, IL-17A, and TNF-α, were produced at much higher levels when Caco-2 cells were cultured with E. lenta than supernatant alone. In addition, TNF receptor–associated factor 6 (involved in T_H17 activation), Sphingosine-1-phosphate receptor 1 (SIPR1) (involved in angiogenesis), and the costimulatory molecule CD28 were expressed higher in the presence of E. lenta compared to culture supernatant of E. lenta. These data suggest that E. lenta directly affects an epithelial cell–induced inflammatory response.

E. lenta treatment reduces T regulatory cells and augments T follicular cells

Previous studies have shown a role of gut microbes in differentiation of T follicular helper and T follicular regulatory cells (11). We ascertained whether alterations in the immune response of E. lenta–treated mice could be due to a change in the ratio of T follicular/T regulatory cells. E. lenta gavage was associated with a decrease in mesenteric lymph node FoxP3⁺ T regulatory cells and an increase in CD4⁺CXCR5⁺ T follicular cells (Fig. 3A; P = 0.03 and P = 0.04; respectively) even though overall there was not a significant increase in CD4⁺ T cells (fig. S5). However, CD4⁺ cells producing IL-17 and IFN-γ only or both were increased in E. lenta–gavaged arthritic mice compared to controls (Fig. 3A; P = 0.02, fig. S5). Overall, there was an increase in cells expressing Bcl6 and CCR5 (Fig. 3A, markers of activated germinal cells (GC), as well as CXCR5⁺ GL-7⁺PD-1⁺ cells (Fig. 3A; P = 0.05).

Fig. 3. — Mice in vivo protocol were used for analyzing immune composition. (A) T regulatory CD4⁺FoxP3⁺ cells and CXCR5⁺ cells as well as IL-17– and IFN-γ–producing CD4 T cells and B cells were enumerated in mesenteric lymph node cells; CD4⁺CD25⁺FoxP3⁺, CD4⁺CXCR5⁺, CD4⁺IL-17⁺IFNγ⁺, CD4⁺Bcl6⁺CXCR5⁺ and CXCR5⁺Gl-7⁺PD-1⁺. (B) Lamina propria cells isolated at the termination of in vivo experiment were analyzed for total CXCR5⁺, CXCR5⁺GL-7⁺PD-1⁺ and CXCR5⁺ICOS⁺PD-1⁺. (C) Cells isolated from synovium were analyzed for CD4⁺T cells and T regulatory, CD4⁺FoxP3⁺, cells. (D) Splenic cells were analyzed for CD19 cells and CD19 cells expressing PNAhi and CXCR5, CD19⁺ cells expressing IL-17 and IFN-γ, and CD11c⁺ cells. Two independent experiments were conducted with cells pooled from 2–3 mice/ experiment, each group had a 40 to 60% of male/female ratio. All comparisons were analyzed by unpaired t test.

To assess the changes locally, we enumerated CXCR5⁺ cells in lamina propria and observed a significant decrease (P < 0.02) in E. lenta–treated mice over the control mice; however, CXCR5⁺GL-7⁺PD-1⁺ cells and regulatory ICOS⁺PD-1⁺ cells were similar in both groups (Fig. 3B), suggesting that CXCR5⁺ cells exit the gut as suggested previously. To evaluate changes in joints, T regulatory cells were analyzed in the synovium of arthritic mice gavaged with E. lenta or not (Fig. 3C). Reduced T regulatory cells were observed in E. lenta–gavaged arthritic mice even though the total numbers of CD4 cells were similar.

We further examined CD19⁺ B cells and GC B cells, CD19⁺PNAhi cells, which could explain an increase in RF in E. lenta–gavaged mice. Treatment with E. lenta led to an increase in CD19⁺PNAhi B cells as well as IL-17– and IFN-γ–producing B cells as compared to controls though total B cells did show any difference between the two groups (Fig. 3D and fig. S5). Because microbial treatment of arthritis has previously shown an increase in myeloid suppressors (12), next, we analyzed the expression of CX3CR1, a marker for migrating cells from the gut (13), on myeloid suppressors, CD11b⁺Gr1⁺ and CD11c⁺Gr-1⁺ cells (13). Although no significant differences between CD11b⁺ myeloid suppressors were observed, CX3CR1⁺ cells were significantly lower in E. lenta–gavaged mice (P = 0.04) (fig. S5). On the other hand, CD11c⁺ DCs were increased significantly in E. lenta–gavaged mice (P = 0.04) though CD11c myeloid suppressors did not show significant differences between the two groups (Fig. 3D and fig. S5).

E. lenta abundance alters microbial composition

We determined the impact of E. lenta colonization by comparing microbial composition before (naïve/day 0) and after (5 weeks after immunization and E. lenta gavage) treatment (Fig. 4 and figs. S6 to S8). The alpha diversity (Shannon index) showed an increase after E. lenta gavage (Fig. 4A). Beta diversity based on Bray-Curtis (BC) distance showed compositional dissimilarity. Dysbiosis was observed in naïve as well as collagen-induced arthritis (CIA) groups after 5 weeks of treatments in a longitudinal follow-up. However, naïve group did not show significant changes in the alpha and beta diversity over 5-week period, demonstrating eubiosis. The principal coordinate analysis (PCoA) plot based on beta diversity revealed that both E. lenta–treated groups (Naïve/EL and CIA/EL) show a similar pattern of separation between the two time points unlike the CIA control group, suggesting specific E. lenta–induced compositional changes. Differential taxa abundance in naïve mice did not show any significant changes except for Turicibacter (figs. S6 to S8) though mice gavaged with E. lenta showed a significant decrease in Firmicutes members such as Allobaculum, Clostridium, Staphylococcus, Jeotgalicoccus, Turicibacter, and Sporosarcina, while genus Arthromitus, Ocillospira, Ruminococcus, Dehalobacterium, and Prevotella were increased. Clostridium and Adlercreutzia were decreased in CIA and CIA‑ E. lenta mice, though CIA group also showed an increase in abundance of Dehalobacterium and Prevotella, similar to Naïve/EL mice.

To understand the impact of E. lenta on microbiome, we also analyzed beta diversity based on BC distance using the endpoint samples (Fig. 4B). The data showed compositional dissimilarity among all the groups, P = 0.001 (Fig. 4B). Differential abundance of taxa at the termination of experiment showed CIA group with an increase in lactobacillus and Clostridium from Firmicutes phylum when compared to naïve control group (Fig. 4C and fig. S8). CII immunization also led to an increase in Adlercreutzia and Odoribacter suggesting disease-associated dysbiosis. However, butyrate-producing bacteria such as Butyrivibrio and mucus-interacting bacteria Mucispirillum were reduced significantly after CII immunization. Comparing naïve mice with unimmunized naïve mice gavaged with E. lenta (Naïve/EL) also showed dysbiosis indicating E. lenta colonization or expansion can cause microbial compositional alterations in the gut (Fig. 4C). Most of the microbial alterations in naïve/EL group were also reflected in CIA group suggesting that E. lenta–induced dysbiosis mimics arthritis-associated changes. Treatment with E. lenta along with CII immunization (CIA/EL) showed lower abundance of Turicibacter with an increase in lactobacillus. Unexpectedly, CIA/EL group showed lower abundance of Adlercreutzia and Clostridium but increased abundance of Mucispirillum when compared to CIA mice suggesting that E. lenta induced microbial alterations.

To confirm the colonization of E. lenta, we analyzed a genus-level relative abundance in all the groups. Eggerthella was not detected in any day 0 naïve conditions (Fig. 4D) and after samples of media-gavaged groups (Naïve and CIA control groups). However, as expected, only E. lenta–gavaged posttreatment samples showed the presence of Eggerthella, confirming that this bacterium is not a commensal for this mouse model.

E. lenta abundance is correlated with decreased amino acid and increased BA levels

Metabolic shifts due to dysbiosis can function as biosensors for microbial functionality. E. lenta–gavaged mice showed a trend toward reduced levels of fecal butyrate, a SCFA (Fig. 5A; P = 0.07). An analysis of SCFAs in fecal samples showed an increase in acetic acid and isovaleric acid in mice gavaged with E. lenta in comparison to nontreated arthritic mice (Fig. 5A; P = 0.008 and 0.03, respectively; fig. S9, A and B). Because E. lenta is known to be involved in ornithine pathway, sera from mice were assessed for citrulline and arginine. Unexpectedly, mice gavaged with E. lenta showed lower citrulline, and arginine levels in sera compared to controls (Fig. 5B; P = 0.003 and P = 0.007, respectively), while fecal samples did not show any significant difference in citrulline (fig. S9). Overall amino acid metabolism, as measured in fecal samples, were significantly reduced in the E. lenta–gavaged mice; fig. S9 and table S1). To determine whether similar phenomenon occurs in humans, we analyzed plasma samples from patients with RA for the presence of arginine and citrulline. The RA cohort showed significantly lower levels of citrulline and arginine as compared to first-degree relatives and random healthy controls (Fig. 5C; P = 0.04 and P = 0.03, respectively).

Fig. 5. — Mice were immunized with CII for 2 weeks and then gavaged with *E. lenta* for 10 weeks. (A) short-chain fatty acids Butyrate, Acetic acid and isovaleric acid measured by LC-MS in fecal samples. Arginine and citrulline were analyzed (B) in sera from mice and (C) plasma samples from patients with RA and healthy controls. (D) Random forest algorithm analysis with the top metabolites in stool samples of *E. lenta*–gavaged mice using nongavaged CII-immunized (CIA) samples as controls. (E) Fecal Trp catabolites, and liver nicotinamide adenine dinucleotide (NAD) in arthritic (CIA) and *E. lenta*–gavaged arthritic (CIA/EL) mice. CIA/EL versus CIA, Trp, Q = 0.23, Indole acetate, Q = 0.11, Xanthurenate, Q = 0.11, indole-3 carboxylate Q = 0.18, and BCAA valine and isoleucine, Q = 0.16 and 0.18, respectively. (F) Fecal primary and secondary bile acids in CIA/EL and CIA mice; deoxycholate (DCA, Q = 0.05), taurocholate (TCA, Q = 0.08), lithocholate (LCA, Q = 0.08), hyodeoxycholate (HDCA, Q = 0.08), 3b-hydroxy-5-cholenoic acid (cholenate, Q = 0.15). Welch’s two sample t test and false discovery rate (Benjamini-Hochberg procedure) was used for multiple testing correction.

Untargeted metabolic profile using fecal samples from mice before and 10 weeks after gavage with E. lenta clearly showed a shift in metabolic profile. A total of 122 metabolites were observed to be altered significantly (P < 0.05) after E. lenta treatment, of which 41 metabolites were increased and 81 were reduced (fig. S10A). PCoA plot and heatmap showed distinct clustering of samples before and after treatment with E. lenta (fig. S10, B and C). Random forest analysis based on the metabolites detected in fecal samples before and 10 weeks after E. lenta gavage showed top metabolites separating the groups, based on their importance (Fig. 5D). The data confirmed an increase in microbial metabolites involved in lipid metabolism and sphingosine as well as increased host-derived metabolites involved in various biochemical pathways. Further, reduced amino acid (fig. S11) was observed in E. lenta–gavaged mice, like that observed in patients with RA (3). There were significant alterations in the Trp, branched-chain amino acids (BCAA) and BA metabolism pathways in E. lenta–gavaged mice (Fig. 5E and fig. S11). Overall, E. lenta led to a decrease in Trp and its cellular metabolites with a significant decrease in xanthurenate (Q = 0.11) as well as microbial catabolites, indole acetic acid (Q = 0.11) and a trend toward reduced Trp, anthranilate, and picolinate (Fig. 5E and fig. S11). On the other hand, an increase in microbial metabolite indole-3 carboxylate (Q = 0.18) with a trend in higher serotonin in fecal samples was observed in mice gavaged with E. lenta as compared to nontreated mice (fig. S11). In addition, nicotinamide adenine dinucleotide (NAD), de novo produced by Trp, was decreased in arthritic mice. Isoleucine and valine were also significantly reduced after E. lenta gavage (Fig. 5E and table S1; Q = 0.16 and Q = 0.18, respectively). The most significant differences observed were the increases in primary BA and microbial-derived secondary BA metabolites in E. lenta–gavaged mice [Fig. 5F and fig. S12; deoxycholate (DCA), Q = 0.05], 3β-hydroxy-5-cholenoic acid (cholenate, Q = 0.15), [Q = taurocholate (TCA) (Q = 0.08), α and β muricholate (Q = 0.1) hyodeoxycholate (HDCA) (Q = 0.08), murideoxycholate (MDCA), (Q = 0.08) and lithocholate (LCA), Q = 0.1].

Because BA and proinflammatory cytokines, IL-6, IL-17, and TNF-α were increased in E. lenta mice, we evaluated the expression of TGR5 in the intestinal cells. Low mRNA expression of TGR5 in the intestines of E. lenta–gavaged mice (fig. S13) supports a protective role of TGR5 in RA.

E. lenta augments sex-specific responses both in patients and humanized mice

Since RA occurs with 3:1 ratio of women to men, we defined if E. lenta abundance has sex-specific impact on CIA. Although all mice eventually developed arthritis and the difference was nonsignificant at week 10 after induction of arthritis, female mice showed an earlier onset (P < 0.05 at week 3 after E. lenta gavage) and more severe arthritis (P = 0.04 at week 4 after E. lenta gavage) (Fig. 6A) than male mice. Intestinal permeability using fluorescein isothiocyanate (FITC)–dextran assay showed a significant increase in females compared to male mice in E. lenta–gavaged group (Fig. 6B; P = 0.01). In addition, at week 4, E. lenta augmented immunoglobulin G (IgG)–rheumatoid factor (RF) in female mice (P < 0.01) while in males no increase of IgG-RF was observed as compared to nongavaged males. Levels of anti-CII antibodies did not differ between the two groups. Males had higher anti-CII antibodies while females produced higher RF irrespective of gavage with E. lenta or not (Fig. 6C, F versus M; anti-CII, P = 0.0001 and RF, P = 0.001). The enhanced antibody response was associated with higher levels of IFN-γ and IL-17 in E. lenta–gavaged female mice as compared to males (P = 0.007 and P = 0.008, respectively) and nongavaged control females (Fig. 6D, CIA versus CIA/EL; P = 0.0001 for both). Males did not produce significantly higher cytokines than control nongavaged mice except IL-23, which was significantly increased in E. lenta–gavaged males as compared to control arthritic males (Fig. 6D; P = 0.008). These data clearly indicate a sex-specific immune response to E. lenta.

Fig. 6. — (A) Arthritis severity and (B) Incidence in male (n = 18) versus female mice (n = 15). Mice in Fig. 1 were used to determine sex-specific response. (B) Comparison of antibodies, anti-collagen antibodies (Anti-CII) and RF between male and female mice. (C) Cytokines in the sera of arthritic mice gavaged with *E. lenta* (CIA/EL) or not (CIA). (D) Gut permeability measured by FITC-dextran method (E) Comparison of abundance of *E. lenta* in patients with RA and healthy controls, Wilcoxon rank sum test was used to compare the relative abundance of *E. lenta* between males and females for patients with RA and controls and (F) patients with seropositive RA, female (F) and male (M) and (G) High RF (>50 U) and low RF (<50 U).

Our previous study showed a correlation between an expansion of E. lenta and RA status. To examine the impact of E. lenta on the immune status in patients with RA, we correlated the abundance of E. lenta with levels of anticitrullinated peptide antibodies (ACPA) and RF, in patients with RA. E. lenta abundance trended higher in females compared to males though the difference was not significant in patients and controls (Fig. 6E). Patients with high levels of RF (>50 U) and /or ACPAs (≥ 200 U) were compared with patients with lower levels of antibodies. Seropositivity and individual presence of RF or ACPA was not significantly correlated to a high abundance of E. lenta associated with sex bias (Fig. 6F and fig. S14). While ACPA⁺RF⁺ female patients showed a high abundance of E. lenta, males were too low in numbers for statistical analysis. However, high RF levels correlated with increased abundance of E. lenta (Fig. 6G; P = 0.007), suggesting that E. lenta influences disease severity by enhancing autoantibody production. The observations in the animal model and patients suggest that E. lenta augments disease severity via antibody response in both, and in the animal model, IL-23 in males, and IFN-γ and IL-17 in females, implicating a sex-specific impact in arthritis.

E. lenta augments preclinical antibody response

To prove that E. lenta affects pathogenesis by augmenting preclinical autoantibodies production, RF and anti-CII antibodies were analyzed in sera from naïve DQ8 mice gavaged with E. lenta. The data showed significant RF production in naïve mice gavaged with E. lenta (Fig. 7A; P = 0.0001), which supports the data from patients with RA (Fig. 6, E and F). For anti-CII antibodies, there was a trend toward higher levels after E. lenta was gavaged (P = 0.08) (Fig. 7B), suggesting that E. lenta might promote activation of B cells, which is also supported by the above data of increased antibodies production when E. lenta proportions are higher (Figs. 1C and 6F). These observations are also in confirmation of our previous data suggesting E. lenta gavage of naïve mice leads to production of T_H17 cytokines (10). Because naïve E. lenta–gavaged mice developed autoantibodies, we examined antigen-specific and nonspecific cellular response by challenging splenic cells with CII, CII-derived peptides, and E. lenta lysate in vitro. Splenic cells isolated from naïve mice, cultured with E. lenta lysate generated a similar response as E. lenta–gavaged mice (Fig. 7C), suggesting that naïve mice harbor T cells reactive to proteins in the E. lenta lysate. To establish whether E. lenta expansion can generate cross-reactive response to CII or its peptides, splenic cells isolated from naïve mice gavaged with E. lenta were cultured in vitro with CII and derived peptides. Naïve mice, gavaged E. lenta or not, do not generate cellular response to CII (fig. S15), but E. lenta–gavaged mice did generate response to CII-derived peptides (Fig. 7D). To determine the kinetics of response augmented by E. lenta, cytokines in sera were measured at varying time points, as indicated, after gavage (Fig. 7E). One week after E. Lenta gavage, mice showed an increase in IL-9 and CXCL5 and granulocyte colony-stimulating factor (G-CSF) followed by an increase in Eotaxin (CCL11) by week 3, and all these cytokines maintained higher levels than naïve state. In addition, gavage with E. lenta of naïve mice led to reduced levels of citrulline and butyric acid, similar to arthritic mice (Fig. 7F; P = 0.001 and P = 0.07, respectively). These data clearly show that E. lenta activates cellular and humoral proinflammatory immune responses and can contribute to RA at the preclinical stage.

Fig. 7. — (A) Antibodies rheumatoid factor (RF) and (B) anti-CII antibodies (CII) in naïve DQ8 mice gavaged with EL (naïve/EL) or not (naïve). Response of splenic cells isolated from (C) naïve, naïve/EL mice and arthritic (CIA) mice to *E. lenta* lysate and (D) CII-derived peptides aa 184–203 and aa 754–773. Response is depicted as stimulation index. (E) Kinetics of serum cytokine production in DQ8 mice, naïve mice gavaged with *E. lenta* (EL) for 1 week (week 1) and for 3 weeks (week 3) (n = 3). (F) Serum citrulline and fecal butyrate in naïve and Naïve/EL mice. All comparisons were analyzed using unpaired t test.

DISCUSSION

On the basis of previous observations of an association of E. lenta (3, 6) with RA status, in this study, using a humanized model of arthritis, we determined the impact of E. lenta abundance on arthritis. Gavaging E. lenta in DQ8 mice increased the expression of SIPR1 in the intestinal epithelium, which is known to augment inflammation via signal transducer and activator of transcription 3 (STAT3) and IFN regulatory factor 4 (IRF4) (14). STAT3, IRF4 as well as IL-17–producing T follicular cells and GC B cells were increased in mice treated with E. lenta. Further, gavaging mice with E. lenta caused microbial/metabolic dysbiosis with an increase in BAs and a decrease in their ligand TGR5 in the gut, which can affect lipid metabolism and contribute to inflammation. The metabolic profile of E. lenta–gavaged mice also showed reduced amino acid and Trp metabolites. These alterations led to preclinical autoreactive cellular and humoral immune response in mice, which augmented arthritis severity. Naïve mice gavaged with E. lenta produced RF even though they did not develop arthritis. Because autoantibodies can be present up to 10 years before the onset of disease in patients with RA, a likely scenario can be envisaged where an expansion of E. lenta contributes to preclinical antibodies production. Present data showing an increased abundance of E. lenta in patients with seropositive RA high RF levels support this concept. In mice, production of autoantibodies could be explained by an increase in germinal B cells. Our data provide evidence that dysbiosis can be responsible for autoantibodies to self-proteins that share sequence similarities with bacterial products. E. lenta shares homology with certain CII peptides, and naïve mice gavaged with E. lenta generate response to those peptides providing a strong support to mimicry theory and further suggesting that an expansion of E. lenta could result in an autoreactive response to CII-derived proteins during normal physiological processes. Further, we show here that E. lenta may be one of the gut bacteria that skews the sex bias in arthritis as both mice and patients with RA showed higher RF in association with E. lenta abundance. Eggerthella in humans has been shown to produce equol, a phytoestrogen, with estrogenic properties and binding affinity to estrogen receptors (15, 16).

Mice after gavage with E. lenta displayed a difference in the alpha diversity with differential abundance of taxa compared to before E. lenta treatment. Naïve mice gavaged with E. lenta showed significant abundance of Candidatus Arthromitus, a segmented filamentous bacteria known to induce a potent T_H17 response, and cause autoimmunity (17), Another taxon that was abundant in naïve E. lenta–gavaged mice, Ruminococcus, has been associated with inflammatory bowel disease (18). Allobaculum, Adlercreutzia, and Turicibacter were recorded as normal microbiota in healthy gut of DQ8 mice in our previous study, where an increase in these three genera correlated to reduced disease severity and partial restoration of eubiosis in CIA model (19). Longitudinal and endpoint analysis showed lactobacillus, and Turicibacter define the impact of E. lenta. Increase in lactobacillus was observed with CIA as well as after E. lenta treatment while lower levels of Turicibacter was specific to E. lenta treatment. Alterations in naïve mice gavaged with E. lenta compared to CIA mice suggest that Eggerthella-induced changes affect gut dysbiosis, which could influence both metabolic and immune changes and generate preclinical response.

We measured metabolic profile to determine microbial functional changes caused by E. lenta. Increasing abundance of E. lenta via gavage led to alterations in metabolic profile with reduced butyrate production. Reduction in butyrate producing taxa, Burtyrivibrio, after E. lenta treatment could be one of the reasons for reduced gut butyrate levels. Butyrate enhances epithelial integrity and generation of T regulatory cells required to keep the colon healthy (20). We have shown previously that Prevotella histicola, upper gut commensal, augments butyrate production and T regulatory cells suppressing disease severity and preserving gut permeability (12, 19). Butyrate also encourages gut homeostasis by stimulating growth of beneficial commensals and suppressing TNF-α production through nuclear factor κB (NF-κB) inhibition in patients with RA (21, 22). Untargeted metabolic profile showed E. lenta gavage led to low amino acids including BCAA and dipeptides supporting previous observations in patients with RA (3, 23, 24). Increased proportion of E. lenta led to a decrease in metabolites, N-acetylglutamine, deoxycarnitine, gamma glutamylmethionine, Trp, N-acetyltryptophan, and methionine, which supports our recent data in patients with RA where an increase in these metabolites was associated with a decrease in disease severity (25). Diet with high methionine has been shown to attenuate arthritic severity in rats (26). Both citrulline and arginine have anti-inflammatory properties and arginine is in phase 2 trial in treatment of RA (27, 28). Reduced levels of both arginine and citrulline suggest alterations in microbial diversity with compensatory microbial changes. Together, our results implicate amino acid metabolism control by microbes in chronic immune activation and arthritis severity.

Another important amino acid, Trp, metabolism was altered in E. lenta–gavaged mice with an increase in microbial derived catabolites, indole 3 carboxylate and serotonin. Serotonin is suggested to be produced by indigenous spore-forming bacteria dominated by clostridia, enterochromaffin cells as well as can be elevated by primary and secondary BA metabolites, cholate and DCA (29, 30). Increased indole 3 carboxylic acid, a microbial catabolite known to be triggered and accumulated in the presence of microbial associated molecular patterns, suggests a dysbiotic microbiome after E. lenta gavage alters the Trp metabolism disrupting the de novo pathway for NAD⁺. However, in the small sample size studied for metabolic profile, there was no significant change in the fecal nicotinamide riboside and nicotinamide levels but a decrease in NAD in livers of E. lenta–gavaged mice was observed.

E. lenta gavage also altered BA metabolism as observed by increased primary and secondary BA including DCA and lithocholate (LCA). E. lenta has enzymes for bile salt hydrolyzation that can hydrolyze via dehydroxylation and epimerization (31) and some strains are able to produce many secondary BA (32, 33). Secondary BA, DCA, and LCA can cause DNA damage (34). In a mouse model, DCA supplementation increased oxidative damage in colonic epithelium (35). Increased secondary BA have also been associated with gut inflammation that can occur via receptors, G-protein–coupled bile receptor 1, TGR5, and sphingosine 1 phosphate receptor 1 (S1PR1). Immune cells express receptors for BA which can influence the inflammatory status (36). S1PR1 augments production of IL-6, TNF-α, and IL-17 via NF-κB and STAT3 (14, 37). E. lenta did enhance SIPR1 expression in epithelial cells. Primary and secondary BA signal via S1PR1 and activate cellular pathways AKT1/2 causing inflammation as well as regulating lipid metabolism. TGR5, decreased in E. lenta–gavaged mice, binds various BA including LCA, DCA, and CA and activates many signaling pathways affecting diverse physiological pathways like inflammation and carbohydrate and lipid metabolism. Increased 3β-hydroxy-5-cholenoic acid in E. lenta–gavaged mice is of interest as a biomarker for abundance of E. lenta as very few bacteria can form 3β forms of BA.

In a recent study using fibroblasts, an increase in BA was observed with senescence in synovial fibroblasts, although senescence associated secretory phenotype was not typical, leading to resolution of arthritis (38). Increase in the expression of G-CSF and CXCL5, observed in naïve mice gavaged with E. lenta, are linked to senescence (39, 40). Accumulation of succinyl carnitine is associated with increased use of amino acids for energy in aged individuals resulting in a decline in amino acids. DNA damage and cellular senescence is characterized by alterations of lipid biosynthesis with an increase in monoglycerols and diglycerols (41, 42). E. lenta gavage in DQ8 mice lead to all these alterations, suggesting that it can contribute to senescence, which is considered a hallmark of RA onset. The data presented here suggest that E. lenta expansion by gavage simulates senescence like changes; an increase in RF, a decrease in amino acids and reduced levels of NAD⁺ (43–46). Age-related microbial changes show reduced gut microbial diversity (47, 48) with an expansion of species in phylum Actinobacteria (43). Increased abundance of E. lenta has been associated with frail microbiota (49).

E. lenta abundance via gavage led to alterations in immune cell profile, decrease in T regulatory cells with a significant increase in CD4⁺ T cells and B cells that produce IL-17 and IFN-γ, which are in confirmation with a recent study showing E. lenta activates T_H17 cells via cardiac glycoside reductase operon, increasing colitis severity (50, 51).The observations suggest that E. lenta reduces myeloid suppressors that regulate immune homeostasis, contributing to production of proinflammatory cytokines augmenting disease severity. An increased intestinal expression of IRF4 in E. lenta–gavaged mice can help T and B cells for GC formation and clonal expansion (52). Preclinical antibody production in E. lenta–gavaged mice could be due to an increase in germinal center B cells as well IL-17 producing B cells. IL-17 can further activate B cells (53), generating a positive feedback loop. While antigen-specific antibodies were not significantly increased, E. lenta led to an increase in RF, more so in females than males. Patients with RA support the mouse data suggesting an abundance of E. lenta is associated with augmentation of antibodies that along with dysbiosis and metabolic shift can precipitate disease. However, we cannot rule out that these effects could be due to alterations in the gut microbial diversity which was altered after gavage with E. lenta.

The study confirms recent observations that E. lenta augments T_H17 production leading to gut inflammation (50) and alter BA (31) and further explains how an abundance of E. lenta affects systemic autoimmune disease. E. lenta abundance increases the expression of S1PR1, STAT3, and IRF4 with a decrease in TGR5 in epithelial cells which augments proinflammatory cytokines and preclinical autoimmune response. The present data present unique observations showing an expansion of E. lenta (i) augments T follicular helper cells, CD4⁺CXCR5⁺ T cells, and IL-17–producing GC B cells, (ii) triggers preclinical autoantibodies production and autoreactive cellular response, and (iii) modulates microbial/metabolic profile with alterations in Trp metabolites leading to reduced levels of NAD as well as other amino acids in CII-immunized mice, mimicking senescence, and exacerbating arthritis. These data confirm a role of gut in systemic inflammation in RA (fig. S16). These conditions are also reported in aged subjects (54–56) suggesting that E. lenta expansion can cause immunosenescence. One can envisage that an expansion of E. lenta can lead to dysbiosis, inflammatory response, and preclinical autoantibodies in patients with RA that contribute to onset/severity of disease following another immune insult. The data suggest that RA and CIA share similarities in immune and metabolic functions with aged individuals. An interesting novel observation is the role of E. lenta in sex bias in T_H17 immunity at preclinical stage in mice. The sex-biased association of E. lenta abundance with antibodies in patients with RA further prove sex-specific impact though further studies are required because of limited numbers. These data emphasize that microbial disease associations need to be studied in the context of sex specificity of the disease.

While our data provide a compelling role of E. lenta in preclinical autoimmunity, further work is warranted to investigate whether targeting E. lenta using antibiotics or specific genes and metabolites can attenuate disease severity or preclinical autoimmunity. In addition, future work will need to determine the enzymatic and structural aspect of E. lenta in immunomodulation of arthritis.

MATERIALS AND METHODS

Research objective

The study was conducted to define the role of Eggerthella in contributing to causation or disease severity of RA. Animal model was used for determining the immune response and metabolic changes caused by E. lenta and its capability to cause arthritis.

Patient data

No patients were enrolled in this study. The patient data collected and analyzed previously were reanalyzed for sex differences in autoantibody profiles (3). Samples from patients with RA (n = 42) and healthy controls consisting of first-degree relatives and random healthy controls (n = 25) collected in previous study were evaluated for amino acids in plasma.

Transgenic mice

Transgenic mice, HLA-DQ8.AEo, were generated as described previously (57). The mice have been characterized, DQ8 mice used in the study lacked endogenous class II molecules (AEo) and expressed DQB1*0302/DQA1*0301 (DQ8.AEo). Mice of both sexes (8 to 12 weeks of age) were used in this study and were bred and maintained in the Department of Comparative Medicine at the Mayo Clinic, Rochester, MN and used in accordance with Animal Resesarch: Reporting of In Vivo Experiments (ARRIVE) guidelines and after the approval from the Institutional Animal Use and Care Committee, protocol number A23015. All experiments used approximately equal numbers of males and females divided in to various groups and were randomized for reproducibility. At least two independent experiments were conducted for all data analysis.

Collagen-induced arthritis

CIA was induced by immunization with CII as previously described (8). Briefly, 100 μl of CII (100 μg of CII emulsified in a 1:1 ratio of Complete Freund’s Adjuvant) was injected intradermally at the base of tail. The onset and progression of arthritis was monitored, and mean arthritic severity was determined using only the arthritic mice; arthritis score was evaluated with a grading system of 0 to 3 for each paw as described previously (8). Arthritis scoring was conducted in a blind fashion without the knowledge of outcome. In vivo experiments were conducted after dividing mice in to various groups and independent experiments for reproducibility. Arthritic mice with and without E. lenta gavage in vivo protocol were chosen randomly for further analysis in experiments for an unbiased approach.

Culture, lysate preparation, and treatment with bacterium

E. lenta ATCC 25559, the typed strain of the species E. lenta and the type species of the genus Eggerthella, was obtained from American Type Culture Collection (ATCC, USA). The late log phase culture was prepared for the treatment using a tryptic soy broth (TSB; Corning, USA) media under strictly anaerobic condition using a Bactron300 anaerobic chamber (SHELDON Manufacturing Inc., USA) at 37°C for 36 hours, with initial media pH of 7.2 ± 0.2. The anaerobic condition of the chamber maintained by the gas mixture containing N₂ (85 to 90%), CO₂ (5 to 10%, and H₂ (5%). The quantity of the bacterium measured every time using serial dilution and plating on to TSB agar plates. Purity of the bacterium qualitatively checked every time of culture preparations using quantitative polymerase chain reaction (qPCR) method with species specific primers (ElenF 5′-ATACTAGGTGTGGGGGGCTCCG-3′ and ElenR 5′-TTTCCCCGGCTTCACGTCCATG-3′). The purity of the bacterium was verified by qPCR.

For oral gavage of transgenic mice, the bacterial concentration was maintained at ∼10⁹ colony-forming units (CFU) ml⁻¹, and each mouse received 100 μl of log phase culture containing approximately ∼10⁸ CFU of live E. lenta cells. The bacteria were administered 7 days before immunization with CII and continued for 4 weeks after immunization on alternative days. Control mice were sham gavaged via administering 100 μl of bacterial media alone. Control mice were sham gavaged via administering 100 μl of bacterial media alone. Four experimental groups were maintained in the study: (i) Naïve control (no CII immunization and no E. lenta gavaged), (ii) Naïve/EL (no CII immunization, but gavaged with E. lenta), (iii) CIA control (no E. lenta gavage and but immunized with CII), and (iv) CIA/EL (E. lenta gavaged and immunized with CII).

Lysate preparation: The lysate was prepared by using 1 ml of culture centrifuged (at 5000 rpm for 10 min) to get the cell pellet. The pellet suspended in to 100 μl of phosphate-buffered saline buffer and vortexed thoroughly. Prepared cells were lysed using a physical method by keeping the tube in water bath for 10 min at 95°C. The lysate was diluted with 900 μl of proliferation media, and 10 μl of lysate was used to challenge splenic/lymph node cells.

Isolation of lamina propria cells

Intestinal tissue was cut longitudinally using a scalpel blade and washed six times using CMF solution (88% 1× Hanks balanced salt solution, 10% Hepes-bicarbonate buffer, and 2% fetal bovine serum). A 1-hour collagenase digestion using Complete RPMI-10/collagenase (1.33 mg/ml) solution released lymphocytes from the intestinal tissue. This mixture was passed through a nylon filter and centrifuged, and the pelleted lamina propria cells were suspended in complete RPMI-10 with gentamycin. Lamina propria cells were pooled from two to three mice for each independent experiment, and experiments were conducted in triplicate.

Isolation of cells from synovium

The synovium around the knee was removed and digested with collagenase as described (58). Single-cell suspension was generated by Ficoll Percoll density gradient centrifugation method.

Cytokine and chemokine expression

Reverse transcription PCR method was used to analyze chemokine expressions. Shortly, RNA was extracted from cells using RNAeasy columns (Qiagen), and cDNA prepared using RNase H-reverse transcriptase (Invitrogen) by standard methods. The expression level of each gene was quantified using the threshold cycle (C_t) method normalized to a housekeeping gene. Qiagen RT2 Profiler PCR microarrays were used as per manufacturer’s instructions. The data were analyzed as per the online resources of the manufacturer from their Data Analysis Center.

Cytokines were measured using the multiplex array system with the mouse cytokine 23-plex panel as per manufacturer’s instructions and analyzed with Bio-Plex manager 2.0 software (Bio-Rad Laboratories, Hercules, CA). Few cytokines were also measured by Capture ELISA using commercial kits (BD biosciences).

FITC-dextran for gut permeability

Alterations in intestinal permeability were determined using 4-kDa FITC-labeled dextran as described previously (3). Mice, arthritic and CIA/E. lenta gavaged, of both sexes were deprived of food for 3 hours and then gavaged with FITC-labeled dextran (0.6 mg/g body weight). FITC-dextran was measured in sera collected 3 hours later.

Metabolomics

Fecal samples collected from CII-immunized mice before and 10 weeks after immunization were stored at −80°C and processed together after all samples were collected. Fecal samples were evaluated for SCFAs by light chromatography mass spectrometry (LC-MS) at Mayo Core facility. Sera collected before and after E. lenta gavage were used to measure citrulline and arginine levels by LC-MS at Mayo Core facility. Stool samples from E. lenta treated and nontreated CII-immunized mice were used for metabolic profile. Untargeted metabolite profiling of fecal samples and analysis was conducted by Metabolon Inc. Random forest was also used to rank the metabolites on the basis of the Mean Decreased Accuracy importance measure. Q value cutoff of 0.2 was used to select the differential metabolites. Table S2 provides the detailed metabolic profile. RA patient and healthy control plasma samples were used for measuring amino acids (3).

Sample collection, 16S sequencing, bioinformatics processing, and statistical analysis

All fecal samples were frozen immediately after collection and 16SrDNA sequencing was conducted together. Microbial DNA was extracted from fecal samples using the MoBio PowerSoil Kit with a bead-beating step and V3-V5 barcoded primers were used for library preparation with Kapa HiFi Hotstart Ready Mix (Kapa Biosystems). 16SrDNA sequencing was performed on one lane of MiSeq using the MiSeq Reagent Kit v2 (Illumina Inc). Hybrid-denovo with default setting was used to process the raw reads into OTUs using information from both paired and unpaired reads (59). Statistical analyses were performed on alpha diversity (Shannon index), beta diversity (BC), and taxa abundances. Reads were rarefied to per sample before alpha and beta diversity analyses. For alpha diversity analysis, paired t test was used. For beta diversity analysis, PERMANOVA within-mouse permutation as well as within groups was used (“vegan” R package, “adonis2” function). PCoA (R “cmdscale” function) was used to visualize the compositional difference on the first principal coordinates (PCs) and between groups using endpoint samples. For differential abundance testing, we used ZicoSeq (“GUniFrac” R package, “ZicoSeq” function) (60), where a permutation-based false discovery rate (FDR) control procedure was used to correct for multiple testing. An FDR-adjusted P value (q value) less than 0.1 was considered significant.

Antibodies

Mice were bled before and after treatment with E. lenta and CII immunization. Titers of sera IgG antibodies against CII and rheumatoid factor were measured by standard ELISA (8) and are shown as optical density.

T cell proliferation assay

For the T cell proliferation assay, mice were immunized with 200 μg of CII emulsified 1:1 in CFA intradermally at the base of the tail and proliferation was done as described (8). In some experiments, CD4⁺ cells (5 × 10⁶) sorted from lymph nodes of CII-primed mice that were treated with E. lenta or not were cultured in vitro in the presence or absence of CD11c⁺ dendritic cells (5 × 10⁵), harvested from spleens, in the presence or absence of the antigen. Stimulation index of 2 or more was taken as a positive response. Peptides derived from CII, aa 184–203 and aa 754–773 were evaluated for T cell proliferation using mesenteric lymph node or splenic cells from mice immunized with CII or CII and E. lenta. For in vitro culture, cells were challenged with 100 μg/ml of the peptide.

Flow cytometry

The expression of DQ in transgenic mice was analyzed by flow cytometry using mAb IVD12 (anti-DQ) for characterization of transgene positive. Conjugated antibodies for various markers, CD3, CD4, CD8, CD11b, CD11c, CD19, CD25, FoxP3, PD-1, GL7, CXCR5, CX3CR1, PNA, Gr-1, and ICOS (R&D Systems, CA) were also used. All experiments were done with cells pooled from 2 mice/ strain and repeated two to three times. Intracellular staining for FoxP3, IFN-γ, IL-17 and IL-10 was performed using specific antibodies obtained (eBioscience, San Diego, CA) as per the manufacturer’s instructions. Phycoerythrin-conjugated (PE) rat IgG2a (eBioscience) was used as the isotype control for FoxP3 staining. Analysis was done using the Cell Quest program (Beckton Dickinson).

Statistical analysis

The numbers of samples for each experiment are detailed in the text and figures. All experiments used approximately equal numbers of male and female mice. Experimental breakdown for various experiments was not needed as DQ8 mice develop robust disease with consistent incidence and disease severity. Sera and plasma were stored from various groups of independent experiments. Experiments were conducted by randomly choosing from various independent experiments for unbiased approach. All experiments were replicated. The difference in the incidence of arthritis between groups, CII-immunized and CII was analyzed using Chi square test. Arthritis onset was analyzed using analysis of variance (ANOVA). Antibody levels, mean scores for arthritic mice, cytokines, proliferation responses and presence of immune cells were compared using unpaired t test. All experiments were conducted in triplicates with at least 2 independent experiments. All statistical analyses were performed in Graph Pad Prism. P value of less than equal to 0.05 was considered significant. Wilcoxon rank sum test was used to compare of the proportion of E. lenta in patients with RA and controls. Untargeted metabolic profile of fecal samples from CII-immunized before and after E. lenta gavage (n = 10) was conducted by Metabolon Inc. and analyzed using Welch’s two sample t test and false discovery rate (Benjamini-Hochberg procedure) was used for multiple testing correction.

Acknowledgments

We acknolwedge support from NIH that was used to perform develop and perform microbiome analysis.

Funding: The work was supported by funds to V.T. from the Department of Defense grants W81XWH-10-1-0257 and W81XWH-15-1-0213 and Mayo Clinic Robert and Arlene Kogod Center of Aging. Microbiome analysis was supported by R01 GM144351 to J.C. J.M.D. has financial relationship with Pfizer and Girihlet.

Author contributions: Conceptualization: V.T., J.M.D., and B.B. Methodology: B.B. and V.T. Experimental investigation: B.B., D.L., and K.W. Data analysis: B.B., D.L., J.C., K.W., and V.T. Supervision: V.T. and B.B. Writing—original draft: B.B., V.T., and J.C. Writing—review and editing: V.T., B.B., J.M.D., J.C., K.W., and D.L.

Competing interests: The authors declare that they have no competing interests. The technology is patented 11,634,744 “Methods and Materials for assessing and treating arthritis”. However, no royalties have been received.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The 16SrDNA data presented in the study are publicly available in NCBI (accession no. PRJNA691623).

Supplementary Materials

This PDF file includes:

Figs. S1 to S16

Table S1

Legend for table S2

Click here for additional data file.^{(2.3MB, pdf)}

Other Supplementary Material for this manuscript includes the following:

Table S2

Click here for additional data file.^{(288.6KB, zip)}

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Associated Data

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Supplementary Materials

Figs. S1 to S16

Table S1

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Table S2

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PERMALINK

Eggerthella lenta augments preclinical autoantibody production and metabolic shift mimicking senescence in arthritis

Baskar Balakrishnan

David Luckey

Kerry Wright

John M Davis

Jun Chen

Veena Taneja

Roles

Abstract

INTRODUCTION

RESULTS

E. lenta enhances arthritis severity by augmenting cellular and humoral response to CII

Fig. 1. E. lenta augments arthritis severity.

Fig. 2. E. lenta augments pro-inflammatory cytokine production.

E. lenta treatment reduces T regulatory cells and augments T follicular cells

Fig. 3. E. lenta reduces T regulatory cells and increases T follicular helper cells and autoreactive B cells.

E. lenta abundance alters microbial composition

Fig. 4. E. lenta alters gut microbial composition.

E. lenta abundance is correlated with decreased amino acid and increased BA levels

Fig. 5. E. lenta causes a decrease in amino acids by altering the metabolic profile.

E. lenta augments sex-specific responses both in patients and humanized mice

Fig. 6. E. lenta has sex-specific impact on arthritis.

E. lenta augments preclinical antibody response

Fig. 7. E. lenta augments pre-clinical antibody production.

DISCUSSION

MATERIALS AND METHODS

Research objective

Patient data

Transgenic mice

Collagen-induced arthritis

Culture, lysate preparation, and treatment with bacterium

Isolation of lamina propria cells

Isolation of cells from synovium

Cytokine and chemokine expression

FITC-dextran for gut permeability

Metabolomics

Sample collection, 16S sequencing, bioinformatics processing, and statistical analysis

Antibodies

T cell proliferation assay

Flow cytometry

Statistical analysis

Acknowledgments

Supplementary Materials

This PDF file includes:

Other Supplementary Material for this manuscript includes the following:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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