ES-62 normalises the gut microbiome in protecting against CIA

ES-62 ameliorates CIA in terms of articular score and frailty, maintaining grip strength at a similar level to that of healthy, Naive (not subjected to CIA) DBA/1 mice (Supplementary Figure 1). Commensal bacteria have increasingly been proposed to contribute to RA pathogenesis9,10 and decline in grip strength during ageing has been associated with changes in the gut microbiome23. In addition to being an indicator of frailty, grip strength is a predictor of a wide range of adverse health outcomes24, e.g., cardiovascular disease, which RA patients are at increased risk of developing25 and that are impacted by the microbiome9,26. Thus, to address whether ES-62-mediated protection reflects modulation of the gut microbiota, a shotgun metagenomic approach was used to profile bacteria populations present in the intestines of CIA mice. Initiation of RA (and CIA) pathogenesis is associated with disruption of the balance of effector:regulatory immune cells and so we characterised the bacterial changes pertaining during established arthritis in the ileum and colon; intestinal sites where the microbiome and the metabolic microenvironment play key roles in shaping Th17 and regulatory immune responses22,27,28. As there is well-documented variation in the microbiome amongst individuals due to a range of environmental factors, we adopted the strategy of pooling samples from mice with representative disease scores to minimise variation due to a factor we could control for, namely disease severity: specifically, we focused on those samples associated with a well-established severe level of disease in CIA mice (articular score: 7.00 ± 0.91) and clear protection against arthritis in ES-62-treated CIA mice (articular score: 0.50 ± 0.33) over three independent experiments.

An overview of the microbiota at the phylum level shows substantial changes between healthy Naive mice and those with arthritis (PBS; Fig. 1a). Firmicutes and Bacteroidetes are the predominant phyla in all groups, but CIA mice in particular, exhibit outgrowths of Firmicutes and Proteobacteria in the ileum, whereas they demonstrate decreased levels of Firmicutes with a compensatory outgrowth of Bacteroidetes, in the colon (Fig. 1a). ES-62 essentially helps maintain the healthy microbiome diversity of Naive mice, which was reduced in CIA mice (Fig. 1a). Deeper analysis illustrates the differential diversity signatures of healthy and arthritic mice, as well as the impact of ES-62 on a global scale (Fig. 1b; Supplementary Table 1). As a consequence of our pooling strategy, we have refrained from discussion of species-level changes as large-scale population studies would be required to address this with complete confidence. Nevertheless, drilling down on the modulation of the Gram-negative Bacteroidetes phylum reveals differential signatures throughout each of the predominant Bacteroides, Porphyromonas and Prevotella genera and the Rikenellaceae family between the colon contents of Naive and CIA mice, identifying those normalised by exposure to ES-62 (Fig. 1c). Similarly, in spite of the fact that differential profiles amongst the groups were observed throughout major genera (e.g., Bacillus, Staphyloccus, Streptococcus, Enterococcus and Clostridium) of Gram-positive Firmicutes (Fig. 1b; Supplementary Table 1), the most dramatic changes with established CIA occurred within the Clostridiales order. In particular, decreases in the Clostridiaceae and the Lachnospiraceae (Fig. 1d) families were noted with ES-62 promoting maintenance of the Ruminococcus, Faecalibacterium and Blauti genera and the family Erysipelotrichaceae in addition to the butyrate-producing genera Dorea and Roseburia, the latter having been implicated in gut health and inflammation homoeostasis29. In terms of the Proteobacteria, CIA was associated with outgrowth of members of the Helicobacter (Epsilonproteobacteria) and Escherichia (Gammaproteobacteria) genera and again this was normalised by ES-62 (Fig. 1e).

Fig. 1 ES-62 normalises the microbiome during CIA towards a Naive phenotype. The composition of bacterial phyla present in the ileum and colon of Naive, PBS and ES-62-treated CIA animals, presenting proportion values as pie charts (a) from a single representative experiment using pooled samples from three mice in each condition. b Heatmap analysis of all bacteria present in the colon of Naive, PBS and ES-62-treated CIA animals from this representative model (articular scores; naive, 0; PBS-CIA, 6.3 ± 2.6; ES-62-CIA, 0; *p < 0.05 for PBS-CIA versus Naive and ES-62-CIA mice) is shown. The profile of bacterial abundance, is provided in Supplementary Table 1 for clarity. c–g Metagenonic analysis was performed on three independent models: in each model, samples from three representative mice/group were pooled in each individual experiment and the mice selected exhibited the following articular scores: Naive, 0; PBS-CIA, 7.00 ± 0.91; ES-62-CIA, 0.50 ± 0.33, where ***p < 0.001 for the PBS-CIA versus Naive and ES-62-CIA cohorts and n = 9 mice for all groups over the three independent models. Statistically significant changes (two-way ANOVA with LSD Fishers multiple comparisons) observed over the three independent experiments (mean ± SEM, n = 3) between the PBS-CIA and ES-62-CIA groups in Bacteroidetes (c; Bacteroidaceae; PBS versus Naive or ES-62, p < 0.05), Firmicutes (d; Clostridiales; PBS versus Naive, p < 0.01 and PBS versus ES-62, p < 0.05) and Proteobacteria (e; Epsilonproteobacteria; PBS versus Naive or ES-62, p < 0.05) in the colon and Firmicutes in the ileum (f; Clostridiaceae; PBS versus ES-62, p < 0.05) as well as functional metagenomics of the colon (g; Phages; Naive versus PBS, p < 0.01, Protein metabolism; PBS versus Naive or ES-62, p < 0.01) are presented as heatmaps with changes in bacterial populations in PBS- and ES-62-treated CIA animals normalised to Naive controls. Ileum (h) and colon (i) faecal matter from Naive or ES-62-treated Naive animals was analysed for changes in bacterial populations in a single experiment using samples pooled from three animals per group and displayed as heatmaps, where the ES-62-treated samples are normalised to the Naive controls Full size image

Perturbation of the microbiome was also observed in the ileum of CIA mice and again, exposure to ES-62 normalised this towards the healthy community (Fig. 1a). ES-62 clearly promoted growth of the Clostridiales, again particularly the Clostridaceae and Lachnospiraceae families, generally even beyond the levels found in healthy mice (Fig. 1f ). In the context of the relative paucity of bacteria in the ileum relative to the colon, this outgrowth perhaps explains why ES-62 increases the overall species diversity observed in the ileum but not the colon of CIA mice (Supplementary Figure 2). In addition, reflecting that CIA perturbs and ES-62 normalises gut bacteria, functional metagenomic analysis showed that ES-62 generally acted to normalise the metabolic capacity of the colonic microbiome in CIA mice (Fig. 1g). Intriguingly, treatment of Naive, healthy mice with ES-62 for the duration of the CIA model also promotes expansion of members of the Clostridiales order, again particularly the Lachnospiraceae, whilst decreasing abundance of Bacilli, the Bacteroidaceae and Porphyromonadaceae families of Bacteroidetes and also, Proteobacteria in the ileum (Fig. 1h). Likewise, ES-62 can also promote depletion of Bacteroidetes and expansion of Ruminococcaceae and Clostridiaceae families and the Clostridia class of Firmicutes in the colon of Naive mice (Fig. 1i). Importantly, ES-62’s modulation of the gut microbiota in the absence of the chronic inflammation associated with CIA suggests that it can act directly, and that there is not an absolute requirement for it to harness immunoregulatory mechanisms to maintain and fine-tune microbiome homoeostasis.

Antibiotics both ameliorate CIA and impact on ES-62 protection

To address whether microbiome perturbation observed in CIA plays a role in the initiation and progression of inflammatory arthritis, we investigated the effect of continuous exposure of mice to a cocktail of broad-spectrum antibiotics (ABX) administered from 1 week prior to initiation of CIA. Such ABX treatment had no obvious effect on overall health as, after a characteristic initial dip, there was no significant difference in body weight amongst the CIA groups at cull (Supplementary Figure 2). Nevertheless, this regimen essentially eliminated the bacterial microbiota, irrespective of treatment group, whilst metagenomic analysis showed the residual gut community to be almost entirely comprising proteobacteria (Supplementary Figure 2). As predicted from previous studies in several inflammatory arthritis models22,30,31, ABX treatment reduced the incidence (PBS, 65.2%; PBS-ABX, 36.3%, as measured by articular score ≥1) and severity of joint pathology in CIA mice, both in terms of articular score (Fig. 2a) and histopathology (Fig. 2b, c). In addition, the protection afforded by ES-62 was reduced in ABX-treated animals (Fig. 2a). Thus, prophylactic administration of ABX resulted in an intermediate phenotype of CIA, irrespective of whether the mice were treated with PBS or ES-62.

Fig. 2 Antibiotic treatment results in an intermediate disease phenotype. a Data are presented as articular scores (mean ± SEM) and are pooled from three independent experiments (PBS; n = 23, ES-62; n = 19, PBS-ABX; n = 22 and ES-62-ABX; n = 22). b H&E staining (scale bars represent 200 µm) of hind paws, from representative mice from each treatment group (clinical articular scores of individual paws were: PBS ≥ 3 (n = 9), ES-62 = 0 (n = 7), PBS-ABX = 0 (n = 3) and ES-62-ABX ≥ 3 (n = 3)) selected to compare joint pathology occurring in the similarly scored ES-62-CIA and PBS-ABX (score 0) and ES-62-ABX and PBS-CIA (articular score ≥ 3) mice. c Blind scoring of pathology exhibited in joint sections imaged in (b). d, e Levels of collagen II (CII)-specific IgG2a (d; PBS; n = 18, ES-62; n = 13, PBS-ABX; n = 16 and ES-62-ABX; n = 14) and IgG1 (e; PBS; n = 15, ES-62; n = 10, PBS-ABX; n = 16 and ES-62-ABX; n = 14) antibodies in serum were determined by ELISA. f Splenic B regulatory cells (CD19+IL-10+ cells) were determined by flow cytometry (Naive; n = 13, PBS; n = 21, ES-62; n = 14, Naive-ABX; n = 8 PBS-ABX; n = 14 and ES-62-ABX; n = 15). g, h IL-10 (g: PBS; n = 6, ES-62; n = 5, PBS-ABX; n = 6 and ES-62-ABX; n = 6) and IL-6 (h: PBS; n = 24, ES-62; n = 10, PBS-ABX; n = 12 and ES-62-ABX; n = 13) concentrations in serum were determined by ELISA. Statistics: all results are presented as mean ± SEM and each symbol represents an individual mouse; data are from one (g) or pooled from two or three independent experiments. Statistical significance was determined using two-way ANOVA (a) or one-way ANOVA (c–h) with LSD Fishers multiple comparisons and significance indicated by asterisks, *p < 0.05 and **p < 0.01 and #p < 0.05 (PBS vs. PBS + ABX [a] and ES-62-ABX vs. Naive [f]) Full size image

ES-62-mediated protection against CIA is associated with restoration of the homoeostatic balance of regulatory:effector B-cell responses via downregulation of aberrant MyD88 signalling2,21. Typically, ES-62 reduces pathogenic anti-collagen type II (CII) IgG2a, but not IgG1 antibody production32,33. Reflecting the ABX-driven amelioration of CIA pathology, anti-CII Ig2a levels in PBS-ABX mice are not significantly different from those in ES-62-CIA mice, whilst those in ES-62-ABX mice are not significantly reduced relative to those in PBS-CIA mice (Fig. 2d). No differences were detected amongst any of the groups in terms of anti-CII IgG1 antibodies (Fig. 2e). Consistent with the effects of ABX on effector B-cell responses, analysis of splenic IL-10+CD19+ Bregs showed that both the decrease occurring during CIA (PBS-CIA) and also the maintenance of healthy levels in ES-62-CIA mice2,21 were lost in ABX-treated animals (Fig. 2f). Moreover, whilst ES-62 increases serum IL-10 levels in CIA mice, this regulatory cytokine is found at similarly low levels in PBS-CIA, PBS-ABX and ES-62-ABX mice (Fig. 2g). At the same time, ES-62-mediated suppression of serum levels of IL-6, a cytokine that promotes B-cell (auto)immunity34, is lost following ABX treatment (Fig. 2h). Collectively, these ABX studies indicate that depletion of the microbiota interferes with generation of inflammatory mediators associated with CIA pathogenesis as well as with the loss of immunoregulatory elements (Bregs) normally contributing to resolution of chronic inflammation. Importantly, they also indicate that ES-62-resetting of the effector:regulatory balance is dependent on an intact gut microbiome and hence suggest that its restoration is a consequence of ES-62-mediated normalisation of the microbiome dysbiosis associated with CIA.

ES-62 also acts in CIA mice to suppress the functional maturation of osteoclasts (OC)20 that directly cause erosive joint damage. Interestingly, changes in the intestinal microbiome have been shown to impact on bone mass35,36 and we therefore investigated whether the intermediate phenotype of joint pathology occurring in ABX-treated CIA mice reflected modulation of osteoclastogenesis resulting from perturbation of the gut microbiota. Exposure of CIA mice to ES-62 restored the numbers of OC differentiated from bone marrow (BM) progenitors ex vivo to Naive levels (Fig. 3a) and blocked their fusion to large, active multinucleated cells (Fig. 3a–c) that resorb bone20. Consistent with its amelioration of CIA pathology, ABX administration resulted in a decrease in large multinucleated OCs and a corresponding increase in total numbers of BM-derived OCs from CIA mice (Fig. 3a–c). Under these conditions of microbiota depletion, the ability of ES-62 to inhibit generation of large multinucleated OCs is lost (Fig. 3b): rather, and perhaps consistent with the increased joint inflammation exhibited by these mice, the ES-62-ABX group exhibits the largest multinucleated cells of the ABX-treated cohorts, displaying significantly lower numbers of OCs but larger, multinucleated cells than Naive-ABX mice. ES-62 rewires osteoclastogenesis by modulating the RANK/OPG bone remodelling axis20 and this is evidenced again here by its ability to significantly decrease RANK expression and increase (albeit not significantly) expression of the decoy receptor, OPG relative to that seen in BM of PBS-CIA mice (Fig. 3d, e). This axis is also targeted by ABX treatment, with the elevated RANK expression observed in PBS-CIA BM being lost following ABX treatment. In addition, the elevated OPG expression observed in ES-62-conditioned OCs was abrogated following ABX treatment (Fig. 3d, e): these changes would result in similar levels of RANKL-driven osteoclastogenesis consistent with the intermediate CIA phenotype observed in PBS- and ES-62-ABX animals. Supporting these ABX-changes in osteoclastogenesis and consequently bone damage, relative to Naive mice, PBS-ABX and ES-62-ABX animals exhibit similar grip strengths (70.8 ± 5.4% and 58.8 ± 6.0%, respectively) that are intermediate to those displayed by PBS- (42.3 ± 6.2%) and ES-62-CIA mice (90.1 ± 9.6%). Collectively, these data not only support a role for the microbiome in osteoclastogenesis but also implicate its involvement in the ability of ES-62 to modulate pathogenic osteoclastogenesis in CIA.

Fig. 3 ES-62 requires the gut microbiome to protect the bone remodelling axis. a–c Osteoclasts (OCs) were differentiated from bone marrow obtained at cull and cultured for 5 days before differentiation, in terms of numbers (a) and size (b) of OCs, was measured using ImageJ analysis software and data were normalised as a percentage of Naive controls with representative images (c) provided (Naive; n = 11, PBS; n = 11, ES-62; n = 6, Naive + ABX; n = 2, PBS + ABX; n = 3, ES-62 + ABX; n = 3). Whole bone marrow was used to quantify RANK (d; PBS; n = 20, ES-62; n = 13, PBS + ABX; n = 15, ES-62 + ABX; n = 15) and OPG (e; PBS; n = 17, ES-62; n = 11, PBS + ABX; n = 16, ES-62 + ABX; n = 14) mRNA levels using qRT-PCR, and fold change was calculated following normalisation to Naive controls. Statistics: all data are presented as mean ± SEM. In a and b, each symbol represents experimental replicates and in d and e each symbol represents individual mice and data are pooled from three independent experiments. Statistical significance was determined using one-way ANOVA with LSD Fishers multiple comparisons and significance indicated by asterisks, *p < 0.05, **p < 0.01 and ***p < 0.001 Full size image

ES-62 protects against gut pathology in CIA

Intestinal dysbiosis has been associated with loss of gut integrity and chronic inflammation in conditions such as obesity that promote autoimmune disorders like RA and cardiovascular comorbidities37,38. Our metagenomic analysis showed that established CIA was associated with gut microbiome changes, in particular with reduction in butyrate-producing bacteria (Fig. 1b, d; Supplementary Table 1) that have been implicated in maintenance of epithelial integrity and gut health29. Strikingly, mice with CIA display severe gut pathology and inflammation, the levels of which directly correlate with severity of CIA (Fig. 4a). Moreover, ES-62 treatment protects against such gut damage, specifically the thickening and generation of stubby villi in the ileum (Fig. 4b, c) and hole-like lesions in the colon (Fig. 4b. d). The physiological relevance of these colon lesions is unclear but they may reflect the suppression of mucus production by goblet cells in response to bacteria39 or alternatively, attachment/effacement lesions induced by pathogenic bacteria, including E. coli40. The gut pathology in PBS-CIA mice was diminished in those administered ABX (Fig. 4b–d), suggesting that the associated reduction in chronic gut (and consequently systemic) inflammation contributes to the amelioration of CIA in PBS-ABX mice. Of note, ES-62-ABX mice displayed increased pathology and inflammatory cell infiltration of the gut tissue compared with ES-62-CIA mice (Fig. 4b), findings again consistent with chronic gut inflammation promoting disease severity.

Fig. 4 CIA is accompanied by microbiome-dependent gut pathology. a Representative H&E images (scale bar representing 50 µm) of ileum sections of Naive and PBS-CIA mice with differential disease severity at cull (mouse articular score as indicated). b Representative H&E images of ileum (top row), colon (middle row) and PAS-stained colon (bottom row) sections displaying scale bar (200 µm) and articular scores of the mice are shown on the images. c Quantitative analysis of ileum villi thickness, where symbols represent mean values of replicate sections as measured using ImageJ analysis software (n = 3/group with 3–5 replicates/animal and data are from two independent experiments). d Lesions were enumerated per colon section of individual mice using ImageJ imaging software (Naive; n = 6, PBS; n = 8, ES-62; n = 7, Naive + ABX; n = 6, PBS + ABX; n = 7, ES-62 + ABX; n = 7). Statistics: data are pooled from two independent experiments. Statistical significance was determined using one-way ANOVA with LSD Fishers multiple comparisons and indicated by asterisks, *p < 0.05, **p < 0.01 and ***p < 0.001, whilst in (c), PBS-ABX is not significantly different from the Naive group Full size image

To further address the role of gut pathology in CIA, we analysed the ileum and colon tissue from Naive mice and those undergoing CIA (treated with PBS or ES-62) at key points during initiation and progression of disease: (i) Naive mice, (ii) the breaking of tolerance and initiation of disease, following immunisation with CII/CFA (≤ day 14), (iii) preclinical (day 21, prior to booster immunisation with CII) and (iv) established disease (≥ day 28; articular score: PBS–5.2 ± 0.8; ES-62–0.9 ± 1.24). This analysis revealed the presence of gut pathology in PBS-CIA mice during the initiation phase with an increase in both ileum villi thickness and colonic lesions following immunisation (Fig. 5a–c). Interestingly, the ileum pathology occurring during the initiation stage in PBS-CIA mice appeared to resolve by the end of the preclinical phase (day 21), although thickening and shortening of the villi was again induced by the booster CII immunisation. This pattern was not the case for the colon lesions, which peaked at day 21 and were maintained throughout active disease in PBS-CIA mice. As opposed to CIA mice, ileum integrity was maintained throughout all phases of disease, whilst the high levels of colon lesions generated by day 21 of the preclinical phase were reduced in ES-62-treated animals (Fig. 5b, c).

Fig. 5 ES-62 protects against gut pathology occurring prior to onset of arthritis. a Representative H&E images (scale bars 200 µm) of ileum and colon of mice culled during the Naive, initiation (post immunisation ≤ d14), preclinical (d21 prior to challenge) and disease (established arthritis d ≥ 28, articular score PBS, 5.2 ± 0.8; ES-62–0.9 ± 1.24) phases of CIA. Changes in villus thickness (b; Initiation; PBS, n = 5, ES-62, n = 6, Pre-Clinical; PBS and ES-62, n = 3, Disease; PBS, n = 4, ES-62, n = 5 mice) and number of colon lesions (c; Initiation; PBS and ES-62, n = 12 images spanning six mice each, Pre-Clinical; PBS, n = 6 [3 mice], ES-62, n = 4 [2 mice]; Disease; PBS, n = 8 [4 mice], ES-62, n = 10 [5 mice]) were quantified using ImageJ analysis software with data normalised to values of Naive mice and presented as mean ± SEM. Statistical significance was determined using two-way ANOVA comparing PBS and ES-62 treatment at each time point where **p < 0.01 and ***p < 0.001. d–f Changes in Bacteroidetes (d), Firmicutes (e) and Butyrivibrio (f) populations in the colon faecal matter of Naive, PBS- and ES-62-treated animals were measured by qPCR, normalised to total bacterial content and presented as % Naive mice. Data are presented as mean ± SEM values from individual animals (Initiation; n = 6, Pre-Clinical; n = 3, Disease; n = 10) and statistical significance determined using two-way ANOVA with LSD Fishers multiple comparisons where *p < 0.05, **p < 0.01 and ***p < 0.001. g Tr1 cells (CD3+IL-10+ cells) were determined by flow cytometric analysis of splenocytes from individual mice (Naive; n = 4, PBS; n = 7, ES-62; n = 4, Naive-ABX; n = 4 PBS-ABX; n = 6 and ES-62-ABX; n = 7) from a single experiment. Statistical significance was determined using one-way ANOVA with LSD Fishers multiple comparisons where *p < 0.05 Full size image

Supporting our metagenomics data, qPCR analysis showed that ES-62 prevents the enrichment of Bacteroidetes and maintains levels of Firmicutes in the colon of mice during established CIA (Fig. 5d, e). However, analysis at the various phases of the disease suggested a more dynamic situation: for example, following the primary immunisation with CII/CFA, there is a significant decrease in levels of both Bacteroidetes and Firmicutes, with the rise in Bacteroidetes evident in established arthritis only occurring following the booster immunisation (Fig. 5d, e). By contrast, ES-62 acts to maintain healthy levels of Firmicutes in CIA mice throughout disease, but most strongly during the initiation and preclinical phases and in addition, inhibits the CIA-induced increase in Bacteroidetes during established disease. Moreover, although ES-62 promoted enrichment of butyrate-producing bacteria (Butyrivibrio) at all stages of disease, this was most evident in the initiation phase (Fig. 5f). Butyrate has been reported to promote the development of regulatory T cells (both Foxp3+ Tregs and IL-10+ Tr1 cells41,42) which by suppressing and resolving chronic inflammation, could contribute to the maintenance of gut barrier integrity. However, whilst we find that the induction of CIA is accompanied by loss of splenic Tr1 cells, the predominant regulatory T-cell subset defective in CIA43, ES-62 does not restore their levels back towards those of Naive mice (Fig. 5g). These data are consistent with our previous findings that protection against CIA afforded by ES-62 is not associated with induction of either Treg or Tr1 cell responses21. Moreover, ABX treatment does not modulate either the CIA-associated depletion of Tr1 cells or the failure of ES-62 to induce their restoration, suggesting their development in this model is not impacted by the status of the microbiome (Fig. 5g). Thus, collectively these data indicate that ES-62 likely promotes Butyrivibrio species to maintain butyrate-mediated protection of intestinal epithelium physiology and barrier integrity44.

The dynamic changes observed in the gut during the initiation phase further questions whether CIA-associated autoimmune (Th17) inflammation is responsible for triggering gut pathology and consequently, perturbation of the microbiome or vice versa. Interestingly, therefore, whilst confirming our findings that dysbiosis and loss of intestinal barrier integrity are established by day 14 in the CIA model, a very recent report45 shows that such breakdown in gut function precedes, and is required for, CIA-associated IL-17 responses as increases in IL-17 can only be determined in the small intestine from day 14 whilst those in the MLN occurred even later, being present during the established disease phase (by day 35, but not detected on day 14), and that ABX-depletion of the microbiota inhibits such IL-17 production and severity of joint disease. Moreover, these effects appeared not to require (any) inflammation for initiation as they could not be replicated by sham immunisation with CFA45. Consistent with this idea that dysbiosis and associated gut pathology precedes and shapes the autoimmune inflammation driving arthritis, we find that CIA-associated perturbation of the colon microbiota is evident as early as day 6 (post CII immunisation): this is broadly normalised by exposure to ES-62, with the worm product modulating levels of potentially pathogenic bacteria (such as the Enterobacteriaceae family of Gamma proteobacteria) to below those found in Naive animals (Fig. 6a). Accompanying the CIA-dysbiosis, there is damage to both the ileum and colon, with shortening and thickening of the ileum villi particularly evident at this early time point and this is protected against by ES-62 (Fig. 6b). These changes in gut function occur in the absence of the systemic inflammation (serum IL-6 cytokine and CII-specific IgG2a antibodies were only present around the limit of detection of the ELISAs employed, with no differences observed amongst Naive, PBS-CIA and ES-62-CIA groups), known to be targeted by ES-62 in CIA (Fig. 2). By contrast, the late microbiota-driven IL-17 responses developed in the MLN in the clinical phase of CIA45 were suppressed by exposure to ES-62 (Fig. 6c). Collectively, these findings indicate that ES-62 primarily acts directly to normalise the CIA-associated gut dysbiosis that both triggers mucosal inflammation and initiates the mesenteric lymph node (MLN) IL-17 responses that drive autoimmunity, rather than indirectly modulating the dysbiosis and gut pathology arising from the systemic inflammation that results from the CIA-immunisation protocol.

Fig. 6 ES-62 modulates the gut-bone marrow axis during the early phases of CIA. a Changes in the indicated bacterial populations in the colon faecal matter of Naive, PBS- or ES-62-treated animals were measured at day 6 post CII/CFA-immunisation by qPCR and data were normalised to total bacterial content and presented as fold change compared to Naive mice (Naive, n = 3; PBS-CIA, n = 3 and ES-62, n = 2). b Representative H&E images of ileum and colon pathology of mice culled at day 6 of CIA. Scale bars are 200 µm. c IL-17+ lymphocytes were determined by flow cytometric analysis of MLN cells from individual mice (naïve, n = 5: PBS, n = 5 and ES-62, n = 5). Statistical significance was determined using one-way ANOVA with LSD Fishers multiple comparisons and significance indicated by asterisks, *p < 0.05 and ***p < 0.001. d Whole bone marrow was used to quantify MyD88 mRNA levels using qRT-PCR and normalised to naive controls. e The proportion of monocytes (CD3-B220-Ter119−Ly6G−Ly6C+) in bone marrow was measured by flow cytometry and normalised to those in Naive control mice. f, g Osteoclasts (OCs) were differentiated from bone marrow obtained at cull and cultured for 5 days and size of OCs (f) was measured using ImageJ analysis software and normalised to those from Naive controls with representative images for each disease stage in both treatment groups displayed (g). Statistics: data are presented as mean ± SEM values from individual animals (d; Naive; n = 4, PBS; n = 8, ES-62; n = 6, Naive + ABX; n = 4, PBS + ABX; n = 8, ES-62 + ABX; n = 8. e Initiation; n = 6, Pre-Clinical; n = 3, Disease; n = 4) or mean ± SD from experimental replicates (f; Initiation; n = 18, Pre-Clinical; n = 9, Disease; PBS: n = 12 and ES-62; n = 15). Statistical significance was determined using two- (e, f) or one-way (d) ANOVA with LSD Fishers multiple comparisons and indicated by asterisks, *p < 0.05, **p < 0.01 and ***p < 0.001 Full size image

Although the precise details of how gut microbiome perturbation occurs and drives chronic autoimmune inflammation and joint destruction in CIA remain to be defined, commensal bacteria play key roles in educating the immune system and BM progenitors and hence, loss of microbiome diversity can impact on both inflammation and bone homoeostasis46,47. Such training involves interactions of Pattern Recognition Receptors (PRR; e.g., TLRs and NODs) with the gut microbiome2,3,48. Consistent with this, ES-62 can rewire BM progenitors and stromal cells from CIA mice to an anti-inflammatory, regulatory or tissue repair phenotype2,16,20,49,50,51 by subverting TLR4 signalling to prevent the upregulation of the key TLR signal transducer, MyD88 observed during chronic inflammation52. Interestingly, therefore, given the increased incidence and severity of CIA in ES-62-ABX mice, we show that ES-62 dampening of aberrant MyD88 expression is abolished by ABX treatment (Fig. 6d).

Commensal bacteria can also dynamically educate OC progenitor (OCP) maturation46,47: as OCs normally act in concert with osteoblasts (OBs) to homeostatically maintain healthy bone, the enhanced functional capacity of OCs elicited by commensal bacteria may actually render hosts more susceptible to bone damage during chronic inflammation, a condition that promotes bone remodelling, particularly as commensal bacteria also appear to act to suppress OB function46,47. Reflecting the dynamic and differential changes in the colonic microbiota, gut inflammation and pathology, there is a strong increase in the monocyte populations containing OCPs during the early inflammatory initiation phase of CIA that has resolved by the end of the preclinical phase only to increase again in the established phase of arthritis following the booster CII immunisation. Consistent with our data that ES-62 does not fundamentally suppress OC differentiation, there are no differences in the percentage of monocytes between the PBS-CIA and ES-62-CIA groups (Fig. 6e). However, following both primary and booster immunisations, BM from CIA mice shows enhanced capacity for functional OC maturation that is suppressed by ES-62 (Fig. 6f, g).