The parasitic worm product ES-62 normalises the gut microbiota bone marrow axis in inflammatory arthritis

The human immune system has evolved in the context of our colonisation by bacteria, viruses, fungi and parasitic helminths. Reflecting this, the rapid eradication of pathogens appears to have resulted in reduced microbiome diversity and generation of chronically activated immune systems, presaging the recent rise of allergic, autoimmune and metabolic disorders. Certainly, gastrointestinal helminths can protect against gut and lung mucosa inflammatory conditions by modulating the microbiome and suppressing the chronic inflammation associated with dysbiosis. Here, we employ ES-62, an immunomodulator secreted by tissue-dwelling Acanthocheilonema viteae to show that helminth-modulation of the gut microbiome does not require live infection with gastrointestinal-based worms nor is protection restricted to mucosal diseases. Specifically, subcutaneous administration of this defined immunomodulator affords protection against joint disease in collagen-induced arthritis, a mouse model of rheumatoid arthritis, which is associated with normalisation of gut microbiota and prevention of loss of intestinal barrier integrity.


Introduction
Parasitic helminths (worms) have evolved the ability to modulate host immune and tissue repair responses in order to promote their survival by limiting the inflammation that would otherwise drive their expulsion and cause pathology 1 . The recent rapid eradication of helminths (and other infectious pathogens) appears to have resulted in over-activated immune systems and this provides a rationale for the increasing prevalence of chronic allergic (e.g. asthma) and autoimmune (e.g. inflammatory bowel disease, type-1 diabetes, multiple sclerosis [MS], systemic lupus erythematosus [SLE] and rheumatoid arthritis [RA]) inflammatory disorders, as well as contributing to the rise in obesity and associated metabolic syndrome co-morbidities including type-2 diabetes and cardiovascular disease [2][3][4][5][6] in developing and urbanised countries. Although genetic studies of patients have identified variants of genes that are associated with these inflammatory diseases in many cases such as RA, these alone do not appear to be strong risk factors. Rather, integration with signals driven by environmental factors (e.g. smoking or diet) is required to trigger disease. Recognition of the impact of environmental factors has focused interest on the role of the microbiota 5,7,8 and hence, on how helminths may regulate this in health and disease 3,9,10 . Indeed, emerging data suggest that commensal bacteria and gastrointestinal (GI) helminths appear to reciprocally regulate the composition of the gut microbiome 11,12 . It appears that such crosstalk has evolved to homeostatically maintain immune system function in health and disease 2 as GI helminths can induce regulatory responses to limit inflammation and promote intestinal barrier integrity while the intestinal bacteria play an essential role in training the immune system by impacting on stem and progenitor cells 13,14 . Consistent with the former, there is increasing evidence from animal models that the protection afforded by GI helminth infection, against inflammatory disorders associated with mucosal tissues like asthma 15 and inflammatory bowel disease 16,17 and coeliac disease 18,19 , involves modulation of the gut microbiota.
Nevertheless, gut, lung or oral dysbiosis has also been implicated in the aetiology of a wide range of systemic and organ-specific autoimmune diseases including musculoskeletal pathologies like RA and SLE [20][21][22][23][24][25][26][27][28][29][30][31] . It is not clear whether the protection afforded by GI helminths against these disorders similarly involves interaction with the microbiome, but infection with helminths like Heligmosomoides polygyrus and Trichuris muris can result in increases in Lactobacillaceae and decreases in Prevotella species 32-35 , commensals that are reported to be dysregulated in RA patients 24,31 . In any case, helminth-mediated protection against autoimmune disease is not limited to GI-tract parasites, with particularly striking examples of this involving filarial nematodes with respect to RA 36 and SLE 37 being reported in India. However, to date it has been unclear whether tissue-resident or blood-borne parasitic worms can mediate these effects via modulation of the host microbiome and if so, which mechanisms they utilise to achieve this protection.
The ability of helminths to ameliorate chronic inflammatory disorders has often been attributed to their capacity to excrete or secrete molecules (ES) that exert immunoregulation to promote immune-evasion and limit host pathology 2 . Amongst the best characterised ES products is ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae that we have shown to prevent initiation and progression of pathology in mouse models of certain allergic (asthma, contact dermatitis) and autoimmune (RA, SLE) inflammatory diseases 1,2,[38][39][40][41][42][43] . Collectively, our studies have identified a unifying mechanism of action that allows effective protection irrespective of the inflammatory phenotype: thus, by subverting TLR4 signalling to downregulate aberrant MyD88-responses, ES-62 acts to homeostatically reset the regulatory:effector immune cell balance, primarily to restore levels of IL-10 + regulatory B cells (Bregs) and suppress pathological IL-17-driven inflammation 1,2,[38][39][40][41][42][43][44] . In experimental models of RA and human disease, perturbation of the microbiota has been shown to disrupt the balance of pathogenic Th17 cells and the counter-regulatory Bregs and Tregs that act to homeostatically resolve inflammation 20,[24][25][26] . Thus, our aim here was to investigate whether the actions of ES-62 reflected an ability to impact on the microbiome. Consistent with this, we now show that whilst joint disease in the collagen-induced arthritis (CIA) mouse model of RA is preceded by disturbance of the gut microbiome with accompanying intestinal inflammation and loss of barrier integrity, ES-62 acts to normalise the microbiome and maintain gut health and function. Further supporting our hypothesis that such local gut pathology and inflammation drives joint disease, prophylactic depletion of the gut microbiota with broad-spectrum antibiotics (ABX) was found to reduce the consequent severity of arthritis in mice undergoing CIA. Such ABX-treatment also reduced the level of protection afforded by ES-62, with these animals exhibiting an intermediate phenotype of disease, comparable to that of the control ABX-treated mice undergoing CIA. These data therefore indicate that a "normalised" microbiome is also required for the full induction of the immunoregulatory actions of ES-62.

Collagen induced Arthritis (CIA)
Male DBA/1 mice were purchased at 6-8 weeks of age (Envigo; Bicester, UK) and then housed and maintained in the Central Research Facility of the University of Glasgow. All experiments were approved by, and conducted in accordance with, the Animal Welfare with PBS or purified endotoxin-free ES-62 (2 µg/injection) subcutaneously on days -2, 0 and 21 and joint inflammation and damage (articular score) determined as described previously 39,45,46 . Grip strength was recorded as per manufacturer's instruction (Ugo basile®, Italy) using a Grip-Strength Meter, which measured the grip strength (peak force and time resistance) of the forelimbs of the mice. The animals were placed over a base plate and gripped a T-shaped grasping bar which was connected to the peak amplifier that automatically detects the animal's response. Three measurements were taken and the average grip strength was calculated. In order to investigate the impact of gut microbiome perturbation on initiation and progression of inflammatory arthritis, animals were given drinking water containing [or not] a cocktail of antibiotics (500 mg/L Vancomycin, 1g/L Neomycin and 1g/L Metronizadole) to eliminate Gram-positive, Gram negative and anaerobic microorganisms 25 7 days prior to the induction of CIA and thereafter continuously throughout the experiment. Blood was sampled using endotoxin-free needles and syringes and the resulting serum isolated and stored at -20 o C in endotoxin-free Eppendorf tubes. Paws, ileum and colon tissue were fixed in 4% paraformaldehyde; ileum and colon faecal contents were collected in sterile RNAlater (Sigma) and stored at -80 o C.

Flow Cytometry
Spleen and bone marrow (BM) cells were suspended in FACS buffer (2.5% BSA; 0.5 mM EDTA, in PBS) following red blood cell-lysis (eBioscience). BM cells were labelled with a cocktail of PE-labelled antibodies specific for CD3, B220 and Ter119 to exclude analysis of lymphocytes and erythroid cell populations using a dump channel, and monocytes were identified by labelling with antibodies against CD11b (FITC), Ly6C (PerCP Cy5. 5) and Ly6G (APC) 43 . Lymphocytes from the spleen were labelled with antibodies specific for CD19 (AF700) and IL-10 (PE or APC). All antibodies were purchased from BioLegend, UK. Fixable viability stain (APC-ef780; ThermoFisher Scientific) was used to select for live cells and for analysis of IL-10 + regulatory B cells (Bregs), lymphocytes were stimulated with PMA, ionomycin, Brefeldin A and LPS as described previously 41,44 .
Data were acquired using a FACS Canto or BD LSRII flow cytometer and populations were gated as described previously using isotype and fluorescence minus one (FMO) controls using FlowJo, LLC analysis software (Tree Star/ BD) 41,44 , 43 .

Histology
Ileum, colon and joint (paw) tissues from individual mice in each treatment group were fixed in 4% paraformaldehyde for 24 hours before gut tissues were embedded in OCT and paw joints were decalcified and subsequently paraffin embedded. Paraffin sections (6 µm) and OCT cryosections (9-10 µm) were prepared and standard H&E histological staining was performed on all tissues for identification of morphological changes 39,43,46 .
Ileum (villi thickness) and colon (number of lesions) pathology was quantitated by Image J analysis. Joint pathology was scored according to the grading system of 0 for no inflammation, 1 for mild inflammation, pannus formation and bone damage, up to a score of 4 representing a high level of inflammation, pannus infiltration and bone and cartilage destruction, as previously described 47 .

Osteoclast differentiation
OCs were differentiated from BM obtained from the hind limbs of experimental animals as previously described 43 . Briefly, following removal of adherent cells, BM cells were cultured in αMEM medium supplemented with 30 ng/ml M-CSF and 50 ng/ml RANKL (Peprotech, London, UK) and then assessed for OC differentiation by TRAP staining (Leukocyte Acid Phosphatase Kit, Sigma-Aldrich, UK) on day 5. Images were obtained using an EVOS FL Auto Cell Imaging System. TRAP+ cells with ≥ 3 nuclei were enumerated and Image J software was used to calculate the average size of multinucleated OCs per field of view (FoV) 43 .

Serum cytokine and antibody ELISAs
Interleukin-6 (IL-6) and IL-10 expression was measured by ELISA according to the manufacturer's instructions (BD Biosciences, Oxford, UK). For determination of collagen type II (CII)-specific IgG1 and IgG2a antibodies in serum 45 , high binding 96 well ELISA plates were coated with CII (5 µg/ml) overnight at 4 o C before washing and blocking with BSA/PBS. Serum was diluted 1:100 and then serially diluted three-fold until 1:218700 and incubated with HRP-conjugated goat anti-mouse IgG1 or IgG2a (1:10,000) in 10% FBS/PBS prior to developing with TMB and 2M sulphuric acid and read at an optical density of 450nm.

qRT-PCR
BM cells (10 6 ) were lysed in RLT Lysis Buffer, while splenic tissue was lysed in Trizol reagent (Sigma), prior to mRNA extraction using RNeasy Plus Mini kit (Qiagen, Germany) or phenol-chloroform extraction, respectively, according to the forward -GAAGATCATCCAAGACATTGAC, reverse -TCCTCCATAAACTGAGTAGC) and βactin (actb; forward -GATGTATGAAGGCTTTGGTC, reverse -TGTGCACTTTTATTGGTCTC). Data were normalised to the reference gene β-actin to obtain the ΔCT values that were used to calculate the fold-change from the ΔΔCT following normalisation to biological control group.

Metagenomics
Genomic DNA from the ileum and colon faecal matter was purified using QIAamp DNA

Statistics
All data were analysed using GraphPad Prism 6 software using One or Two-Way ANOVA with Fishers LSD post-tests for parametric data or Kruskal-Wallis test and Dunn's posttest for non-parametric data. Unsupervised hierarchical clustering with Euclidean distance was performed on the colon samples using the heatmap.2 function of the gplots package in R. Supervised heatmaps were generated using GraphPad Prism 7 software. Indicators of significance include * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

ES-62 protection against CIA is associated with normalisation of the gut microbiome
As reported previously, ES-62 ameliorates CIA in terms of articular score (Fig. 1a) and reflecting this protection against joint morbidity, we now show that it prevents the resulting low grip strength found in CIA-mice (Fig. 1b). Indeed, treatment with ES-62 maintains grip strength in CIA-mice at a similar level to that found in healthy, Naive (not subjected to CIA) DBA/1 mice (Fig. 1b). Commensal bacteria have increasingly been proposed to play a role in RA pathogenesis (reviewed in [49][50][51] ) and the decline in grip strength during ageing has been associated with changes in the gut microbiome 52 .
Interestingly, in addition to being an indicator of frailty, grip strength has been shown to be a predictor of a wide range of adverse health outcomes 53 , e.g., cardiovascular disease, which RA patients are at increased risk of developing 54 and that are impacted by the microbiome 49,55 . Thus, to address whether the protection afforded by ES-62 is associated modulation of the gut microbiota, a metagenomic approach was used to profile the bacterial populations present in the intestines of CIA-mice, exposed or not to ES-62. Initiation of RA (and CIA) pathogenesis is associated with disruption of the balance of effector:regulatory immune system 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 (ileum-associated) and regulatory (colon-associated) immune responses 25,56,57 .
An overview of the microbiota of the ileum and colon at the phylum level shows substantial changes between healthy Naive DBA/1 mice and those with established arthritic disease (PBS; Fig. 1c). Firmicutes and Bacteriodetes are the predominant phyla in all treatment groups but with respect to CIA-mice, whereas they exhibit outgrowths of Firmicutes and Proteobacteria in the ileum, by contrast they exhibit decreased levels of Firmicutes with a compensatory outgrowth of Bacteriodetes in the colon (Fig. 1c). ES-62 essentially helps to maintain the healthy diversity of the microbiota observed in Naive mice, which was reduced in the CIA-mice (Fig. 1c). Deeper analysis illustrates the differential diversity signatures exhibited by healthy and arthritic mice, as well as the impact of ES-62 treatment (Fig. 1d): drilling down on the modulation of the Gram -ve Bacteroidetes phylum reveals some key differential signatures throughout each of the predominant Bacteroides, Porphyromonas and Prevotella genera between the colon contents of Naive and CIA mice, and identifying those normalised by exposure to ES-62 ( Fig. 1e). Strikingly, at the species level, B. vulgatus was found to be significantly increased in the colon of CIA relative to Naive or ES-62-CIA mice (Fig. 1e). By contrast abundance of members of the Rikenellaceae family and specifically, Alistipes putredinis, were reduced in the colon of CIA mice when compared to Naive and ES-62-treated CIA mice (Fig. 1e). Similarly, in spite of the fact that differential profiles amongst the treatment groups were observed throughout major genera (e.g. Bacillus, Staphyloccus, Streptococcus, Enterococcus and Clostridium) of Gram +ve Firmicutes, the most dramatic changes observed with established CIA occurred within the Clostridiales order. In particular, decreases in the Clostridiaceae (Fig. 1f) and the Lachnospiraceae (Fig. 1f) families were noted with ES-62 promoting maintenance of the Ruminococcus, Faecalibacterium, Blauti and Erysipelotrichaceae genera and butyrate-producing Dorea and Roseburia species, the latter of which have been implicated in gut health and inflammation homeostasis 58 . In terms of the Proteobacteria, CIA was associated with outgrowth of members of the Helicobacter, specifically Helicobacter hepaticus (Epsilonproteobacteria) and Escherichia, particularly Escherichia coli (Gammaproteobacteria) genera (Fig. 1g) and again this was normalised by ES-62.
Perturbation of the microbiome was also observed in the ileum of CIA mice and again, exposure to ES-62 acted to normalise this towards the healthy community (Fig. 1c). In terms of protective signatures, despite the relative paucity of bacteria in the ileum relative to the colon, ES-62 clearly promoted growth of the Clostridiales, again particularly Clostridaceae and Lachnospiraceae species but in this case generally promoting their outgrowth beyond the levels found in healthy mice (Fig. 1h). This presumably reflects that whilst treatment of naive, healthy DBA/1 mice with ES-62 for the duration of the CIA model had little effect on the colon microbiota, it promoted expansion of Clostridiales species in the ileum ( Fig. S1a and b), perhaps explaining the increase in species diversity observed in the ileum (Fig. S1c) but not the colon (Fig. S1d) of CIA-mice treated with ES-62. Additionally, reflecting the ability of CIA to perturb, and ES-62 to normalise, the gut bacteria, functional metagenomic analysis also showed that ES-62 generally acted to normalise the metabolic capacity of the colonic microbiome ( Fig. 1i).

Perturbation of the gut microbiome with broad-spectrum antibiotics both ameliorates CIA and impacts on the protection afforded by ES-62
To address whether the perturbation of the microbiome 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; 500 mg/L Vancomycin, 1g/L Neomycin and 1g/L Metronizadole) administered in their drinking water from one week prior to initiation of CIA. Such ABX treatment had no obvious effect on the overall health of the animals as, after a characteristic initial dip, there was no significant difference in body weight amongst the CIA groups at cull (Fig.   S2a). Nevertheless, this regimen essentially eliminated the bacterial microbiota of all animals irrespective of treatment group (Fig. S2b), whilst metagenomic analysis showed the residual gut community to be almost entirely comprised of proteobacteria ( Fig. S2c and S2d). As predicted from previous studies in a variety of inflammatory arthritis models 25,59-61 , 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 and c). In addition, the protection afforded by ES-62 was reduced in ABX-treated animals such that the articular score of such mice was not significantly different from PBS-CIA mice receiving ABX or not (Fig 2a). ABX mice are no longer significantly reduced relative to those in PBS-CIA mice (Fig 2d).
No differences were detected amongst any of the group 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 + "regulatory" B cells showed that both the decrease in these cells occurring during CIA (PBS-CIA) and also the maintenance of healthy levels in ES-62-CIA mice 2,44 were lost in ABX-treated animals, with the levels in ES-62-ABX mice mirroring those reduced levels found in PBS-CIA animals (Fig. 2f). Moreover, whilst ES-62 acts to increase 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)immunity 63,64 , is lost following ABX treatment such that ES-62-ABX mice display the high levels of this cytokine found in PBS-CIA mice (Fig. 2h).
In addition to maintaining immune system homeostatic regulation and promoting resolution of inflammation that becomes dysregulated in CIA mice, ES-62 also acts to suppress the functional maturation of osteoclasts (OC) 43 that directly cause erosive joint damage. Interestingly, changes in the intestinal microbiome have been shown to impact on bone mass 65-67 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. As shown previously, exposure to ES-62 in vivo had no effect per se on the numbers of OC differentiated from bone marrow (BM) progenitors ex vivo (Fig. 3a) but rather, it blocked their fusion to large, active multinucleated cells (Fig. 3a -c) that resorb bone 43 .
Consistent with its amelioration of CIA pathology, ABX administration resulted in a decrease in large multinucleated OC and a corresponding increase in the total numbers of OCs detected in BM from CIA mice (Fig. 3a -c). ES-62 rewires osteoclastogenesis by modulating the RANK/OPG bone remodelling axis 43 and this is evidenced again here by its ability to significantly decrease expression of RANK and increase (albeit not significantly) expression of the decoy receptor, OPG in BM relative to that seen in BM from PBS-CIA mice (Fig. 3d and e)

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 are known to promote autoimmune disorders like RA and cardiovascular comorbidities 69,70 . Our metagenomic analysis showed that established CIA was associated with changes to the gut microbiome, particularly with respect to a reduction in butyrate-producing species (Fig. 1g, i, j and S1) that have been implicated in the maintenance of epithelial integrity and gut health 50,58 . Strikingly, we have found that mice with CIA display severe gut pathology and inflammation, the levels of which directly correlate with severity of CIA (Fig. 4a).
Moreover, treatment with ES-62 protects against such gut damage, specifically the thickening and generation of stubby villi in the ileum (Fig. 4b and c) and hole-like "lesions" appearing in the colon (Fig. 4b and d). The physiological relevance of these colon "lesions" is unclear but may reflect the widely-established suppression of mucus production by goblet cells in response to bacteria 71,72 or alternatively, they may be attachment/effacement lesions induced by pathogenic bacteria including E. coli 73  This analysis revealed the presence of gut pathology in PBS-CIA mice during the initiation phase with an increase in ileum villi thickness and a significant increase in the number of colonic lesions following immunisation (Fig. 5a -c). Interestingly, the ileum pathology occurring during the initiation stage in PBS-CIA mice appeared to resolve in these animals by the end of the pre-clinical 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 in ES-62-CIA-mice whilst the high levels of colon lesions generated by day 21 of the pre-clinical phase were reduced in ES-62treated animals ( Fig. 5b and c).
Validating our metagenomic analysis, qPCR analysis showed that ES-62 acts to prevent the enrichment of Bacteriodetes and maintain levels of Firmicutes in the colon of mice during established CIA ( Fig. 6a and b). However, analysis at the various pre-clinical phases of the disease showed a more dynamic situation: for example, following the primary immunisation with CII/CFA, there is a significant decrease in the levels of both Bacteroidetes and Firmicutes, with the rise in Bacteriodetes evident in established arthritis only occurring following the booster immunisation ( Fig. 6a and b). In contrast, ES-62 acts to maintain "healthy" levels of Firmicutes in CIA-mice throughout but most strongly during the initiation and preclinical phase where it also acted to maintain the lowest levels of Bacteroidetes ( Fig. 6a and b). Moreover, although ES-62 treatment promoted enrichment of butyrate-producing bacteria (Butyrivibrio) at all stages of disease, this was most evident in the initiation phase of disease, and presumably this promotes and maintains gut integrity (Fig. 6c).  (Fig. 6d).
Commensal bacteria can also dynamically educate OC progenitor (OCP) maturation 68,74 : 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 function 68,74 . Reflecting the dynamic and differential changes in 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, BM from CIA mice following both primary and booster immunisations, shows enhanced capacity for functional OC maturation that is suppressed by in vivo exposure to ES-62 ( Fig. 6f and g).

Discussion
This study is the first to demonstrate that a defined, systemically-acting parasitic wormderived product can impact on, and harness, the microbiome to exert its therapeutic effects against chronic inflammation in target organs distal to the gut such as the joints.
Collectively, our data suggest that ES-62-mediated protection against inflammatory joint disease is associated with normalisation of the gut dysbiosis observed in arthritic mice towards the "healthy" microbiome observed in naive mice. This "normalisation" is highly reminiscent of that seen following treatment of RA patients with DMARDs 80 and lends further weight to increasing evidence that changes in microbiome status may contribute to pathogenesis in this multifactorial disease 31,49,74,81,82 . Indeed, perturbation of the microbiota has been shown to influence both the generation of pathogenic Th17 cells and also the homeostatic induction of inflammation-resolving Bregs and Tregs in experimental models of RA and human disease 20,[24][25][26] . Moreover, and consistent with the idea that microbiome status can promote disease in susceptible individuals, development of arthritis in a number of animal models is lost under "Germ-Free" (GF) conditions or following depletion of the microbiota by treatment with broad-spectrum ABX 25,49,59,61 .
Certainly, potentially pathogenic changes in the gut microbiome have been reported following analysis of stool faeces from patients with new-onset disease as well as those with established arthritis. However, differential, and even contradictory, associations have been reported that perhaps reflect the difficulties in standardising stool samples or their failure to fully recapitulate the contents of the gut microbiota and the specific communities shaped by the intestinal microenvironment 31,[83][84][85] . Nevertheless, with respect to the Bacteroidetes, whose levels we found to be generally elevated during established CIA, Prevotella copri has been reported to be enriched in stool samples of new-onset patients 24,86 . Moreover, transfer of such faecal matter containing Prevotella copri into GF arthritis-prone SKG mice leads to enhanced levels of zymosan-stimulated intestinal Th17 cells and more severe arthritis 86 . However, SKG mice are increasingly perceived to be a model of Spondyloarthritis rather than RA 31 and perhaps reflecting this, although we found Prevotellaceae species to be upregulated in the colon of mice with established arthritis, this was neither restricted to P. copri nor was it reversed by treatment with ES-62. Moreover, P. histocola has been reported to be protective against CIA in mice transgenic for the RA-risk gene HLA-DQ8 87 20 . However, as also reported for the IL1rn -/model 60 , we were unable to detect any OTUs that could be identified as SFB (e.g. Candidatus arthromitus) in our metagenomic analysis: moreover, we detected no significant differences by qPCR in the levels of SFB found in either the colon and ileum between PBS-and ES-62-treated CIA mice at any phase of disease (data not shown). Our findings therefore resonate with the report that despite being present in all cages, SFB could only be detected in 2/10 mice undergoing CIA prior to the booster CII immunisation 61 and of relevance to RA, does not appear to be present in the gut metagenomic analysis of the large numbers of adult human faecal samples sequenced for the Human Microbiome Project 85 . Indeed, and further questioning the role for SFB in the K/BxN model, a recent study reports that IL-17 actually appears dispensable for arthritis in K/BxN mice and proposes that the gut microbiota regulates joint disease via impacting on T follicular helper (Tfh) rather than Th17 cells 90 .
Further faecal profiling of Firmicutes has linked changes in the Clostridiaceae, Coriobacteriaceae and Lachnospiraceae families to patients with established disease 80,82 .
Our metagenomic analysis of luminal colon and ileum faecal matter shows the protective actions of ES-62 to be most strongly associated with maintenance of the Clostridiaceae, Lachnospiraceae and Ruminococcaceae families, particularly those associated with butyrate production (Blautia, Roseburia, Dorea and Butyrvibrio and Ruminococcus). Interestingly, Ruminococcus species were found to be depleted in the IL1rn -/model of inflammatory arthritis supporting the suggestion that maintenance and/or enrichment of butyrate-producing bacteria are protective 91 . Reflecting this, administration of butyrate was found to ameliorate severity of CIA, notably in terms of reduced inflammatory cell infiltration of the joint, pannus formation and cartilage and bone destruction 91 . In contrast, administration of butyrate exacerbated antibodyinduced arthritis, a model wherein the administration of serum from K/BxN mice (containing anti-glucose-6-phosphate isomerase autoantibodies) to C57BL/6 mice bypasses the initiation and adaptive immunity phases of disease. These apparently conflicting data can be reconciled by the proposal that butyrate needs to act during the preclinical phases of disease to exhibit its protective actions 91 . Perhaps consistent with this, whilst ES-62 protects against the depletion of butyrate-producing species observed in mice with established disease, we also find Butyrivibrio to be most enriched by ES-62 in the initiation phase of CIA. Collectively, these data suggest that depletion of butyrateproducing bacteria associated with the onset of CIA may contribute to the gut pathology promoting and perpetuating the inflammation that drives the breakdown of immune tolerance and consequent autoimmune joint disease.
Certainly, we find gut pathology to precede onset of joint disease, being detectable hepaticus, the latter of which has also been reported to be enriched in the IL1rn -/model 60 ] and accompanying gut pathology reminiscent of the suppression of goblet cell mucus production and attachment/effacement lesions resulting from pathological enteric infections, including E. coli [71][72][73] . Perhaps reflecting the loss of diversity in the microbiota and the breakdown of intestinal homeostasis in RA, in addition to their increased faecal abundance of P. copri, early onset RA patients also exhibit P. copri in their synovial fluid 24,95 . Moreover, E. coli and other infectious microorganisms have been reported to commonly colonise RA patients and may exacerbate disease [96][97][98] .
The normalisation of the gut microbiota by ES-62 may actually result in a dual pronged mechanism by which butyrate, in addition to its local gut-protecting actions, could also impact systemically to protect more directly against joint pathology in CIA. Consistent with this idea, butyrate has been reported to suppress osteoclastogenesis 99

Acknowledgements
The work was funded by an award to MMH, WH and PAH from Arthritis Research UK (21133).

Conflicts of Interest
The authors have no conflicts of interest.  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), oneway ANOVA (c -h) with LSD Fishers multiple comparisons and significance indicated by asterisks, * = p < 0.05 and ** = p < 0.01.   significance is denoted as * = p < 0.05 and *** = p < 0.001.  a, b and d). Statistical significance was determined using one-way ANOVA with Dunn's multiple comparisons comparing Naïve, PBS or ES-62-treated groups to their respective ABX-treated control and indicated by asterisks ** = p < 0.01.