Cindy Boer

Microbiome Composition, Joint Pain and Inflammation | 239 5.2 data analysis are not done in the exact same way[34]. We sought replication in the LLD cohort, which has a different study population and data preparation method than our cohort does[27]. Despite these differences, we could replicate our association in LLD for all taxonomic levels, class, order, family and for the genus of Streptococcus. Also, in the meta-analysis of RS and LLD, Streptococcus spp. was significantly associated with knee WOMAC pain. Obesity-mediated gastrointestinal-microbiome changes are postulated to affect low-grade systemic and local inflammation in OA[23-25]. Nevertheless, in our study the effect of Streptococcus spp. on knee WOMAC pain is not fully driven by BMI. This suggests a direct role for the gastrointestinal microbiome in OA-related knee pain and inflammation. We postulate that greater Streptococcus spp. abundance leads to higher knee WOMAC pain through local joint inflammation. This is in line with our observation that Streptococcus spp. abundance was significantly associated with effusion severity in the knee joints. This leads to believe that Streptococcus spp. might also be involved in other inflammatory joint pain disorders. This is not unlikely since several Streptococcus spp. have been linked to osteomyelitis[35,36], rheumatic fever[37,38] and, post-strep- tococcal reactive arthritis[39,40]. The last two are disorders in which due to molecular mimicry with group A Streptococcus, cross-reactive antibodies are produced against joint tissues, leading to rheumatic joint inflammation and damage[38]. However, these disorders involvemainly pathogenic species, such as S. pyogenes . Yet, most Streptococcus spp. are commensal species and have been found throughout the human oral-gastro-in- testinal microbiome[41-43], still, these can produce immunogenic bacterial products. Several Streptococcus spp. have been shown to constitutively produce MVs[18]. These MVs may present Streptococcus spp. epitopes and/or may contain immunogenic prod- ucts[17,24]. Such bacterial products can trigger macrophage activation through TLR pathways. This type of macrophage activation is predominantly seen in OA-related joint inflammation[16,21] and is thought to be related to pain[3-7]. We therefore propose that greater Streptococcus spp. abundance may lead to an increase of bacterial products in the circulation through increased production of metabolites that pass the gut-blood barrier or through immunogenic products that prime local or systemic macrophages. We have summarized this hypothetical model in Figure 2 . Our results show a difference in microbiome β-diversity in individuals with higher knee WOMAC-pain scores, which is driven by greater abundancy of Streptococ- cus spp. This association is highly robust. Moreover, we replicated our findings in an independent cohort. However, our study has some limitations. First, a cross-sectional study design was used and cannot unequivocally establish causality. For this, longitudi- nal studies are required. Second, other follow-up studies are needed to better elucidate