Prevotella copri in individuals at risk for rheumatoid arthritis.

OBJECTIVES: Rheumatoid arthritis (RA) has been associated with a relative expansion of faecal Prevotellaceae. To determine the microbiome composition and prevalence of Prevotella spp. in a group of individuals at increased risk for RA, but prior to the development of the disease. METHODS: In an ongoing cohort study of first-degree relatives (FDRs) of patients with RA, we identified 'FDR controls', asymptomatic and without autoantibodies, and individuals in pre-clinical RA stages, who had either developed anticitrullinated peptide antibodies or rheumatoid factor positivity and/or symptoms and signs associated with possible RA. Stool sampling and culture-independent microbiota analyses were performed followed by descriptive statistics and statistical analyses of community structures. RESULTS: A total of 133 participants were included, of which 50 were categorised as 'FDR controls' and 83 in 'pre-clinical RA stages'. The microbiota of individuals in 'pre-clinical RA stages' was significantly altered compared with FDR controls. We found a significant enrichment of the bacterial family Prevotellaceae, particularly Prevotella spp., in the 'pre-clinical RA' group (p=0.04). CONCLUSIONS: Prevotella spp. enrichment in individuals in pre-clinical stages of RA, before the onset of RA, suggests a role of intestinal dysbiosis in the development of RA.

Experimental selection of long-term intracellular mycobacteria.

Some intracellular bacteria are known to cause long-term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long-term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and also with the specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.

Size-dependent mechanism of cargo sorting during lysosome-phagosome fusion is controlled by Rab34.

Phagosome maturation is an essential part of the innate and adaptive immune response. Although it is well established that several Ras-related proteins in brain (Rab) proteins become associated to phagosomes, little is known about how these phagosomal Rab proteins influence phagosome maturation. Here, we show a specific role for Rab34 and mammalian uncoordinated 13-2 (Munc13-2) in phagolysosome biogenesis and cargo delivery. Rab34 knockdown impaired the fusion of phagosomes with late endosomes/lysosomes and high levels of active Rab34 promoted this process. We demonstrate that Rab34 enhances phagosome maturation independently of Rab7 and coordinates phagolysosome biogenesis through size-selective transfer of late endosomal/lysosomal cargo into phagosomes. More importantly, we show that Rab34 mediates phagosome maturation through the recruitment of the protein Munc13-2. Finally, we report that the alternative maturation pathway controlled by Rab34 is critical for mycobacterial killing because Rab34 silencing resulted in mycobacterial survival, and Rab34 expression led to mycobacterial killing. Altogether, our studies uncover Rab34/Munc13-2 as a critical part of an alternative Rab7-independent phagosome maturation machinery and lysosome-mediated killing of mycobacteria.

Internalization, phagolysosomal biogenesis and killing of mycobacteria in enucleated epithelial cells.

Bacterial and parasitic intracellular pathogens or their secreted products have been shown to induce host cell transcriptional responses, which may benefit the host, favour the microorganism or be unrelated to the infection. In most instances, however, it is not known if the host cell nucleus is proximately required for the development of an intracellular infection. This information can be obtained by the infection of artificially enucleated host cells (cytoplasts). This model, although rather extensively used in studies of viral infection, has only been applied to few bacterial pathogens, which do not include Mycobacterium spp. Here, we investigate the internalization, phagosome biogenesis and survival of M. smegmatis in enucleated type II alveolar epithelial cells. Cytoplasts were infected with M. smegmatis, but the percentage of infection was significantly lower than that of nucleated cells. Scanning electron microscopy indicated that in both cells and cytoplasts, bacteria were internalized by a phagocytosis-like mechanism. Interestingly, phagosome fusion with lysosomes and mycobacterial killing were both more efficient in enucleated than in nucleated cells, a finding that may be correlated with the increased number of autophagic vesicles developed in cytoplasts. We provide evidence that although quantitative changes were observed, the full development of the infection, as well as mycobacterial killing did not require the presence of the host cell nucleus.

Perturbation of the gut microbiome by Prevotella spp. enhances host susceptibility to mucosal inflammation.

Diverse microbial signatures within the intestinal microbiota have been associated with intestinal and systemic inflammatory diseases, but whether these candidate microbes actively modulate host phenotypes or passively expand within the altered microbial ecosystem is frequently not known. Here we demonstrate that colonization of mice with a member of the genus Prevotella, which has been previously associated to colitis in mice, exacerbates intestinal inflammation. Our analysis revealed that Prevotella intestinalis alters composition and function of the ecosystem resulting in a reduction of short-chain fatty acids, specifically acetate, and consequently a decrease in intestinal IL-18 levels during steady state. Supplementation of IL-18 to Prevotella-colonized mice was sufficient to reduce intestinal inflammation. Hence, we conclude that intestinal Prevotella colonization results in metabolic changes in the microbiota, which reduce IL-18 production and consequently exacerbate intestinal inflammation, and potential systemic autoimmunity.

Induction of IL-22-Producing CD4+ T Cells by Segmented Filamentous Bacteria Independent of Classical Th17 Cells.

The intestinal microbiota modulates IL-22 production in the intestine, including the induction of IL-22-producing CD4+ T helper cells. Which specific bacteria are responsible for the induction of these cells is less well understood. Here, we demonstrate through the use of novel gnotobiotic knock-in reporter mice that segmented filamentous bacteria (SFB), which are known for their ability to induce Th17 cells, also induce distinct IL-17A negative CD4+ T cell populations in the intestine. A subset of these cells instead produces IL-22 upon restimulation ex vivo and also during enteric infections. Furthermore, they produce a distinct set of cytokines compared to Th17 cells including the differential expression of IL-17F and IFN-γ. Importantly, genetic models demonstrate that these cells, presumably Th22 cells, develop independently of intestinal Th17 cells. Together, our data identifies that besides Th17, SFB also induces CD4+ T cell populations, which serve as immediate source of IL-22 during intestinal inflammation.

Distinct Polysaccharide Utilization Determines Interspecies Competition between Intestinal Prevotella spp.

Prevotella spp. are a dominant bacterial genus within the human gut. Multiple Prevotella spp. co-exist in some individuals, particularly those consuming plant-based diets. Additionally, Prevotella spp. exhibit variability in the utilization of diverse complex carbohydrates. To investigate the relationship between Prevotella competition and diet, we isolated Prevotella species from the mouse gut, analyzed their genomes and transcriptomes in vivo, and performed competition experiments between species in mice. Diverse dominant Prevotella species compete for similar metabolic niches in vivo, which is linked to the upregulation of specific polysaccharide utilization loci (PULs). Complex plant-derived polysaccharides are required for Prevotella spp. expansion, with arabinoxylans having a prominent impact on species abundance. The most dominant Prevotella species encodes a specific tandem-repeat trsusC/D PUL that enables arabinoxylan utilization and is conserved in human Prevotella copri strains, particularly among those consuming a vegan diet. These findings suggest that efficient (arabino)xylan-utilization is a factor contributing to Prevotella dominance.

Shaping of Intestinal Microbiota in Nlrp6- and Rag2-Deficient Mice Depends on Community Structure.

Contradicting observations have been made regarding the relative contributions of immune sensors to shaping the microbiome, yet the reasons for these discrepancies are not fully understood. Here, we investigated the contribution of environmental factors in shaping the microbiome in mice deficient in adaptive immunity (Rag2-/-) and Nlrp6, an immune sensor proposed to be involved in regulation of microbiota composition. In conventionally housed Nlrp6-/- mice, familial transmission has a significant effect on microbiota composition, complicating the analysis of genotype-dependent effects. Notably, after rederivation into standardized specific pathogen-free (SPF) conditions devoid of pathobionts, microbiota composition was indistinguishable between WT, Rag2-/-, and Nlrp6-/- mice. However, upon reintroduction of a pathobiont-containing community host, genotype-dependent differences reappear, specifically affecting the relative abundance of pathobionts such as Helicobacter spp. Our results show that the impact of Nlrp6 and also of adaptive immunity on microbiota composition depends on community structure and primarily influences pathobionts but not commensals.

The proneurotrophin receptor sortilin is required for Mycobacterium tuberculosis control by macrophages.

Sorting of luminal and membrane proteins into phagosomes is critical for the immune function of this organelle. However, little is known about the mechanisms that contribute to the spatiotemporal regulation of this process. Here, we investigated the role of the proneurotrophin receptor sortilin during phagosome maturation and mycobacterial killing. We show that this receptor is acquired by mycobacteria-containing phagosomes via interactions with the adaptor proteins AP-1 and GGAs. Interestingly, the phagosomal association of sortilin is critical for the delivery of acid sphingomyelinase (ASMase) and required for efficient phagosome maturation. Macrophages from Sort1(-/-) mice are less efficient in restricting the growth of Mycobacterium bovis BCG and M. tuberculosis. In vivo, Sort1(-/-) mice showed a substantial increase in cellular infiltration of neutrophils in their lungs and higher bacterial burden after infection with M. tuberculosis. Altogether, sortilin defines a pathway required for optimal intracellular mycobacteria control and lung inflammation in vivo.

Molecular characterization of an interleukin-4-inducing factor from Schistosoma mansoni eggs.

The eggs of the parasitic trematode Schistosoma mansoni are powerful inducers of a T helper type 2 (Th2) immune response and immunoglobulin E (IgE) production. S. mansoni egg extract (SmEA) stimulates human basophils to rapidly release large amounts of interleukin (IL)-4, the key promoter of a Th2 response. Here we show purification and sequence of the IL-4-inducing principle of S. mansoni eggs (IPSE). Stimulation studies with human basophils using SmEA fractions and natural and recombinant IPSE as well as neutralization and immunodepletion studies using antibodies to recombinant IPSE demonstrate that IPSE is the bioactive principle in SmEA leading to activation of basophils and to expression of IL-4 and IL-13. Regarding the mechanism of action, blot analysis showed that IPSE is an IgE-binding factor, suggesting that it becomes effective via cross-linking receptor-bound IgE on basophils. Immunohistology revealed that IPSE is enriched in and secreted from the subshell area of the schistosome egg. We conclude from these data that IPSE may be an important parasite-derived component for skewing the immune response toward Th2.