Faculty Bibliography

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-kappaB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

BACKGROUND: Airway abnormalities and lung tissue citrullination are found in both rheumatoid arthritis (RA) patients and individuals at-risk for disease development. This suggests the possibility that the lung could be a site of autoimmunity generation in RA, perhaps in response to microbiota changes. We therefore sought to test whether the RA lung microbiome contains distinct taxonomic features associated with local and/or systemic autoimmunity. METHODS: 16S rRNA gene high-throughput sequencing was utilized to compare the bacterial community composition of bronchoalveolar lavage fluid (BAL) in patients with early, disease-modifying anti-rheumatic drugs (DMARD)-naive RA, patients with lung sarcoidosis, and healthy control subjects. Samples were further assessed for the presence and levels of anti-citrullinated peptide antibodies (including fine specificities) in both BAL and serum. RESULTS: The BAL microbiota of RA patients was significantly less diverse and abundant when compared to healthy controls, but similar to sarcoidosis patients. This distal airway dysbiosis was attributed to the reduced presence of several genus (i.e., Actynomyces and Burkhordelia) as well as reported periodontopathic taxa, including Treponema, Prevotella, and Porphyromonas. While multiple clades correlated with local and systemic levels of autoantibodies, the genus Pseudonocardia and various related OTUs were the only taxa overrepresented in RA BAL and correlated with higher disease activity and erosions. CONCLUSIONS: Distal airway dysbiosis is present in untreated early RA and similar to that detected in sarcoidosis lung inflammation. This community perturbation, which correlates with local and systemic autoimmune/inflammatory changes, may potentially drive initiation of RA in a proportion of cases.

BACKGROUND: Seronegative joint diseases are characterized by a lack of well-defined biomarkers since autoantibodies are not elevated. Calprotectin (S100A8/A9) is a damage-associated molecular pattern (DAMP) which is released by activated phagocytes, and high levels are found in seronegative arthritides. In this study, we investigated the biomarker potential of systemic and local levels of these S100 proteins to assess joint inflammation and joint destruction in an experimental model for seronegative arthritis. METHODS: Serum levels of S100A8/A9 and various cytokines were monitored during disease development in interleukin-1 receptor antagonist (IL-1Ra)-/- mice using ELISA and multiplex bead-based immunoassay, and were correlated to macroscopic and microscopic parameters for joint inflammation, bone erosion, and cartilage damage. Local expression of S100A8 and S100A9 and matrix metalloproteinase (MMP)-mediated cartilage damage in the ankle joints were investigated by immunohistochemistry. In addition, local S100A8 and activated MMPs were monitored in vivo by optical imaging using anti-S100A8-Cy7 and AF489-Cy5.5, a specific tracer for activated MMPs. RESULTS: Serum levels of S100A8/A9 were significantly increased in IL-1Ra-/- mice and correlated with macroscopic joint swelling and histological inflammation, while serum levels of pro-inflammatory cytokines did not correlate with joint swelling. In addition, early serum S100A8/A9 levels were prognostic for disease outcome at a later stage. The increased serum S100A8/A9 levels were reflected by an increased expression of S100A8 and S100A9 within the ankle joint, as visualized by molecular imaging. Next to inflammatory processes, serum S100A8/A9 also correlated with histological parameters for bone erosion and cartilage damage. In addition, arthritic IL-1Ra-/- mice with increased synovial S100A8 and S100A9 expression showed increased cartilage damage that coincided with MMP-mediated neoepitope expression and in vivo imaging of activated MMPs. CONCLUSIONS: Expression of S100A8 and S100A9 in IL-1Ra-/- mice strongly correlates with synovial inflammation, bone erosion, and cartilage damage, underlining the potential of S100A8/A9 as a systemic and local biomarker in seronegative arthritis not only for assessing inflammation but also for assessing severity of inflammatory joint destruction.

Background/Purpose: Despite significant advances in the therapeutics of inflammatory arthritides, methotrexate (MTX) remains the mainstay of treatment for rheumatoid arthritis (RA) and related conditions worldwide. However, it has a hihgly variable inter-individual bioavailabity (ranging from 20 to 80%) and there is currently no mechanism to predict efficacy. One of the fundamental roles of the intestinal microbiome is to metabolize xenobiotics and synthetic drugs. Here we characterize the effects of oral methotrexate on the gut community composition of patients with RA and the potential role of baseline human microbiota in predicting response to MTX therapy. Methods: Demographic characteristics, drug use and disease activity scores-28 (DAS28) were recorded from new-onset rheumatoid arthritis (NORA) patients (n=33). For each participant, fecal samples were collected at baseline and at pre-established intervals for at least 3 months after initiation of oral methotrexate (range 3-48 months). 16S rDNA was extracted per protocol (MoBio, USA) and amplicons targeting the hypervariable V4 region were sequenced using 454 and MiSeq (Illumina) platforms to define the microbiota composition. The obtained 16S rRNA sequences were analyzed using the Quantitative Insights into Microbial Ecology (QIIME) pipeline. Taxonomic relative abundance at all hierarchical levels was determined to establish baseline microbiota composition prior to MTX initiation. Two-tailed Wilcoxon non-parametric test was applied to identify significant microbiota taxonomic changes that occur after MTX therapy. The False Discovery Rate (FDR) approach was applied to adjust for multiple hypothesis testing. Changes with a P<0.05 and FDR<0.2 were considered significant.Spearman correlations between baseline relative composition of intestinal microbiota and clinical response to MTX at each time point were also applied. Results: To quantify microbiota similarities among fecal samples we used unweighted UniFrac and hierarchical clustering. Samples from MTX-treated patients clustered with their respective baseline samples, indicating that the gut microbiota is stable with inter-individual taxonomic differences maintained for at least 6 months. NORA patients receiving MTX developed minimal changes over time. Intriguingly, however, baseline microbiome signatures in these patients predicted clinical response to MTX at 3 and 6 months, including the overabundance of unclassified Coriobacteriaceae (r=-0.756; P <0.01) and a Coprococcus-related OTU (r=-0.755; P =0.022). Conclusion: Although oral methotrexate does not induce significant changes in the over all structure of the human intestinal microbiota of NORA patients, the abundance of several taxa at baseline correlate with a significant improvement in clinical disease activity 3 and 6 months into therapy. Whether specific intestinal commensals can modulate the pharmacokinetics and bioavailability of methotrexate (and other DMARDs), remains to be elucidated. Better understanding of MTX pharamcomicrobiomics will be necessary to achieve precision medicine strategies in RA and related conditions

The role of the gut microbiome in animal models of inflammatory and autoimmune disease is now well established. The human gut microbiome is currently being studied as a potential modulator of the immune response in rheumatic disorders. However, the vastness and complexity of this host-microorganism interaction is likely to go well beyond taxonomic, correlative observations. In fact, most advances in the field relate to the functional and metabolic capabilities of these microorganisms and their influence on mucosal immunity and systemic inflammation. An intricate relationship between the microbiome and the diet of the host is now fully recognized, with the microbiota having an important role in the degradation of polysaccharides into active metabolites. This Review summarizes the current knowledge on the metabolic role of the microbiota in health and rheumatic disease, including the advances in pharmacomicrobiomics and its potential use in diagnostics, therapeutics and personalized medicine.

In the past few years, new developments have been reported on the role of Toll-like receptors (TLRs) in chronic inflammation in rheumatic diseases. The inhibitory function of TLR10 has been demonstrated. Receptors that enhance the function of TLRs, and several TLR inhibitors, have been identified. In addition, the role of the microbiome and TLRs in the onset of rheumatic diseases has been reported. We review novel insights on the role of TLRs in several inflammatory joint diseases, including rheumatoid arthritis, systemic lupus erythematosus, gout and Lyme arthritis, with a focus on the signalling mechanisms mediated by the Toll-IL-1 receptor (TIR) domain, the exogenous and endogenous ligands of TLRs, and the current and future therapeutic strategies to target TLR signalling in rheumatic diseases.

Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Dendritic cells (DCs) and macrophages (Mfs) integrate microbial signals and direct adaptive immunity. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. Th17 cells are critical for mucosal immune protection and at steady state are induced by commensal bacteria, such as segmented filamentous bacteria (SFB). Here, we examined the roles of mucosal DCs and Mfs in Th17 induction by SFB in vivo. We show that Mfs, and not conventional CD103(+) DCs, are essential for the generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.

BACKGROUND: Infection with Coxiella burnetii can lead to acute and chronic Q fever. Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR6, nucleotide-binding oligomerization domain receptor 1 (NOD1), NOD2, and the mitogen-activated protein kinases are central in the innate immune response against microorganisms, but little is known about their role in the recognition of C. burnetii in humans. METHODS: Human peripheral blood mononuclear cells (PBMCs) were stimulated with C. burnetii Nine Mile and the Dutch outbreak isolate C. burnetii 3262. TLRs were inhibited using specific antibodies or antagonists. Additionally, the influence of human polymorphisms in TLRs and Nod-like receptors (NLRs) on C. burnetii-induced cytokine production was assessed. RESULTS: Inhibition of TLR2, p38, JNK, and ERK led to decreased cytokine responses in C. burnetii-stimulated human PBMCs. Humans with polymorphisms in TLR1 and NOD2 had reduced cytokine production, compared with humans with wild-type genotypes, after stimulation. Interestingly, polymorphisms in TLR6 led to decreased cytokine production after C. burnetii 3262 stimulation but not after C. burnetii Nine Mile stimulation. CONCLUSIONS: The TLR1/TLR2 heterodimer and NOD2 are important recognition receptors for the induction of cytokine responses against C. burnetii in humans. Furthermore, an interesting finding was the divergent recognition of C. burnetii Nine Mile and C. burnetii 3262.

In autoimmune diseases, a disturbance of the balance between T helper 17 (Th17) and regulatory T cells (Tregs) is often observed. This disturbed balance is also the case in rheumatoid arthritis (RA). Genetic predisposition to RA confers the presence of several polymorphisms mainly regulating activation of T lymphocytes. However, the presence of susceptibility factors is neither necessary nor sufficient to explain the disease development, emphasizing the importance of environmental factors. Multiple studies have shown that commensal gut microbiota is of great influence on immune homeostasis and can trigger the development of autoimmune diseases by favoring induction of Th17 cells over Tregs. However the mechanism by which intestinal microbiota influences the Th cell balance is not completely understood. Here we review the current evidence supporting the involvement of commensal intestinal microbiota in rheumatoid arthritis, along with a potential role of Toll-like receptors (TLRs) in modulating the relevant Th cell responses to trigger autoimmunity. A better understanding of TLR triggering by intestinal microbiota and subsequent T cell activation might offer new perspectives for manipulating the T cell response in RA patients and may lead to the discovery of new therapeutic targets or even preventive measures.