Microbiota-dependent indole production stimulates the development of collagen-induced arthritis in mice.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model, we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease and that indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colonic lymphocytes to indole increased the expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.

Microbiota-dependent indole production is required for the development of collagen-induced arthritis.

Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identify alterations in tryptophan metabolism, and specifically indole, that correlate with disease. We demonstrate that both bacteria and dietary tryptophan are required for disease, and indole supplementation is sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1ß; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a novel therapeutic pathway for RA and SpA.

Distinct Role of TNFR1 and TNFR2 in Protective Immunity Against Orientia tsutsugamushi Infection in Mice.

Infection with Orientia tsutsugamushi, an obligate intracellular bacterium, can cause mild or severe scrub typhus. Some patients develop acute lung injury, multi-organ failure, and fatal infection; however, little is known regarding key immune mediators that mediate infection control or disease pathogenesis. Using murine models of scrub typhus, we demonstrated in this study the requirement of TNF-TNFR signaling in protective immunity against this infection. Mice lacking both TNF receptors (TNFR1 and TNFR2) were highly susceptible to O. tsutsugamushi infection, displaying significantly increased tissue bacterial burdens and succumbing to infection by day 9, while most wild-type mice survived through day 20. This increased susceptibility correlated with poor activation of cellular immunity in inflamed tissues. Flow cytometry of lung- and spleen-derived cells revealed profound deficiencies in total numbers and activation status of NK cells, neutrophils, and macrophages, as well as CD4 and CD8 T cells. To define the role of individual receptors in O. tsutsugamushi infection, we used mice lacking either TNFR1 or TNFR2. While deficiency in either receptor alone was sufficient to increase host susceptibility to the infection, TNFR1 and TNFR2 played a distinct role in cellular responses. TNF signaling through TNFR1 promoted inflammatory responses and effector T cell expansion, while TNFR2 signaling was associated with anti-inflammatory action and tissue homeostasis. Moreover, TNFRs played an intrinsic role in CD8+ T cell activation, revealing an indispensable role of TNF in protective immunity against O. tsutsugamushi infection.

Orientia tsutsugamushi selectively stimulates the C-type lectin receptor Mincle and type 1-skewed proinflammatory immune responses.

Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.

Microbial Influences of Mucosal Immunity in Rheumatoid Arthritis.

PURPOSE OF REVIEW: This review will summarize recent data defining the relationship between rheumatoid arthritis (RA) and the microbiome at mucosal sites throughout the body. It will highlight what is known, what is speculated, and current knowledge gaps regarding the microbiome in RA. RECENT FINDINGS: An extensive relationship between the microbiome and immune cell function can influence RA-related inflammation and T cell and B cell biology. Studies are beginning to characterize microbial changes in individuals who are at risk for RA, which is a critical element needed to understand the influence of the microbiome on RA pathogenesis. Expanding our understanding of the microbiome in RA beyond the bacteria at the gut and oral mucosae into the lung and urogenital surfaces, including viral and fungal components, and establishing the relationship across mucosal sites will be critical in future work. Importantly, approaches to manipulate the microbiome could lead to novel therapeutic and preventive strategies.

Polarized lung inflammation and Tie2/angiopoietin-mediated endothelial dysfunction during severe Orientia tsutsugamushi infection.

Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1 and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (MΦ) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.

Scrub Typhus Pathogenesis: Innate Immune Response and Lung Injury During Orientia tsutsugamushi Infection.

Scrub typhus is an understudied, potentially lethal disease caused by infection with Orientia tsutsugamushi. Despite causing an estimated 1 million cases per year and an increasing global presence, mechanisms of scrub typhus pathogenesis remain unclear. One of the most life-threatening conditions that can arise in scrub typhus patients is acute respiratory distress syndrome (ARDS). The development of ARDS is a complex process; some of its pathological hallmarks, including prolonged recruitment of inflammatory immune cells to the lung and vasculature damage, have been observed in humans and/or animal models of O. tsutsugamushi infection. Although different cell types and mechanisms may contribute to ARDS development during O. tsutsugamushi infection, this review highlights our current evidence of pulmonary endothelial activation and damage, the potential roles of neutrophils and macrophages in the lung, and the knowledge gaps in this field. Continued investigation of the lung microenvironment and cellular interactions will help elucidate disease pathogenesis and possible treatment during scrub typhus.

Type 1-skewed neuroinflammation and vascular damage associated with Orientia tsutsugamushi infection in mice.

BACKGROUND: Scrub typhus is a life-threatening disease, due to infection with O. tsutsugamushi, a Gram-negative bacterium that preferentially replicates in endothelial cells and professional phagocytes. Meningoencephalitis has been reported in scrub typhus patients and experimentally-infected animals; however, the neurological manifestation and its underlying mechanisms remain poorly understood. To address this issue, we focused on Orientia tsutsugamushi Karp strain (OtK), and examined host responses in the brain during lethal versus self-healing scrub typhus disease in our newly established murine models. PRINCIPLE FINDINGS: Following inoculation with a lethal dose of OtK, mice had a significant increase in brain transcripts related to pathogen-pattern recognition receptors (TLR2, TLR4, TLR9), type-1 responses (IFN-γ, TNF-α, CXCL9, CXCR3), and endothelial stress/damage such as angiopoietins, but a rapid down-regulation of Tie2. Sublethal infection displayed similar trends, implying the development of type 1-skewed proinflammatory responses in infected brains, independent of time and disease outcomes. Focal hemorrhagic lesions and meningitis were evident in both infection groups, but pathological changes were more diffuse and frequent in lethal infection. At 6-10 days of lethal infection, the cortex and cerebellum sections had increased ICAM-1-positive staining in vascular cells, as well as increased detection of CD45+ leukocytes, CD3+ T cells, IBA1+ phagocytes, and GFAP+ astrocytes, but a marked loss of occludin-positive tight junction staining, implying progressive endothelial activation/damage and cellular recruitment in inflamed brains. Orientia were sparse in the brains, but readily detectable within lectin+ vascular and IBA-1+ phagocytic cells. These CNS alterations were consistent with type 1-skewed, IL-13-suppressed responses in lethally-infected mouse lungs. SIGNIFICANCE: This is the first report of type 1-skewed neuroinflammation and cellular activation, accompanied with vascular activation/damage, during OtK infection in C57BL/6 mice. This study not only enhances our understanding of the pathophysiological mechanisms of scrub typhus, but also correlates the impact of immune and vascular dysfunction on disease pathogenesis.

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice.

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33-/- mice. Importantly, IL-33-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.