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 PLOS NEGLECTED TROPICAL DISEASES

BackgroundScrub 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 findingsFollowing 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.SignificanceThis 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.

Background Aberrant hippocampal neurogenesis is an important pathological feature of sepsis-associated encephalopathy. In the current study, we examined the potential role of the long noncoding RNA (lncRNA) sex-determining region Y-box 2 (SOX2) overlapping transcript (SOX2OT), a known regulator of adult neurogenesis in sepsis-induced deficits in hippocampal neurogenesis and cognitive function. Methods Sepsis was induced in adult C57BL/6 J male mice by cecal ligation and perforation (CLP) surgery. Randomly selected CLP mice were transfected with short interfering RNAs (siRNAs) against SOX2OT or SOX2, or with scrambled control siRNA. Cognitive behavior was tested 8–12 days post-surgery using a Morris water maze. Western blotting and RT-qPCR were used to determine expression of SOX2, Ki67, doublecortin (DCX), nestin, brain lipid-binding protein, and glial fibrillary acidic protein (GFAP) in the hippocampus. The number of bromodeoxyuridine (BrdU)+/DCX+ cells, BrdU+/neuronal nuclei (NeuN)+ neurons, and BrdU+/GFAP+ glial cells in the dentate gyrus were assessed by immunofluorescence. Results CLP mice showed progressive increases in SOX2OT and SOX2 mRNA levels on days 3, 7, and 14 after CLP surgery, accompanied by impaired cognitive function. Sepsis led to decrease in all neuronal markers in the hippocampus, except GFAP. Immunofluorescence confirmed the decreased numbers of BrdU+/DCX+ cells and BrdU+/NeuN+ neurons, and increased numbers of BrdU+/GFAP+ cells. SOX2OT knockdown partially inhibited the effects of CLP on levels of SOX2 and neuronal markers, neuronal populations in the hippocampus, and cognitive function. SOX2 deficiency recapitulated the effects of SOX2OT knockdown. Conclusion SOX2OT knockdown improves sepsis-induced deficits in hippocampal neurogenesis and cognitive function by downregulating SOX2 in mice. Inhibiting SOX2OT/SOX2 signaling may be effective for treating or preventing neurodegeneration in sepsis-associated encephalopathy.

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27 of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-0435 28 #d Current Address: Department of Physiology, OUHSC, 940 Stanton Young Blvd., Oklahoma 29 City, OK 73104-5042 30 31 32 33 * Corresponding Authors: Jinjun Liu: jupet@163.com; Lynn Soong: lysoong@utmb.edu 34 35 36 Abstract 38Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; 39 however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated 40 pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. 41 Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological 42 characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1, 43 VEGFR2, and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial 44 cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-45 cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the 46 transcriptional and functional levels. In vitro infection of primary human EC cultures and serum 47 52 (CD206 + CD64 + CD11b + Ly6G -) cells. In vitro studies indicated extensive bacterial replication in 53 M2-type, but not M1-type, MΦs, implying the protective and pathogenic roles of M1-skewed 54 responses. This is the first detailed investigation of lung cellular immune responses during acute 55 O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-56 skewed inflammatory responses, highlighting future therapeutic research for the control of this 57 neglected tropical disease.58 59 4 Author Summary 60Scrub typhus is a life-threatening disease, infecting an estimated one million people yearly. 61Acute lung injury is the most common clinical observation; however, its pathogenic biomarkers 62 and mechanisms of progression remain unknown. Here, we used a lethal infection mouse model 63 that parallels certain aspects of severe scrub typhus, primary human endothelial cell cultures, and 64 patient sera to define pathogenic biomarkers following Orientia tsutsugamushi infections. We 65 found a significant increase in the levels of endothelial activation/stress markers (angiopoietins 66 and ICAM-1) in infected mouse lungs and in patient sera, but a progressive loss of endothelium-67 specific Tie2 receptor and junction proteins (VE-cadherin), at severe stages of disease. These 68 signs of vasculature disruption positively correlated with the timing and magnitude of 69 recruitment/activation of proinflammatory MΦ subsets in infected lungs. Bacterial growth in 70 vitro was favored in M2-like, but not in M1-like, MΦs. This study, for the first time, reveals 71 endothelial malfunction and dysregulated inflammatory responses, suggesting potential 72 therapeutic targets to ameliorate tissue damage and pathogenesis. 73 74 Scrub typhus is a febrile and potentially lethal illness that infects an estimated one million 75 individuals per year [1]. The disease is ca...

Background Perioperative neurocognitive disorders (PNDs) occur frequently after surgery and worsen patient outcome. How C-X-C motif chemokine (CXCL) 13 and its sole receptor CXCR5 contribute to PNDs remains poorly understood. Methods A PND model was created in adult male C57BL/6J and CXCR5−/− mice by exploratory laparotomy. Mice were pretreated via intracerebroventricular injection with recombinant CXCL13, short hairpin RNA against CXCL13 or a scrambled control RNA, or ERK inhibitor PD98059. Then surgery was performed to induce PNDs, and animals were assessed in the Barnes maze trial followed by a fear-conditioning test. Expression of CXCL13, CXCR5, and ERK in hippocampus was examined using Western blot, quantitative PCR, and immunohistochemistry. Levels of interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) in hippocampus were assessed by Western blot. Results Surgery impaired learning and memory, and it increased expression of CXCL13 and CXCR5 in the hippocampus. CXCL13 knockdown partially reversed the effects of surgery on CXCR5 and cognitive dysfunction. CXCR5 knockout led to similar cognitive outcomes as CXCL13 knockdown, and it repressed surgery-induced activation of ERK and production of IL-1β and TNF-α in hippocampus. Recombinant CXCL13 induced cognitive deficits and increased the expression of phospho-ERK as well as IL-1β and TNF-α in hippocampus of wild-type mice, but not CXCR5−/− mice. PD98059 partially blocked CXCL13-induced cognitive dysfunction as well as production of IL-1β and TNF-α. Conclusions CXCL13-induced activation of CXCR5 may contribute to PNDs by triggering ERK-mediated production of pro-inflammatory cytokines in hippocampus.

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Acute lung injury (ALI) leading to acute respiratory distress syndrome is a prevailing pathologic manifestation during O. tsutsugamushi-induced severe scrub typhus in humans. In this study, we tested a hypothesis that lung pathology in scrub typhus is due in part to dysregulated activation of inflammatory responses, leading to vascular malfunction and ALI. Following infection with a lethal dose of O. tsutsugamushi in mice, lung tissues had a significant increase in ICAM1, VEGFR2, and angiopoietin-2 (Ang2), as measured by flow cytometry and immunofluorescence. Several unique features were also observed. First, a progressive decrease or loss of endothelial functional markers (Ang1 and Tie2) and CD41+ platelets, even after the peak of bacterial replication (around day 6), implying continued endothelial cell stress and tissue damage. Second, a sustained neutrophil influx and activation as disease progressed, and lung-recruited neutrophils became highly activated in releasing azurophilic granules or myeloperoxidase around day 10 (prior to host death). Finally, lung-derived macrophages were highly polarized to an M1 phenotype (CD80+CD64+CD11b+Ly6G−) at day 6-day 10, with no signs of M2 activation (CD206+CD64+CD11b+Ly6G−). This study reveals specific biomarkers for vascular stress/dysfunction and uncovers a type 1-skewed, but type 2-suppressed, immune responses in the lungs of lethally-infected mice. More importantly, it furthers findings from human patients and cells, implying an immunopathogenic role in ALI development during severe scrub typhus. Understanding of leukocyte effector molecules and endothelial stress pathways triggered by bacterial vs. host factors will help control this neglected tropical disease.