Thrombospondin-1 Contributes to Mortality in Murine Sepsis through Effects on Innate Immunity

BACKGROUND:Thrombospondin-1 (TSP-1) is involved in many biological processes, including immune and tissue injury response, but its role in sepsis is unknown. Cell surface expression of TSP-1 on platelets is increased in sepsis and could activate the anti-inflammatory cytokine transforming growth factor beta (TGFβ1) affecting outcome. Because of these observations we sought to determine the importance of TSP-1 in sepsis. METHODOLOGY/PRINCIPAL FINDINGS:We performed studies on TSP-1 null and wild type (WT) C57BL/6J mice to determine the importance of TSP-1 in sepsis. We utilized the cecal ligation puncture (CLP) and intraperitoneal E. coli injection (i.p. E. coli) models of peritoneal sepsis. Additionally, bone-marrow-derived macrophages (BMMs) were used to determine phagocytic activity. TSP-1-/- animals experienced lower mortality than WT mice after CLP. Tissue and peritoneal lavage TGFβ1 levels were unchanged between animals of each genotype. In addition, there is no difference between the levels of major innate cytokines between the two groups of animals. PLF from WT mice contained a greater bacterial load than TSP-1-/- mice after CLP. The survival advantage for TSP-1-/- animals persisted when i.p. E. coli injections were performed. TSP-1-/- BMMs had increased phagocytic capacity compared to WT. CONCLUSIONS:TSP-1 deficiency was protective in two murine models of peritoneal sepsis, independent of TGFβ1 activation. Our studies suggest TSP-1 expression is associated with decreased phagocytosis and possibly bacterial clearance, leading to increased peritoneal inflammation and mortality in WT mice. These data support the contention that TSP-1 should be more fully explored in the human condition

Spontaneously opening GABA receptors play a significant role in neuronal signal filtering and integration

Acknowledgements This work was supported by The Rosetrees Trust Research Grant A1066, RS MacDonald Seedcorn Award and Wellcome Trust—UoE ISSF Award to S.S. The authors thank Prof. David Wyllie (University of Edinburgh) and Prof. Dmitri Rusakov (UCL) for their valuable suggestions on paper preparation.Peer reviewedPublisher PD

Environmental effects of ozone depletion, UV radiation and interactions with climate change : UNEP Environmental Effects Assessment Panel, update 2017

Peer reviewe

Thrombospondin-1–Deficient Mice Are Not Protected from Bleomycin-Induced Pulmonary Fibrosis

Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-β (TGF-β). Because active TGF-β is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1–deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1–deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-β. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF

Caspase-7 activation by the Nlrc4/Ipaf inflammasome restricts Legionella pneumophila infection.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4(-/-) (Ipaf(-/-)), and caspase-1(-/-) derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7(-/-) macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium

Important Roles for Macrophage Colony-stimulating Factor, CC Chemokine Ligand 2, and Mononuclear Phagocytes in the Pathogenesis of Pulmonary Fibrosis

Rationale: An increase in the number of mononuclear phagocytes in lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) worsens prognosis. Chemokines that recruit mononuclear phagocytes, such as CC chemokine ligand 2 (CCL2), are elevated in bronchoalveolar lavage (BAL) fluid (BALF) from patients with IPF. However, little attention is given to the role of the mononuclear phagocyte survival and recruitment factor, macrophage colony-stimulating factor (M-CSF), in pulmonary fibrosis

Serum pro-inflammatory cytokine analysis.

<p>Data represent median (25^th–75^th percentile). n = 9 for WT or TSP deficient animals at 12 hours after CLP; all pg/ml except HMGB1 in ng/ml.</p

TSP-1 deficient mice are resistant to surgical sepsis-induced mortality, and have a lower peritoneal bacterial load following induction of surgical peritonitis.

<p>WT and TSP-1−/− mice were subjected to either CLP or sham surgery as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019654#s4" target="_blank">Materials and Methods</a> on Day 0. A. Mice were monitored for a course of 7 days for survival (p<0.001 by Kaplan-Meier, n = 20 WT CLP, n = 20 TSP-1−/− CLP). All mice receiving sham surgeries survived until study end (7 days) (n = 5 WT sham, n = 6 TSP-1−/−sham). B. Mice underwent CLP surgery at hour 0 and were euthanized at various time points after surgery. Sterile peritoneal lavage was performed and colony-forming unit counts were performed on the lavage fluid from mice 3, 6, 12, and 24 hours after the CLP procedure. Data represents 3 mice per time point, and error bars are +/−SEM (*p<0.05, for 6, 12 and 24 hours after CLP, ANOVA). C. Spleens were harvested under sterile conditions from WT and TSP-1−/−mice 3 hours post-CLP. Organs were then crushed under sterile conditions. Data represents colony-forming unit counts from total organ protein of 3 mice per genotype and error bars are +/−SEM (*p = 0.04).</p

TSP-1 deficient and WT mice exhibit similar amounts of wound healing markers after CLP.

<p>CLP surgery was performed on WT and TSP-1−/− mice, and cecums were harvested 3, 6, and 12 hours afterwards and immediately processed for protein and RNA assessment [CTGF (A) and VEGF (B) and collagen I (E)]. [For all panels, WT (black bars) and TSP-1−/− (white bars); error bars represent ±SEM] A. Cecal CTGF transcript is shown for each timepoint after surgery. Data represents n = 6 for WT and TSP-1−/− at each timepoint. B. Cecal VEGF transcript is shown for each timepoint after surgery. Data represents n = 5 for 3 hours, n = 6 for 6 hours and n = 3 for 12 hours. C. Cecal protein lysates were assessed via ELISA for active TGFβ1. Data represents active TGFβ1 in 50 µg total protein/sample (n = 6). D. Cecums were harvested at 12 and 24 hours after CLP and collagen measured via Sircoll Assay. Data represents n = 3 for all timepoints. E. Cecal collagen I transcript is shown for each timepoint after surgery. Data represents n = 6 for background and timepoint. F. WT and TSP-1 −/− fibroblasts were compared in their ability to close a mechanical scratch <i>in vitro</i> by scratch closure assay. The mechanical defect was photographed hourly for 48 hours, and unclosed scratch area (“wound” size) was determined using Photoshop. Data points represent the “wound” size at each time point for each background [n = 4 for WT (black squares) and n = 5 for TSP-1−/− (white squares)].</p