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

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

Mice containing functional TSP-1 protein have decreased phagocytic capacity as compared to mice lacking functional TSP-1, and show increased survival in a non-surgical model of sepsis.

<p>BMMs were co-incubated with IgG-opsonized, labeled sheep red blood cells and phagocytic index was determined via blinded counting of ingested red blood cells per 100 macrophages. Phase contrast images of BMMs overlayed with images of SRBCs ingested are shown for WT mice (A) and TSP-1−/− mice (B). C. The quantification of phagocytic capacity is indicated by phagocytic index. Images and Data represent n = 3 for WT <u>animals </u>and n = 3 for TSP-1−/− <u>animals</u>, and error bars are +/– SEM (*p<0.01 by student's t test). D. WT and TSP-1−/− mice were subjected to either <i>E.coli</i> or PBS injection 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. Mice were monitored for a course of 7 days for survival (p<0.001 by Kaplan-Meier, n = 40 WT CLP, n = 40 TSP-1−/− <i>e.coli</i> injection). All mice receiving sham surgeries lived the course of 7 days (n = 5 WT PBS, n = 5 TSP-1−/− PBS).</p

Peritoneal lavage fluid cell counts after CLP.

<p>Data represent mean ± SEM. n = 3 for WT at 3,6,12 hours, n = 3 for TSP-1−/− at 3,6,12 hours.</p