Genetic analysis of potential regulators of HIF-1 in C. elegans

Oxygen is necessary for most life on our planet, and the ability to sense and adapt to changes in oxygen concentration is required for life to persist. Hypoxia is defined as the deficiency of oxygen reaching body tissues. In mammals, tissue response to hypoxia is integral to a wide variety of processes, including the building of vasculature, heart disease, and tumor angiogenesis. The transcriptional response to hypoxia is largely regulated by hypoxia inducible factors (HIFs). These HIFs are heterodimeric, consisting of an oxygen-sensing alpha subunit and a non-oxygen-sensing beta subunit. When oxygen levels are normal, HIF-1 alpha is hydroxylated by EGLN/PHD prolyl hydroxylases. This modification targets HIF-1alpha for degradation via the von Hippel Lindau tumor suppressor protein (VHL). Oxygen is an essential substrate for EGLN/PHD activity, and during hypoxia, the HIF-1 alpha subunit is not hydroxylated, and is, therefore, stable. HIF-1 alpha translocates to the nucleus, where it regulates the transcription of downstream genes. This pathway is evolutionarily conserved between mammals and Caenorhabditis elegans, where genes encode homologs of HIF-1 alpha, PHD/EGLN and VHL as HIF-1, EGL-9 and VHL-1, respectively. Working on C. elegans, our lab is attempting to find negative regulators of the HIF-1 pathway by performing a forward genetic screen and identifying mutations that cause increased expression of a HIF-1-responsive reporter gene. I have shown progress in mapping these potential positive regulator mutations and in attempting to prioritize mutations obtained in the screen by molecular analysis of various RNA transcripts and protein levels. In addition, I tested a hypothesis that a novel ubiquitin-degradation pathway that is independent of VHL-1 contributed to HIF-1 regulation

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

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation

Genetic analysis of potential regulators of HIF-1 in C. elegans

Oxygen is necessary for most life on our planet, and the ability to sense and adapt to changes in oxygen concentration is required for life to persist. Hypoxia is defined as the deficiency of oxygen reaching body tissues. In mammals, tissue response to hypoxia is integral to a wide variety of processes, including the building of vasculature, heart disease, and tumor angiogenesis. The transcriptional response to hypoxia is largely regulated by hypoxia inducible factors (HIFs). These HIFs are heterodimeric, consisting of an oxygen-sensing alpha subunit and a non-oxygen-sensing beta subunit. When oxygen levels are normal, HIF-1 alpha is hydroxylated by EGLN/PHD prolyl hydroxylases. This modification targets HIF-1alpha for degradation via the von Hippel Lindau tumor suppressor protein (VHL). Oxygen is an essential substrate for EGLN/PHD activity, and during hypoxia, the HIF-1 alpha subunit is not hydroxylated, and is, therefore, stable. HIF-1 alpha translocates to the nucleus, where it regulates the transcription of downstream genes. This pathway is evolutionarily conserved between mammals and Caenorhabditis elegans, where genes encode homologs of HIF-1 alpha, PHD/EGLN and VHL as HIF-1, EGL-9 and VHL-1, respectively. Working on C. elegans, our lab is attempting to find negative regulators of the HIF-1 pathway by performing a forward genetic screen and identifying mutations that cause increased expression of a HIF-1-responsive reporter gene. I have shown progress in mapping these potential positive regulator mutations and in attempting to prioritize mutations obtained in the screen by molecular analysis of various RNA transcripts and protein levels. In addition, I tested a hypothesis that a novel ubiquitin-degradation pathway that is independent of VHL-1 contributed to HIF-1 regulation.</p

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

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

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