image_1_Integrin αDβ2 (CD11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection.pdf

<p>β₂ integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin α_Dβ₂ in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of α_Dβ₂ results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in α_Dβ₂ had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of α_Dβ₂ in bacterial infection.</p

image_5_Integrin αDβ2 (CD11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection.pdf

<p>β₂ integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin α_Dβ₂ in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of α_Dβ₂ results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in α_Dβ₂ had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of α_Dβ₂ in bacterial infection.</p

image_2_Integrin αDβ2 (CD11d/CD18) Modulates Leukocyte Accumulation, Pathogen Clearance, and Pyroptosis in Experimental Salmonella Typhimurium Infection.pdf

<p>β₂ integrins are critical in host defense responses to invading pathogens and inflammation. Previously, we reported that genetic deficiency of integrin α_Dβ₂ in mice altered outcomes in experimental systemic infections including accelerated mortality in animals infected with Salmonella enterica serovar Typhimurium. Here, we show that deficiency of α_Dβ₂ results in impaired accumulation of leukocytes in response to peritoneal infection by S. Typhimurium, impaired pathogen clearance in vivo, defective bacterial elimination by cultured peritoneal macrophages, and enhanced pyroptosis, a cell death process triggered by Salmonella. Salmonella-infected animals deficient in α_Dβ₂ had increased levels of peritoneal cytokines in addition to other markers of pyroptosis, which may contribute to inflammatory injury and increased mortality in the context of impaired bacterial killing. These observations indicate important contributions of leukocyte integrins to the host response in experimental Salmonella infection and reveal previous activities of α_Dβ₂ in bacterial infection.</p

LPC activates JNK and p38, but not ERK, in macrophages.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of different concentrations of LPC mix (Sigma) for 20 min at 37 °C in a 5% CO₂ atmosphere, and the cytoplasm content was homogenized and assayed as follows. The Phospho-MAPK array was used for analysis of enzymatic activation (<b>A</b>). The reaction was visualized with the enhanced chemiluminescent system and subjected to densitometric analysis (***, p< 0.001, ANOVA). Protein levels of the phosphorylated MAPKs JNK (<b>B</b>), p38 (<b>C</b>) and ERK (<b>D</b>) were determined by Western blot. Data is the mean ± S.E. of two different experiments.</p

LPC triggers IL-8 production through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected and stimulated as described on Figure 1. After 20 hours of incubation, IL-8 production was measured by the ELISA assay. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC triggers NF-қB activation through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected in three different groups. Groups A and B received expression constructs for TLR4 (<b>A</b>) or TLR2 and TLR1 (<b>B</b>). Both also received MD-2, CD14, and CD36 constructs and the ELAM-1-firefly luciferase and β-actin-<i>Renilla</i> luciferase reporter plasmids. The third group (<b>C</b>) received only the empty vector pDisplay and the luciferase reporter plasmids. Groups A and B were separately stimulated with 0.1, 1, 10, 100 and 200 µM of different types of LPC (Sigma; C14:0, C16:0, C18:0, and C18:1), 100 ng/mL of LPS and 1 nM of Pam3CSK4 (P3C). Group C was stimulated with LPS, Pam3Cys or 0.1, 1, 10 and 100 µM of LPC (C16:0). The agonists were diluted in DMEM medium with 10% bovine fetal serum. After 4 h of incubation, luciferase activity was measured and expressed as the ratio of NF-қB-dependent firefly luciferase activity to the control <i>Renilla</i> luciferase activity. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits NF-қB translocation, iNOS expression, and NO production in LPS-stimulated macrophages.

<p>Peritoneal macrophages from BALB/cmice were incubated in the absence or presence of 1 µg/mL LPS and different concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere. After 1 h of incubation, NF-қB translocation (<b>A</b>) was assayed by Western blot analysis. After 24 hours, NO production (<b>B</b>) was assayed by measuring the amount of nitrite in the culture supernatant using the Griess reagent, and iNOS expression (<b>C</b>) was determined by Western blot analysis followed by densitometry (lower panel). Data is the mean ± S.D. of three different experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits LPS-induced ERK activation.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of 1 µg/mL LPS or in the presence or absence of the indicated concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere (<b>A</b>, <b>D</b>, <b>E</b>). In parallel HEK 293A cells with TLR constructs as indicated (<b>B</b>, <b>C</b>). Each group received expression constructs for TLR4 (<b>B</b>) or both TLR2 and TLR1 (<b>C</b>), as well as MD-2, CD14 and CD36 plasmids. The cells were then incubated in the absence or presence of 100 ng/mL LPS or 1 nM Pam3CSK4 (P3C) and 10 or 100 µM of LPC, for 40 min at 37 °C in a 5% CO₂ atmosphere. After incubation either macrophages or HEK cells were homogenized, the protein levels was determined and samples evaluated by Western blot with the use of antibodies against p-ERK (<b>A</b>, <b>B</b>, <b>C</b>), p-JNK (<b>D</b>) and p-P38 (<b>E</b>). Loading controls were run with the use of antibodies raised towards actin. Experiments were performed at least two times with different animals and samples.</p