Uncoupling scavenger receptor A-mediated phagocytosis of bacteria from endotoxic shock resistance

Abstract

Unresolved infection by gram-negative bacteria can result in the potentially lethal condition known as endotoxic shock, whereby uncontrolled inflammation can lead to multiple organ failure and death of the infected host. Previous results have demonstrated that animals deficient in class A scavenger receptor (SRA), a trafficking receptor for bacteria and bacterium-derived molecules, are more susceptible to endotoxic shock. This has been proposed to be a result of impaired SRA-dependent phagocytic clearance of bacteria resulting in stronger proinflammatory stimuli. In this report, we test the hypothesis that there is an obligate reciprocal relationship between SRA-mediated phagocytosis of bacteria and susceptibility to endotoxic shock. Here, we demonstrate that both SRA-dependent and -independent gram-negative bacterial strains elicit SRA-dependent increased cytokine production in vitro and in vivo and increased susceptibility to endotoxic shock in SRA-deficient mice. This is the first evidence showing that SRA-mediated clearance of LPS is functionally distinct from the role of SRA in bacterial phagocytosis and is a formal demonstration that the SRA-dependent cytokine responses and the resultant endotoxic shock are not coupled to SRA-mediated clearance of bacteria.

FIG. 1.

SRA^+/− BMDCs exhibit elevated cytokine production in response to E. coli DH5α. A total of 5 × 10⁵ WT, SRA^+/−, TLR4^+/−, SRA^+/− TLR4^+/−, SRA^−/−, or SRA^−/− TLR4^−/− BMDCs were cocultured with 5 × 10⁵ DH5α bacteria for 6 hours. Cell supernatants were collected and analyzed by ELISA for TNF-α (A) or IL-6 (B) production. n ≥ 6 for all genotypes; means and standard deviations are shown. Statistical significance (P ≤ 0.05) for values higher than WT (*) and lower than WT (**) is indicated.

FIG. 2.

SRA-independent gram-negative bacteria elicit increased cytokines in vitro in the absence of SRA. (A) WT and SRA^−/− BMDCs were assessed for relative phagocytosis of E. coli DH5α and K-12 and P. aeruginosa PA14 bacteria by gentamicin protection assay. Data are normalized as the percentage CFU of WT bacterial uptake. n ≥ 9 for all groups. (B and C) A total of 5 × 10⁵ WT, SRA^+/−, or SRA^−/− BMDCs were cocultured with 5 × 10⁵ CFU of the indicated bacteria, or LPS as a positive control, for 6 hours. Supernatants from these cultures were collected and analyzed by ELISA for TNF-α (B) and IL-6 (C). n ≥ 9; means and standard deviations are shown. Statistical significance (P ≤ 0.05) from WT values is indicated (*).

FIG. 3.

SRA dependence for phagocytosis varies among different bacterial strains in vivo. (A) WT, SRA^+/−, or SRA^−/− mice were injected i.p. with 5 × 10⁶ live DH5α E. coli cells. Peritoneal phagocytes were subsequently harvested by lavage, and relative phagocytosis was assessed by gentamicin protection assay. SRA^−/− mice were found to be significantly impaired in phagocytosis of DH5α (P ≤ 0.01) from both WT and SRA^+/−mice. (B and C) No statistically significant difference was observed between WT and SRA^−/− mice injected with 5 × 10⁶ live K-12 E. coli (B) or PA14 P. aeruginosa (C) bacteria. (D) Total cell harvests from peritoneal lavage samples and total macrophage numbers were quantified. No statistical differences among the groups were found. For phagocytosis assays, data are normalized as the percentage CFU of WT bacterial uptake. n ≥ 8 for all groups. The mean and individual mouse values are represented on the graphs. Statistical significance (P ≤ 0.05) from WT values is indicated (**).

FIG. 4.

SRA^−/− mice produce increased cytokines in vivo to both SRA-dependent and -independent bacteria. WT and SRA^−/− mice were injected i.p. with 1 × 10⁹ live DH5α bacteria (A), 1 × 10⁹ live K-12 bacteria (B), or 5 × 10⁷ live PA14 bacteria (C). After 3 hours, serum and peritoneal lavage samples were collected and analyzed by ELISA for TNF-α levels. n ≥ 4; means and standard deviations are shown. Statistical significance (P ≤ 0.05) from WT values is indicated (*).

FIG. 5.

SRA^−/− mice are more susceptible to lethal endotoxic shock induced by both SRA-dependent and -independent bacteria. (A) As a positive control, WT and SRA^−/− mice were challenged i.p. with 400 μg LPS. (B to D) Survival plots of WT and SRA^−/− mice challenged i.p. with 1 × 10⁹ CFU live DH5α bacteria (B), 1 × 10⁹ CFU live K-12 bacteria (C), or 1 × 10⁶ CFU live PA14 bacteria (D). For all graphs, n ≥ 9. Under every condition, SRA^−/− mice were found to be significantly more susceptible to the indicated challenge than WT mice. Statistical significance (P ≤ 0.05) from WT values is indicated (*).

FIG. 6.

High-dose bacterial challenge-induced lethality is dependent on TLR4 and MyD88 expression. Survival plot of WT, SRA^−/−TLR4^−/−, and MyD88^−/− mice challenged i.p. with 2 × 10⁹ CFU live DH5α bacteria. At lethal doses for WT animals, SRA^−/−TLR4^−/− and MyD88^−/− mice were completely resistant to endotoxic shock. n ≥ 4. Statistical significance (P ≤ 0.05) from WT values is indicated (*).