Leukotriene B4 enhances innate immune defense against the puerperal sepsis agent Streptococcus pyogenes

Abstract

Puerperal sepsis is a leading cause of maternal mortality worldwide. Streptococcus pyogenes [group A Streptococcus; (GAS)] is a major etiologic agent of severe postpartum sepsis, yet little is known regarding the pathogenesis of these infections. Tissue macrophages provide innate defense against GAS, and their actions are highly regulated. The intracellular second messenger cAMP can negatively regulate macrophage actions against GAS. Because leukotriene (LT) B(4) has been shown to suppress intracellular cAMP in macrophages, we hypothesized that it could enhance innate defenses against GAS. We assessed the capacity of LTB(4) to modulate antistreptococcal actions of human macrophages, including placental and decidual macrophages and used a novel intrauterine infection model of GAS in mice lacking the 5-lipoxygenase enzyme to determine the role of endogenous LTs in host defense against this pathogen. Animals lacking 5-lipoxygenase were significantly more vulnerable to intrauterine GAS infection than were wild-type mice and showed enhanced dissemination of bacteria out of the uterus and a more robust inflammatory response than did wild-type mice. In addition, LTB(4) reduced intracellular cAMP levels via the BLT1 receptor and was a potent stimulant of macrophage phagocytosis and NADPH oxidase-dependent intracellular killing of GAS. Importantly, interference was observed between the macrophage immunomodulatory actions of LTB(4) and the cAMP-inducing lipid PGE(2), suggesting that interplay between pro- and anti-inflammatory compounds may be important in vivo. This work underscores the potential for pharmacological targeting of lipid mediator signaling cascades in the treatment of invasive GAS infections.

Figure 1. Leukotrienes are important determinants of survival and containment of intrauterine GAS infection in mice

(A) Wild-type C57BL/6 or 5LO^−/− mice(n = 10 per group) were inoculated with 10⁴ CFU of GAS in the right uterine horn on day 0 and survival was recorded over time. Vehicle (PBS)-treated mice did not die (n = 10; data not shown). Data are expressed as mean ± SEM followed by Log-rank (Mantel-Cox) Test; ***P < 0.001. (B) Nonpregnant mice C57BL/6 or 5LO^−/− (n = 10 per group) were inoculated with 10⁴ CFU of GAS in the right uterine horn and then euthanized 24 h after infection. Bacterial loads from blood, spleen and uterus were determined as described in Materials and Methods. Data shown are from one representative experiment from two independent experiments with similar results. Data are expressed as mean ± SEM followed by unpaired Student t test; *P < 0.05. (C) After 24 h infection, enzyme immunoassay quantification of LTB₄ concentrations were performed on uterine homogenates obtained from mice that had received either an intrauterine PBS injection (uninfected) or GAS. Data are presented as the mean ± SEM followed by one-way ANOVA and are representative of two independent experiments with similar results (n = 10 mice per group). ***P<0.001 compared with wild-type uninfected control. (D) Inflammatory mediators in uterine homogenates of C57BL/6 or 5LO^−/− mice 24 h after inoculation with 10⁴ CFU of GAS. 24 h after intrauterine inoculation with GAS cytokines and chemokines were measured by ELISA.***P < 0.001 by Student t test (n = 10 mice per group from 2 combined experiments with similar results). IL, interleukin; MCP, monocyte chemotactic protein-1.

Figure 2. LTB₄ increases leukocyte phagocytosis of GAS

Macrophages were exposed for 15 min to LTB₄ before challenge with FITC-labeled HK GAS and phagocytosis was determined as described in Materials and Methods. Data shown are from (A) PMA-treated human THP-1 cells, (B) human peripheral blood monocytes, (C) human decidual macrophages, (D) human placental macrophages, and (E) C57BL/6 mouse peritoneal macrophages. All data are expressed as a percentage of the phagocytic activity of untreated cells. *P < 0.05, **P<0.01 vs untreated as determined by one-way ANOVA for A-D and t-test for E.

Figure 3. LTB₄ enhances GAS phagocytosis in THP-1 cells via the BLT-1 receptor

(A) PMA-treated THP-1 cells were incubated with the BLT1 receptor antagonist U75302 (10 μM) or LTB₄ (10 nM) for 15 min before challenge with FITC-labeled HK GAS as described in Materials and Methods. Data shown are mean ± SEM of four independent experiments. (B) PMA-treated THP-1 cells were incubated with 0.001–1 μM of BLT2 agonist (12-HHT) for 15 min before challenge with HK FITC-GAS at 150:1. Phagocytosis was quantified after 3h by fluorometry as detailed in Materials and Methods. *P<0.05 vs untreated and ^#P<0.05 vs LTB₄ as determined by one-way ANOVA.

Figure 4. Enhancement of phagocytosis by LTB₄ depends on Gα_i-receptor activation

(A) PMA-treated THP-1 cells were cultured for 0, 15, 60, 300, 600 and 900 sec with LTB₄ (10 nM) and intracellular cAMP was measure by ELISA. Mean +/− SEM data from five independent experiments were combined with n = 3 per each condition per experiment. ***P<0.001 (B) PMA-treated THP-1 cells were cultured overnight with 3 μg/mL of PTX and the 18 h later the cells were incubated for 15 min with LTB₄ (10 nM) before GAS phagocytosis was quantified as detailed in Materials and Methods. *P<0.05 vs untreated cells. (C) FLUO-4NM-loaded, PMA-treated THP-1 cells were exposed to LTB₄ (10 nM) and A23187 (1μM) and changes in intracellular Ca²⁺ were measured as described in Materials and Methods. Data are expressed as a percentage of relative to untreated cells. *P<0.05 vs untreated cells. .

Figure 5. LTB₄ enhances macrophage killing of GAS through activation of reactive oxygen species production

(A) PMA-treated THP-1 cells were exposed to apocynin (100 μM) for 30 min and/or LTB₄ (10 nM) for 15 min prior to inoculation with live unopsonized GAS (10 bacteria per cell). Phagocytosis was determined as detailed in Materials and Methods, after 30 min THP-1 cells were washed to remove extracellular bacteria and lysed to release intracellular bacteria, which were enumerated on solid agar. Data are expressed as the percent of bacterial CFU relative to the untreated control and are presented as the mean ± SEM of five independent experiments. *P< 0.05 compared to untreated control as determined by one-way ANOVA. (B) PMA-treated THP-1 cells were incubated with H2DCFDA (10 μM) for 30 min followed by LTB₄ (10 nM) and/or live unopsonized GAS (10 bacteria per cell) for another 30 min and H₂O₂ production was measured as described. Data are expressed relative to untreated cells and display the mean ± SEM of eight independent experiments. *P< 0.05 compared to untreated cells; ^#P<0.05 compared to LTB₄ as determined by one-way ANOVA.

Figure 6. Effects of exogenous PGE₂ and LTB₄ on phagocytosis and cAMP production in macrophages

(A) PMA-treated THP-1 cells were exposed for 1 min to LTB₄ (10 nM) or vehicle followed by PGE₂ (1 μM) for 3 min. Intracellular cAMP was determined as described in the text. Data are expressed as the mean ± SEM from seven independent experiments. ***P<0.001; **P<0.01 compared to untreated cells; #P<0.001 vs. PGE₂ treatment. (B) PMA-treated THP-1 cells were cultured with LTB₄ (100 nM) and PGE₂ (10 μM) for 15 min before challenge with HK FITC-GAS. Phagocytosis was quantified after 3 h as detailed in Materials and Methods. Data are expressed as the mean ± SEM relative to untreated cells for four independent experiments. ** P<0.01 compared to untreated and #P<0.0001 compared to PGE₂ as determined by one-way ANOVA.

Figure 7. Summary of immunoregulation of macrophages by LTB₄ during Streptococcus pyogenes infection

Ligation of BLT1 by LTB₄ results in the activation of Gα_i protein, with a subsequent decrease in intracellular cAMP levels. This, in turn, limits the activation of protein kinase A (PKA), an endogenous suppressor of both phagocytosis and NADPHox-dependent reactive oxygen intermediate (ROI) generation. These actions oppose those of the endogenous lipid mediator prostaglandin E₂ (PGE₂), which stimulates cAMP production via EP2 and/or EP4 receptors in macrophages. Compounds used in our studies to modulate signaling are shown. AC, adenylate cyclase; APY, apocynin; PTX, pertussis toxin.