Lipid mediators in innate immunity against tuberculosis: opposing roles of PGE2 and LXA4 in the induction of macrophage death

Abstract

Virulent Mycobacterium tuberculosis (Mtb) induces a maladaptive cytolytic death modality, necrosis, which is advantageous for the pathogen. We report that necrosis of macrophages infected with the virulent Mtb strains H37Rv and Erdmann depends on predominant LXA(4) production that is part of the antiinflammatory and inflammation-resolving action induced by Mtb. Infection of macrophages with the avirulent H37Ra triggers production of high levels of the prostanoid PGE(2), which promotes protection against mitochondrial inner membrane perturbation and necrosis. In contrast to H37Ra infection, PGE(2) production is significantly reduced in H37Rv-infected macrophages. PGE(2) acts by engaging the PGE(2) receptor EP2, which induces cyclic AMP production and protein kinase A activation. To verify a role for PGE(2) in control of bacterial growth, we show that infection of prostaglandin E synthase (PGES)(-/-) macrophages in vitro with H37Rv resulted in significantly higher bacterial burden compared with wild-type macrophages. More importantly, PGES(-/-) mice harbor significantly higher Mtb lung burden 5 wk after low-dose aerosol infection with virulent Mtb. These in vitro and in vivo data indicate that PGE(2) plays a critical role in inhibition of Mtb replication.

Figure 1.

LXA₄ and COX2 production of human Mφ infected with H37Rv and H37Ra. (A) LC-MS-MS of endogenous PGE₂ (top) and LXA₄ (bottom) produced by H37Ra- (MOI 10) and H37Rv- (MOI 10) infected human Mφ, respectively, at 24 h. The spectrum is a representative LC-MS-MS (n = 3). The prominent ions and relative intensity matched with authentic PGE₂ and LXA₄ under these LC-MS-MS conditions. (B) LXA₄ production in human Mφ infected with H37Rv and H37Ra (MOI 10:1) at 0–48 h. Differences in LXA₄ concentrations in supernatants from H37Ra- and H37Rv-infected Mφ are statistically significant (*, P < 0.002; n = 3). (C) LXA₄ accumulation at 48 h in Mφ supernatants infected with H37Ra and H37Rv (MOI 2, 5, and 10:1). The differences in LXA₄ production induced by H37Ra and H37Rv are statistically significant at all MOIs (P < 0.01; n = 3). (D) COX2 mRNA accumulation in Mφ infected with H37Ra or H37Rv (MOI 10:1; uninfect, uninfected). (E) Production of COX2 protein in Mφ infected with H37Ra and H37Rv (MOI 10:1) at different time points. In all studies, n represents the number of independent experiments and the error bars represent SE. Black lines indicate that intervening lanes have been spliced out.

Figure 2.

Prostanoid production of Mφ infected with Mtb. (A) PGE₂ production of human Mφ 0–48 h after infection with H37Ra and H37Rv (MOI 10:1). Differences in PGE₂ concentrations in supernatants from H37Ra- and H37Rv-infected Mφ are statistically significant (*, P < 0.002; n = 3). (B) PGE₂ production by Mφ infected with H37Ra and H37Rv (MOI 2:1–10:1). The differences in PGE₂ production induced by H37Ra in comparison to H37Rv are statistically significant at all MOIs (*, P < 0.01; n = 3). (C) Quantification of PGE₂, PGF_2α, and PGD₂ in 48-h supernatants from human Mφ (5 × 10⁶ /ml) infected with H37Ra and H37Rv (MOI 10:1; *, P < 0.002; n = 4). Data are presented as means ± SE. In all studies, n represents the number of independent experiments.

Figure 3.

Effect of exogenous prostanoids produced by Mtb-infected Mφ on H37Rv-induced mitochondrial cationic dye release. (A) PGE₂ blocks DiOC₆(3) release from mitochondria of human Mφ infected with H37Rv (MOI 5:1; 48 h). A fluorescence-activated cell sorter diagram is shown. (B) Down-regulation of cationic dye release by increasing concentrations of PGE₂ (2–10 μM) is statistically significant at all PGE₂ concentrations (P < 0.01; n = 6). (C) Effect of 1 μM of various prostanoids on H37Rv-induced mitochondrial DiOC₆(3) release of Mφ. Only PGE₂ inhibition of cationic dye release from the mitochondria is statistically significant (*, P < 0.007; n = 3). PGF_2α has borderline activity. The concentration of LTB₄ is 0.1 μM. (D) H37Ra (MOI 5:1)-induced DiOC₆(3) release from mitochondria of WT and PGES^−/− mouse Mφ infected in the absence and presence of 1 μM PGE₂. At 48 h, cationic dye release from the mitochondria was measured (*, P < 0.006; n = 5). (E) 1 μM PGE₂ does not alter H37Ra- and H37Rv-induced cytochrome c release from the mitochondria (not significant; n = 3). Data are presented as means ± SE. In all studies, n represents the number of independent experiments.

Figure 4.

Effect of LXA₄ on Mtb-infected Mφ. (A, left) addition of 10⁻⁹ M LXA₄ to H37Ra-infected human Mφ (MOI 10:1) enhances mitochondrial cationic dye release (*, statistically significant difference; n = 3; P = 0.04). (A, right) LXA₄ by itself is ineffective. Addition of 10⁻⁹ and 10⁻¹⁰ M LXA₄ to PGES^−/− mouse Mφ infected with H37Ra (MOI 5:1) does not affect mitochondrial cationic dye release (not significant; n = 3). (B, left) Production of COX2 protein by H37Ra (MOI 10:1)-infected Mφ is inhibited by the addition of 10⁻⁹ M LXA₄. Western analysis of Mφ extracts. The actions of LXA₄ were mimicked by its metabolic stable analogue (not depicted). (B, right) PGE₂ production by Mφ (*, P < 0.001; n = 3). Black lines indicate that intervening lanes have been spliced out. (C and D) Targeted silencing (t) of the 5-LO gene (C, ∼70% inhibition at t 50) abrogates production of LXA₄ by H37Rv-infected Mφ compared with nontargeted (nt) silencing (D, left, 50 nM siRNA; P = 0.001; n = 3) and reduces mitochondrial cationic dye release after H37Rv infection (D, right; nt, nontargeted; P < 0.04; n = 3). Black lines indicate that intervening lanes have been spliced out. (E) Targeted silencing of the 5-LO gene reconstitutes production of PGE₂ after infection of Mφ with H37Rv, indicating that block of PGE₂ production is caused by the action of LXA₄ (P < 0.003; n = 3). Data are presented as means ± SE. In all studies, n represents the number of independent experiments (*, statistically significant).

Figure 5.

EP2 mediates PGE₂-dependent protection against cationic mitochondrial dye release. Data are presented as means ± SE. (A) EP1, EP2, EP3, and EP4 are constitutively expressed in human Mφ. Their expression is not increased by either H37Ra or H37Rv infection. (B) EP2^−/− Mφ fail to respond to PGE₂ by down-regulating DiCO₆(3) release from the mitochondria infected with H37Rv (top) or with H37Ra (bottom), indicating that EP2 mediates the protective function of PGE₂. Mφ from EP1, EP3, and EP4 ^−/− mice were equally responsive to 1 μM PGE₂ (*, statistically significant; P < 0.01; n = 5). (C) The cAMP-dependent PKA inhibitor KT5720 abrogates inhibition of mitochondrial cationic dye release by PGE₂ (black columns; P < 0.01; n = 3). Addition of KT5720 to H37Ra-infected (MOI 10:1) Mφ enhanced Mφ necrosis (gray columns; *, P < 0.01; n = 3). (D) The PI3K inhibitor LY294002 does not abrogate inhibition of mitochondrial cationic dye release by PGE₂ (not significant; n = 3). In all studies, n represents the number of independent experiments.

Figure 6.

Mycobacterial burden of PGES^−/− and WT Mφ in vitro and in vivo. (A) Mycobacterial burden after 4 h (inoculum), 4 d, and 7 d of PGES^−/− and WT Mφ in vitro infected with H37Rv (MOI 10:1). The difference in the bacterial burden was significant at 4 and 7 d after infection (*, P < 0.03). (B) 5 wk after aerosol infection, CFUs were determined by plating of homogenized lung tissue as indicated on the ordinate. The difference in mycobacterial burden in the lungs of PGES^−/− versus WT mice is statistically significant (*, P = 0.002; five mice per group). (C and D) Induction of LXA₄ and PGE₂ production during the course of mycobacterial infection. WT mice were infected by aerosol exposure with virulent (Erdmann) or avirulent (H37Ra). LXA₄ (C) and PGE₂ (D) measured by ELISA in the sera at 7, 14, and 35 d after infection (*, P < 0.01; three mice per time points). These results are representative of two independent experiments. The error bars represent SE.

Figure 7.

Infection of Mφ with the virulent H37Rv predominantly induces LXA₄ production and block of COX2 and PGE₂ production, which might lead to necrotic cell lysis and spread of the infection. In contrast, Mφ infected with avirulent H37Ra produce larger amounts of PGE₂ which blocks LXA₄ production and supports Mφ apoptosis and containment of the Mtb. Predominant production of either PGE₂ or LXA₄ by Mφ infected with H37Ra or H37Rv, respectively, is supported by the fact that PGE₂ inhibits LXA₄ production and vice versa.