Lack of PPARγ in Myeloid Cells Confers Resistance to Listeria monocytogenes Infection

The peroxisomal proliferator-activated receptor γ (PPARγ) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPARγ. The relevance of PPARγ in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPARγ. This prompted us to investigate whether PPARγ in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPARγ (LysMCre×PPARγflox/flox). Loss of PPARγ in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFNγ TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPARγ enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6Chi monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPARγ was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPARγ and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPARγ activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection

Enhanced effector functions following abelation of PPARγ from myeloid cells.

<p>(A) BMDM from LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected <i>in vitro</i> with <i>L.monocytogenes</i> at a MOI 10. Expression of IFNγ, TNF, IL-1β, IL-6 and IL-12 was determined 6 hrs post infection in cell culture supernatant by ELISA. The data shown are means ± SE of three independent experiments. (B) Looking at the paracrine effects of PPARγ ablation in myeloid cells, we infected BMDM from wild type C57BL/6 mice with <i>Listeria</i> at an MOI of 10 and incubated them with sterile filtered conditioned medium from PPARγ^WT or PPARγ^KO macrophages infected previously for 18 hrs with <i>L. monocytogenes</i>; at the indicated time points intracellular <i>Listeria</i> growth was determined as CFU/ml in the lysates of infected cells. Experiments were performed in triplicates. One representative out of four experiments is shown. The <i>p</i> value for titers of wt vs. ko at 4 hrs post infection were <0.05.</p

Enhanced recruitment of inflammatory monocytes to the site of infection in LysM-PPARγ^KO mice.

<p>LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected i.p. with <i>Listeria</i> (2×10⁴ CFU). (A) at indicated time points total numbers of CD11b⁺ Ly6C^high monocytes in the peritoneal cavity (PEC), liver and spleen were determined by FACS analysis (B–C) Expression of CCL2, CCL7 and CCR2 in the liver (B) and spleen (C) of infected mice as determined by qRT-PCR. (D) LSEC isolated from wild type C57BL/6 mice were incubated <i>in vitro</i> with sterile filtered supernatant peritoneal macrophages from LysM- PPARγ^WT and LysM- PPARγ^KO mice that were either infected (18 hrs) or left non-infected. The fold increase in expression of CD54 on the surface of LSEC determined by increase in mean fluorescence intensity by flow cytometry in response to contact with supernatant from infected vs non-infected peritoneal macrophages was determined after 24 hrs.</p

Enhanced resistance of LysM- PPARγ^KO mice to lethal infection with <i>Listeria monocytogenes</i>.

<p>(A) Survival of LysM- PPARγ^KO mice and LysM- PPARγ^WT littermates after intraperitoneal infection with 2×10⁴ (LD 50) 5×10⁵ (LD 80), 2×10⁶ (100× LD50) CFU of <i>Listeria</i>. (n = 10, one representative out of three experiments is shown ). LD 50: p = 0.04; LD 80; 0.01; 100× LD50: not significant difference. (B, C) Colony forming units of <i>Listeria monocytogenes</i> in liver (B) and spleen (C) of LysM- PPARγ^WT or LysM- PPARγ^KO mice infected i.p. with 2×10⁴ CFU. (n = 9); significance ** p = 0.005.</p

PPARγ expression is induced in bone-marrow derived monocytes after infection with <i>Listeria monocytogenes</i>.

<p>(A) Time course of PPARγ expression in human monocytes following <i>Listeria monocytogenes</i> infection detected by Western blot. (B) PPARγ target genes (n = 80) with the most significant differential expression in human macrophages post infection. Gene expression differences (log scale) are visualized as a heat map following hierarchical clustering of rows and columns (red = increased expression). (C,D) Time course of PPARγ expression in bone marrow derived macrophages (BMDM) post infection detected by Western blot or immunohistochemistry. (blue = DAPI staining the cell nucleus and <i>Listeria</i> DNA; green = PPARγ).</p

Similar phagocytic capacity in myeloid cells from LysM- PPARγ^WT and LysM- PPARγ^KO mice.

<p>Bone marrow derived macrophages (BMDM) and peritoneal exudates cells (PEC) from LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected with FITC-labelled <i>Listeria</i> at MOI 2 or MOI 5). (A) Mean fluorescence intensity (MFI) of FITC positive cells was assessed by flow cytometry. and (B) Percentage of <i>Listeria</i> containing macrophages (FITC positive). (C) BMDM or PEC from LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected with <i>Listeria</i> (MOI 10) and intracellular <i>Listeria</i> growth was determined as CFU/ml in the lysates of infected cells. The <i>p</i> values for titres of WT vs. KO at 4 hrs p.i. were <0.05 and <0.01 for BMDM and PEC, respectively. (D–F) BMDM from LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected with <i>Listeria</i> (MOI of 10). The capacity to produce reactive oxygen species (ROS) in <i>Listeria</i> infected macrophages was determined by the OxyBURST reagent (D). At 24 hrs p.i. the concentration of NO2- as surrogate marker for the production of reactive nitrogen intermediates was determined with Griess reagent (E). Expression of iNOS was analyzed by quantitative RT-PCR (F).</p

Enhanced expression of inflammatory cytokines in LysM-PPARγ^KO mice during infection with <i>Listeria in vivo</i>.

<p>(A and B) LysM- PPARγ^WT and LysM- PPARγ^KO mice were infected i.p. with 2×10⁴ CFU of <i>Listeria</i>. At indicated time points, expression of IFNγ, TNF, IL-1β, IL-6 and IL-12 was determined in the liver (A) and spleen (B) of mice (n = 5) by qRT-PCR. The data shown are means ± SE of three independent experiments.</p