Coxiella burnetii lipopolysaccharide blocks p38α-MAPK activation through the disruption of TLR-2 and TLR-4 association

Abstract

To survive in macrophages, Coxiella burnetii hijacks the activation pathway of macrophages. Recently, we have demonstrated that C. burnetii, via its lipopolysaccharide (LPS), avoids the activation of p38α-MAPK through an antagonistic engagement of Toll-like receptor (TLR)-4. We investigated the fine-tuned mechanism leading to the absence of activation of the p38α-MAPK despite TLR-4 engagement. In macrophages challenged with LPS from the avirulent variants of C. burnetii, TLR-4 and TLR-2 co-immunoprecipitated. This association was absent in cells challenged by the LPS of pathogenic C. burnetii. The disruption makes TLRs unable to signal during the recognition of the LPS of pathogenic C. burnetii. The disruption of TLR-2 and TLR-4 was induced by the re-organization of the macrophage cytoskeleton by C. burnetii LPS. Interestingly, blocking the actin cytoskeleton re-organization relieved the disruption of the association TLR-2/TLR-4 by pathogenic C. burnetii and rescued the p38α-MAPK activation by C. burnetii. We elucidated an unexpected mechanism allowing pathogenic C. burnetii to avoid macrophage activation by the disruption of the TLR-2 and TLR-4 association.

Keywords: Coxiella burnetii; TLR-2; TLR-4; cytoskeleton; macrophages.

Figure 1

Activation of p38α MAPKs. BMDMs from wild type mice, were challenged with C. burnetii vLPS and avLPS (1 μg/ml) for different periods (up to 120 min). The phosphorylation of p38α MAPK was determined using phospho-p38α MAPK cell-based ELISA. The results are expressed as normalized RFU and represent the mean ± SD (n = 3).

Figure 2

TLR-2 and TLR-4 distribution and colocalization. BMDMs from wild type mice were challenged for 5 min with C. burnetii LPS (1 μg/ml). The distribution of (A) TLR-2 and (B) TLR-4 at the BMDMs surface was determined by confocal microscopy. The scale bar indicates 5 μm. The number of TLRs signal detected (C) and the area (D) were quantified using ImageJ software. The results are expressed as the mean ± SD (n = 3, ^*p < 0.05). (E) The colocalization of TLR-2 with TLR-4 was determined using confocal microscopy. The colocalization of TLR-2 with TLR-4 was quantified using ImageJ software. The results are expressed as the mean ± SD (n = 3, ^*p < 0.05). The scale bar indicates 5 μm. (F) BMDMs in non-starved conditions were either left untreated or treated with vLPS or avLPS (1 μg/ml) for 5 min, then TLR-2 was immunoprecipitated and coimmunoprecipitated with TLR-4 was visualized by immunoblotting. The blot shown is representative of three experiments.

Figure 3

Cytoskeleton remodeling induced by C. burnetii LPS impairs TLRs signaling. BMDMs were challenged with (A) vLPSs or (B) avLPS at 1 μg/ml for 5 min, then F-actin was labeled with phalloidin alexa-488. Macrophages were examined by confocal microscopy. Representative cells are shown, the scale bar indicates 5 μm. (C) The percentage of BMDMs showing filopodia was evaluated. For some experiments macrophages were treated with cytochalasin-D. The results are expressed as the mean ± SD (n = 3 ^*p < 0.05). (D) BMDMs in non-starved conditions were either left untreated or treated with vLPS (1 μg/ml) for 5 min in presence or not of cytochalasin-D, then TLR-2 was immunoprecipitated and coimmunoprecipitated with TLR-4 was visualized by immunoblotting. The blot shown is representative of three experiments. (E) BMDMs from wild type mice were challenged with vLPS (1 μg/ml) for different periods (min) in presence or not of cytochalasin-D, and the phosphorylation of p38α MAPK was determined using phospho-p38α MAPK cell-based ELISA. The results are expressed as normalized RFU and represent the mean ± SD (n = 3).