Mycobacteria Manipulate G-Protein-Coupled Receptors to Increase Mucosal Rac1 Expression in the Lungs

Abstract

Mycobacterium bovis bacille Calmette-Guérin (BCG) is currently the only approved vaccine against tuberculosis (TB). BCG mimics M. tuberculosis (Mtb) in its persistence in the body and is used as a benchmark to compare new vaccine candidates. BCG was originally designed for mucosal vaccination, but comprehensive knowledge about its interaction with epithelium is currently lacking. We used primary airway epithelial cells (AECs) and a murine model to investigate the initial events of mucosal BCG interactions. Furthermore, we analysed the impact of the G-protein-coupled receptors (GPCRs), CXCR1 and CXCR2, in this process, as these receptors were previously shown to be important during TB infection. BCG infection of AECs induced GPCR-dependent Rac1 up-regulation, resulting in actin redistribution. The altered distribution of the actin cytoskeleton involved the MAPK signalling pathway. Blocking of the CXCR1 or CXCR2 prior to infection decreased Rac1 expression, and increased epithelial transcriptional activity and epithelial cytokine production. BCG infection did not result in epithelial cell death as measured by p53 phosphorylation and annexin. This study demonstrated that BCG infection of AECs manipulated the GPCRs to suppress epithelial signalling pathways. Future vaccine strategies could thus be improved by targeting GPCRs.

Keywords: Bacille Calmette-Guérin; G-protein-coupled receptors; Pulmonary tuberculosis.

Fig. 1

Mycobacteria increase Rac1 expression. The impact of BCG infection, CXCR1 and CXCR2 on modulated epithelial Rac1 expression was studied in a murine model and by Western blot. a, b Mycobacterial infection significantly altered Rac1 expression in vivo as detected by immunofluorescence microscopy compared to uninfected mice. c, d BCG infection increased epithelial Rac1 expression, but CXCR1/2 blocking prior to infection decreased Rac1. Data are presented as representative images or as mean ± SEM of 3 separate experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig. 2

Mycobacteria up-regulate actin distribution. a, b BCG-induced increased actin expression was detected in BCG-infected AECs. CXCR2 blocking or the addition of Rac1 inhibitor prior to infection decreased the actin expression. c In vivo, the infection increased actin distribution in the BCG-infected mice compared to the uninfected controls, visualised by immunofluorescence microscopy. d The images were further analysed by ImageJ software. Data are presented as representative images or as mean ± SEM of 3 separate experiments. ** p < 0.01; *** p < 0.001.

Fig. 3

Mycobacteria activate the epithelial MAPK pathway. We used BCG to investigate epithelial kinase modulation using the human phospho-kinase array. Mycobacterial infection led to increased epithelial phosphorylation of PYK2, p38 kinase, and MEK1/2 (MAPK1/2) compared to medium control 6 and 72 h after infection. JNK had decreased after 6 h but increased after 72 h compared to the control. Data are presented as mean ± SEM of 3 experiments. *** p < 0.001.

Fig. 4

Mycobacteria modulate epithelial p53 and STAT pathways. Mycobacterial influence on epithelial p53 and STAT was investigated with the phosphorylation assay. BCG infection of primary AECs increased the phosphorylation of p53 at the regulatory S319 and apoptotic S46 domains, 6 and 72 h after infection. In contrast, the phosphorylation at S15, which activates transcription and cell survival, was not induced by the infection. STAT1 (Y701), STAT5b (Y699), and STAT6 (Y641) were suppressed 6 h after infection, while BCG induced phosphorylation of STAT1 (Y701) and STAT5b (Y699) 72 h after infection. STAT6 (Y641) levels remained reduced during the study. Data are presented as mean ± SEM of 3 separate experiments. * p < 0.05, ** p < 0.01; *** p < 0.001.

Fig. 5

Mycobacteria regulate NF-κB and c-Jun through CXCR1 and CXCR2. We determined the impact of CXCR1 and CXCR2 blockage on mycobacteria-induced NF-κB and c-Jun regulation. BCG infection did not affect epithelial NF-κB and AP-1 activation. However, the blockage of CXCR1 or CXCR2 prior to infection increased NF-κB and c-Jun protein levels compared to uninfected cells. Data are presented as mean ± SEM of 3 separate experiments. * p < 0.05; *** p < 0.001.

Fig. 6

Controlled epithelial cytokine secretion. Mycobacterial control of transcriptional factors was analysed as epithelial cytokine secretion. BCG infection of AECs induced significant IL-6 and IL-10 secretion. Blockage of CXCR1/2 prior to infection increased IL-6 secretion even further. IL-10 levels were decreased significantly by CXCR2 blockage, but not by CXCR1 antibodies. Data are presented as mean ± SEM of 3 separate experiments. *** p < 0.001.