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Mycobacterium tuberculosis -triggered Hippo pathway orchestrates CXCL1/2 expression to modulate host immune responses

View Article: PubMed Central - PubMed

ABSTRACT

Mycobacterium tuberculosis (Mtb) pathogenesis encompasses a plethora of finely regulated alterations within the host which eventually coin the outcome of infection. Chemokines are important components in directing immune cell recruitment to the site of infection, and shaping the disease progression. Here, we demonstrate that Hippo (mammalian sterile 20–like 1 and 2 kinases, MST1/2, in mammals), is activated during mycobacterial infection in a toll-like receptor (TLR) 2-interleukin receptor-1 associated kinases (IRAK1/4)-dependent manner. Mtb-triggered Hippo signaling modulates the expression and secretion of chemokines (CXCL1 and CXCL2); as silencing MST1/2 compromised the ability of Mtb to furnish the same. Further insight into the mechanism of Hippo-mediated regulation of chemokines revealed the role for a non-canonical Hippo effector interferon (IFN) regulatory factor (IRF) 3 in the process and marked the effect to be independent of LATS1. Alongside their ability to guide directed recruitment of immune cells, we have uncovered a paracrine role for Hippo-mediated secretion of CXCL1 and CXCL2 in the production of anti-microbial peptides (beta-defensins), iNOS, NOX2 and pro-inflammatory molecules during mycobacterial infection of the host. This study highlights the involvement of TLR2-IRAK1/4-MST1/2-IRF3 axis in Mtb-triggered modulation of chemokines and identifies Hippo signaling as a novel regulator of host-mycobacterial interactions.

No MeSH data available.


Related in: MedlinePlus

MST1/2 are involved in the production of CXCL1 and CXCL2 upon mycobacterial infection of macrophages.(a) Murine peritoneal macrophages were infected with Mtb for 12 h and the transcript levels of the indicated chemokines were assessed by qRT-PCR. (b) RAW264.7 macrophages were transfected with MST1/2 KD constructs for 36 h followed by infection with Mtb for 12 h to analyze the transcript levels of the indicated chemokines by qRT-PCR. (c and d) Macrophages were infected with Mtb for the indicated time points to detect the levels of CXCL1 (c) or CXCL2 (d) in culture supernatants by ELISA. (e–h) Macrophages were transfected with MST1/2 KD constructs (e,f) or Stk4/Stk3 siRNAs (g and h) for 36 h followed by infection with Mtb for 12 h and culture supernatants were analyzed for CXCL1 (e,g) and CXCL2 (f,h) by ELISA. (i) Validation of MST1/2 knocked down in RAW264.7 macrophages by immunoblotting. All the data represent mean ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova or t test). KD, kinase dead; Med, medium; pg, picogram; siRNA, small interfering RNA; h, hour; ns, not significant; T, total; NT, non-targeting.
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f4: MST1/2 are involved in the production of CXCL1 and CXCL2 upon mycobacterial infection of macrophages.(a) Murine peritoneal macrophages were infected with Mtb for 12 h and the transcript levels of the indicated chemokines were assessed by qRT-PCR. (b) RAW264.7 macrophages were transfected with MST1/2 KD constructs for 36 h followed by infection with Mtb for 12 h to analyze the transcript levels of the indicated chemokines by qRT-PCR. (c and d) Macrophages were infected with Mtb for the indicated time points to detect the levels of CXCL1 (c) or CXCL2 (d) in culture supernatants by ELISA. (e–h) Macrophages were transfected with MST1/2 KD constructs (e,f) or Stk4/Stk3 siRNAs (g and h) for 36 h followed by infection with Mtb for 12 h and culture supernatants were analyzed for CXCL1 (e,g) and CXCL2 (f,h) by ELISA. (i) Validation of MST1/2 knocked down in RAW264.7 macrophages by immunoblotting. All the data represent mean ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova or t test). KD, kinase dead; Med, medium; pg, picogram; siRNA, small interfering RNA; h, hour; ns, not significant; T, total; NT, non-targeting.

Mentions: Chemokines serve critical function in directing immune cells to inflammatory site; which might be decisive in controlling the severity of disease3839. In this regard, it is important to study the regulatory mechanisms involved in the production of chemokines. A screen for 11 inflammatory chemokines9 identified CCL2, CCL3, CCL7, CXCL1, CXCL2 and CXCL15 to be induced upon mycobacterial infection (Fig. 4a). Further, the possible role of Hippo signaling in regulating Mtb-induced chemokines expression was implicated by over expressing kinase dead forms of MST1/2 in RAW264.7 macrophages. This marked CXCL1 and CXCL2 to be MST1/2-responsive as Mtb failed to induce these chemokines in presence of catalytically inactive MST1/2 (Fig. 4b). In agreement with the above observations, it was found that there was infection-induced secretion of CXCL1 and CXCL2 into the culture supernatants (Fig. 4c,d), which was compromised in the presence of MST1/2 KD constructs (Fig. 4e,f) or Stk4/Stk3 siRNA (Fig. 4g,h). The knock down efficiency of MST1 and MST2 by Stk4/Stk3 siRNA is shown in Fig. 4i. These observations clearly revealed the novel role of MST1/2 in Mtb-induced production of CXCL1 and CXCL2.


Mycobacterium tuberculosis -triggered Hippo pathway orchestrates CXCL1/2 expression to modulate host immune responses
MST1/2 are involved in the production of CXCL1 and CXCL2 upon mycobacterial infection of macrophages.(a) Murine peritoneal macrophages were infected with Mtb for 12 h and the transcript levels of the indicated chemokines were assessed by qRT-PCR. (b) RAW264.7 macrophages were transfected with MST1/2 KD constructs for 36 h followed by infection with Mtb for 12 h to analyze the transcript levels of the indicated chemokines by qRT-PCR. (c and d) Macrophages were infected with Mtb for the indicated time points to detect the levels of CXCL1 (c) or CXCL2 (d) in culture supernatants by ELISA. (e–h) Macrophages were transfected with MST1/2 KD constructs (e,f) or Stk4/Stk3 siRNAs (g and h) for 36 h followed by infection with Mtb for 12 h and culture supernatants were analyzed for CXCL1 (e,g) and CXCL2 (f,h) by ELISA. (i) Validation of MST1/2 knocked down in RAW264.7 macrophages by immunoblotting. All the data represent mean ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova or t test). KD, kinase dead; Med, medium; pg, picogram; siRNA, small interfering RNA; h, hour; ns, not significant; T, total; NT, non-targeting.
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f4: MST1/2 are involved in the production of CXCL1 and CXCL2 upon mycobacterial infection of macrophages.(a) Murine peritoneal macrophages were infected with Mtb for 12 h and the transcript levels of the indicated chemokines were assessed by qRT-PCR. (b) RAW264.7 macrophages were transfected with MST1/2 KD constructs for 36 h followed by infection with Mtb for 12 h to analyze the transcript levels of the indicated chemokines by qRT-PCR. (c and d) Macrophages were infected with Mtb for the indicated time points to detect the levels of CXCL1 (c) or CXCL2 (d) in culture supernatants by ELISA. (e–h) Macrophages were transfected with MST1/2 KD constructs (e,f) or Stk4/Stk3 siRNAs (g and h) for 36 h followed by infection with Mtb for 12 h and culture supernatants were analyzed for CXCL1 (e,g) and CXCL2 (f,h) by ELISA. (i) Validation of MST1/2 knocked down in RAW264.7 macrophages by immunoblotting. All the data represent mean ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova or t test). KD, kinase dead; Med, medium; pg, picogram; siRNA, small interfering RNA; h, hour; ns, not significant; T, total; NT, non-targeting.
Mentions: Chemokines serve critical function in directing immune cells to inflammatory site; which might be decisive in controlling the severity of disease3839. In this regard, it is important to study the regulatory mechanisms involved in the production of chemokines. A screen for 11 inflammatory chemokines9 identified CCL2, CCL3, CCL7, CXCL1, CXCL2 and CXCL15 to be induced upon mycobacterial infection (Fig. 4a). Further, the possible role of Hippo signaling in regulating Mtb-induced chemokines expression was implicated by over expressing kinase dead forms of MST1/2 in RAW264.7 macrophages. This marked CXCL1 and CXCL2 to be MST1/2-responsive as Mtb failed to induce these chemokines in presence of catalytically inactive MST1/2 (Fig. 4b). In agreement with the above observations, it was found that there was infection-induced secretion of CXCL1 and CXCL2 into the culture supernatants (Fig. 4c,d), which was compromised in the presence of MST1/2 KD constructs (Fig. 4e,f) or Stk4/Stk3 siRNA (Fig. 4g,h). The knock down efficiency of MST1 and MST2 by Stk4/Stk3 siRNA is shown in Fig. 4i. These observations clearly revealed the novel role of MST1/2 in Mtb-induced production of CXCL1 and CXCL2.

View Article: PubMed Central - PubMed

ABSTRACT

Mycobacterium tuberculosis (Mtb) pathogenesis encompasses a plethora of finely regulated alterations within the host which eventually coin the outcome of infection. Chemokines are important components in directing immune cell recruitment to the site of infection, and shaping the disease progression. Here, we demonstrate that Hippo (mammalian sterile 20&ndash;like 1 and 2 kinases, MST1/2, in mammals), is activated during mycobacterial infection in a toll-like receptor (TLR) 2-interleukin receptor-1 associated kinases (IRAK1/4)-dependent manner. Mtb-triggered Hippo signaling modulates the expression and secretion of chemokines (CXCL1 and CXCL2); as silencing MST1/2 compromised the ability of Mtb to furnish the same. Further insight into the mechanism of Hippo-mediated regulation of chemokines revealed the role for a non-canonical Hippo effector interferon (IFN) regulatory factor (IRF) 3 in the process and marked the effect to be independent of LATS1. Alongside their ability to guide directed recruitment of immune cells, we have uncovered a paracrine role for Hippo-mediated secretion of CXCL1 and CXCL2 in the production of anti-microbial peptides (beta-defensins), iNOS, NOX2 and pro-inflammatory molecules during mycobacterial infection of the host. This study highlights the involvement of TLR2-IRAK1/4-MST1/2-IRF3 axis in Mtb-triggered modulation of chemokines and identifies Hippo signaling as a novel regulator of host-mycobacterial interactions.

No MeSH data available.


Related in: MedlinePlus