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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.

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Mycobacterium tuberculosis (Mtb)-driven TLR2 signaling activates Hippo pathway.(a) Mouse primary peritoneal macrophages were infected with Mtb H37Rv for 1 h to assess the phosphorylated forms of MST1/2, LATS1 and the total levels of MST1, MST2 and LATS1 by immunoblotting. (b) Hippo pathway activation was assessed by immunoblotting in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (c) Hippo pathway was assessed in lung, lymph node and spleen of WT and Tlr2 KO mice infected with Mtb H37Rv by aerosol route. (d) Myd88 was knocked down in RAW264.7 and THP1 macrophages followed by infection with Mtb H37Ra for 1 h to check the status of Hippo pathway by immunoblotting. β-ACTIN was used as loading control for immunoblotting experiments on total cell lysates. Blots are representatives of three independent experiments. siRNA, small interfering RNA; WT, wild type; NT, non-targeting; BMDM, bone marrow derived macrophages; PM, peritoneal macrophages; THP1, human leukemia monocytic cell line; h, hour; KO, knock out.
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f1: Mycobacterium tuberculosis (Mtb)-driven TLR2 signaling activates Hippo pathway.(a) Mouse primary peritoneal macrophages were infected with Mtb H37Rv for 1 h to assess the phosphorylated forms of MST1/2, LATS1 and the total levels of MST1, MST2 and LATS1 by immunoblotting. (b) Hippo pathway activation was assessed by immunoblotting in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (c) Hippo pathway was assessed in lung, lymph node and spleen of WT and Tlr2 KO mice infected with Mtb H37Rv by aerosol route. (d) Myd88 was knocked down in RAW264.7 and THP1 macrophages followed by infection with Mtb H37Ra for 1 h to check the status of Hippo pathway by immunoblotting. β-ACTIN was used as loading control for immunoblotting experiments on total cell lysates. Blots are representatives of three independent experiments. siRNA, small interfering RNA; WT, wild type; NT, non-targeting; BMDM, bone marrow derived macrophages; PM, peritoneal macrophages; THP1, human leukemia monocytic cell line; h, hour; KO, knock out.

Mentions: Cell fate decisions modulating immunological homeostasis during pathogenic mycobacterial infections often play critical role in overall outcome of the infection. In this perspective, the Hippo pathway, a key regulator of immunological homeostasis appears to influence significant steps during initiation or progression of various patho-physiological conditions31. Interestingly, role of Hippo signaling during mycobacterial infection of the host immune cells and subsequent manifestations towards disease progression is not known. In this regard, we found activation of Hippo signaling in Mtb H37Rv infected mouse peritoneal macrophages (PM) (Fig. 1a) as evaluated by increase in the phosphorylation status of canonical Hippo pathway components; MST1/2, LATS1. However, the total pool of MST1, MST2 and LATS1 did not alter during mycobacterial infection as indicated in (Fig. 1a). Hippo pathway activation by Mtb was also confirmed in bone marrow derived macrophages (BMDM), RAW264.7 and THP1 macrophages (Fig. 1b). Induced phosphorylation of MST1/2 and LATS1 clearly suggest Hippo pathway activation in Mtb-infected macrophages. A recent report suggests the ability of TLR1, TLR2 and TLR4 to substantially activate MST1 and MST2 when treated with specific TLR agonists34. Further, earlier understanding from our and others’ work indicate that of all other PRRs, mycobacteria-specific responsiveness of TLR2 results in the vigorous activation of various signaling pathways in macrophages12131435. In this perspective, the ability of Mtb to activate effectors of Hippo signaling was significantly compromised in lungs, spleen and lymph node of tlr2 knockout mice (Fig. 1c). Altogether, the above results suggest an essential role for Mtb specific TLR2 signaling in regulating Hippo pathway activation.


Mycobacterium tuberculosis -triggered Hippo pathway orchestrates CXCL1/2 expression to modulate host immune responses
Mycobacterium tuberculosis (Mtb)-driven TLR2 signaling activates Hippo pathway.(a) Mouse primary peritoneal macrophages were infected with Mtb H37Rv for 1 h to assess the phosphorylated forms of MST1/2, LATS1 and the total levels of MST1, MST2 and LATS1 by immunoblotting. (b) Hippo pathway activation was assessed by immunoblotting in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (c) Hippo pathway was assessed in lung, lymph node and spleen of WT and Tlr2 KO mice infected with Mtb H37Rv by aerosol route. (d) Myd88 was knocked down in RAW264.7 and THP1 macrophages followed by infection with Mtb H37Ra for 1 h to check the status of Hippo pathway by immunoblotting. β-ACTIN was used as loading control for immunoblotting experiments on total cell lysates. Blots are representatives of three independent experiments. siRNA, small interfering RNA; WT, wild type; NT, non-targeting; BMDM, bone marrow derived macrophages; PM, peritoneal macrophages; THP1, human leukemia monocytic cell line; h, hour; KO, knock out.
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Related In: Results  -  Collection

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f1: Mycobacterium tuberculosis (Mtb)-driven TLR2 signaling activates Hippo pathway.(a) Mouse primary peritoneal macrophages were infected with Mtb H37Rv for 1 h to assess the phosphorylated forms of MST1/2, LATS1 and the total levels of MST1, MST2 and LATS1 by immunoblotting. (b) Hippo pathway activation was assessed by immunoblotting in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (c) Hippo pathway was assessed in lung, lymph node and spleen of WT and Tlr2 KO mice infected with Mtb H37Rv by aerosol route. (d) Myd88 was knocked down in RAW264.7 and THP1 macrophages followed by infection with Mtb H37Ra for 1 h to check the status of Hippo pathway by immunoblotting. β-ACTIN was used as loading control for immunoblotting experiments on total cell lysates. Blots are representatives of three independent experiments. siRNA, small interfering RNA; WT, wild type; NT, non-targeting; BMDM, bone marrow derived macrophages; PM, peritoneal macrophages; THP1, human leukemia monocytic cell line; h, hour; KO, knock out.
Mentions: Cell fate decisions modulating immunological homeostasis during pathogenic mycobacterial infections often play critical role in overall outcome of the infection. In this perspective, the Hippo pathway, a key regulator of immunological homeostasis appears to influence significant steps during initiation or progression of various patho-physiological conditions31. Interestingly, role of Hippo signaling during mycobacterial infection of the host immune cells and subsequent manifestations towards disease progression is not known. In this regard, we found activation of Hippo signaling in Mtb H37Rv infected mouse peritoneal macrophages (PM) (Fig. 1a) as evaluated by increase in the phosphorylation status of canonical Hippo pathway components; MST1/2, LATS1. However, the total pool of MST1, MST2 and LATS1 did not alter during mycobacterial infection as indicated in (Fig. 1a). Hippo pathway activation by Mtb was also confirmed in bone marrow derived macrophages (BMDM), RAW264.7 and THP1 macrophages (Fig. 1b). Induced phosphorylation of MST1/2 and LATS1 clearly suggest Hippo pathway activation in Mtb-infected macrophages. A recent report suggests the ability of TLR1, TLR2 and TLR4 to substantially activate MST1 and MST2 when treated with specific TLR agonists34. Further, earlier understanding from our and others’ work indicate that of all other PRRs, mycobacteria-specific responsiveness of TLR2 results in the vigorous activation of various signaling pathways in macrophages12131435. In this perspective, the ability of Mtb to activate effectors of Hippo signaling was significantly compromised in lungs, spleen and lymph node of tlr2 knockout mice (Fig. 1c). Altogether, the above results suggest an essential role for Mtb specific TLR2 signaling in regulating Hippo pathway activation.

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