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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.


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MST1/2 activate IRF3 during mycobacterial infection of macrophages.(a) Immunoblotting was performed to assess IRF3 activation in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (b) IRF3 activation was assessed by immunoblotting in cells isolated from spleen, lymph node and lung of mice infected with Mtb H37Rv. (c) RAW264.7 and THP1 macrophages were transfected with MST1/2 KD constructs followed by infection with Mtb H37Ra for 1 h and IRF3 activation was assessed by immunoblotting. (d) MST1/2 were knocked down in RAW264.7 and THP1 macrophages by Stk4/Stk3 siRNA followed by infection with Mtb H37Ra for 1 h to assess the IRF3 activation by immunoblotting. (e) Immuno-pulldown experiment was performed in BMDM, PM, RAW264.7 and THP1 macrophages to analyze the Mtb-driven interaction of pMST1/2 with pIRF3. (f) Immuno-pulldown experiment was performed to assess the interaction of pMST1/2 with pIRF3 in lung, spleen and lymph node of mice infected with Mtb H37Rv. Blots are representatives of three independent experiments. p, phospho; T, total; BMDM, bone marrow derived macrophage; PM, peritoneal macrophage; NT, non-targeting; siRNA, small interfering RNA; h, hour; IB, immunoblotting; IP, immuno-pulldown; IgG, immunoglobulin G; α, anti.
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f6: MST1/2 activate IRF3 during mycobacterial infection of macrophages.(a) Immunoblotting was performed to assess IRF3 activation in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (b) IRF3 activation was assessed by immunoblotting in cells isolated from spleen, lymph node and lung of mice infected with Mtb H37Rv. (c) RAW264.7 and THP1 macrophages were transfected with MST1/2 KD constructs followed by infection with Mtb H37Ra for 1 h and IRF3 activation was assessed by immunoblotting. (d) MST1/2 were knocked down in RAW264.7 and THP1 macrophages by Stk4/Stk3 siRNA followed by infection with Mtb H37Ra for 1 h to assess the IRF3 activation by immunoblotting. (e) Immuno-pulldown experiment was performed in BMDM, PM, RAW264.7 and THP1 macrophages to analyze the Mtb-driven interaction of pMST1/2 with pIRF3. (f) Immuno-pulldown experiment was performed to assess the interaction of pMST1/2 with pIRF3 in lung, spleen and lymph node of mice infected with Mtb H37Rv. Blots are representatives of three independent experiments. p, phospho; T, total; BMDM, bone marrow derived macrophage; PM, peritoneal macrophage; NT, non-targeting; siRNA, small interfering RNA; h, hour; IB, immunoblotting; IP, immuno-pulldown; IgG, immunoglobulin G; α, anti.

Mentions: IRF3 is a transcription factor known for its role in defending the host against viral infections by stimulating the production of interferons414243. It has been known to be responsive to LPS-induced TLR4 signaling44 and has been implicated in host responses during mycobacterial infection4546 and also in the modulation of chemokines during viral and certain bacterial infections454748. Interestingly, a recent report showed that MST1/2 is capable of regulating IRF3 in response to viral infection49. However, the regulation of IRF3 activity by MST1/2 during mycobacterial infection of macrophages is not known. This premise led us to investigate the role for MST1/2 in regulating IRF3 activity in mycobacteria-infected macrophages. To this end the phosphorylation status of IRF3 was analysed in Mtb-infected BMDM, PM, RAW264.7 and THP1 macrophages (Fig. 6a) and in lung, lymph node and spleen (Fig. 6b) of Mtb aerosolized mice; which clearly showed the activation of IRF3 without changing its total cellular pool. In order to assess the role for MST1/2 in regulating IRF3 activity, RAW264.7 and THP1 macrophages transiently transfected with MST1/2 kinase dead construct, were infected with Mtb (Fig. 6c). Interference with MST1/2 function clearly showed reduction in the Mtb-induced IRF3 activation. These results clearly suggest that catalytic activities of MST1/2 are essential for IRF3 activation during mycobacterial infection. Further, knock down of MST1/2 by Stk4/Stk3 siRNA in RAW264.7 and THP1 macrophages also led to a significant loss in infection-driven IRF3 activation (Fig. 6d). Next we assessed the mechanism by which MST1/2 regulate the activity of IRF3. To this end immuno-pulldown experiments clearly showed the interaction of phospho MST1/2 with phospho IRF3 in Mtb-infected BMDM, PM, RAW264.7, THP1 macrophages (Fig. 6e) and in lung, lymph node and spleen of Mtb aerosolized mice (Fig. 6f). Altogether, these results clearly suggest infection-induced MST1/2 interaction-dependent activation of IRF3.


Mycobacterium tuberculosis -triggered Hippo pathway orchestrates CXCL1/2 expression to modulate host immune responses
MST1/2 activate IRF3 during mycobacterial infection of macrophages.(a) Immunoblotting was performed to assess IRF3 activation in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (b) IRF3 activation was assessed by immunoblotting in cells isolated from spleen, lymph node and lung of mice infected with Mtb H37Rv. (c) RAW264.7 and THP1 macrophages were transfected with MST1/2 KD constructs followed by infection with Mtb H37Ra for 1 h and IRF3 activation was assessed by immunoblotting. (d) MST1/2 were knocked down in RAW264.7 and THP1 macrophages by Stk4/Stk3 siRNA followed by infection with Mtb H37Ra for 1 h to assess the IRF3 activation by immunoblotting. (e) Immuno-pulldown experiment was performed in BMDM, PM, RAW264.7 and THP1 macrophages to analyze the Mtb-driven interaction of pMST1/2 with pIRF3. (f) Immuno-pulldown experiment was performed to assess the interaction of pMST1/2 with pIRF3 in lung, spleen and lymph node of mice infected with Mtb H37Rv. Blots are representatives of three independent experiments. p, phospho; T, total; BMDM, bone marrow derived macrophage; PM, peritoneal macrophage; NT, non-targeting; siRNA, small interfering RNA; h, hour; IB, immunoblotting; IP, immuno-pulldown; IgG, immunoglobulin G; α, anti.
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f6: MST1/2 activate IRF3 during mycobacterial infection of macrophages.(a) Immunoblotting was performed to assess IRF3 activation in BMDM, PM, RAW264.7 and THP1 macrophages infected with Mtb H37Ra for 1 h. (b) IRF3 activation was assessed by immunoblotting in cells isolated from spleen, lymph node and lung of mice infected with Mtb H37Rv. (c) RAW264.7 and THP1 macrophages were transfected with MST1/2 KD constructs followed by infection with Mtb H37Ra for 1 h and IRF3 activation was assessed by immunoblotting. (d) MST1/2 were knocked down in RAW264.7 and THP1 macrophages by Stk4/Stk3 siRNA followed by infection with Mtb H37Ra for 1 h to assess the IRF3 activation by immunoblotting. (e) Immuno-pulldown experiment was performed in BMDM, PM, RAW264.7 and THP1 macrophages to analyze the Mtb-driven interaction of pMST1/2 with pIRF3. (f) Immuno-pulldown experiment was performed to assess the interaction of pMST1/2 with pIRF3 in lung, spleen and lymph node of mice infected with Mtb H37Rv. Blots are representatives of three independent experiments. p, phospho; T, total; BMDM, bone marrow derived macrophage; PM, peritoneal macrophage; NT, non-targeting; siRNA, small interfering RNA; h, hour; IB, immunoblotting; IP, immuno-pulldown; IgG, immunoglobulin G; α, anti.
Mentions: IRF3 is a transcription factor known for its role in defending the host against viral infections by stimulating the production of interferons414243. It has been known to be responsive to LPS-induced TLR4 signaling44 and has been implicated in host responses during mycobacterial infection4546 and also in the modulation of chemokines during viral and certain bacterial infections454748. Interestingly, a recent report showed that MST1/2 is capable of regulating IRF3 in response to viral infection49. However, the regulation of IRF3 activity by MST1/2 during mycobacterial infection of macrophages is not known. This premise led us to investigate the role for MST1/2 in regulating IRF3 activity in mycobacteria-infected macrophages. To this end the phosphorylation status of IRF3 was analysed in Mtb-infected BMDM, PM, RAW264.7 and THP1 macrophages (Fig. 6a) and in lung, lymph node and spleen (Fig. 6b) of Mtb aerosolized mice; which clearly showed the activation of IRF3 without changing its total cellular pool. In order to assess the role for MST1/2 in regulating IRF3 activity, RAW264.7 and THP1 macrophages transiently transfected with MST1/2 kinase dead construct, were infected with Mtb (Fig. 6c). Interference with MST1/2 function clearly showed reduction in the Mtb-induced IRF3 activation. These results clearly suggest that catalytic activities of MST1/2 are essential for IRF3 activation during mycobacterial infection. Further, knock down of MST1/2 by Stk4/Stk3 siRNA in RAW264.7 and THP1 macrophages also led to a significant loss in infection-driven IRF3 activation (Fig. 6d). Next we assessed the mechanism by which MST1/2 regulate the activity of IRF3. To this end immuno-pulldown experiments clearly showed the interaction of phospho MST1/2 with phospho IRF3 in Mtb-infected BMDM, PM, RAW264.7, THP1 macrophages (Fig. 6e) and in lung, lymph node and spleen of Mtb aerosolized mice (Fig. 6f). Altogether, these results clearly suggest infection-induced MST1/2 interaction-dependent activation of IRF3.

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