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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.


Regulation of innate immune responses by CXCL1 and CXCL2 are dependent on Mtb induced MST1/2 activity.(a–g) Fresh macrophages were treated with equal volumes of cell culture supernatants derived from MST1/2 knocked down macrophages which were infected for 12 h with Mtb or left uninfected to study gene expression of Defb1, Defb2, Defb3, Defb14 (a), Nox1, Nox2, Nox3, Nox4 (b), Nos2 (c) and Lyz (d) by qRT-PCR or ELISA was performed to detect the levels of secreted TNF-α, IL-12p40 and IL-1β (e) in the culture supernatants and protein expression of iNOS (f,g) was analyzed by immunoblotting. (h–j) Supernatant obtained from MST1/2 knocked down Mtb infected macrophages were treated with recombinant CXCL1 and CXCL2 or left untreated. These supernatants were used to treat fresh macrophages and ELISA was performed to detect TNF-α (h), IL-12p40 (i) and IL-1 β (j). Blots are representatives of three independent experiments. All the data represent means ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova). Med, medium; pg, picogram; sup, supernatant; KD, kinase dead; siRNA, small interfering RNA; NT, non-targeting; ns, not significant.
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f10: Regulation of innate immune responses by CXCL1 and CXCL2 are dependent on Mtb induced MST1/2 activity.(a–g) Fresh macrophages were treated with equal volumes of cell culture supernatants derived from MST1/2 knocked down macrophages which were infected for 12 h with Mtb or left uninfected to study gene expression of Defb1, Defb2, Defb3, Defb14 (a), Nox1, Nox2, Nox3, Nox4 (b), Nos2 (c) and Lyz (d) by qRT-PCR or ELISA was performed to detect the levels of secreted TNF-α, IL-12p40 and IL-1β (e) in the culture supernatants and protein expression of iNOS (f,g) was analyzed by immunoblotting. (h–j) Supernatant obtained from MST1/2 knocked down Mtb infected macrophages were treated with recombinant CXCL1 and CXCL2 or left untreated. These supernatants were used to treat fresh macrophages and ELISA was performed to detect TNF-α (h), IL-12p40 (i) and IL-1 β (j). Blots are representatives of three independent experiments. All the data represent means ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova). Med, medium; pg, picogram; sup, supernatant; KD, kinase dead; siRNA, small interfering RNA; NT, non-targeting; ns, not significant.

Mentions: Since, CXCL1 and CXCL2 production were regulated by MST1/2; the role of Mtb-activated MST1/2 in regulating the CXCL1- and CXCL2- induced innate immune functions of macrophages was assessed. We found that the capacity of CXCL1 and CXCL2 to induce the concerned immune effectors was dependent on MST1/2 activity; as macrophages treated with culture supernatants derived from Mtb-infected MST1/2-knocked-down cells showed significant reduction in the production of Defb (Fig. 10a), Nox (Fig. 10b), Nos2 (Fig. 10c,f,g), Lyz (Fig. 10d), and pro-inflammatory molecules (Fig. 10e). Further, to specifically validate the requirement of CXCL1 and CXCL2 in inducing innate immune regulators, culture supernatants derived from Mtb-infected MST1/2 knocked down macrophages were exogenously treated with recombinant CXCL1 and CXCL2. Naïve macrophages treated with this supernatant could significantly rescue the level of pro-inflammatory cytokines i.e. TNF-α (Fig. 10h), IL-12p40 (Fig. 10i) and IL-1β (Fig. 10j). Taken together, our observations reveal that CXCL1 and CXCL2 secreted from Mtb-infected macrophages in an MST1/2-dependent manner, initiate paracrine signaling in neighboring macrophages to contribute towards the production of innate immune effectors like defensins, NOX, iNOS and pro-inflammatory cytokines as part of host defense upon mycobacterial infection (Fig. S27).


Mycobacterium tuberculosis -triggered Hippo pathway orchestrates CXCL1/2 expression to modulate host immune responses
Regulation of innate immune responses by CXCL1 and CXCL2 are dependent on Mtb induced MST1/2 activity.(a–g) Fresh macrophages were treated with equal volumes of cell culture supernatants derived from MST1/2 knocked down macrophages which were infected for 12 h with Mtb or left uninfected to study gene expression of Defb1, Defb2, Defb3, Defb14 (a), Nox1, Nox2, Nox3, Nox4 (b), Nos2 (c) and Lyz (d) by qRT-PCR or ELISA was performed to detect the levels of secreted TNF-α, IL-12p40 and IL-1β (e) in the culture supernatants and protein expression of iNOS (f,g) was analyzed by immunoblotting. (h–j) Supernatant obtained from MST1/2 knocked down Mtb infected macrophages were treated with recombinant CXCL1 and CXCL2 or left untreated. These supernatants were used to treat fresh macrophages and ELISA was performed to detect TNF-α (h), IL-12p40 (i) and IL-1 β (j). Blots are representatives of three independent experiments. All the data represent means ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova). Med, medium; pg, picogram; sup, supernatant; KD, kinase dead; siRNA, small interfering RNA; NT, non-targeting; ns, not significant.
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f10: Regulation of innate immune responses by CXCL1 and CXCL2 are dependent on Mtb induced MST1/2 activity.(a–g) Fresh macrophages were treated with equal volumes of cell culture supernatants derived from MST1/2 knocked down macrophages which were infected for 12 h with Mtb or left uninfected to study gene expression of Defb1, Defb2, Defb3, Defb14 (a), Nox1, Nox2, Nox3, Nox4 (b), Nos2 (c) and Lyz (d) by qRT-PCR or ELISA was performed to detect the levels of secreted TNF-α, IL-12p40 and IL-1β (e) in the culture supernatants and protein expression of iNOS (f,g) was analyzed by immunoblotting. (h–j) Supernatant obtained from MST1/2 knocked down Mtb infected macrophages were treated with recombinant CXCL1 and CXCL2 or left untreated. These supernatants were used to treat fresh macrophages and ELISA was performed to detect TNF-α (h), IL-12p40 (i) and IL-1 β (j). Blots are representatives of three independent experiments. All the data represent means ± SE (N = 3), *p < 0.05, **p < 0.005, ***p < 0.0001 (One way Anova). Med, medium; pg, picogram; sup, supernatant; KD, kinase dead; siRNA, small interfering RNA; NT, non-targeting; ns, not significant.
Mentions: Since, CXCL1 and CXCL2 production were regulated by MST1/2; the role of Mtb-activated MST1/2 in regulating the CXCL1- and CXCL2- induced innate immune functions of macrophages was assessed. We found that the capacity of CXCL1 and CXCL2 to induce the concerned immune effectors was dependent on MST1/2 activity; as macrophages treated with culture supernatants derived from Mtb-infected MST1/2-knocked-down cells showed significant reduction in the production of Defb (Fig. 10a), Nox (Fig. 10b), Nos2 (Fig. 10c,f,g), Lyz (Fig. 10d), and pro-inflammatory molecules (Fig. 10e). Further, to specifically validate the requirement of CXCL1 and CXCL2 in inducing innate immune regulators, culture supernatants derived from Mtb-infected MST1/2 knocked down macrophages were exogenously treated with recombinant CXCL1 and CXCL2. Naïve macrophages treated with this supernatant could significantly rescue the level of pro-inflammatory cytokines i.e. TNF-α (Fig. 10h), IL-12p40 (Fig. 10i) and IL-1β (Fig. 10j). Taken together, our observations reveal that CXCL1 and CXCL2 secreted from Mtb-infected macrophages in an MST1/2-dependent manner, initiate paracrine signaling in neighboring macrophages to contribute towards the production of innate immune effectors like defensins, NOX, iNOS and pro-inflammatory cytokines as part of host defense upon mycobacterial infection (Fig. S27).

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.