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. 2020 Dec;11(1):39-48.
doi: 10.1080/21505594.2019.1706305.

Metformin Promotes Innate Immunity Through a Conserved PMK-1/p38 MAPK Pathway

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Free PMC article

Metformin Promotes Innate Immunity Through a Conserved PMK-1/p38 MAPK Pathway

Yi Xiao et al. Virulence. .
Free PMC article

Abstract

Metformin, as the first-line oral drug for type 2 diabetes, has proven benefits against aging, cancer and cardiovascular diseases. But the influence of metformin to the immune response and its molecular mechanisms remain obscure. Metformin increases resistance to not only the Gram-negative pathogens Pseudomonas aeruginosa and Salmonella enterica but also the Gram-positive pathogens Enterococcus faecalis and Staphylococcus aureus. Meanwhile, metformin protects the animals from the infection by enhancing the tolerance to the pathogen infection rather than by reducing the bacterial burden. Through the screening of classical immune pathways in C. elegans, we find metformin enhances innate immunity through p38 MAPK pathway. Furthermore, activated p38/PMK-1 by metformin acts on the intestine for innate immune response. In addition, metformin-treated mice have increased resistance to P. aeruginosa PA14 infection and significantly increased the levels of active PMK-1. Therefore, promoted p38/PMK-1-mediated innate immunity by metformin is conserved from worms to mammals. Our work provides a conserved mechanism by which metformin enhances immune response and boosts its therapeutic application in the treatment of pathogen infection.

Keywords: C. elegans; Metformin; innate immunity; p38 MAPK pathway.

Figures

Figure 1.
Figure 1.
Metformin enhances pathogen resistance. (a) Metformin promotes innate immune response to P. aeruginosa PA14 compared to WT in a dose-dependent manner (*P< 0.05, log-rank test). (b) metformin (50 mM) did not delay the proliferation of P. aeruginosa PA14. (c) Metformin (50 mM) did not affect the colony-forming units (CFUs) of bacteria in WT worms after P. aeruginosa PA14 infection. These results are mean ± SD of three independent experiments, each involving 15 parallel groups. NS, no significance.
Figure 2.
Figure 2.
Metformin promotes innate immunity through the p38 MAPK pathway. (a–i) PMK-1/p38 MAPK is involved in metformin-mediated innate immunity. Metformin (50 mM) enhanced resistance to P. aeruginosa PA14 in WT (N2) (a), daf-2(e1370) (c), mpk-1(n2521) (d), egl-30(n686) (e), dkf-2(ok1704)(f), fshr-1(ok778)mutants (g), but not in pmk-1(km25) mutants (b). *P< 0.05 versus worms + metformin (log-rank test). (h and i) Mutations in the components of the p38 MAPK pathway suppressed metformin-mediated resistance of worms to PA14. (h) nsy-1(ag3); (i) sek-1(ag1).
Figure 3.
Figure 3.
Metformin activates p38 MAPK signaling in C. elegans. (a) The phosphorylation of p38 MAPK was elevated in WT worms (N2) exposed to metformin (50 mM). (b) The right panel shows quantification of phosphorylated p38 MAPK levels. These results are mean ± SD of three independent experiments performed in triplicate. *P< 0.05 vs E. coliOP50 (one-way ANOVA followed by a Student-Newman-Keuls test). (c) The mRNA levels of three p38 MAPK targets K08D8.5, lys-2, and F35E12.5 in worms exposed to metformin (50 mM). These results are mean ± SD of three independent experiments performed in triplicate. *P< 0.05 versus OP50 (one-way ANOVA followed by a Student-Newman-Keuls test). (d) Expression of K08D8.5p::GFP was up-regulated in WT worms, but not in worms subjected to pmk-1(km25) mutants, exposed to metformin (50 mM). (e) The right panel shows quantification of fluorescence intensity. These results are mean ± SD of three independent experiments performed in triplicate. *P< 0.05 versus OP50 (one-way ANOVA followed by a Student-Newman-Keuls test).
Figure 4.
Figure 4.
Intestinal PMK-1 enhances resistance to pathogen infection after metformin treatment. (a) metformin (50 mM) did not increase resistance to P. aeruginosa PA14 infection in the intestine knockdown of pmk-1worms. However, RNAi of pmk-1 in hypodermis (b), muscle (c), and neuron (d), respectively, after metformin treatment did not prevent metformin action on promoting pseudomonas resistance. (e) Expression of pmk-1 under the intestinal-specific vha-6 promoter (AY102) restored resistance against P. aeruginosa PA14 infection in pmk-1(km25) mutants after treatment with metformin (50 mM) .*P< 0.05 versus worms + metformin (log-rank test). (f) Pre-treatment of AY102 worms with metformin (50 mM) restored the level of active PMK-1. (g) The right panel shows quantification of phosphorylated p38 MAPK levels. These results are mean ± SD of three independent experiments performed in triplicate. *P< 0.05 versus OP50 (one-way ANOVA followed by a Student-Newman-Keuls test).
Figure 5.
Figure 5.
Metformin protected mice against P. aeruginosa infection and increased p-p38 in the lung. (a) Metformin (200 mg/kg body weight) treated mice increases the resistance to P. aeruginosa PA14 infection compared with control mice. *P< 0.05 (log-rank test). (b) The p38 inhibitor SB202190 increased the susceptibility to P. aeruginosa PA14 infection compared with control mice *P< 0.05 (log-rank test) and suppressed the enhanced resistance to P. aeruginosa PA14 upon metformin (200 mg/kg body weight) treatment P =0.0512 (log-rank test). (c) Metformin (200 mg/kg body weight) significantly increased the levels of active PMK-1 in the lung. (d) The right panel shows quantification of phosphorylated p38 MAPK levels. These results are mean ± SD of three independent experiments performed in triplicate. *P< 0.05 versus control (one-way ANOVA followed by a Student-Newman-Keuls test).

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Grant support

This work was supported by Science & Technology Talent Support Project of the Educational Department of Guizhou Province (KY[2018]055), Science & Technology Foundation for the Excellent Youth Scholars of Guizhou Province (KY[2015]12), Science & Technology Plan of Zun yi [(2018) 16], Key Lab Construction Project of the Educational Department of Guizhou Province (KY[2014]212), The Xin miao Foundation of Zun yi Medical University [(2017)5733-025].

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