Involvement of skeletal muscle gene regulatory network in susceptibility to wound infection following trauma

Abstract

Despite recent advances in our understanding the pathophysiology of trauma, the basis of the predisposition of trauma patients to infection remains unclear. A Drosophila melanogaster/Pseudomonas aeruginosa injury and infection model was used to identify host genetic components that contribute to the hyper-susceptibility to infection that follows severe trauma. We show that P. aeruginosa compromises skeletal muscle gene (SMG) expression at the injury site to promote infection. We demonstrate that activation of SMG structural components is under the control of cJun-N-terminal Kinase (JNK) Kinase, Hemipterous (Hep), and activation of this pathway promotes local resistance to P. aeruginosa in flies and mice. Our study links SMG expression and function to increased susceptibility to infection, and suggests that P. aeruginosa affects SMG homeostasis locally by restricting SMG expression in injured skeletal muscle tissue. Local potentiation of these host responses, and/or inhibition of their suppression by virulent P. aeruginosa cells, could lead to novel therapies that prevent or treat deleterious and potentially fatal infections in severely injured individuals.

Figure 1. Highly virulent P. aeruginosa actively restricts skeletal muscle gene induction within 1 h post-inoculation.

Relative expression ratio levels of 20 fly genes that encode proteins with essential functions of thorax skeletal muscle, under different conditions: 1, 6, and 12 h after thoracic injury only (A), thoracic injury and PA14 inoculation (B), or thoracic injury and CF5 inoculation (C); 1 h following thoracic injury and co-infection with 100∶100 and 200∶100 PA14:CF5 CFUs/fly (D); or following abdominal injury alone or together with inoculation with PA14 or CF5 (E). The relative expression ratio levels of selected SMGs for each condition were calculated versus naïve. The statistical evaluation of differences in expression values between PA14 and CF5 inoculated flies is presented in Table S1.

Figure 2. Skeletal muscle structural gene function contributes to host hypersusceptibility to infection.

Survival kinetics of mutants for the flight muscle genes act88F, hdp, or Tm2 following local PA14 infection of the thorax (A) or the abdomen (B) relative to wild-type flies. Detailed statistical evaluations of the survival kinetics are presented in Table S2.

Figure 3. Loss of muscle specific expression of TpnC41C and Gst2 increases fly susceptibility to infection.

Survival kinetics of flies carrying the RNAi control gene yuri (blue diamonds), versus muscle specific TpnC41C RNAi transgenic flies (red triangles), following thoracic (A) or abdominal (B) PA14 infection. (C) Homozygous flies bearing two distinct loss-of-function Gst2^06253/04227alleles, presented as Gst2(−/−), versus the corresponding heterozygous Gst2^06253/+ flies, presented as Gst2(−/+). (D) Wild-type flies, versus Gst2^GS2160 loss of function flies, presented as Gst2(GS), and overexpression of the fly muscle specific gene Gst2 in the Gst2^GS2160 loss of function background flies presented as Gst2(GS);Gst2 muscle following local PA14 infection of the thorax. Detailed statistical evaluations of the survival kinetics are presented in Table S2.

Figure 4. hep mediates increased resistance to thoracic, but not abdominal or systemic, infection.

Survival kinetics of (A, C, E) and bacterial proliferation per fly in (B, D, F) wild-type and hep¹ flies, following local PA14 infection of the thorax (A, B), or the abdomen (C, D), or systemic injector-pumping PA14 infection (E, F). Error bars indicate Standard Deviation of the mean (C, D, F) and * indicate t-test P-values of ≤0.05 (B). The difference in mortality kinetics between wild type and mutant flies is statistically significant. Detailed statistical evaluations of the survival kinetics are presented in Table S2.

Figure 5. JNK inhibition increases P. aeruginosa proliferation in mouse muscle.

(A) “Infection” and “Infection + JNK inhibitor” mice were respectively injected with empty micelles and micelles filled with the JNK inhibitor SP600125, and then inoculated with bioluminescent P. aeruginosa cells. The inhibitor did not perturb bacterial growth in vitro. Luminescence was assessed 0, 1, and 3 d post-treatment. The luminescence signal is magnified 3X in the lower panels. (B) Average luminescence from eight control versus eight JNK inhibitor mice. *P≤0.05 for days 1, 2, and 3 based on t- and Wilcoxon tests. RLU, relative luminescence units. (C) Diagram depicting the proposed role of JNK pathway in host defense and the ability of P. aeruginosa to affect SMGs expression.