Muscles provide protection during microbial infection by activating innate immune response pathways in Drosophila and zebrafish

Abstract

Muscle contraction brings about movement and locomotion in animals. However, muscles have also been implicated in several atypical physiological processes including immune response. The role of muscles in immunity and the mechanism involved has not yet been deciphered. In this paper, using Drosophila indirect flight muscles (IFMs) as a model, we show that muscles are immune-responsive tissues. Flies with defective IFMs are incapable of mounting a potent humoral immune response. Upon immune challenge, the IFMs produce anti-microbial peptides (AMPs) through the activation of canonical signaling pathways, and these IFM-synthesized AMPs are essential for survival upon infection. The trunk muscles of zebrafish, a vertebrate model system, also possess the capacity to mount an immune response against bacterial infections, thus establishing that immune responsiveness of muscles is evolutionarily conserved. Our results suggest that physiologically fit muscles might boost the innate immune response of an individual.

Keywords: Anti-microbial peptides; Drosophila; Immunity; Infection; Muscle; Zebrafish.

Fig. 1.

IFM mutants are susceptible to bacterial infection. (A) Hematoxylin-stained hemithoraces of the fly strains used in the study, alongside a representative schematic, show a spectrum of defects in the IFMs. CS: the wild-type fly strain Canton-S. (B,C) Survival curves of the muscle mutants post-infection with (B) Salmonella and (C) Bacillus subtilis by septic injury. A log-rank test was done for estimating the significance of the survival curves. (B) Salmonella infected versus uninfected: Canton-S, P=0.0094; up¹, P=0.0034; up¹⁰¹, P<0.0001; hdp², P<0.0001; hdp³, P<0.0001; TM2³, P=0.0006; Act88F^KM88, P<0.0001. Mutant versus wild type: up¹, P=0.1171; up¹⁰¹, P<0.0001; hdp², P<0.0001; hdp³, P<0.0001; TM2³, P=0.0364; Act88F^KM88, P<0.0001. (C) Bacillus infected versus uninfected: All fly strains, P<0.0001. Mutant versus wild type: up¹, P=0.0139; up¹⁰¹, P=0.0002; hdp², P=0.0346; hdp³, <0.0001; TM2³, P=0.1646; Act88F^KM88, P=0.0009. n>80. Open circles, IFM-specific mutants; dashed lines, uninfected controls; U, uninfected; I, infected; dpi, days post-infection; hpi, hours post-infection.

Fig. 2.

Induction of AMPs in muscle mutants upon infection is sub-optimal. (A,B) qRT-PCR analyses for estimation of induction of gene expression for anti-microbial peptides (A) Drosocin and (B) Cecropin upon infection. The mRNA was extracted from whole flies 6 h after infection with Salmonella. The values represented are mean±s.e.m. of three independent replicates. *, significant difference in expression of infected versus uninfected; #, significant difference in induction levels of infected mutant versus infected wild type. ns, P>0.05; */#, P<0.05; **/##, P<0.001; ***/###, P<0.0001. CS, the wild-type fly strain Canton-S; u, uninfected; i, infected.

Fig. 3.

IFMs induce AMPs upon infection. (A,A′) Infected AMP reporter fly strains show increased expression of AMPs in their IFMs compared with naïve IFMs. Drs-GFP (Drosomycin) flies were infected with Gram-positive Staphylococcus aureus. Dro-GFP (Drosocin), Dipt-LacZ (Diptericin) and Cec-LacZ (Cecropin) flies were infected with Gram-negative Enterobacter cloacae. Flies were visualized 12 h post-infection. Scale bar: 100 µm. (B,C) Change in expression of the seven Drosophila AMPs in the IFMs of wild-type flies when infected with (B) Salmonella or (C) Bacillus subtilis. The mRNA was isolated from IFMs 6 hpi. Solid columns represent uninfected controls for each of the genes tested. The values represented are mean±s.e.m. of three independent replicates. *, significant difference in expression of infected versus uninfected. *P<0.05; **P<0.001; ***P<0.0001.

Fig. 4.

IFM-mediated immune response is essential for survival upon bacterial infection. (A,A′) Expression of UH3-Gal4 is restricted to IFMs in 2-day-old adults. Pattern of expression was visualized with UAS-GFP as a reporter. Yellow dotted line represents the body outline of the fly. White asterisks represent the dorsal longitudinal muscles and arrowheads indicate the dorso-ventral muscles, which together constitute the IFM. Also see Fig. S4. (B,C) Survival of male flies (n>50) infected with (B) Salmonella and (C) Bacillus subtilis. A log-rank test was done for estimating the significance of the survival curves. (B) Salmonella infected versus uninfected: UH3-Gal4/+ (Gal4 control), P=0.2207; Rel-RNAi/+ (UAS control), P=0.0640; UH3>Rel-RNAi (test), P<0.0001. Test versus infected Gal4 control, P=0.0003. Test versus infected UAS control, P=0.0009. (C) Bacillus infected versus uninfected: All fly strains, P<0.0001. Test (UH3>Dif-RNAi) versus infected Gal4 control (UH3-Gal4/+), P=0.0381. Test versus infected UAS control (Dif-RNAi/+),  P=0.0046. Solid lines, infected flies; dashed lines, uninfected controls; KD, knockdown; U, uninfected; I, infected, (B′,B″,C′,C″) AMP gene induction in the IFMs upon infection as tested by qRT-PCR of tissues isolated at 6 hpi. Induction of (B′) Diptericin and (B″) Drosocin was entirely lost in flies with altered IFM-Imd signaling. Similarly increased expression of (C′) Drosomycin and (C″) Metchnikowin was entirely lost in flies with altered IFM-Toll signaling. The values represented are mean±s.e.m. of three replicates. *, significant difference in expression of infected versus uninfected. ns, P>0.05; ***P<0.0001.

Fig. 5.

Vertebrate skeletal muscles are immune responsive. (A,C-C‴) Adult zebrafish myotomes induce cytokine expression upon infection. (A) Agarose gel images of expression level of cytokine genes assessed by RT-PCR. qRT-PCR analysis was performed to estimate the induction in gene expression of the zebrafish cytokine: TLR4a (C), TNFα (C′), IL10α (C″) and IL1β (C‴). (B) Induction of Hepcidin expression, a vertebrate AMP, upon infection with Salmonella. NTC, no template control; UP, unpricked; UI, uninfected with sterile injury; Inf, infected. The values represented are mean±s.e.m. of three independent replicate experiments. *, significant difference in expression of infected versus unpricked; #, infected versus uninfected. **/##, P<0.001; ***/###, P<0.0001.