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. 2017 Oct 25:9:ecurrents.md.8a7e35c50fa2b48156799d3c39788175.
doi: 10.1371/currents.md.8a7e35c50fa2b48156799d3c39788175.

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy

Michelle Goody, Denise Jurczyszak  1 , Carol Kim, Clarissa Henry  2
Affiliations

Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy

Michelle Goody et al. PLoS Curr. .

Abstract

Introduction: Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases.

Methods: We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD.

Results: We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic.

Discussion: These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases. Elucidating the mechanisms of immune-mediated muscle damage will not only apply to DMD and IAV, but also to other conditions where the immune system, inflammation, and muscle tissue are known to be affected, such as autoimmune diseases, cancer, and aging.

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Figures

Human IAV infects zebrafish muscle cells and causes muscle fiber damage
Human IAV infects zebrafish muscle cells and causes muscle fiber damage
All embryo images are side mounts, dorsal top, anterior left. Panels A-D are phalloidin stained to visualize F-actin. White arrowheads denote retracted fibers. (A) PBS-injected control at 24 hpi (3 dpf). (B) PBS-injected control at 48 hpi (4 dpf). (C) IAV-infected embryo at 24 hpi. (D) IAV-infected embryo at 48 hpi. (E) Quantification of the proportion of muscle segments per embryo with damaged fibers in IAV-infected embryos over developmental time. Fiber damage is more frequently observed at 48 hpi than at 24 hpi. (F) Spatial location of damaged fibers along the anterior-posterior axis of IAV-infected embryos at 24 hpi. The frequency of damaged fibers peaks in segments 5-9, which is in the anterior of the fish near the Duct of Cuvier (the site of injection). (G) PBS-injected control at 24 hpi (3 dpf). (H) NS1-GFP-injected zebrafish at 24 hpi. Note the punctate green fluorescence in infected cells throughout the body. (I) NS1-GFP-injected zebrafish at 24 hpi. Higher magnification view of a GFP-positive, infected muscle fiber. (I1) Merged panel of NS1-GFP and brightfield images.
IAV infection compromises the sarcolemma
IAV infection compromises the sarcolemma
All embryo images are side mounts, dorsal top, anterior left. (A) Tg(fli1:GFP) zebrafish embryo with labeled endothelial cells (green) DC-injected with PBS plus EBD (red). EBD remains in the vasculature. White arrowheads point to EBD in an intersomitic vessel (top left), the dorsal aorta (middle), and the caudal vein (bottom right). (B-C) Wild-type zebrafish injected with PBS plus EBD. (B) Cropped EBD panel. (B1) Cropped EBD and brightfield panels merged. (C) EBD panel. Note that muscle fibers are impermeable to EBD in PBS-injected zebrafish. (D) Tg(fli1:GFP) zebrafish embryo DC-injected with IAV plus EBD (red). EBD leaked out of the vasculature and penetrated muscle fibers (black arrowheads). (E-F) Wild-type zebrafish injected with IAV plus EBD. (E) Cropped EBD panel. (E1) Cropped EBD and brightfield panels merged. (F) EBD panel. Note the uptake of EBD by long (black arrowheads) and retracted fibers (white arrowheads) indicative of sarcolemma damage.
IAV infection disrupts muscle fiber-ECM adhesion
IAV infection disrupts muscle fiber-ECM adhesion
All embryo images are side mounts, dorsal top, anterior left of zebrafish at 24 hpi. Lettered panels show phalloidin staining for F-actin in red. Panels numbered 1 show immunohistochemistry for beta-Dystroglycan, Dystrophin, or Paxillin proteins in green. Panels numbered 2 are merged images of phalloidin and antibody staining. (A-A2) Phalloidin and beta-Dystroglycan staining in a PBS-injected zebrafish. White arrow in A1 denotes MTJ localized beta-Dystroglycan and white arrowhead in A1 points to neuromuscular junction localized beta-Dystroglycan. (B-B2) Phalloidin and beta-Dystroglycan staining in an IAV-injected zebrafish. White arrow in B points to a retracted fiber and white arrowheads in B1-2 highlight beta-Dystroglycan staining at the unattached end of a retracted fiber. (C-C2) Phalloidin and Dystrophin staining in an IAV-injected zebrafish. White arrow in C points to a retracted fiber and white arrowheads in C1-2 highlight Dystrophin staining at the unattached end of a retracted fiber. (D-D2) Phalloidin and Paxillin staining in an IAV-injected zebrafish. White arrow in D points to a retracted fiber and white arrowheads in D1-2 highlight Paxillin staining at the unattached end of a retracted fiber. These results showing that some retracted fibers retain the localization of ECM adhesion proteins suggest that muscle fibers-ECM adhesion can be disrupted external to the sarcolemma.
Molecular and cellular markers of inflammation are present in the muscle tissue of IAV-infected zebrafish
Molecular and cellular markers of inflammation are present in the muscle tissue of IAV-infected zebrafish
All embryo images are side mounts, dorsal top, anterior left of zebrafish at 24 hpi. (A) PBS-injected Tg(NFkB:GFP) zebrafish embryo which expresses GFP in cells where NFkB transcription factor-dependent gene expression is occurring. NFkB signaling is active in the lateral line system, but not in muscle cells. Inset panel is a merge of fluorescence and brightfield imaging. (B) IAV-injected Tg(NFkB:GFP) zebrafish embryo. NFkB-dependent gene transcription is turned on in muscle cells in response to IAV infection. Inset panel is a merge of fluorescence and brightfield images. (C) Quantification of pro-inflammatory cytokine mRNA expression in IAV-infected zebrafish compared to PBS-injected zebrafish at 24 hpi. Expression of interleukin 1, beta (11.1 +/- 5.2-fold increase; 3 biological replicates; 3 independent experiments) and interleukin 8 (3.7 +/- 1.4-fold increase; 3 biological replicates; 3 independent experiments) increases in response to IAV while tumor necrosis factor a expression remains unchanged (1.1 +/- 0.5-fold increase; 2 biological replicates; 2 independent experiments). (D-E2) Lettered panels show phalloidin staining (red), panels numbered 1 show GFP-positive neutrophils, and panels numbered 2 are merged. (D-D2) PBS-injected Tg(mpx:GFP) zebrafish. Note that not many neutrophils are present in muscle tissue. (E-E2) IAV-infected Tg(mpx:GFP) zebrafish. Note the retracted muscle fibers and the infiltration of muscle tissue by neutrophils. White arrowheads in E2 point to neutrophils localized to the unanchored ends of retracted muscle fibers.
Muscle damage caused by IAV infection is exacerbated in the zebrafish model of DMD
Muscle damage caused by IAV infection is exacerbated in the zebrafish model of DMD
All embryo images are side mounts, dorsal top, anterior left of zebrafish at 24 hpi stained with phalloidin to visualize F-actin. White arrowheads point to foci of muscle damage. (A) Anterior muscle segments of a PBS-injected wild-type sibling embryo. (B) Posterior muscle segments of a PBS-injected wild-type sibling embryo. Note the lack of muscle damage present. (C) Anterior muscle segments of a PBS-injected sapje/dmd mutant embryo. (D) Posterior muscle segments of a PBS-injected sapje/dmd mutant embryo. Note that certain muscle segments in the anterior and the posterior have foci of muscle damage. (E) Anterior muscle segments of an IAV-injected wild-type sibling embryo. (F) Posterior muscle segments of an IAV-injected wild-type sibling embryo. Note the damaged fibers in the anterior muscle segments. (G) Anterior muscle segments of an IAV-injected sapje/dmd mutant embryo. (H) Posterior muscle segments of an IAV-injected sapje/dmd mutant embryo. Note the presence of damaged fibers in every imaged muscle segment of this embryo. (I) Quantification of the average number of muscle segments with damaged fibers per embryo at 24 hpi. Note that the prevalence of fiber damage in IAV-infected sapje/dmd mutants is more than would be predicted from adding the prevalence of damaged fibers of IAV-infected zebrafish and sapje/dmd mutants together. (J) Plot tracking survival for 5 days post injection. All PBS-injected wild-type siblings and sapje/dmd mutants lived for 5 dpi (blue lines). Most mortalities were observed in IAV-infected wild-type siblings between 4 and 5 dpi (green line). IAV-infected sapje/dmd mutants succumbed to the infection earlier than their wild-type siblings with more mortalities occurring on the first and second days post infection. Survival curves from individual experiments representative of three replicates.
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References

    1. Crum-Cianflone NF. Bacterial, fungal, parasitic, and viral myositis. Clin Microbiol Rev. 2008 Jul;21(3):473-94. PubMed PMID:18625683. - PMC - PubMed
    1. Bassett DI, Bryson-Richardson RJ, Daggett DF, Gautier P, Keenan DG, Currie PD. Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo. Development. 2003 Dec;130(23):5851-60. PubMed PMID:14573513. - PubMed
    1. Dumont NA, Wang YX, von Maltzahn J, Pasut A, Bentzinger CF, Brun CE, Rudnicki MA. Dystrophin expression in muscle stem cells regulates their polarity and asymmetric division. Nat Med. 2015 Dec;21(12):1455-63. PubMed PMID:26569381. - PMC - PubMed
    1. Borgatta B, Pérez M, Rello J, Vidaur L, Lorente L, Socías L, Pozo JC, Pozo J, Garnacho-Montero J, Rello J. Elevation of creatine kinase is associated with worse outcomes in 2009 pH1N1 influenza A infection. Intensive Care Med. 2012 Jul;38(7):1152-61. PubMed PMID:22527080. - PubMed
    1. Lorenzoni PJ, Kay CS, Scola RH, Carraro Júnior H, Werneck LC. Muscle biopsy features in critical ill patients with 2009 influenza A (H1N1) virus infection. Arq Neuropsiquiatr. 2012 May;70(5):325-9. PubMed PMID:22618784. - PubMed

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