Influenza a Virus Infection Induces Muscle Wasting Via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1

This information is current as Kathryn A. Radigan, Trevor T. Nicholson, Lynn C. Welch, of September 27, 2021. Monica Chi, Luciano Amarelle, Martín Angulo, Masahiko Shigemura, Atsuko Shigemura, Constance E. Runyan, Luisa Morales-Nebreda, Harris Perlman, Ermelinda Ceco, Emilia Lecuona, Laura A. Dada, Alexander V. Misharin, Gokhan M. Mutlu, Jacob I. Sznajder and G. R. Scott Budinger Downloaded from J Immunol 2019; 202:484-493; Prepublished online 7 December 2018; doi: 10.4049/jimmunol.1701433 http://www.jimmunol.org/content/202/2/484 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inﬂuenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1

Kathryn A. Radigan,*,1 Trevor T. Nicholson,*,1 Lynn C. Welch,*,1 Monica Chi,* Luciano Amarelle,*,† Martı´n Angulo,*,† Masahiko Shigemura,* Atsuko Shigemura,* Constance E. Runyan,* Luisa Morales-Nebreda,* Harris Perlman,* Ermelinda Ceco,* Emilia Lecuona,* Laura A. Dada,* Alexander V. Misharin,* Gokhan M. Mutlu,‡ Jacob I. Sznajder,*,2 and G. R. Scott Budinger*,2

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration–approved Ab antagonist Downloaded from to the IL-6R (tocilizumab) attenuated the severity of influenza A–induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1+/2 and atrogin-12/2 mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome. The Journal of Immunology, 2019, 202: 484–493. http://www.jimmunol.org/

nfluenza A pneumonia is the most common cause of mor- ARDS, muscle weakness results in substantial morbidity and long- bidity from an infectious agent, responsible for at least term reductions in quality of life (8–11). I 20,000–50,000 deaths in the United States yearly (1, 2). The As survival from ARDS continues to improve (12, 13), it is lung epithelium is the primary target of the influenza A virus increasingly important to prevent muscle dysfunction and other (IAV) infection, and the resulting diffuse damage to the epithelium long-term sequelae of the syndrome (14). Our understanding of can impair gas exchange, resulting in severe pneumonia and de- the pathophysiology of muscle weakness associated with critical velopment of acute respiratory distress syndrome (ARDS). About illness is incomplete. Some investigators have argued that skeletal by guest on September 27, 2021 30–40% of patients with ARDS die, and most survivors often muscle dysfunction in ARDS is largely attributable to the pro- suffer multiple morbidities related to their prolonged critical ill- longed immobility associated with critical illness (14, 15). Others ness (3, 4). Muscle weakness is evident in up to 50% of ARDS have shown that the ubiquitin proteasome system is activated in survivors, in whom it is associated with prolonged lengths of stay skeletal muscle within hours or days of the onset of critical illness, in the intensive care unit and the development of the multiple- suggesting a role for active signaling processes that promote organ dysfunction syndrome (5–7). In patients who survive muscle dysfunction (7, 16). Maintenance of muscle mass requires an equilibrium between the *Division of Pulmonary and Critical Care Medicine, Department of Medicine, Feinberg synthesis and breakdown of myofiber proteins. Muscle atrophy School of Medicine, Northwestern University, Chicago, IL 60611; †Departamento occurs when there is a net loss of muscle mass, causing shrinkage of de Fisiopatologıá, Facultad de Medicina, Universidad de la Repu´blica, Montevideo 11600, Uruguay; and ‡Section of Pulmonary and Critical Care Medicine, Univer- the myofibers. There are several different types of skeletal muscle sity of Chicago, Chicago, IL 60637 atrophy, including sarcopenia, disuse atrophy, and cachexia (9, 10, 1K.A.R., T.T.N., and L.C.W. are first coauthors. 17). The forkhead box O (FoxO) class of transcription factors has 2J.I.S. and G.R.S.B. are cocorresponding authors. been implicated in the control of muscle degradation (18, 19). ORCIDs: 0000-0003-3187-1493 (T.T.N.); 0000-0003-1666-3696 (L.C.W.); 0000- Specifically, FoxO1 and FoxO3 upregulate the muscle-specific 0001-7767-6584 (M.S.); 0000-0003-0474-718X (L.A.D.); 0000-0003-2879- ubiquitin ligases: muscle atrophy F-box protein (MAFbx/atrogin- 3789 (A.V.M.); 0000-0002-2089-4764 (J.I.S.). 1) and muscle RING finger 1 (MuRF1), which target muscle pro- Received for publication October 12, 2017. Accepted for publication November 5, teins for degradation by the ubiquitin proteasome system (20, 21). 2018. The proinflammatory cytokine IL-6 has been reported to regulate This work was supported by grants from the National Institute on Aging (PO1-AG- 49665) and the National Institutes of Health (HL-71643, T32-HL-076139). atrogin-1 and thus promote muscle atrophy (22–24). Cellular sig- Address correspondence and reprint requests to Dr. Jacob I. Sznajder and Dr. G. R. Scott naling induced by IL-6 has been shown to activate JAK/STAT3 (25). Budinger, Northwestern University, 240 E. Huron Street, McGaw M300, Chicago, IL Phosphorylation of STAT3 leads to the translocation of FoxO3 to 60611. Email addresses: [email protected] (J.I.S.) and s-buding@northwestern. the nucleus, leading to upregulation of atrogin-1 (19, 26). edu (G.R.S.B.) In animal models and healthy volunteers infected with IAV, IL-6 Abbreviations used in this article: ARDS, acute respiratory distress syndrome; BAL, a bronchoalveolar lavage; CSA, cross-sectional area; dpi, day postinfection; EDL, and TNF- are rapidly detected in bronchoalveolar lavage (BAL) extensor digitorum longus; FoxO, forkhead box O; IAV, influenza A virus; fluid and nasal washings (27, 28). IL-6 is also increased in the MAFbx/atrogin-1, muscle atrophy F-box protein; MOI, multiplicity of infection; serum of patients with lung injury (29, 30) and in mouse models of MuRF1, muscle RING finger 1; Scramb; scrambled; siRNA, small interfering RNA. influenza infection (28). Increased levels of IL-6 and other in- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 flammatory cytokines are associated with prolonged mechanical www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701433 The Journal of Immunology 485 ventilation (29, 30) and increased mortality (29–31). In this study, Cell culture, small interfering RNA transfection, drug we sought to determine whether IL-6–mediated activation of treatment, myotube analysis, and viral plaque assay STAT3/atrogin-1 was necessary for the development of muscle C2C12 mouse myoblasts (CRL-1772; American Type Culture Collection, dysfunction in a murine model of influenza A infection. Manassas, VA) were cultured and differentiated as we have previously described (34). For experimental procedures, myoblasts were grown to 90– 95% confluence, and then media was switched to 2% horse serum DMEM Materials and Methods (differentiation media) and renewed daily for optimal myotube formation. Reagents For small interfering RNA (siRNA) transfection experiments, 90–95% confluent C2C12 myoblasts were switched to differentiation media (day 0). All cell culture reagents were from Corning Life Sciences (Tewksbury, On day 1 of differentiation, the media was replaced with antibiotic-free MA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, differentiation media, and the transfection protocol was performed 12 h MO). later. On day 2 of differentiation, cells were transfected with 60 pmol of Mice either mouse STAT3, FKHRL1, FKHR, or control siRNA (scrambled [Scramb]) pools (Santa Cruz Biotechnology, Dallas, TX) using Lipofect- Adult male (12–16 wk) C57BL/6 wild-type mice were obtained from The amine RNAiMax transfection reagent (Thermo Fisher Scientific, Waltham, Jackson Laboratory (Bar Harbor, ME). Age-matched male atrogin-12/2 MA) according to the manufacturer’s instructions. On day 4 of differen- and atrogin-1+/2 mice as well as wild-type littermates (atrogin-1+/+)ona tiation, cells were treated with 10 ng/ml of recombinant mouse IL-6 (BD 129S/C57BL/6 background have been described previously and were ob- Biosciences, San Jose, CA) for 16 h. For the dose-response treatment of tained from Regeneron Pharmaceuticals and S. Lecker at Beth Israel IL-6, cells were treated on day 4 of differentiation with various concen- Deaconess Medical Center and Harvard Medical School (20). Mice were trations of IL-6 (2.5, 5, and 10 ng/ml) for 16 h. For experiments with provided with food and water ad libitum, maintained on a 14-h light/10-h tocilizumab, cells were treated with 10 mg/ml on day 3 for 24 h followed dark cycle, and handled according to National Institutes of Health and by treatment on day 4 of differentiation with 10 ng/ml of recombinant Downloaded from Northwestern University Institutional Animal Care and Use Committee mouse IL-6 for 16 h. C2C12 myotubes were treated on day 4 with a guidelines. Northwestern University’s Institutional Animal Care and Use multiplicity of infection (MOI) of 2 of IAV (A/WSN/33 [H1N1]) for 16 h. Committee approved all animal experimental protocols. On day 5 of differentiation, cells were imaged, and then cell lysates were obtained for Western blot analysis. Images were acquired using a Nikon IAV and infection Eclipse TE2000-U inverted scope with a 403 objective. Myotube diam- ∼ IAV (A/WSN/33 [H1N1]), a mouse-adapted virus, was provided by R. Lamb, eters were quantified by one blinded operator measuring 375–679 tube diameters (5–10 measurements per fiber) from five random fields from a Ph.D., Sc.D., Northwestern University, Evanston, IL. Mice were anesthetized http://www.jimmunol.org/ with isoflurane, their lungs were intubated with a 20-gauge angiocath (32, 33), minimum of three independent experiments using MetaMorph Software and two 25-ml sterile aliquots of PBS (control) or IAV (A/WSN/33 [H1N1]) (Molecular Devices, Sunnyvale, CA; version 6.3) as previously described (1500 or 150 PFU/mouse) were instilled through the catheter as we have (34). Madin–Darby canine kidney cells (CCL-34; American Type Culture Collection) were maintained as described previously and used to perform a previously described (28). We continuously observed mice infected with IAV viral plaque assay with homogenized soleus muscle or lungs from mice for signs of distress (slowed respiration, failure to respond to cage tapping, lack of grooming, huddling, and fur ruffling). Mice that developed these infected with influenza (37). symptoms were sacrificed, and the death was recorded as an influenza A– Western blot analysis induced mortality. Weight was measured at infection time and prior to harvest 7 d postinfection (dpi) and recorded as percentage of weight loss from C2C12 myotubes were washed with ice-cold PBS and then lysed with 23 baseline. Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA). Lysates were centrifuged at 20,000 3 g and the supernatant was collected. Soleus and by guest on September 27, 2021 Administration of tocilizumab EDL muscles were lysed with ice-cold lysis buffer (Nonidet P-40 1%, glycerol 10%, NaCl 137 mM, Tris-HCl [pH 7.5] 20 mM) containing Tocilizumab is a humanized mAb against the IL-6R; for our studies, we used protease (1 cOmplete Mini, EDTA-free tablet, Roche) and phosphatase a mouse-adapted form of tociluzimab (Genentech, South San Francisco, (sodium fluoride 30 mM, b-glycerophospate 250 mM, sodium orthova- CA). Briefly, adult male C57BL/6 mice were anesthetized with isoflurane nadate 1 mM) inhibitors (38). Tissue was then homogenized with a Tissue- and were administered tocilizumab (8 mg/kg) or PBS alone by a retro- m Tearor (BioSpec Products, Bartlesville, OK) for 1 min. Samples were orbital injection in a total volume of 150 l. After 24 h, the mice were centrifuged at 15,000 rpm for 10 min at 4˚C, and the supernatant was infected with IAV or PBS as described above. collected. Protein concentrations were determined with Protein Assay Dye Measurement of muscle dysfunction (Bio-Rad, Hercules, CA) using the Bradford method (39). Equal amounts of protein were loaded on an SDS-PAGE apparatus, and Western blot Immediately prior to muscle harvest, forelimb skeletal muscle strength was analysis was performed as described previously (40). Incubation with assessed using a digital grip strength meter (Columbus Instruments, Co- primary Abs was performed overnight at 4˚C. Immunoblots were quanti- lumbus, OH) as described previously (34, 35). Grip strength was measured fied by densitometry using Image J 1.46r (National Institutes of Health) or in each animal six successive times, and the average of the highest four Image Studio Software (LI-COR Biosciences, Lincoln, NE) (41). The values for each mouse was used. The same operator performed all tests. following Abs were used: rabbit monoclonal to Fbx32 (catalog: ab168372; The mice were then terminally anesthetized with Euthasol (pentobarbital 1:1000; Abcam, Cambridge, U.K.), rabbit polyclonal to MuRF1 (catalog: sodium/phenytoin sodium). The soleus and extensor digitorum longus MP3401; 1:1000; ECM Biosciences, Versailles, KY), rabbit monoclonal to (EDL) muscles were excised, and the tendon was trimmed under a mi- GAPDH (D16H11) (catalog: 5174s; 1:1000; Cell Signaling Technology), croscope to ensure optimal accuracy for weight measurement. The muscles rabbit monoclonal to STAT3 (79D7) (catalog: 4904s; 1:1000; Cell Sig- were then blotted dry and weighed. Muscles were either frozen in liquid naling Technology), rabbit monoclonal to FoxO3a (75D8) (catalog: 2497s; nitrogen–cooled isopentane for cryosectioning or snap frozen in liquid 1:1000; Cell Signaling Technology), and rabbit polyclonal to FoxO1 (L27) nitrogen for protein extraction. (catalog: 9454s; 1:1000; Cell Signaling Technology). Primary Abs were detected with HRP-conjugated secondary Abs, including anti-rabbit IgG, Immunohistochemistry and fiber size assessment HRP linked (catalog: 7074s; 1:2000 dilution; Cell Signaling Technology) and goat anti-rabbit IgG, HRP conjugate (catalog: 170-6515; 1:16,000 m Soleus and EDL serial transverse cryosections (8 m) were obtained from dilution; Bio-Rad). the Northwestern University Mouse Histology and Phenotyping Labora- tory and mounted on glass slides. Sections were fixed in 4% formaldehyde, BAL fluid and blood collection for measurement of cell count, permeabilized, and blocked. Immunostaining was performed with laminin protein, and IL-6 primary Ab (catalog: L9393; 1:50; Sigma-Aldrich) followed by Alexa Fluor 568–conjugated secondary Ab (catalog: A-11011; 1:200; Life Animals were euthanized with Euthasol (pentobarbital sodium/phenytoin Technologies, Carlsbad, CA). Images were acquired with a Zeiss LSM 510 sodium) prior to BAL and blood collection. A midline dissection confocal microscope using a 403 objective (Northwestern University through the abdominal cavity up to the thoracic cavity was performed, Center for Advanced Microscopy) and analyzed using Fiji (National In- followed by a small incision at the edge of the diaphragm, which resulted in stitutes of Health) (36). Fiber size and cross-sectional area (CSA) were a fatal pneumothorax. Then, a right ventricular puncture for the collection of determined as previously described (34). All analysis was performed by blood was performed. Blood was collected in a microvette microtube with one blinded operator. EDTA (Kent Scientific), kept on ice, and then spun at 2000 rpm. Serum was 486 IL-6 MEDIATES MUSCLE WASTING DURING INFLUENZA A INFECTION collected and used for IL-6 determination by ELISA (KMC0062; Thermo grip strengths were markedly decreased, but there was no difference Fisher Scientific) (37). BAL fluid was obtained through a 20-gauge between control (PBS-treated) and IAV-treated mice, suggesting that angiocath ligated into the trachea through a tracheostomy. A total of 1 ml lower levels of atrogin-1 are protective during influenza infection of PBS instilled into the lungs was aspirated three times to collect BAL +/+ fluid for cell counts (Cellometer K2; Nexcelom Bioscience), protein (Fig. 2D). Whereas atrogin-1 mice had significantly decreased wet quantification (Bradford assay), and IL-6 determination by ELISA. muscle weight for soleus and EDL after IAV infection, wet muscle weight after influenza A infection was similar to uninfected mice in Statistics 2 2 2 the atrogin-1 / mice (Fig. 2E, 2F). The haploinsufficient atrogin-1+/ Results are shown as mean 6 SEM. Data were analyzed and statistical mice showed levels of wet muscle weight loss that were inter- differences were compared by a Student t test between two groups and by a mediate between atrogin-1+/+ and atrogin-12/2 (Fig. 2E, 2F). The one-way ANOVA with Dunnett or Tukey post hoc corrections for three or more groups using GraphPad Prism version 7.0 for Windows (GraphPad mean fiber CSA for the soleus and EDL muscles were decreased +/+ Software, La Jolla, CA). All in vitro experiments were repeated a mini- in the atrogin-1 IAV-infected mice as compared with control mum of three times. We considered specific data points outliers if they (PBS-treated) mice, but the IAV-induced decrease in CSA was deviated by more than two times the SD from the mean, and they were attenuated in atrogin-1+/2 and atrogin-12/2 mice (Fig. 2G–P). excluded automatically from the statistical analysis. Results were consid- Each of the atrogin-1 genotypes infected with IAV lost body ered significant when the p value was ,0.05. weight as compared with the PBS-treated mice, but the atrogin-1+/2 and atrogin-12/2 weight loss was slightly less severe (Fig. 3A). Results Additionally, IL-6 levels were measured in serum and BAL fluid. C57BL/6 wild-type mice lose muscle weight and strength after Total protein and cell count were also determined in the BAL fluid. 2 2 influenza infection IL-6 levels were markedly increased in the IAV-infected atrogin-1 / Downloaded from We infected C57BL/6 wild-type mice with either a low dose (150 mice in both serum and BAL fluid as compared with IAV-infected +/2 2/2 PFU/mouse) or high dose (1500 PFU/mouse) of IAV (A/WSN/33 atrogin-1 and atrogin-1 mice (Fig. 3B, 3C). There was no [H1N1]) and assessed total body weight, forelimb grip strength, difference in total protein and cell count of the BAL fluid among the soleus and EDL muscle wet weight, and fiber CSA. Compared with different atrogin-1 genotypes infected with IAV (Fig. 3D, 3E). control (PBS-treated) mice, IAV-infected mice experienced sig- The IL-6R Ab tocilizumab attenuates influenza-induced nificant weight loss after both low-dose and high-dose IAV in- http://www.jimmunol.org/ muscle dysfunction fection (Fig. 1A). Forelimb grip strength was significantly decreased in the IAV-infected mice (Fig. 1B). In addition, mice To investigate the role of IL-6 in mediating influenza A–induced infected with either low- or high-dose influenza had significantly muscle atrophy in vivo, we treated C57BL/6 mice with tocilizumab, decreased soleus and EDL wet muscle weight (Fig. 1C, 1D). which is a mAb with specificity for the IL-6R that prevents IL-6 from Fig. 1E and 1H show representative images of soleus and EDL binding to both the soluble and membrane-bound IL-6R through laminin-stained cryosections that were used to determine the mean competitive blockade (42). C57BL/6 mice were administered tocili- CSA. Mice infected with high-dose IAV had significantly de- zumab (8 mg/kg) or PBS (control) by retro-orbital injection 24 h creased mean soleus and EDL CSA (Fig. 1F, 1I), with the histo- prior to infection with IAV (1500 PFU/mouse) or PBS, and then 7 grams showing a left shift of frequency distribution indicating a dpi, weight loss and grip strength were assessed. There was no dif- by guest on September 27, 2021 predominance of thinner fibers in IAV-infected mice (Fig. 1G, 1J). ference in total-body IAV-induced weight loss between the PBS- and To determine that IAV infection was restricted to the lung, we tocilizumab-treated mice (Fig. 4A). The reduction of grip strength measured by plaque assay the viral load in lungs and soleus muscle observed in IAV-infected mice treated with vehicle was attenuated in of mice infected with IAV for 7 d. As expected, the viral load was the IAV-infected and tocilizumab-treated mice (Fig. 4B). Consistent almost undetectable in soleus muscle homogenates compared with with these findings, soleus and EDL wet muscle weights were sig- lung homogenates of wild-type IAV-infected mice (soleus: 38.33 nificantly reduced in influenza-infected compared with uninfected PFU/ml 6 6.009 SEM; lung: 1.25 3 107 PFU/ml 6 8.3660 3 105 wild-type mice, whereas there was no significant decrease in either SEM). A Student t test between two groups was used to show the soleus or EDL wet muscle weight in uninfected compared with statistical differences, with p , 0.0001 (n = 3–5 mice per group). tocilizumab-treated mice (Fig. 4C, 4D). The induction of atrogin-1 protein by influenza A was attenuated in the soleus muscle Influenza-induced muscle dysfunction is attenuated in mice of tocilizumab-treated mice (Fig. 4D). Similarly, the influenza A– with genetic loss of atrogin-1 induced reductions in soleus and EDL CSA were attenuated in The ubiquitin proteasome system regulates muscle degradation via mice that had been pretreated with tocilizumab compared with the E3 ubiquitin ligases MAFbx/atrogin-1 and MuRF1 (20, 21). vehicle, as was the left shift of muscle fiber size frequency dis- We set out to determine if one or both of these muscle-specific E3 tribution for both the soleus and EDL (Fig. 4F–M). ubiqutin ligases were upregulated in our murine model of IAV infection. C57BL/6 wild-type mice were infected with IAV (1500 IL-6 acts via STAT3, FoxO3a, and atrogin-1 to induce skeletal PFU/mouse), and 7 d dpi, the mice were sacrificed and the soleus muscle atrophy muscles were excised. We found that atrogin-1 protein expression To obtain insight into the mechanisms by which IL-6 induces muscle was significantly increased, whereas MuRF1 levels remained un- atrophy, we treated differentiated C2C12 myotubes with various changed (Fig. 2A, 2B). To further explore the role of atrogin-1 in doses of rIL-6 (2.5, 5, and 10 ng/ml) or with 1 MOI of IAVor media mediating muscle protein loss with influenza, we used mice with alone, and blinded measurements of myotube diameters were per- heterozygous or homozygous deletion of the MAFBx/atrogin-1 formed 16 h later. Treatment of C2C12 cells with IL-6 resulted in a gene (designated as atrogin-1 throughout the paper) (20). Com- dose-dependent reduction in myotube diameter, whereas treatment pared with their wild-type (atrogin-1+/+) littermates, levels of with IAV had no effect on fiber diameter (Fig. 5A). To determine if atrogin-1 were significantly lower in the soleus muscle of atrogin- STAT3 was involved in the IL-6–induced reduction in myotube di- 1+/2 and atrogin-12/2 mice (Fig. 2C). Phenotypic differences in the ameter, we transfected well-differentiated C2C12 cells with Scramb animals were also evident. Basally, atrogin-1+/2 mice had slightly siRNA or siRNA specific for STAT3 followed by treatment with rIL-6, reduced grip strength, but this was not statically different as compared and then we measured myotube diameters. Silencing of STAT3 with the PBS-treated atrogin-1+/+ mice. Atrogin-12/2 mice baseline prevented the decrease in diameter seen with IL-6 treatment The Journal of Immunology 487 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Wild-type mice lose muscle weight and strength after influenza infection and have reduced CSA after influenza infection. Adult male C57BL/6 mice were infected with 150 or 1500 PFU/mouse of IAVor PBS (control), and 7 dpi, (A) total body weight (n = 6 mice per group) and (B) forelimb grip strength were determined (n = 4–9 mice per group). (C)Soleus(n = 11–12 mice per group) and (D)EDL(n = 5–13 mice per group) wet muscle weights were determined after muscles were excised, frozen, and cryosectioned. (E–J) Soleus and EDL CSAs were determined using cross-sections immunostained for laminin. (E and H) Representative images are shown (original magnification 340). Scale bars, 20 mm. (F) Mean soleus fiber CSA (n = 3–6 mice per group) and (G) a histogram depicting the fiber size distribution. (I) Mean EDL fiber CSA (n = 4–6 mice per group) and (J) histogram depicting the fiber size distribution. Bars represent means 6 SEM. n $ 3 of at least three independent experiments. A one-way ANOVAwith Dunnett post hoc corrections for comparison with more than three groups and a Student t test between two groups were used to show statistical differences. All groups are compared with the PBS-treated control, and p values are shown.

(Fig. 5B). To determine whether STAT3 acts through FoxO1 or develop skeletal muscle weakness that is associated with pro- FoxO3a to reduce myotube diameter, we compared the effect of IL-6 longed duration of mechanical ventilation and with increased on myotube diameter in control and FoxO1- and FoxO3a-silenced short-term and long-term mortality (43, 44). In patients who sur- cells. Silencing of FoxO3a attenuated the decline in myotube di- vive ARDS, muscle dysfunction may persist for years after hos- ameter induced by IL-6 (Fig. 5C), but silencing of FoxO1 did not pital discharge, impairing quality of life and affecting survival prevent the decrease in myotube diameter (Fig. 5D). Consistent with (1, 2, 9, 10, 17). our in vivo findings, pretreatment of myotubes with tocilizumab Using a murine model of influenza A infection, we show that prevented the IL-6–induced decrease in myotube diameter as com- IL-6 released from the influenza A–infected lung upregulates pared with the myotubes that received the mock treatment and IL-6 atrogin-1 to promote the active degradation of muscle proteins via (Fig. 5E). Moreover, treatment of myotubes with IL-6 increased the the ubiquitin proteasome system. Muscle mass and function could expression of atrogin-1 in vehicle-treated cells, and this effect was be preserved during influenza A infection through genetic deletion attenuated in cells pretreated with tocilizumab (Fig. 5F). Accordingly, of atrogin-1 or through pharmacologic blockade of the IL-6R. The we provide evidence that IL-6 released from the lungs as a result of fact that mice infected with IAV had negligible viral load detected influenza infection leads to the upregulation of the E3 ubiquitin ligase in the soleus muscle homogenates and that differentiated C2C12 atrogin-1, which results in muscle degradation (Fig. 6). mouse myotubes have reduced muscle fiber size when treated with IL-6 and not when incubated with IAV suggest an endocrine Discussion mechanism for the muscle loss observed in influenza A–infected Severe infection with IAV is an important cause of lung injury and mice. The effects of IL-6 on fiber size were attenuated by ARDS (1, 2). A significant proportion of patients with ARDS knockdown of STAT3 or FoxO3a and by pharmacologic inhibition 488 IL-6 MEDIATES MUSCLE WASTING DURING INFLUENZA A INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Inﬂuenza-induced muscle dysfunction is attenuated in mice with genetic loss of atrogin-1. (A and B) Adult male C57BL/6 mice were infected with 1500 PFU/mouse of IAVor PBS (control), and 7 dpi, soleus muscles were excised and atrogin-1 (n = 6 mice per group) and MuRF1 (n = 3 mice per group) expression was determined by Western blot. b-Actin was used as loading control. A Student t test between two groups was used to show statistical differences. (C) Soleus muscle from atrogin-1 (+/+, +/2, and 2/2) mice was excised, and atrogin-1 expression was determined by Western blot. GAPDH was used as loading control. (n = 3–4 mice per group). A one-way ANOVAwith Dunnett post hoc corrections for comparison with more than three groups was used to show statistical differences. (D–P) Atrogin-1 (+/+, +/2, and 2/2) mice were infected with 1500 PFU/mouse of IAVor PBS (control), and 7 dpi, (D) forelimb grip strength was determined. (n = 4–10 mice per group). A one-way ANOVA with Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. (E) Soleus (n = 4–9 mice per group) and (F) EDL (n = 3–9 mice per group) wet muscle weights were determined after muscles were excised, frozen, and cryosectioned. A one-way ANOVA with Tukey post hoc (Figure legend continues) The Journal of Immunology 489 Downloaded from http://www.jimmunol.org/

FIGURE 3. IL-6 levels were still increased in both serum and BAL fluid in the IAV-infected mice with genetic loss of atrogin-1.(A) Adult male atrogin-1 (+/+, +/2, and 2/2) mice were infected with 1500 PFU/mouse of IAVor PBS (control), and 7 dpi, total body weight was assessed (n = 6–8 mice per group). (B) IL-6 was measured by ELISA in the serum. (C) IL-6 was measured by ELISA in the BAL fluid. (D) Proteins in the BAL fluid were measured by Bradford assay. (E) Live cells in BAL fluid were measured by trypan blue exclusion (n = 3–5 mice per group). Bars represent means 6 SEM, and p values are shown. A one-way ANOVAwith Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. n $ 3 by guest on September 27, 2021 from at least three independent experiments. of the IL-6R but not by knockdown of FoxO1. These data suggest in the atrogin-1 2/2 mice, the lack of atrogin-1 in the muscle is that muscle loss during influenza A infection is an active process enough to protect against muscle atrophy. initiated by endocrine signals from the injured lung. Although our studies demonstrate a deleterious role for IL-6 Our data in mice are potentially relevant for humans. IL-6 and during IAV-induced lung injury, IL-6 can also be produced by TNF-a are the first detectable cytokines after influenza infection muscle fibers during exercise, where it acts as a myokine (46). For (27, 37) and are increased in the serum of patients with lung injury example, with extreme exercise, IL-6 may be sufficiently pro- (29–31). In humans with influenza A infection, IL-6 levels peak duced by working muscle to increase circulating levels up to 100- within 2 d of infection and remain elevated for up to 7 d, with fold (47) without signs of muscle tissue damage (48) or a change levels that correlate with symptom severity (27). In patients with in IL-6–expressing immune cells (49). During exercise, IL-6 is ARDS, the activation of proteolytic pathways follows a similar released from muscle in response to low glycogen stores (50) or time course, with increases in ubiquitination of muscle proteins activation of AMPK, and it acts through autocrine and paracrine detected within hours of onset of critical illness that remain active mechanisms to increase fat oxidation and enhance insulin- for up to 1 wk following discharge from the intensive care unit stimulated glucose uptake through GLUT4 translocation (51). (7, 45). Our in vivo measurements were performed at 1 wk fol- As a myokine, IL-6 also provides an endocrine signal to enhance lowing infection with IAV (A/WSN/33 [H1N1]), administered hepatic glucose production during exercise (52). Importantly, IL-6 intratracheally, at which point proteolytic pathways are active and is produced as the predominant myokine in response to exercise, muscle weakness is evident. In atrogin-1 mice, we observed an and yet, as we observed, it can also induce muscle dysfunction as increase in IL-6 in both serum and BAL fluid after IAV infection part of the cytokine storm associated with IAV infection. We in all the genotypes, but even though it was profoundly increased speculate that differences in the kinetics of IL-6 release and its

corrections for comparison with more than three groups was used to show statistical differences. (G–P) Soleus (n = 3–8 mice per group) and EDL (n = 3–8 mice per group) CSAs were determined using muscle cross-sections immunostained for laminin. A one-way ANOVA with Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. (G and H) Mean soleus and EDL fiber CSAs are shown. (I and J) Representative images are shown (original magnification 340). Scale bars, 20 mm. (K–P) Histograms depicting the fiber size distribution for soleus muscle of (K) atrogin-1+/+ (L), atrogin-1+/2, and (M) atrogin-12/2 mice and EDL muscle of (N) atrogin-1+/+ (O), atrogin-1+/2, and (P) atrogin-12/2 mice. Bars represent means 6 SEM, and p values are shown. n $ 3 from at least three independent experiments. 490 IL-6 MEDIATES MUSCLE WASTING DURING INFLUENZA A INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. The IL-6R Ab tocilizumab attenuates influenza-induced muscle dysfunction. Adult male wild-type C57BL/6 mice were pretreated with the IL-6R Ab tocilizumab (8 mg/kg), 24 h later they were infected with 1500 PFU/mouse of IAVor PBS (control), and 7 dpi, (A) total body weight (n = 4–8 mice per group) and (B) forelimb grip strength were determined (n = 7–8 mice per group). (C) Soleus (n = 9–12 mice per group) and (D) EDL (n = 10–12 mice per group) wet muscle weights were determined after muscles were excised, frozen, and cryosectioned. For (A)–(D), a one-way ANOVA with Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. The p values are shown. (E) Quantification and representative Western blot of atrogin-1 expression in the mouse soleus muscle of PBS- and tocilizumab-treated mice (n = 3 mice per group). A one-way ANOVA with Dunnett post hoc corrections for comparison with more than three groups was used to show statistical differences. All groups are compared with the PBS-treated control, and p values are shown. (F–M) Muscle cross-sections immunostained for laminin Ab were used to determine the mean soleus and EDL fiber CSAs. (F and J) Representative images are shown (original magnification 340). Scale bars, 20 mm. (G) Soleus (n = 4 mice per group) and (K) EDL (n = 3–6 mice per group) mean fiber CSAs are shown. A one-way ANOVA with Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. (H, I, L, and M) Histograms depicting the fiber size distribution are shown for (H and I) soleus and (L and M) EDL of mice treated with PBS or tocilizumab prior to influenza infection. Bars represent means 6 SEM, and p values are shown. n $ 3 of at least three independent experiments. association with other proinflammatory cytokines may underlie we saw no change in total body weight in mice treated with the differential response of muscle to IL-6. After exercise, IL-6 tocilizumab. Nevertheless, in health, IL-6 has an important role levels peak rapidly and then fall (53, 54), whereas sustained ele- in maintenance of muscle bulk, modulating muscle carbohy- vations are present for days after influenza infection (27). Further, drate and lipid metabolism (47, 58–60). The differing roles for IL-6 generated during exercise is not accompanied by an increase IL-6 signaling in the muscle during health and disease are in other cytokines elevated during influenza A infection (55). Our poorly understood and suggest that the effects of IL-6 in the data are consistent with those of others who have shown that muscle might be modulated by other factors present in the prolonged exposure to rIL-6 can induce muscle atrophy (22, 56) muscle microenvironment during disease. Furthermore, influ- and with observations from others that high levels of IL-6 are enza A–induced muscle loss was only partially inhibited by associated with the age-related decline in muscle function due to tocilizumab, suggesting that other cytokines released in re- sarcopenia (57). sponse to influenza A infection (e.g., IL-1b, IL-18, and others), We found that pharmacologic inhibition of the IL-6R using the changes in inflammatory cell populations (e.g., regulatory Food and Drug Administration–approved drug tocilizumab at- T cells), or nutrient deprivation might independently contribute tenuated muscle loss during influenza A infection. We did not to muscle dysfunction (61–66). Influenza A infection in mice see adverse effects of tocilizumab in the muscle as there was no provides an excellent model system to further explore these difference in muscle mass of the soleus and EDL. Furthermore, possibilities. The Journal of Immunology 491 Downloaded from http://www.jimmunol.org/

A FIGURE 5. IL-6 acts via STAT3, FoxO3a, and atrogin-1 to induce skeletal muscle dysfunction. ( ) C2C12 myotubes were treated with IL-6 (2.5, 5, by guest on September 27, 2021 10 ng/ml) or infected with two MOI IAVon day 4 of differentiation for 16 h. Phase contrast images were acquired on day 5 of differentiation. Graph depicts average myotube diameters (∼375 measurements per group from at least four independent experiments). A one-way ANOVA with Dunnett post hoc corrections for comparison with more than three groups was used to show statistical differences. All groups are compared with the control (CT), and p values are shown. (B–D) Myotubes were transfected with Scramb, STAT3 (∼425 measurements per group from at least three independent experiments), FoxO3a (∼625 measurements per group from at least five independent experiments), or FoxO1 (∼679 measurements per group from at least four independent experiments) siRNA on day 2 of differentiation followed by IL-6 treatment (10 ng/ml) on day 4. Phase contrast images were obtained and cell lysates were analyzed on day 5 of differentiation. Upper panel shows graph depicting average myotube diameter and lower panel depicts Western blot showing knockdown efficiency. GAPDH is shown as loading control. A one-way ANOVA with Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. (D and E) C2C12 myotubes were treated with tocilizumab (10 mg/ml) on day 3 of differentiation for 24 h followed by treatment with IL-6 for 16 h. (E) Graph depicts average myotube diameter (∼475 measurements per group from at least four independent experiments). A one-way ANOVA with Tukey post hoc corrections for comparison with more than three groups was used to show statistical differences. (F) Quantification and representative Western blot of atrogin-1 expression in C2C12 myotubes. GAPDH is shown as loading control (n = 3 mice per group). Bars represent means 6SEM, and p values are shown.

We observed increased levels of atrogin-1 in muscle following to underlie the development of multiple-organ dysfunction syn- influenza A infection, and heterozygous and homozygous loss of drome. Although our study does not answer this question, we saw atrogin-1 resulted in a dose-dependent inhibition of influenza A– no worsening in weight loss or mortality in influenza A–infected induced muscle degradation. In cultured myotubes, the induction atrogin-1 compared with wild-type controls, nor did we observe of atrogin-1 by IL-6 required the activation of STAT3 and FoxO3a any impact of tocilizumab on these parameters. However, tocili- but not FoxO1. These data suggest inhibiting atrogin-1 directly or zumab has been associated with transient neutropenia, increased the pathways required for its induction may preserve muscle mass body weight, and elevated cholesterol in clinical trials (68, 69). during influenza A infection. These results should be interpreted There are a number of limitations to this study. First, because with some caution, however, as others have observed increased the expression of atrogin-1 is largely limited to the muscle, the expression of mRNA for both atrogin-1 and MuRF1 in the skeletal effects of atrogin-1 loss in other cells is less of a concern. muscle following influenza infection (67). Further, it is not clear However, it is possible that partial or complete developmental whether the IL-6– and atrogin-1–mediated degradation of muscle loss of atrogin-1 might activate pathways in atrogin-1+/2 and proteins is an adaptive process during influenza A infection. For atrogin-12/2 mice, respectively, which protect against influenza example, the degradation of muscle proteins might provide met- A–induced muscle loss. Further studies using inducible knock- abolic substrates to fuel inflammatory cell responses to pathogen outs or pharmacologic inhibitors could address these concerns. challenge. Alternatively, the systemic release of IL-6 might be a Additionally, our model system is limited to infection with a maladaptive consequence of the excessive inflammation thought single mouse-adapted IAV. Studies using other strains of IAV or 492 IL-6 MEDIATES MUSCLE WASTING DURING INFLUENZA A INFECTION

FIGURE 6. In a murine model of influenza A infection, the release of IL-6 leads to the upregulation of the E3 ligase atrogin-1, leading to skeletal muscle dysfunction. Downloaded from administering tocilizumab to pigs, which are susceptible to cir- Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009. N. Engl. J. Med. 361: 1935–1944. culating IAVs, could address this limitation. Finally, although 5. Stevens, R. D., D. W. Dowdy, R. K. Michaels, P. A. Mendez-Tellez, IL-6 levels are often elevated in patients with ARDS secondary P. J. Pronovost, and D. M. Needham. 2007. Neuromuscular dysfunction acquired to other causes, the importance of IL-6 in muscle degradation in in critical illness: a systematic review. Intensive Care Med. 33: 1876–1891. these conditions is unknown. 6. Walsh, C. J., J. Batt, M. S. Herridge, and C. C. Dos Santos. 2014. Muscle wasting and early mobilization in acute respiratory distress syndrome. Clin. Chest Med. 35: 811–826. http://www.jimmunol.org/ In summary, we provide evidence that influenza infection re- 7. Puthucheary, Z. A., J. Rawal, M. McPhail, B. Connolly, G. Ratnayake, P. Chan, sults in the release of IL-6 from the injured lung, which activates N. S. Hopkinson, R. Phadke, T. Dew, P. S. Sidhu, et al. 2013. Acute skeletal muscle wasting in critical illness. [Published erratum appears in 2014 JAMA 311: STAT3 and FoxO3a in the muscle to induce the expression of 625.] JAMA 310: 1591–1600. atrogin-1. Atrogin-1 is an E3 ligase that targets key muscle 8. Herridge, M. S., J. Batt, and C. D. Santos. 2014. ICU-acquired weakness, proteins for active degradation via the ubiquitin proteasome morbidity, and death. Am. J. Respir. Crit. Care Med. 190: 360–362. 9. Herridge, M. S., C. M. Tansey, A. Matte´, G. Tomlinson, N. Diaz-Granados, system. Thus, as depicted in Fig. 6, IL-6 acts as an endocrine A. Cooper, C. B. Guest, C. D. Mazer, S. Mehta, T. E. Stewart, et al; Canadian signal to promote active degradation of the skeletal muscle Critical Care Trials Group. 2011. Functional disability 5 years after acute re- during influenza A pneumonia. Inhibition of the IL-6R and ge- spiratory distress syndrome. N. Engl. J. Med. 364: 1293–1304. 10. Herridge, M. S., A. M. Cheung, C. M. Tansey, A. Matte-Martyn, N. Diaz-Granados, netic deletion of atrogin-1 attenuates the loss of muscle mass and F. Al-Saidi, A. B. Cooper, C. B. Guest, C. D. Mazer, S. Mehta, et al; Canadian Critical by guest on September 27, 2021 function associated with influenza A infection, suggesting these Care Trials Group. 2003. One-year outcomes in survivors of the acute respiratory distress syndrome. N. Engl. J. Med. 348: 683–693. pathways might be therapeutically targeted to preserve muscle 11. Ceco, E., S. E. Weinberg, N. S. Chandel, and J. I. Sznajder. 2017. Metabolism and mass in patients with influenza A–induced ARDS. skeletal muscle homeostasis in lung disease. Am. J. Respir. Cell Mol. Biol. 57: 28–34. 12. Esteban, A., F. Frutos-Vivar, A. Muriel, N. D. Ferguson, O. Pen˜uelas, V. Abraira, K. Raymondos, F. Rios, N. Nin, C. Apezteguıá, et al. 2013. Evolution of mortality Acknowledgments over time in patients receiving mechanical ventilation. Am. J. Respir. Crit. Care Med. 188: 220–230. We thank Angela Pantell and Marina Casalino Matsuda for technical assistance. 13. Lilly, C. M., S. Cody, H. Zhao, K. Landry, S. P. Baker, J. McIlwaine, We are grateful to Regeneron Pharmaceuticals for providing the atrogin-1 mice. M. W. Chandler, and R. S. Irwin, University of Massachusetts Memorial Critical Histology services were provided by the Northwestern University Mouse Care Operations Group. 2011. Hospital mortality, length of stay, and preventable Histology and Phenotyping Laboratory, which is supported by National Cancer complications among critically ill patients before and after tele-ICU reengin- eering of critical care processes. JAMA 305: 2175–2183. 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