Therapeutic exercise attenuates neutrophilic lung injury and skeletal muscle wasting

Abstract

Early mobilization of critically ill patients with the acute respiratory distress syndrome (ARDS) has emerged as a therapeutic strategy that improves patient outcomes, such as the duration of mechanical ventilation and muscle strength. Despite the apparent efficacy of early mobility programs, their use in clinical practice is limited outside of specialized centers and clinical trials. To evaluate the mechanisms underlying mobility therapy, we exercised acute lung injury (ALI) mice for 2 days after the instillation of lipopolysaccharides into their lungs. We found that a short duration of moderate intensity exercise in ALI mice attenuated muscle ring finger 1 (MuRF1)-mediated atrophy of the limb and respiratory muscles and improved limb muscle force generation. Exercise also limited the influx of neutrophils into the alveolar space through modulation of a coordinated systemic neutrophil chemokine response. Granulocyte colony-stimulating factor (G-CSF) concentrations were systemically reduced by exercise in ALI mice, and in vivo blockade of the G-CSF receptor recapitulated the lung exercise phenotype in ALI mice. Additionally, plasma G-CSF concentrations in humans with acute respiratory failure (ARF) undergoing early mobility therapy showed greater decrements over time compared to control ARF patients. Together, these data provide a mechanism whereby early mobility therapy attenuates muscle wasting and limits ongoing alveolar neutrophilia through modulation of systemic neutrophil chemokines in lung-injured mice and humans.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. Intratracheal instillation of LPS causes lung injury in mice

(A) Representative hematoxylin and eosin (H&E)–stained left lung sections of sham and ALI mice are shown and were evaluated for parenchymal inflammation using digital imaging software at low (left panels) or high (middle and right panels) magnification at day 3. In overlay images, normal alveolar space (blue), airways (red), bronchial epithelium (yellow), and injured areas (green) can be identified. Scale bar, 3 mm (left panels); 300 µm (middle and right panels). (B) Quantification of the percentage of lung inflammation based on digital morphometry images. (C) Bronchoalveolar total cell counts (left axis) and protein levels (right axis) in sham and ALI mice on day 3. n = 4 to 6 per group. Data were analyzed using the Student’s two-tailed t test.

Fig. 2. Physical activity is reduced in ALI mice

(A and B) Traveling (A) and rearing (B) activity was measured 2 days before and 3 days after instillation of intratracheal LPS (i.t.LPS). A.U., arbitrary unit. (C to E) Sham and ALI mice underwent hindlimb immobilization at day 0 and were harvested at day 3. MuRF1 mRNA (C) and protein (D and E) levels were quantified in the tibialis anterior muscle of sham and ALI control (C) and immobilized (I) muscles. (F) Experimental scheme of most exercise experiments detailed within this article. Deviations from this scheme are noted within the text. Values represent means ± SEM. n = 4 per group (A and B); n =3 to 4 per group (C to E). (A and B) *P = 0.00002, **P = 0.0003, and ***P = 0.00006 with Bonferroni correction at all time points compared to time 0 using the Student’s two-tailed t test.

Fig. 3. Therapeutic exercise attenuates ALI-induced muscle atrophy and improves muscle performance

(A) Type I (light) and II (dark) myofibers of the soleus and the diaphragm were identified by adenosine triphosphatase (ATPase) and laminin (green) staining. Scale bar, 100 µm. (B and C) Muscle fiber cross-sectional area (CSA) was quantified in sham, ALI, and ALI + Ex mice. (D to F) MuRF1 mRNA (D) and protein (E and F) levels normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) in gastrocnemius (GAS), diaphragm (DIA), and soleus (SOL) muscles. (G and H) Phosphorylated and total p65 protein from gastrocnemius nuclear extracts. D2 (day 2). (I to L) Ex vivo isolated soleus (I and J) absolute and specific twitch and (K and L) tetanic contractile force measurements. Values represent means ± SEM. All experimental time points are at day 3, other than (G) and (H), which are at day 2. n = 4 to 5 per group (A to D); n = 6 to 8 per group of two combined experiments (F); n = 3 per group (G and H); n = 4 animals and 8 muscles per group (I to L). Data were analyzed using the Student’s two-tailed t test or analysis of variance (ANOVA) for group differences with multiple time points.

Fig. 4. Therapeutic exercise reduces alveolar neutrophilia

(A and B) BAL total leukocyte (A) and differential cell counts (B) in sham, ALI, and ALI + Ex mice at specified time points. (C and D) Total blood leukocytes (C) and differential cell counts (D) were measured in sham, ALI, and ALI + Ex mice at day 3. n = 3 to 6 per group. Data were analyzed using the Student’s two-tailed t test.

Fig. 5. Blockade of G-CSF activity limits lung injury but does not attenuate muscle atrophy

(A to D) G-CSF, IL-17A, IL-17F, and IL-23 protein quantification in the plasma of sham, ALI, and ALI + Ex mice. (E) BAL cell counts were quantified at day 3 after systemic administration of isotype antibody (ALI + isotype Ab), exercise (ALI + Ex + vehicle), or G-CSFR–blocking antibody (ALI + G-CSFR Ab) 1 day after i.t.LPS administration. (F) Type I (light) and II (dark) myofibers of the soleus were identified by ATPase and laminin (green) staining. (G) Cross-sectional area was quantified in ALI + isotype Ab, ALI + Ex + vehicle, and ALI + G-CSFR Ab mice. Scale bar, 100 µm. (H and I) Soleus muscle lysates were probed for MuRF1 protein and normalized to GAPDH in sham, ALI + isotype Ab, ALI + Ex + vehicle, and ALI + G-CSFR Ab mice (H) and quantified by densitometry (I). n = 3 to 7 per group. Data were analyzed using the Student’s two-tailed t test.

Fig. 6. Early mobility therapy reduces plasma G-CSF over time in patients with ARF

(A) G-CSF concentrations measured by enzyme-linked immunosorbent assay (ELISA) at specified time points on plasma samples from patients in a randomized controlled study of early mobility therapy. (B) G-CSF concentrations normalized to the mean baseline concentration of G-CSF of each group at specific time points. Group differences over time were analyzed using the generalized estimating equation (GEE) method, and time point differences in (B) were compared using the Mann-Whitney test. ex, exercise.