Interleukin-4 administration improves muscle function, adult myogenesis, and lifespan of colon carcinoma-bearing mice

Abstract

Background: Anorexia, body wasting, inflammation, muscle, and adipose tissue loss are hallmarks of cancer cachexia, a syndrome that affects the majority of cancer patients, impairing their ability to endure chemotherapeutic therapies and reducing their lifespan. In the last 10 years, alterations of protein turnover and impairment of adult myogenesis have been proposed as major contributing factors.

Methods: Muscle stem cells, including satellite cells, mesoangioblasts, and fibroadipogenic progenitors, were isolated and characterized from C26 colon carcinoma-bearing (C26) mice. Circulating levels of interleukin-4/13 (IL4/IL13) were analysed by ELISA, and the effects of IL4 on muscle mass and function, protein synthesis, muscle regeneration, and myogenic progenitor cell number were analysed at both functional (treadmill and grip test) and molecular levels (qRT-PCR, immunofluorescence analysis, surface sensing of translation, and western blot). The Kaplan-Meier test was used to analyse the survival curve of IL4-treated and IL4-untreated C26 mice.

Results: The administration of IL4 to C26 mice rescued muscle mass by increasing protein synthesis. The IL4 treatment improved performances and prolonged survival of C26 mice. IL4 administration re-established both number and function of satellite cells and fibroadipogenic progenitors without affecting mesoangioblasts in C26 mice, rescuing myogenesis. Upon IL4 treatment, a high number of cytotoxic lymphocytes and type II macrophages were observed with a subsequent increase in necrotic areas of C26 tumours.

Conclusions: The results here presented shed new light on IL4 signalling during muscle wasting and early stages of muscle regeneration that explain the beneficial effect observed in IL4-treated C26 mice. These findings might aid to develop therapeutic approaches to improve mobility and quality of life in cachectic patients.

Keywords: Cancer-induced skeletal muscle atrophy; Interleukin-4; Muscle function; Muscle regeneration; Protein synthesis; Survival curve.

Figure 1

Characterization of control (C) and C26 muscle stem cells. (A) Body weight loss of C (n = 6) and C26 mice (n = 8) during 14 days of tumour growth, expressed as percentage of initial body weight. (B) Tibialis anterior (TIB; C: 98 ± 5.9 mg/100 g i.b.w.), gastrocnemius (GSN; C: 280.5 ± 19 mg/100 g i.b.w.), and quadriceps (QD; C: 396 ± 29 mg/100 g i.b.w.) muscle weight from C and C26, expressed as percentage of the initial body weight. (C) BF images (on the left) and quantification (on the right) of satellite cells (SCs) extracted from C‐specific and C26‐specific muscles. (D) Representative SCs clones (on the left) and quantification (on the right) from C and C26, 4 days after seeding. (E) IF of SCs from C and C26 stained for PAX7 (red) and MyoD (green; column on the left = Day 0 of differentiation), or MyHC (red) and MyoD (green; column on the right = Day 2 of differentiation). (F) BF images of MABs isolated (column on the left) from C and C26 muscle explants after 7 days of organ culture and stained for ALP (column on the right). Quantification of C‐MABs and C26‐MABs (graph on the right). (G) GFP⁺ C‐MABs and C26‐MABs (column on the left), BF overlapped with fluorescent images of the same cells with C2C12 in co‐culture (middle column). IF for MyHC (red) and GFP (green) on GFP⁺ C‐MABs and C26‐MABs in co‐culture with C2C12 after 5 days of differentiation medium (column on the right). (H) Transversal sections of TIB from αSG‐null mice injected with GFP⁺C‐MABs or GFP⁺C26‐MABs (column on the right) or with the same cells pretreated during 48 h with dorsomorphin (5 μM, DM), before the injection. Quantification (on the right) of the percentage of GFP⁺ αSG⁺ fibres in the injected muscles. (I) C and C26 populations of adipocyte‐like cells were stained with Oil‐Red‐O (column on the left) and perilipin (PLIN1; column on the right). Nuclei were stained with HOECHST (blue). Plasma levels for (J) IL4 (pg/mL) and (K) IL13 (pg/mL) cytokines in C and C26 mice. Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. C. Scale bar: 500 μm.

Figure 2

IL4 treatment counteracts muscle weight loss and fibre CSA, restores muscle performances, and prolongs survival of tumour‐bearing mice. (A) Percentage of body weight during 14 days after tumour injection in C26 (n = 6) and IL4‐treated tumour‐bearing mice (C26 + IL4, n = 6) was compared with control (C, n = 4) and IL4‐treated (IL4, n = 4) mice. (B) Plasma levels of IL4 (pg/mL) from C, C26, and daily treated IL4 and C26 + IL4 mice. (C) H&E staining (column on the left) of TIB muscles from the different groups with insets at a higher magnification (column on the right). (D) TIB (C: 105 ± 4 mg/100 g i.b.w.), GSN (C: 269 ± 7 mg/100 g i.b.w.), expressed as percentage of the initial body weight. (E) Fibre size distribution of TIB muscles. (F) Grip test analysis, (G) running time, and (H) distance measured by treadmill exhaustion assay for C26 and C26 + IL4 and their controls group, C and IL4. (I) Kaplan–Meier test for the significance of the difference between survival curve from C26 and C26 + IL4 attributed a P = 0.001 (n = 10 per group). Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 vs. C; ⁺ P < 0.05, ⁺⁺ P < 0.01, ⁺⁺⁺ P < 0.001, ⁺⁺⁺⁺ P < 0.0001 vs. IL4; ^$ P < 0.05, ^$$ P < 0.01, ^$$$ P < 0.001, ^$$$$ P < 0.0001 vs. C26.

Figure 3

Tumour mass and flow cytometric analysis of tumour‐associated immune cells. (A) H&E staining, (B) quantifications of the necrotic area (eosin‐positive area—white) on the cell‐dense area (haematoxylin‐positive area—grey) of tumour masses from C26 and C26 + IL4 mice (P < 0.01 vs. C26). (C) Representative flow cytometry analysis and quantifications of the tumour digested cells from C26 and C26 + IL4 mice for the main markers of lymphocytes. (D) IF analysis of F4/80 and CD206 markers and (E) flow cytometry analysis of the tumour digested cells from C26 and C26 + IL4 mice for the same macrophage markers and quantifications. Significance of the differences: ^$ P < 0.05, ^$$ P < 0.01 vs. C26.

Figure 4

IL4 treatment is associated to rescue of muscle synthesis. (A) WB for puromycin (Puro) incorporation in newly synthetized muscle proteins from C, IL4, C26, and C26 + IL4 muscles was normalized on the levels of tubulin (aTub) levels and (B) quantified. (C) WB and (D) quantifications of P‐AKT, (E) P‐p70, (F) P‐JNK, and (G) P‐ERK levels normalized for the total protein levels and on aTub expression levels. Significance of the differences is reported as ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. C, ^**** P < 0.0001; ⁺ P < 0.05, ⁺⁺ P < 0.01, ⁺⁺⁺⁺ P < 0.0001 vs. IL4; ^$ P < 0.05, ^$$ P < 0.01, ^$$$$ P < 0.0001 vs. C26.

Figure 5

IL4 treatment counteracts muscle SC accumulation and decreases the levels of FAPs. After sacrifice and tissue digestion, muscle stem cells were analysed by flow cytometry for the expression of different markers of SCs (Itga7 and CD34) (A) and consequently (B, D, F) quantified. (C) The levels of PAX7 and myogenin (Myog) were analysed by WB, and (D, E) correspondent quantifications are reported on the right. (F) Protein levels of F4/80 and CD206 and (G, H) correspondent quantifications. (I) Flow cytometry analysis for the expression of markers for MABs (ALP); (I, J) quantification or for FAPs (Sca1) (K) and (L) quantification in C, IL4, C26, and C26 + IL4 muscles. (M) Populations of adipocyte‐like cells were stained with Oil‐Red‐O (red) and perilipin (PLIN1; green). Nuclei were stained with HOECHST (blue). Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001, ^**** P < 0.0001 vs. C; ⁺ P < 0.05, ⁺⁺ P < 0.01, ⁺⁺⁺ P < 0.001, ⁺⁺⁺⁺ P < 0.0001 vs. IL4; ^$ P < 0.05, ^$$ P < 0.01 vs. C26.

Figure 6

IL4 treatment induces a higher number of precursors to be activated after cardiotoxin (CTX)‐induced injury. In regeneration conditions, after 10 days from CTX‐induced muscle injury, (A) TIB (C: 97 ± 6 mg/100 g i.b.w.), (B) GSN (C: 262 ± 12 mg/100 g i.b.w.), and (C) QD (C: 369 ± 14 mg/100 g i.b.w.) muscle weights, expressed as percentage of the initial body weight. (D) H&E staining (column on the left) with insets from the different muscles (column on the right). (E) Representative IF muscle staining for Lam (purple) and eMyHC (green). Nuclei were stained with HOECHST (blue). (F) Fibre size distribution of TIB muscles from the different groups. (G) WB for eMyHC and (H) quantification. (I) PAX7, Myog protein levels with a representative Gapdh and their (J, K) quantifications. (L) Protein levels of F4/80 and CD206 in the same muscles have been as well quantified in (M) and (N). Significance of the differences is reported as ^*** P < 0.001, ^**** P < 0.0001 vs. C; ⁺⁺ P < 0.01, ⁺⁺⁺⁺ P < 0.0001 vs. IL4; ^$ P < 0.05, ^$$ P < 0.01, ^$$$$ P < 0.0001 vs. C26; ^£ P < 0.05 vs. CTX IL4; ^# P < 0.05, ^## P < 0.01 vs. CTX C26; ^{^} P < 0.05, ^{^^} P < 0.01, ^{^^^} P < 0.001 vs. C26 + IL4.

Figure 7

IL4 on C2C12 reduces the TNFα‐induced proliferative effect and is essential for myotube differentiation. (A) IF, (B) flow cytometry analysis, and (C) quantification of Ki67⁺ C2C12 cells treated with 10 ng/mL IL4, 100 ng/mL TNFα, or a combination of the two cytokines (TNFα + IL4) during 48 h of proliferating medium. (D) WB and (E) quantifications of the protein levels of MyHC and IL4R1a normalized for aTub after 10 ng/mL IL4 or 100 ng/mL TNFα or a combination of the two (TNFα + IL4) treatments during all C2C12 differentiation period (5 days differentiating medium). (F) IF for MyHC (red) on C2C12 during the three last days of differentiation (24 h = Day 3 of differentiation, 48 h = Day 4 of differentiation, 72 h = Day 5 of differentiation) after the double IL4Ra and IL13Ra silencing. HOECHST (blue) was used to stain nuclei. (G) WB and (H, I) quantifications for MyHC and IL4Ra protein levels normalized for aTub after double IL4Ra and IL13Ra silencing. (J) IF, (K) WB, and (L) quantification for the levels of ESGP (Myomerger) normalized on the ones of Gapdh that were analysed in the same cells silenced for IL4Ra and IL13Ra. Significance of the differences: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. C; ⁺ P < 0.05, ⁺⁺ P < 0.01, ⁺⁺⁺ P < 0.001 vs. IL4.