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. 2022 Jul 28:13:947267.
doi: 10.3389/fimmu.2022.947267. eCollection 2022.

Left ventricle- and skeletal muscle-derived fibroblasts exhibit a differential inflammatory and metabolic responsiveness to interleukin-6

Isabell Matz  1   2 , Kathleen Pappritz  1   2 , Jochen Springer  1   2 , Sophie Van Linthout  1   2
Affiliations

Left ventricle- and skeletal muscle-derived fibroblasts exhibit a differential inflammatory and metabolic responsiveness to interleukin-6

Isabell Matz et al. Front Immunol. .

Abstract

Interleukin-6 (IL-6) is an important player in chronic inflammation associated with heart failure and tumor-induced cachexia. Fibroblasts are salient mediators of both inflammation and fibrosis. Whereas the general outcome of IL-6 on the heart's function and muscle wasting has been intensively studied, the influence of IL-6 on fibroblasts of the heart and skeletal muscle (SM) has not been analyzed so far. We illustrate that SM-derived fibroblasts exhibit higher basal mRNA expression of α-SMA, extracellular matrix molecules (collagen1a1/3a1/5a1), and chemokines (CCL2, CCL7, and CX3CL1) as compared to the left ventricle (LV)-derived fibroblasts. IL-6 drives the transdifferentiation of fibroblasts into myofibroblasts as indicated by an increase in α-SMA expression and upregulates NLRP3 inflammasome activity in both LV- and SM-derived fibroblasts. IL-6 increases the release of CCL7 to CX3CL1 in the supernatant of SM-derived fibroblasts associated with the attraction of more pro(Ly6Chi) versus anti(Ly6Clo) inflammatory monocytes as compared to unstimulated fibroblasts. IL-6-stimulated LV-derived fibroblasts attract less Ly6Chi to Ly6Clo monocytes compared to IL-6-stimulated SM-derived fibroblasts. In addition, SM-derived fibroblasts have a higher mitochondrial energy turnover and lower glycolytic activity versus LV-derived fibroblasts under basal and IL-6 conditions. In conclusion, IL-6 modulates the inflammatory and metabolic phenotype of LV- and SM-originated fibroblasts.

Keywords: IL-6; NLRP3 inflammasome; fibroblasts; left ventricle; skeletal muscle.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Skeletal muscle (SM)-derived fibroblasts show a higher gene expression of α-SMA, components of the extracellular matrix (ECM), and chemokines compared to left ventricle (LV)-derived fibroblasts. mRNA expression of (A) α-SMA, ECM components, and modulators (Col1a1, Col3a1, Col5a1, LOX, and LOXL2) and (B) chemokines (CCL2, CCL7, Cx3CL1) normalized to GAPDH of LV-derived (black) and SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle). Mean ± 95% CI; n = 36, N = 6; Mann–Whitney/Welch’s test with post-hoc Benjamini–Hochberg correction; adjusted p-value: *p < 0.05,***p < 0.001.
Figure 2
Figure 2
IL-6 stimulation leads to differential regulation of α-SMA in left ventricle (LV)- and skeletal muscle (SM)-derived fibroblasts. mRNA expression of α-SMA, ECM components, and modulators (Col1a1, Col3a1, Col5a1, LOX, and LOXL2) normalized to GAPDH and the respective basal condition of (A) LV-derived (black) and (B) SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle); n = 36, N = 6, Mann–Whitney/Welch’s test. (C) Percentage of gated α-SMA+ cells (flow cytometry data) of LV-derived (black) and SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle); n = 18, N = 3, Kruskal–Wallis/one-way ANOVA. (D) Cell number assessed via Crystal Violet assay and absorption at 595 nm from LV-derived (black) and SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle). Total collagen content of LV-derived (black) and SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle) measured via Sirius Red assay and absorption at 540 nm (E) unnormalized and (F) normalized to cell number (Crystal Violet assay data); n = 30, N = 3, Kruskal–Wallis/one-way ANOVA; all data are presented as mean ± 95% CI and tested with post-hoc Benjamini–Hochberg correction; adjusted p-values: **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
IL-6 stimulation leads to secretion of anti- and pro-inflammatory chemokines in left ventricle- and skeletal muscle-derived fibroblasts. mRNA expression of CCL2, CCL7 and CX3CL1 normalized to GAPDH and the respective basal condition of (A) LV-(black) and (B) SM- (red) derived fibroblasts after 24 h culture with basal medium without IL-6 (basal condition; hollow circle) or with10 ng/ml IL-6 (filled circle); n=36, N=6, Mann-Whitney/Welch’s test; Protein expression of (C) CCL2, (D) CCL7 and (E) CX3CL1 in the supernatant and ratio of (F) CCL2 and CX3CL1 and (G) CCL7 and CX3CL1 normalized to total protein content measured via bicinchoninic acid assay and respective basal condition of LV (black)- and SM (red)-derived fibroblasts after 72 h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml IL-6 (dark filled circle); n=24, N=4, Kruskal-Wallis/One-Way ANOVA; All data are presented as mean ± 95 % CI and tested with post hoc Benjamini-Hochberg correction; adjusted p-values: *p < 0.05.
Figure 4
Figure 4
IL-6 stimulation leads to attraction of different monocyte subsets to left ventricle (LV)- versus skeletal muscle (SM)-derived fibroblasts. Percentage of gated (A) CD11b+CD115+Ly6Chi and (B) CD11b+CD115+Ly6Clo monocytes and (C) ratio of Ly6Chi versus Ly6Clo monocytes attracted by the medium of LV-derived (black) and SM-derived (red) fibroblasts after 72-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle), n = 18, N = 3; Kruskal–Wallis/one-way ANOVA. All data are presented as mean ± 95% CI and tested with post-hoc Benjamini–Hochberg correction; adjusted p-values: *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
IL-6 stimulation leads to upregulation of NLRP3 inflammasome activity in left ventricle (LV)- and skeletal muscle (SM)-derived fibroblasts. Percentage of gated (A) NLRP3+, Caspase-1+, and IL-1β+ cells in the global population; (B) NLRP3+, Caspase-1+, and IL-1β+ cells in the α-SMA+ subpopulation; and (C) NLRP3+, Caspase-1+, and IL-1β+ cells in the α-SMA− subpopulation of LV-derived (black) and SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle), n = 18, N = 3. All data are presented as mean ± 95% CI and tested with post-hoc Benjamini–Hochberg correction; adjusted p-values: **p < 0.01,***p < 0.001.
Figure 6
Figure 6
IL-6 stimulation leads to differential regulated mitochondrial and glycolytic disturbances in left ventricle (LV)- versus skeletal muscle (SM)-derived fibroblasts. Mitochondrial stress test measuring (A) oxygen consumption rate (OCR) describing basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory and glycolytic stress test assessing (B) extracellular acidification rate (ECAR) describing glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification normalized to protein amount in μg of LV-derived (black) and SM-derived (red) fibroblasts after 4-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle); n = 15, N = 3. All data are presented as mean ± 95% CI and tested with post-hoc Benjamini–Hochberg correction; adjusted p-values: * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 7
Figure 7
IL-6 trans-signaling is differently regulated in left ventricle (LV)- versus skeletal muscle (SM)-derived fibroblasts. (A) IL6R mRNA expression normalized to GAPDH and respective control of LV-derived (black) and SM-derived (red) fibroblasts after 24-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle), n = 18, N = 3, Kruskal–Wallis/one-way ANOVA. (B) Protein expression of gp130 in the supernatant normalized to total protein content measured via bicinchoninic acid assay of LV-derived (black) and SM-derived (red) fibroblasts after 72-h culture with basal medium without IL-6 (basal condition; bright hollow circle) or with 10 ng/ml of IL-6 (dark filled circle). All data are presented as mean ± 95% CI and tested with post-hoc Benjamini–Hochberg correction.
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