Implication of the NLRP3 Inflammasome in Bovine Age-Related Sarcopenia

Abstract

Sarcopenia is defined as the age-related loss of skeletal muscle mass, quality, and strength. The pathophysiological mechanisms underlying sarcopenia are still not completely understood. The aim of this work was to evaluate, for the first time, the expression of NLRP3 inflammasome in bovine skeletal muscle in order to investigate the hypothesis that inflammasome activation may trigger and sustain a pro-inflammatory environment leading to sarcopenia. Samples of skeletal muscle were collected from 60 cattle belonging to three age-based groups. Morphologic, immunohistochemical and molecular analysis were performed to assess the presence of age-related pathologic changes and chronic inflammation, the expression of NLRP3 inflammasome and to determine the levels of interleukin-1β, interleukin-18 and tumor necrosis factor alpha in muscle tissue. Our results revealed the presence of morphologic sarcopenia hallmark, chronic lymphocytic inflammation and a type II fibers-selective NLRP3 expression associated to a significant decreased number of immunolabeled-fibers in aged animals. Moreover, we found a statistically significant age-related increase of pro-inflammatory cytokines such as interleukin-1β and interleukin-18 suggesting the activation of NLRP3 inflammasome. Taken together, our data suggest that NLRP3 inflammasome components may be normally expressed in skeletal muscle, but its priming and activation during aging may contribute to enhance a pro-inflammatory environment altering normal muscular anabolism and metabolism.

Keywords: NLRP3 inflammasome; immunosenescence; inflammaging; sarcopenia.

Figure 1

Representative histological sections of skeletal muscle from aged, adult and young cows. Aged cow (group A): Hematoxylin and eosin (HE) stain shows moderate variability in muscle fiber size, with atrophic fibers occasionally showing angular profile and a mild, chronic, lymphopcytic inflammatory infiltrate within the endomysium. Engel trichrome (ET) stain shows muscle fibers showing subsarcolemmal mitochondrial accumulations (ragged red fibers). Nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) stain shows deep blue ragged blue fiber and “moth-eaten” fibers displaying moderate to severe irregular disruption of myofibrillar network. Succinate dehydrogenase (SDH) stain shows ragged blue fiber deeply stained in blue. COX stain shows a cytochrome oxidase (COX)–negative fiber and many others showing a detectable decrease of COX activity. Nonspecific esterase (NSE) stain shows muscle fibers presenting small, pale, and multisegmented neuromuscular junctions. Adult cow (group B): Hematoxylin and eosin (HE) shows mild to moderate variability in muscle fiber size and multiple disseminated atrophic fibers. Engel trichrome (ET) shows mild to moderate subsarcolemmal mitochondrial accumulations (ragged red fibers). Nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) stain shows a mild irregular disruption of myofibrillar network. deeply stained in blue. Succinate dehydrogenase stain (SDH) stain shows few ragged blue fibers with mild mitochondrial accumulation. COX stain shows mild detectable decrease of COX activity. Nonspecific esterase (NSE) stain shows muscle fibers displaying small, pale, and multisegmented neuromuscular junctions. Young cow (group C): Normal muscle fibers with no remarkable lesions.

Figure 2

Immunohistochemical staining for CD4, CD8, MHC-I and MHC-II in skeletal muscle of aged, adult and young cows. Aged cows (Group A): there is a mild to moderate inflammatory infiltrate in the endomysium consisting mostly in CD8+, rare CD4+ T cells. Scattered muscle fibers show sarcolemmal immunolabeling for MHC-I and MHC-II antibodies. HRP method with Mayer’s hematoxylin counterstain. Adult cows (Group B): there is a mild inflammatory infiltrate in the endomysium consisting mostly in CD8+, rare CD4+ T cells. Scattered muscle fibers show sarcolemmal immunolabeling for MHC-I, but not for MHC-II antibody. HRP method with Mayer’s hematoxylin counterstain. Young cows (Group C): no inflammation is detectable and there is absence of MHC-I and MHC-II immunolabeling on muscle fibers. HRP method with Mayer’s haematoxylin counterstain.

Figure 3

Immunohistochemical expression of NLRP3 in skeletal muscle in cows. (A) Aged cows (Group A), (B) Adult cows (Group B), (C) Young cows (Group C). HRP method with Mayer’s hematoxylin counterstain. (D) Immunoreactivity score for NLRP3 expression. There is a statistically significant negative association between age and the presence of NLRP3 immunolabeled muscle fibers. Each value is the mean ± SEM (* p < 0.05; *** p < 0.001).

Figure 4

Selective expression of NLRP3 inflammasome in type II muscle fiber. Serial sections of skeletal muscle show a selective expression of NLRP3 inflammasome in type II muscle fibers. Moreover, aged animals (Group A) show severe muscular atrophy restricted to type II fibers stained white (ATPase pH 4.3) or dark brown (ATPase pH 9.4). In adult animals (Group B) the selective atrophy is mild, and it is absent in muscle of young animals (Group C). Immunohistochemistry was performed with HRP method and Mayer’s hematoxylin counterstain.

Figure 5

Western blot analysis for NLRP3 inflammasome expression in bovine muscle during aging. Skeletal muscle tissue from young, adult and aged bovine was homogenized and the protein lysates were analyzed by Western blot using antibodies for NLPR3 and for GAPDH as control. The blots were detected by ECL and autoradiography. In skeletal muscle of young bovine there is an increase of immunoreactivity for NLPR3 inflammasome compared to adult and aged animals. Data are shown as mean ±SD and asterisks denote statistically differences (* p < 0.05; ** p < 0.01).

Figure 6

Changes of TNF-α, IL-1β and IL-18 levels in the skeletal muscle of cows measured with RT-PCR analysis. Our results showed that TNF-α, IL-1β and IL-18 were differently expressed in the three groups. (A) TNF-α levels were very low in young cows (Group C) and increasingly higher in adult to aged animals (** p < 0.01; *** p < 0.001 vs. control). (B) IL-1β levels were increasingly higher from young cows (Group C) to adult and aged animals (Group B and A, respectively) (* p < 0.05 vs. control). (C) IL-18 levels were very low in young cows (Group C) and increasingly higher in adult to aged animals (Group B and A, respectively) (* p < 0.05; *** p < 0.001 vs. control).