NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis

Abstract

Background: Inclusion body myositis (IBM) is the most prevalent muscle disease in adults for which no current treatment exists. The pathogenesis of IBM remains poorly defined. In this study, we aimed to explore the interplay between inflammation and mitochondrial dysfunction in IBM.

Methods: The study population consisted of 38 IBM patients and 22 age- and sex-matched controls without a myopathy. Mean age was 62.9 years (SD = 9) in IBM group and 59.7 (10) in controls. Bulk RNA sequencing, Meso Scale Discovery electrochemiluminescence (ECL), western blotting, histochemistry and immunohistochemistry were performed on frozen muscle samples from the study participants.

Results: We demonstrated activation of the NLRP3 inflammasome in IBM muscle samples, with the NLRP3 inflammasome being the most upregulated pathway on RNA sequencing, along with increased expression of NLRP3 and ASC proteins in IBM group. NLRP3 RNA levels most strongly correlated with TLR7 (correlation coefficient ρ = 0.91) and complement activation-related genes, and inversely correlated with several mitochondria-related genes among others. On muscle histopathology, there was increased NRLP3 immunoreactivity in both inflammatory cells and muscle fibres. Mitophagy is critical for removing damaged mitochondria and preventing the formation of a vicious cycle of mitochondrial dysfunction-NLRP3 inflammasome activation. Herein, we showed altered mitophagy, as witnessed by the elevated levels of p-S65-Ubiquitin, a mitophagy marker, in muscle lysates from IBM patients compared to controls (median of 114.3 vs. 81.25 ECL units, p = 0.005). The p-S65-Ubiquitin levels were most significantly elevated in IBM males compared to male controls (136 vs. 83.5 ECL units; p = 0.013), whereas IBM females had milder nonsignificant elevation compared to female controls (97.25 vs. 69 ECL units, p = 0.31). On muscle histopathology, p-S65-Ubiquitin aggregates accumulated in muscle fibres that were mostly Type 2 and devoid of cytochrome-c-oxidase reactivity. NLRP3 RNA levels correlated with p-S65-Ubiquitin levels in both sexes (males: ρ = 0.48, females: ρ = 0.54) but with loss of muscle strength, as reflected by the manual motor test score, only in males (males: ρ = 0.62, females: ρ = -0.14). Lastly, we identified sex-specific molecular pathways in IBM. Females had upregulation of pathways related to response to stress, which could conceivably offset some of the pathomechanisms of IBM, while males had upregulation of pathways related to cell adhesion and migration.

Conclusions: There is activation of the NLRP3 inflammasome in IBM, along with altered mitophagy, particularly in males, which is of potential therapeutic significance. These findings suggest sex-specific mechanisms in IBM that warrant further investigation.

Keywords: autophagy; inclusion body myositis; inflammasome; mitochondrial dysfunction; mitophagy.

FIGURE 1

Inclusion body myositis transcriptome. Transcriptomic analysis of muscle tissue from 38 patients with inclusion body myositis and 22 controls. (A) PCA (to the left) and heatmap (middle) showing clear separation between IBM patients and controls. Volcano plot (to the right) representing differentially expressed genes (blue). (B) Top 10 upregulated and top 10 downregulated pathways in IBM. The NLRP3 inflammasome and mitophagy pathways are detailed in heatmaps, with downregulated genes shown in blue and upregulated genes shown in red. Transcriptomic sex differences are shown in C and D. (C) Volcano plot showing the signed ‐log₁₀ (p value) for each gene in females on the x‐axis and in males on the y‐axis. Differentially expressed genes that are consistent in both sexes are shown in black, female‐unique genes are shown in red, male‐unique genes are shown in blue and genes with reverse trends are shown in purple. Genes that did not reach fold change or p value cutoffs in either sex are shown in grey (not significant). (D) The top 10 upregulated and downregulated pathways for sex‐specific genes.

FIGURE 2

NLRP3 inflammasome activation in inclusion body myositis. (A) Western blot of muscle lysates from IBM patients and controls showing increased NLRP3 and ASC protein expression in samples from both males and females with IBM. The Mann–Whitney test was performed for group comparisons. Western blot results comparing the IBM group to the control group were as follows (Mann–Whitney U, two‐tailed p value): NLRP3 for both sexes (20, p = 0.0009), NLRP3 for males (6, p = 0.045), NLRP3 for females (3, p = 0.011), ASC for both sexes (4, p < 0.0001), ASC for males (3, p = 0.0047) and ASC for females (0, p = 0.0007). (B) NLRP3 immunohistochemistry showing increased NLRP3 immunoreactivity in scattered muscle fibres (examples shown as white arrows) as well as in inflammatory cells (examples shown as black arrowheads). (C) Radar maps displaying top coexpressed and inversely coexpressed genes with NLRP3 according to Spearman rank correlation analysis. The correlation coefficient corresponds to the distance from the centre. All represented genes had a p value less than 0.0001.

FIGURE 3

Altered mitophagy in inclusion body myositis patients. (A) Measurement of p‐S65‐Ub levels via sandwich ELISA in muscle samples from IBM patients and controls. The results comparing the IBM samples to the controls were as follows (Mann–Whitney U, two‐tailed p value): both sexes (43, p = 0.0054), males only (7, p = 0.0136) and females only (16, p = 0.31). MSD‐ECL:Meso Scale Discovery‐Electrochemiluminescence. (B and C) Frozen muscle sections from IBM samples and controls were reacted to p‐S65‐Ub, NLRP3 and ATPase at pH 4.3 (Type 1 fibres: dark brown, Type 2 fibres: pink) and at pH 4.6 (Type 1 fibres: dark brown; Type 2a fibres: light brown; Type 2b fibres: in between shade), and cytochrome c oxidase. (B) Top row: muscle samples from controls; p‐S65‐Ub shows clear differentiation of fibre types, reflecting the difference in baseline mitophagy and mitochondrial content between fibre types. Type 1 fibres, dark brown on ATPase 4.3, had increased p‐S65‐Ub and CCO reactivity. Middle and bottom rows: muscle samples from male and female IBM patients, respectively, demonstrating p‐S65‐Ub‐positive aggregates in scattered muscle fibres (examples shown as arrows). These fibres were almost exclusively Type 2 fibres (ATPase stains) that were also devoid of CCO activity (CCO‐negative fibres). Additionally, all CCO‐negative fibres, with or without observable abnormal p‐S65‐Ub reactivity, were Type 2 fibres. Scale bar for Panel B: top row: 50 μm; middle row: 50 μm; bottom row: 200 μm. (C) Muscle samples from IBM patients were reacted for NLRP3 and p‐S65‐Ub, demonstrating muscle fibres with increased NLRP3 and p‐S65‐Ub reactivity (arrows). Scale bar: top row 50 μm, bottom row 20 μm.

FIGURE 4

Correlation of NLRP3 inflammasome activation with altered mitophagy and muscle weakness. Pearson correlation plot demonstrating correlation coefficient values for the NLRP3 RNA level, p‐S65‐Ub level, manual muscle test (MMT) score and disease duration by sex. Both sexes: NLRP3 and p‐S65‐Ub, p‐value = 0.059; NLRP3 and MMT score, p = 0.072; NLRP3 and disease duration, p = 0.543; p‐S65‐Ub and MMT score, p = 0.58; p‐S65‐Ub and disease duration, p = 0.699. Males: NLRP3 and p‐S65‐Ub, p = 0.116; NLRP3 and MMT score, p = 0.032; NLRP3 and disease duration, p = 0.699; p‐S65‐Ub and MMT score, p = 0.359; p‐S65‐Ub and disease duration, p = 0.49. Females: NLRP3 and p‐S65‐Ub, p = 0.106; NLRP3 and MMT score, p = 0.706; NLRP3 and disease duration, p = 0.781; p‐S65‐Ub and MMT score, p = 0.642; p‐S65‐Ub and disease duration, p = 0.898.

FIGURE 5

The vicious cycle of inflammasome activation‐mitochondrial dysfunction/altered mitophagy in inclusion body myositis. The release of mitochondrial damage‐associated molecular patterns (DAMPs) results in the activation of the NLRP3 inflammasome. Under normal conditions, damaged mitochondria and the NRLP3 inflammasome are both subsequently removed by mitophagy and autophagy, reestablishing cellular homeostasis. In IBM, mitophagy and autophagy are altered, establishing a feedforward loop in which the inflammatory milieu results in additional oxidative stress and mitochondrial dysfunction with further release of mitochondrial DAMPs and subsequent aberrant NLRP3 inflammasome activation.