Targeting interleukin-1 for reversing fat browning and muscle wasting in infantile nephropathic cystinosis

Abstract

Background

Ctns^−/− mice, a mouse model of infantile nephropathic cystinosis, exhibit hypermetabolism with adipose tissue browning and profound muscle wasting. Inflammatory cytokines such as interleukin (IL)-1 trigger inflammatory cascades and may be an important cause for cachexia. We employed genetic and pharmacological approaches to investigate the effects of IL-1 blockade in Ctns^−/− mice.

Methods

We generated Ctns^−/− Il1β^−/− mice, and we treated Ctns^−/− and wild-type control mice with IL-1 receptor antagonist, anakinra (2.5 mg/kg/day, IP) or saline as vehicle for 6 weeks. In each of these mouse lines, we characterized the cachexia phenotype consisting of anorexia, loss of weight, fat mass and lean mass, elevation of metabolic rate, and reduced in vivo muscle function (rotarod activity and grip strength). We quantitated energy homeostasis by measuring the protein content of uncoupling proteins (UCPs) and adenosine triphosphate in adipose tissue and skeletal muscle. We measured skeletal muscle fiber area and intramuscular fatty infiltration. We also studied expression of molecules regulating adipose tissue browning and muscle mass metabolism. Finally, we evaluated the impact of anakinra on the muscle transcriptome in Ctns^−/− mice.

Results

Skeletal muscle expression of IL-1β was significantly elevated in Ctns^−/− mice relative to wild-type control mice. Cachexia was completely normalized in Ctns^−/− Il1β^−/− mice relative to Ctns^−/− mice. We showed that anakinra attenuated the cachexia phenotype in Ctns^−/− mice. Anakinra normalized UCPs and adenosine triphosphate content of adipose tissue and muscle in Ctns^−/− mice. Anakinra attenuated aberrant expression of beige adipose cell biomarkers (UCP-1, CD137, Tmem26, and Tbx1) and molecules implicated in adipocyte tissue browning (Cox2/Pgf2α, Tlr2, Myd88, and Traf6) in inguinal white adipose tissue in Ctns^−/− mice. Moreover, anakinra normalized gastrocnemius weight and fiber size and attenuated muscle fat infiltration in Ctns^−/− mice. This was accompanied by correction of the increased muscle wasting signalling pathways (increased protein content of ERK1/2, JNK, p38 MAPK, and nuclear factor-κB p65 and mRNA expression of Atrogin-1 and Myostatin) and the decreased myogenesis process (decreased mRNA expression of MyoD and Myogenin) in the gastrocnemius muscle of Ctns^−/− mice. Previously, we identified the top 20 differentially expressed skeletal muscle genes in Ctns^−/− mice by RNAseq. Aberrant expression of these 20 genes have been implicated in muscle wasting, increased energy expenditure, and lipolysis. We showed that anakinra attenuated 12 of those top 20 differentially expressed muscle genes in Ctns^−/− mice.

Conclusions

Anakinra may provide a targeted novel therapy for patients with infantile nephropathic cystinosis.

Introduction

Cystinosis is a genetic disorder characterized by the accumulation of cystine in different tissues and organs. Infantile nephropathic cystinosis (INC) is the most common and severe form of cystinosis.¹ INC patients exhibit signs and symptoms of the renal Fanconi syndrome and chronic kidney disease (CKD) in early childhood.² Metabolic abnormalities such as cachexia are common complications in patients with INC, which are associated with poor quality of life and mortality, and for which there is no current therapy increased expression of inflammatory cytokines, including interleukin (IL)-1β, has been implicated in the aetiology of cachexia and muscle wasting associated with different disease processes. Consistent with this finding, patients with cystinosis had higher concentrations of circulating IL-1β compared with controls.¹ Peripheral blood mononuclear cells from patients with cystinosis revealed a significant increase in IL-1β transcript levels compared with controls.³ Recent evidence in preclinical models suggested that blockade of IL-1 signalling may be a logical therapeutic target for chronic disease-associated muscle wasting because IL-1β activates nuclear factor-κB (NF-κB) signalling and induces expression of muscle catabolic gene IL-6 and atrogin-1 in C2C12 myocytes.^4,5 Anakinra is an IL-1 receptor antagonist that blocks both IL-1α and IL-1β.⁶ Duchenne muscular dystrophy is an X-linked muscle disease characterized by muscle inflammation that is associated with increased circulating serum levels of IL-1β. Subcutaneous administration of anakinra normalized muscle function in a mouse model of Duchenne muscular dystrophy.⁷ Similarly, serum IL-1β is elevated in haemodialysis patients, and a 4 week treatment with anakinra was shown to be safe in these patients while significantly reducing markers of systemic inflammation such as C-reactive protein (CRP) and IL-6.⁸ In this study, we utilized Ctns^−/− mice as an established model of INC in which we have previously shown hypermetabolism with adipose tissue browning and profound muscle wasting by 12 months of age.⁹ We explored the role of inflammatory cytokines in Ctns^−/− mice and evaluated the efficacy of IL-1 targeted therapy on adipose tissue browning and muscle wasting. Anakinra was FDA approved for the treatment of rheumatoid arthritis in 2001 and is safe and an effective therapeutic option in a variety of diseases including diseases involving muscle. It may be repurposed as novel therapy for cachexia in INC.

Methods Study design

All animal work was conducted in compliance and approved by the Institutional Animal Care and Use Committee at the University of California, San Diego. Wild-type (WT), Il6^−/− and Il1β^−/−, Ctns^−/−, Ctns^−/− Il6^−/−, and Ctns^−/− Il1β^−/− mice were on the same c57BL/6 genetic background. Ctns^−/− mice were kindly provided by Professor Corinne Antignac, and Ctns^−/− Il6^−/− and Ctns^−/− Il1β^−/− mice were generated by crossing Ctns^−/− mice with Il6^−/− and Il1β^−/− mice, respectively. Twelve-month-old male mice were used for all of the following five studies. Study 1: We evaluated the gastrocnemius muscle IL-1β and IL-6 mRNA and protein content in Ctns^−/− mice and WT mice. Results were presented in Figure 1A–1D. Study 2: We evaluated the effects of genetic deletion of Il1β and Il6 in Ctns^−/− mice. We compared ad libitum food intake and weight change in WT, Ctns^−/−, Ctns^−/− Il6^−/−, and Ctns^−/− Il1β^−/− mice. The study period was 6 weeks. Results were shown in Figure 1E and 1F. Study 3: We evaluated the beneficial effects of genetic deletion of Il1β and Il6 in Ctns^−/− mice beyond nutritional effects by employing a pair-feeding strategy. The study period was 6 weeks. Ctns^−/− mice were fed ad libitum, and then WT, Ctns^−/− Il6^−/−, and Ctns^−/− Il1β^−/− mice were fed the same amount of rodent diet based on the recorded food intake of Ctns^−/− mice. Results were shown in Figure 1G–1N. Study 4: We evaluated the effects of anakinra in Ctns^−/− mice. WT and Ctns^−/− mice were given anakinra (2.5 mg/kg/day, IP) or vehicle (normal saline), respectively. The study period was 6 weeks. All mice were fed ad libitum. We compared food intake and weight change in all groups of mice. Results were shown in Figure 2A and 2B. Study 5: We evaluated the metabolic effects of anakinra in Ctns^−/− mice beyond nutritional stimulation by employing a pair-feeding strategy. WT and Ctns^−/− mice were given anakinra (2.5 mg/kg/day, IP) or vehicle (normal saline), respectively. The study period was 6 weeks. Vehicle-treated Ctns^−/− mice were fed ad libitum while all other groups of mice were fed the same amount of rodent diet based on the recorded food intake of vehicle-treated Ctns^−/− mice. Results were shown in Figures 2E–2G and 3–6 as well as Supporting Information, Figures S1 and S2.

Serum and blood chemistry

Blood urea nitrogen and serum concentration of bicarbonate was measured (Table S1). Serum creatinine was analysed by liquid chromatography-tandem mass spectrometry method.¹⁰

Body composition, metabolic rate, and in vivo muscle function

Body composition (for lean and fat content) of mice was measured by quantitative magnetic resonance analysis (EchoMRI-100™, Echo Medical System) twice, prior to the initiation of the study as well as at the end of the study, and change of total body fat and lean mass in individual mouse was calculated. Twenty-four-hour metabolic rate (VO₂) of mice was measured using Oxymax indirect calorimetry (Columbus Instrument) at the end of the study. At the end of the study, muscle function (grip strength and rotarod activity) in mice was assessed using a grip strength metre (Model 47106, UGO Basile) and rotarod performance tool (model RRF/SP, Accuscan Instrument), respectively.^9,11

Protein assay for muscle and adipose tissue

Mice were sacrificed at the end of the study, and gastrocnemius muscle, inguinal white adipose tissue (WAT), and intercapsular brown adipose tissue were dissected and processed in a homogenizer tube (USA Scientific, catalogue 1420-9600) containing ceramic beads (Omni International, catalogue 19-646) using a Bead Mill Homogenizer (Omni International). Protein concentration of tissue homogenate was assayed using Pierce BAC Protein Assay Kit (Thermo Scientific, catalogue 23227). Uncoupling protein (UCP) content and adenosine triphosphate (ATP) concentration in adipose tissue and muscle homogenates were assayed. Protein concentration of phospho-ERK 1/2 (Thr202/Ty2r204) and total ERK 1/2, phospho-JNK (The183/Tyr185) and total JNK, phospho-p38 MAPK (Thr180/Tyr182) and total p38 MAPK, NF-κB p65 (phospho-Ser536) and total NF-κB p65, and IL-6 and IL-1β in muscle homogenates was assayed (Table S1).

Gastrocnemius wet weight, fiber size, and fatty infiltration

The left gastrocnemius muscle of mice was dissected at the end of the study. Wet weight of the left gastrocnemius muscle was recorded immediately after tissue excision and utilized for subsequent protein and mRNA studies. Freshly excised right gastrocnemius muscle was frozen in isopentane/liquid nitrogen and stored in −80°C before further processing. Muscle cryosections were used for muscle fiber cross-sectional area assessment as well as Oil Red O staining. We measured muscle fiber cross-sectional area of gastrocnemius muscle, using ImageJ software (https://rsbweb.nih.gob/ij/).^9,11 In addition, portions of the dissected right gastrocnemius muscle samples were incubated with Oil Red O (Oil Red O Solution, catalogue number O0625, Sigma Aldrich). Detailed procedures for Oil Red O staining were in accordance with published protocol.¹² We followed a recently established protocol to quantify muscle fat infiltration. Acquisition and quantification of images were analysed using ImageJ software.¹³

Muscle RNAseq analysis

Previously, we performed RNAseq analysis on gastrocnemius muscle mRNA in 12-month-old Ctns^−/− mice versus age-appropriate WT mice.¹¹ Detailed procedures for mRNA extraction, purification, and subsequent construction of cDNA libraries as well as analysis of gene expression were published. We then performed Ingenuity Pathway Analysis enrichment tests for those differentially expressed muscle genes in Ctns^−/− mice versus WT mice, focusing on pathways related to energy metabolism, skeletal and muscle system development and function, and organismal injury and abnormalities. We identified the top 20 differentially expressed muscle genes in Ctns^−/− versus WT mice.¹¹ In this study, we performed qPCR analysis for those top 20 differentially expressed gastrocnemius muscle genes in the different experimental groups.

Quantitative real-time PCR

Total RNA from adipose and gastrocnemius muscle samples was isolated using TriZol (Life Technology) and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen). Quantitative real-time RT-PCR of target genes was performed using KAPA SYBR FAST qPCR kit (KAPA Biosystems). Expression levels were calculated according to the relative 2^−ΔΔCt method.¹¹ All primers are listed (Table S2).

Statistical analysis

Continuous variables are expressed as mean ± standard error of the mean. We assessed the statistical significance of differences between groups using two-sample t-tests. All tests were two-sided. A P value less than 0.05 was considered significant. Statistical analyses were performed using SPSS software Version 16.0 for Macintosh. ^# represents a statistically significant (P < 0.05) difference from WT control mice. Bars with specific P values between groups represent a statistical significance from Ctns^−/− mice.

Results Increased muscle interleukin-6 and interleukin-1β mRNA and protein content in Ctns^−/− mice

We chose to study Ctns^−/− mice at 12 months of age based on our previous data demonstrating a significant cachexia phenotype. As expected, all of the mice were uraemic and had elevated creatine, but normal bicarbonate levels serum (Table S3). In order to investigate the role of inflammatory cytokines in cachexia and muscle wasting, we measured gastrocnemius muscle mRNA and protein content of IL-6 and IL-1β in 12-month-old Ctns^−/− mice and age-matched WT controls. Gastrocnemius mRNA and protein content of both IL-6 and IL-1β were significantly elevated in Ctns^−/− mice relative to WT mice (Figure 1A–1D).

Genetic deletion of Il1β normalizes cachexia in Ctns^−/− mice

We then employed a genetic approach to determine whether IL6 and I1β deficiency would affect the cachexia phenotype in Ctns^−/− mice. Twelve-month-old Ctns^−/−, Ctns^−/− Il6^−/−, and Ctns^−/− Il1β^−/− mice were all uraemic and had elevated creatinine levels but normal bicarbonate levels (Table S4). All mice were fed ad libitum for 6 weeks, and average daily food intake and final weight gain of the mice were recorded. We showed that genetic deletion of Il1β completely corrected anorexia and normalized weight gain in Ctns^−/− mice whereas deletion of Il6 only partially rescued the phenotype in Ctns^−/− mice (Figure 1E and 1F). Furthermore, to investigate the potential metabolic effects of genetic deficiency of Il1β and Il6 beyond its nutritional effects, we employed a pair-feeding strategy. Ctns^−/− mice were fed ad libitum, and then all other groups of mice were fed the same amount of rodent diet based on the recorded food intake of the Ctns^−/− group (Figure 1G). Despite receiving the same amount of total calorie intake as other groups of mice, cardinal features of cachexia phenotype such as decreased weight gain, decreased fat mass content and hypermetabolism (manifested as elevated oxygen consumption), decreased lean mass content and gastrocnemius weight, and reduced muscle function (decreased rotarod and grip strength) were still prominent in Ctns^−/− mice (Figure 1H–1N). Importantly, parameters of cachexia phenotype were normalized relative to WT mice or significantly improved in Ctns^−/− Il1β^−/− mice compared with Ctns^−/− mice. However, Ctns^−/− Il6^−/− mice had only minimal non-significant improvement relative to Ctns^−/− mice.

Anakinra attenuates cachexia and muscle wasting in Ctns^−/− mice

In order to better model a real-world approach in patients, we used a pharmacological approach to test the effects of IL-1β blockade in pre-established INC-associated cachexia and muscle wasting. Anakinra, an IL-1 receptor antagonist that blocks both IL-1α and IL-1β function, was used to treat 12-month-old Ctns^−/− mice and WT controls for 6 weeks compared with vehicle. All mice were fed ad libitum. Food intake and weight gain were normalized in anakinra-treated Ctns^−/− mice (Figure 2A and 2B). We also investigated the beneficial effects of anakinra in Ctns^−/− mice beyond appetite stimulation and its consequent body weight gain through the utilization of a pair-feeding approach. Daily ad libitum food intake for Ctns^−/− mice treated with vehicle was recorded. Following that, anakinra-treated Ctns^−/− mice and WT mice treated with anakinra or vehicle were food restricted such that the mice ate an equivalent amount of food as vehicle-treated Ctns^−/− mice (Figure 2C). Serum and blood chemistry of mice were measured (Table S5). Anakinra normalized weight gain, and metabolic rate and significantly improved or normalized fat and lean mass content, gastrocnemius weight, and muscle function (grip strength and rotarod activity) in Ctns^−/− mice (Figure 2D–2J).

Anakinra normalizes energy homeostasis in adipose tissue and skeletal muscle in Ctns^−/− mice

Adipose tissue (inguinal WAT and intercapsular brown adipose tissue) and gastrocnemius protein content of UCPs was significantly increased in Ctns^−/− mice compared with WT mice (Figure 3A, 3C, and 3E). In contrast, ATP content in adipose tissue and skeletal muscle was significantly decreased in Ctns^−/− mice compared with WT mice (Figure 3B, 3D, and 3F). Anakinra normalized UCPs and ATP content of adipose tissue and muscle in Ctns^−/− mice.

Anakinra ameliorates browning of adipose tissue in Ctns^−/− mice

Beige adipose cell surface markers (CD137, Tmem26, and Tbx1) mRNA levels were elevated in inguinal WAT in Ctns^−/− mice relative to WT mice (Figure 4A–4C). This was accompanied by an increased UCP-1 protein content in inguinal WAT shown in Figure 3A, which is a biomarker for beige adipocytes, and usually undetectable in WAT. Anakinra normalized the elevated protein content of inguinal WAT UCP-1 as well as increased mRNA expression of CD137, Tmen, and Tbx1 in Ctns^−/− mice (Figures 3A and 4A–4C). Anakinra also ameliorated expression of important signature molecules implicated in WAT. Activation of the Cox2/Pgf2α signalling pathway and chronic inflammation stimulate biogenesis of WAT browning. Significantly increased inguinal WAT mRNA expression of Cox2 and Pgf2α was found in Ctns^−/− mice compared with WT mice (Figure 4D and 4E). Moreover, inguinal WAT levels of toll-like receptor 2 (Tlr2) as well as myeloid differentiation primary response 88 (MyD88) and tumour necrosis factor receptor-associated factor 6 (Traf6) were up-regulated in Ctns^−/− mice compared with WT mice (Figure 4F–4H), and anakinra significantly reduced inguinal WAT gene expression of Cox2/Pgf2α as well as genes in the toll-like receptor pathway in Ctns^−/− mice.

Anakinra improves muscle fiber size and attenuates muscle fat infiltration in Ctns^−/− mice

We evaluated the effect of anakinra on skeletal muscle morphology in Ctns^−/− mice. Representative results of muscle sections are illustrated (Figure 5A–5D). Anakinra significantly improved average cross-sectional area of gastrocnemius fibers in Ctns^−/− mice (Figure 5E). We also evaluated fat infiltration of the gastrocnemius muscle in Ctns^−/− mice. Representative results of Oil Red O staining of muscle section are shown (Figure 5F–5I). We quantified intensity of muscle fat infiltration in mice and observed that anakinra attenuated muscle fat infiltration in Ctns^−/− mice (Figure 5J).

Anakinra attenuates signalling pathways implicated in muscle wasting in Ctns^−/− mice

Muscle relative phospho-ERK 1/2 (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), and phospho-p38 MAPK protein content (Thr180/Tyr182) were significantly increased in Ctns^−/− mice compared with WT mice (Figure 6A–6C). In addition, the ratio of muscle phospho-NF-κB p65 (phosphorylated S536) relative to total NF-κB p65 protein content was elevated in Ctns^−/− mice relative to WT mice (Figure 6D). Importantly, anakinra attenuated or normalized phosphorylation of muscle ERK 1/2, JNK, p38 MAPK, and NF-κB p65 protein content in Ctns^−/− mice. Administration of anakinra improved muscle regeneration and myogenesis by decreasing mRNA expression of negative regulators of skeletal muscle mass (Atrogin-1 and Myostatin) (Figure 6E and 6F) as well as increasing muscle mRNA expression of pro-myogenic factors (MyoD and Myogenin) in Ctns^−/− mice (Figure 6G and 6H).

Molecular mechanism by RNAseq analysis

Recently, we profiled differential expression of gastrocnemius mRNA between 12-month-old Ctns^−/− mice and WT mice using RNAseq analysis.¹¹ Ingenuity Pathway Analysis enrichment tests identified the top 20 differentially expressed muscle genes in Ctns^−/− mice versus WT mice. The top 15 up-regulated genes were Ankdr2, Csrp3, Cyfip2, Fhl1, Ly6a, Mup1, Myl2, Myl3, Nlrc3, Sell, Sln, Spp1, Tnnc1, Tnni1, and Tpm3 whereas the top 5 down-regulated genes were Atf3, Cidea, Fos, Sncg, and Tbc1d1 in Ctns^−/− mice relative to WT mice. We performed qPCR analysis for those top 20 differentially expressed muscle genes in the present study. Importantly, anakinra significantly reduced (Ankdr2, Csrp3, Cyfip2, Fhl1, Ly6a, Nlrc3, Tnnc1, and Tpm3) and significantly increased (Atf3, Cidea, Sncg, and Tbc1d1) muscle gene expression in Ctns^−/− mice relative to WT mice (Figures S1 and S2). Functional significance of each of these 12 genes is listed (Table 1). Non-significant changes were observed in Mup1, Myl2, Myl3, Sell, Sln, Spp1, Tnni1, and Fos.

Table 1 Anakinra normalized or attenuated expression of important muscle genes that have been implicated in muscle wasting in Ctns^−/− mice

Up-regulated DEG	Functional significance and reference
Ankrd2	Implicated in mechanical stretch of skeletal muscle14,15
Csrp3	Associated with skeletal muscle dystrophy16
Cyfip2	Associated with muscle wasting17
Fhl1	Activates myostatin signalling and promotes atrophy in skeletal muscle18
Ly6a	Associated with remodelling of the extracellular matrix during skeletal muscle regeneration19
Nlrc3	Implicated in skeletal muscle wasting by inhibiting cell proliferation and promoting cell apoptosis20
Tnnc1	Regulates striated muscle contraction21
	Implicated in cardiomyopathy pathogenesis and age-related skeletal muscle wasting21
Tpm3	Promotes slow myofibre hypotrophy and associated with generalized muscle weakness22
Down-regulated DEG	Functional significance
Atf3	Impairs motor neuron survival and muscle innervation23
Cidea	Increases metabolic rates, lipolysis in brown adipose tissue, and higher core temperature24
Sncg	Increases energy expenditure, particularly in brown and white adipose tissues25
	Associated with cancer cachexia through the TGF-β signalling pathway25
Tbc1d1	Impairs glucose transport in skeletal muscle26
	Associated with follistatin-induced muscle hypertrophy26

Previously, we studied differential expression of gastrocnemius mRNA between Ctns^−/− mice and WT mice using RNAseq analysis.¹¹ We focus on pathways related to energy metabolism, skeletal and muscular system development and function, nervous system development and function, and organismal injury and abnormalities. In the present study, we performed qPCR analysis for top 20 differentially expressed muscle genes determined by our previous studies. Importantly, anakinra normalized (Ankdr2, Csrp3, Ly6a, Nlrc3, Tnnc1 and Tpm3, Atf3, Cidea, Sncg, and Tbc1d1) and attenuated (Cyfip2 and Fhl1) muscle gene expression in Ctns^−/− mice relative to WT mice. Functional significance of each of these differentially expressed muscle genes is listed. DEG, differential expressed genes.

Discussion

Previously, we described cachexia characterized by adipose tissue browning and muscle wasting in Ctns^−/− mice, an established mouse model of INC, but the aetiology was not clear. Increased expression of inflammatory cytokines such as IL-6 and IL-1 have been implicated in the aetiology of cachexia and muscle. Our studies using specific cytokine-deficient mice and IL-1 targeted therapy suggest that IL-1 is a critical cytokine in INC-associated cachexia. The novel findings of this study are (i) anakinra corrects anorexia, normalizes weight gain, and attenuates hypermetabolism. (ii) Anakinra attenuates the expression of markers of adipose tissue browning as well as perturbed energy expenditure and thermogenesis. (iii) Anakinra attenuates muscle wasting, improved muscle function, and corrected perturbations of aberrant expression of molecules implicated in muscle wasting. Together, our results suggest that anakinra may be an effective targeted treatment approach for cachexia in patients with INC.

Interleukin-1β suppresses food intake, activates energy metabolism, and reduces weight gain in experimental animals.^27,28 IL-1β signals through appetite-regulating neuropeptides, such as leptin resulting in appetite suppression.²⁹ We have demonstrated that elevated circulating level of leptin through the activation of melanocortin receptor 4 induces CKD-associated cachexia.³⁰ IL-1 and IL-6 are produced not only by circulating immune cells but also by various cells in tissues such as muscle and adipose tissue. For this present study, we showed that skeletal muscle IL-1β and IL-6 mRNA and protein level was significantly increased in Ctns^−/− mice, and Ctns^−/− Il1β^−/− mice had normalized parameters of cachexia phenotype relative to control mice (Figure 1). Furthermore, we showed that anakinra improved anorexia and normalized weight gain in Ctns^−/− mice relative to control mice (Figure 2). Our results further highlight the beneficial effects of anakinra beyond food stimulation and accompanied weight gain. In pair-fed studies in which anakinra-treated Ctns^−/− mice and anakinra-treated WT mice were fed the same amount of food, cachexia was attenuated in anakinra-treated Ctns^−/− mice relative to control mice (Figure 2).

The basal metabolic rate accounts for up to 80% of the daily calorie expenditure by individual.³¹ Skeletal muscle metabolism is a major determinant of resting energy expenditure.^32,33 IL-1β increases basal metabolic rate (as represented by an increase in resting oxygen consumption) in a dose-dependent manner.³⁴ In our study, anakinra normalized the increased 24 h metabolic rate in Ctns^−/− mice (Figure 2F). Adipose tissue UCP-1 expression is essential for adaptive adrenergic non-shivering thermogenesis, and muscle UCP-3 level controls body metabolism.³⁵ The energy generated when dissipating the proton gradient via up-regulation of UCPs is not used for cellular ATP production or other biochemical processes but instead to produce heat.^36,37 We showed that anakinra normalized muscle and adipose tissue UCPs and ATP content in Ctns^−/− mice (Figure 3). Blockade of IL-1 receptor signalling may also mitigate the metabolic dysfunction through leptin signalling. Infusion of leptin increased UCPs expression in skeletal muscle and adipose tissue.^38,39

Perhaps our most novel finding is that anakinra reduced adipose tissue browning in Ctns^−/− mice. New evidence suggests a maladaptive role of adipose tissue browning in the context of cachexia.⁴⁰ Activation of adipose tissue browning is associated with profound energy expenditure and weight loss in cachectic patients. Browning of adipose tissue increases energy expenditure and takes place before skeletal muscle atrophy in murine models of cancer and CKD-associated muscle wasting.^41,42 We previously demonstrated adipose tissue browning in Ctns^−/− mice (as evidenced by the detection of inguinal WAT UCP-1 protein and increased expression of beige adipose cell markers CD137, Tmem, and Tbx1).^9,11 Cox2 is a downstream effector of β-adrenergic signalling and induces biogenesis of beige cells in WAT depots.⁴³ We showed that anakinra normalized expression of inguinal WAT Cox2, Pgf2α, and important inflammatory molecules (Tlr2, MyD88, and Trap6) in Ctns^−/− mice and normalized key inflammatory molecules (Tlr2 and MyD88) involved in adipose tissue browning in Ctns^−/− mice (Figure 4). Recent data suggest that IL1β signalling mediates adipocyte browning via regulating of mitochondrial oxidative responses in both cultured human and animal adipocytes.⁴⁴

We studied the impact of anakinra on muscle wasting in Ctns^−/− mice. Anakinra normalized lean mass content, gastrocnemius wet weight, and muscle function in Ctns^−/− mice relative to WT mice (Figure 2G–2J). Furthermore, anakinra normalized average gastrocnemius cross-sectional area as well as muscle fat infiltration of gastrocnemius muscle in Ctns^−/− mice (Figure 5). Muscle fat infiltration is a significant predictor of both muscle function and mobility function across a wide variety of co-morbid conditions such as diabetes, spinal cord injury, and kidney disease.^45–47 Muscle adipose tissue may release pro-inflammatory cytokines within the muscle and impair the local muscle environment, impair blood flow, or increase the rate of lipolysis within skeletal muscle resulting in an increased concentration of glucose within the skeletal muscle itself followed by insulin resistance.^48,49

We investigated the impact of anakinra on signalling molecules that modulate muscle mass regulation in Ctns^−/− mice. Systemic inflammation decreases muscle regeneration and increases muscle catabolism that eventually leads to muscle wasting in CKD. Inhibition of IL-1 reduced systemic inflammation (decreased serum CRP and IL-6) in CKD patients.^8,50 A recent study also showed that blockade of IL-1 signalling significantly improved inflammatory status (a decrease in plasma concentration of IL-6 and TNF) and antioxidative properties in CKD patients.⁵¹ Systemic inflammation, as assessed by serum concentration of CRP, is a strong and independent risk factor for skeletal muscle wasting in CKD patients.⁵² IL-1 has been shown to stimulate the expression of catabolic genes.^6,53,54 MAPK are a family of protein phosphorylating enzymes that regulate a diverse aspect of cellular responses including skeletal muscle regeneration and differentiation.^55–57 Activation of the NF-κB signalling pathway leads to severe muscle wasting in mice.⁵⁸ IL-1α and IL-1β, both isoforms of IL-1, stimulated catabolism in C2C12 myotubes via activation of NF-κB signalling and atrogin-1 expression.⁴ Importantly, anakinra attenuated or normalized phosphorylation of muscle ERK 1/2, JNK, p38 MAPK, and NF-κB p65 protein content in Ctns^−/− mice. In addition, anakinra improved muscle regeneration and myogenesis by decreasing mRNA expression of negative regulators of skeletal muscle mass (Atrogin-1 and Myostatin) as well as increasing muscle mRNA expression of pro-myogenic factors (MyoD and Myogenin) in Ctns^−/− mice (Figure 6). Our findings were in agreement with a recent report demonstrating that IL-1β administration inhibiting muscle mRNA expression of atrogin-1.²⁷

Finally, we studied the impact of anakinra on muscle transcriptomics in Ctns^−/− mice. We showed that anakinra normalized or attenuated muscle expression of 12 out of 20 top differentially expressed genes in Ctns^−/− mice.¹¹ Detailed functional significance of each of these 12 differentiated expressed muscle genes is listed in Table 1. Anakinra normalized (Csrp3, Ly6a, Nlrc3, Tnnc1 and Tpm3, Atf3, Cidea, Sncg, and Tbc1d1) and attenuated (Cyfip2 and Fhl1) muscle gene expression in Ctns^−/− mice relative to WT mice (Figures S1 and S2). Increased expression of Csrp3, Cyfip2, Fhl1, Nlcr3, Tnnc1, and Tpm3 have been implicated in muscle atrophy and muscle weakness.^{16–18,20–22} Increased expression of Ly6a has been associated with aberrant remodelling of the extracellular matrix during skeletal muscle regeneration.¹⁹ In addition, increased expression of Ankrd2 as well as decreased expression of Atf3 impairs muscle function and impairs survival of motor neuron and muscle innervation.^14,15,23 Recent data also suggest that decreased expression of Cidea and Sncg are associated with higher energy expenditure as well as accelerated lipolysis of adipose tissue.^24,25 Furthermore, decreased expression of Tbc1d1 impairs glucose transport in skeletal muscle and has been implicated in follistatin-induced muscle hypertrophy.²⁶

Conclusion

We employed genetic and pharmacological approaches and demonstrated significant beneficial effects of IL-1 blockade on cachexia in Ctns^−/− mice. We report that anakinra attenuates adipose tissue browning and muscle wasting in Ctns^−/− via multiple cellular mechanisms (Figure 7). IL-1 receptor antagonist may represent a novel targeted treatment for cachexia in patients with INC by attenuating adipose tissue browning and muscle wasting.

Acknowledgements

All authors of this manuscript certify that they complied with the ethical guidelines for authorship and publishing in the Journal of Cachexia, Sarcopenia and Muscle update 2017.⁵⁹

Conflict of interest

None declared.

Funding

This investigation was supported by National Institutes of Health (RO1 DK12581). This investigation was also supported in whole or in part by a grant from the Cystinosis Research Foundation. R.M. is funded by grants from the NIH (RO1 DK125811, R01 HD095547, U01 DK066143) and the California Institute of Regenerative Medicine (CLIN2-11478). H.H. is funded by NIH awards R01 DK113592, R01 HL140898, R01 AI134030 and a UC collaborative grant. P.Z. was supported by ‘Spring Sunlight Program’ cooperative research project of Ministry of Education (HLJ2019023) and Research Fund for Young & Middle-Aged Innovative Science of the Second Affiliated Hospital of Harbin Medical University (CX2016-03).