The role of muscle in Spondyloarthritis

A espondiloartrite axial (axSpA) é uma doença reumática inflamatória, caracterizada principalmente pelo envolvimento da coluna e articulações sacroilíacas, e geralmente apresenta-se como dor crónica nas costas e rigidez. À medida que a doença progride, a mobilidade da coluna vertebral e a função física são prejudicadas podendo afetar as atividades da vida diária. A genética e os fatores ambientais (microbiota e microtrauma) são as causas conhecidas da suscetibilidade e progressão da doença. Esta tese teve como objetivo melhorar o nosso conhecimento atual da fisiopatologia da axSpA, caracterizando as propriedades musculares axiais e periféricas e identificando biomarcadores genéticos e proteicos que possam explicar tais propriedades. Realizamos um estudo transversal com 54 participantes: 27 pacientes com axSpA e 27 controles saudáveis (HC), pareados por idade, sexo e nível de atividade física. Dados epidemiológicos, clínicos e de caracterização muscular (propriedades físicas musculares, força, massa e desempenho) foram registados e comparados entre pacientes com axSpA e HC. Foi ainda colhido sangue periférico para abordagens ómicas. A transcriptómica e a proteómica foram realizadas por sequenciação de RNA e tecnologias de espectrometria de massa, respectivamente. Os nossos resultados indicam que pacientes com axSpA (idade média de 36,5 (DP 7,5) anos, 67% do sexo masculino e duração média da doença de 6,5 (3,2) anos) não apresentaram diferença significativa na rigidez muscular segmentar em comparação com os HC, apesar de apresentarem uma discreta menor rigidez lombar. Pacientes com axSpA, comparados com os HC, apresentaram menor força total, bem como menor força nos membros superiores e inferiores, independentemente das propriedades físicas dos músculos. Os pacientes também apresentaram velocidades de marcha significativamente menores do que o HC. As características da marcha podem representar um potencial biomarcador em pacientes com axSpA. A análise de enriquecimento de genes expressos diferencialmente permitiu revelar vias metabólicas significativas (como sinalização de IL6 e vias do sistema imunológico) com papel patológico para esse grupo de pacientes. Salienta-se ainda que níveis séricos aumentados de várias citocinas pró-inflamatórias em pacientes com axSpA foram observados em correlação adequada com os parâmetros de atividade da doença. Além disso, foram identificados genes expressos diferencialmente associados ao músculo (como NACA, FRG1 e ARPC5L) desempenham papeis no desenvolvimento de miotubos e montagem de actina, afetando eventualmente a força muscular em pacientes com axSpA. Finalmente, a integração dos resultados da transcriptómica e da proteómica mostra que a análise dos genes e proteínas diferencialmente expressos permite obter uma clara discriminação entre pacientes com axSpA e HCs, possibilitando ainda avançar no diagnóstico, prognóstico e eventuais opções terapêuticas para esse grupo de pacientes. Globalmente, os resultados aqui obtidos permitem oferecer algumas possibilidades interessantes para explicar o papel do músculo na patogénese da axSpA.Axial spondylarthritis (axSpA) is an inflammatory rheumatic disease, characterized primarily by the involvement of the spine and sacroiliac joints, and usually presenting as chronic back pain and stiffness. As the disease progresses, impaired spinal mobility and physical function may impact activities of daily living. Genetics and environmental factors (microbiota and microtrauma) are the known causes of disease susceptibility and progression. This thesis aimed to improve our current knowledge of axSpA physiopathology by characterizing axial and peripheral muscle properties and identifying genetic and protein biomarkers that might explain such properties. We performed a cross-sectional study on 54 participants: 27 patients with axSpA and 27 healthy controls (HC), matched by age, gender, and level of physical activity. Epidemiological, clinical, and muscle characterization (muscle physical properties, strength, mass, and performance) data were registered and compared between patients with axSpA and HC. Peripheral blood was collected for omics approaches. Transcriptomics and proteomics were performed by RNA-sequencing, RT q-PCR, and mass spectrometry technologies, respectively. Our results indicate patients with axSpA (mean age 36.5 (SD 7.5) years, 67% males, and mean disease duration of 6.5 (3.2) years) had no significant difference in segmental muscle stiffness compared with the HC, despite showing a slight numerically higher lower lumbar stiffness. Patients with axSpA, compared to the HC, had lower total strength as well as lower strength in the upper and lower limbs, independently of muscle physical properties. Patients also had significantly lower gait speeds than the HC. Gait characteristics may represent a potential biomarker in patients with axSpA. Enrichment analysis of differentially expressed genes reveals significant pathways (such as IL6 signaling and immune system pathways) with a pathological role for this group of patients. Notably, increased serum levels of various pro-inflammatory cytokines in patients with axSpA have been observed in proper correlation with disease activity parameters. Moreover, differentially muscle-associated expressed genes (such as NACA, FRG1, and ARPC5L) play roles in the development of myotubes and actin assembly, eventually affecting muscle strength in patients with axSpA. Worth of note, the integration of transcriptomic’s and proteomic’s results shows number of genes and proteins causes a clear discrimination between patients with axSpA and HCs that may advance diagnosis, prognosis, and therapeutic options for this group of patients. This work, taken together, provides some interesting possibilities to explain the role of muscle in the pathogenesis of axSpA

The Effect of ACTN3 and VDR Polymorphisms on Skeletal Muscle Performance in Axial Spondyloarthropathies

Funding Information: This study was supported by the funding through project MyoSpA, from iNOVA4 health. PM was supported by the National Institute for Health Research (NIHR) University College London Hospitals (UCLH) Biomedical Research Centre (BRC). Publisher Copyright: © Copyright © 2021 Pimenta, Mateus, Rodrigues-Manica, Pinheiro-Torres, Neto, Domingues, Lage Crespo, Sardoo, Machado, Branco, Silva and Pimentel-Santos.PBackground: Spondyloarthritis (SpA) are the most common group of chronic inflammatory rheumatic diseases affecting about 1.5% of the adult Caucasian population. Low back pain is the most common symptom. The aetiopathogenesis of SpA is multifactorial, with well-known genetic and environmental contributions. Furthermore, muscle properties might also be involved in the pathophysiological process and these could be modulated by the genetic background. Alpha-actinin-3 (ACTN3) and Vitamin D receptor (VDR) genes are well-known genes related with muscle performance. Our aim was to analyze four SNPs of these genes and to evaluate their influence in axial SpA (axSpA) susceptibility, phenotype and muscle properties. Methods: We performed a pilot study based on case-control approach involving 56 participants: 28 axSpA patients and 28 healthy controls matched by age, gender and levels of physical activity. Clinical, epidemiological and muscle characterization data—muscle physical properties (stiffness, tone, and elasticity), strength, mass, and performance, were collected. Two different muscles were considered for analysis, the Multifidus and Gastrocnemius. Four SNPs of ACTN3 (rs1815739) and VDR (rs2228570, rs731236, and rs7975232), were selected, analyzed and correlated with clinical, epidemiological and muscle characterization data. Results: In total, 51 individuals (27 axSpA patients and 24 matched controls) were eligible for further genetic analysis, 66.7% being male and with a mean age of 36 years. Muscle physical properties, muscle strength and muscle mass were similar in both groups; however, axSpA patients showed a decrease in muscle performance. None of the studied SNPs were associated with disease susceptibility/phenotype, muscle physical properties, muscle strength or muscle mass. However, ACTN3 rs1815739 and VDR rs2228570 were shown to be associated with muscle performance. Conclusion: Our results suggest an association between ACTN3 and VDR polymorphisms and muscle performance in axSpA.publishersversionpublishe

MULTI-OMICS TEMPORAL PROFILING OF AXIAL SPONDYLOARTHRITIS PATIENTS REVEALS AN ASSOCIATION OF THERAPEUTIC RESPONSE TO ADALIMUMAB WITH DISEASE ACTIVITY AND INNATE / ADAPTIVE IMMUNITY

ABSTRACT Background Axial Spondyloarthritis can lead to significant disability and impairment in quality of life. TNF inhibitors are recommended to patients enduring active disease despite conventional treatment. Nonetheless, up to 40% of patients of patients fail to respond to TNF inhibitors. In this context, it is important to identify as early as possible patients highly likely to respond. This study aims at identifying, among axial spondyloarthritis patients undergoing treatment with the TNF inhibitor adalimumab, early molecular biomarkers differentiating good responders from non-responders after 14 weeks of treatment, as measured by ASAS20. Methods Peripheral blood RNA sequencing and serum proteins measured by mass spectrometry were evaluated in a cohort of biologic naïve axial spondyloarthritis patients (n = 35), before (baseline) and after (3-5 days, 2 weeks and 14 weeks) treatment with adalimumab. Results from differential expression analysis were used in combination with clinical data to build logistic regression models and random forest models to predict response to adalimumab at baseline. Results Responders to adalimumab presented higher levels of markers of innate immunity at baseline, mostly related with neutrophils, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP and AFF3, the top differentially expressed gene between responders and non-responders at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC=0.96), with random forest models suggesting 80% predictive accuracy. A treatment-associated signature suggests a reduction in inflammatory activity, with C-reactive protein and Haptoglobin showing strong and early decrease in the serum of axial spondyloarthritis patients, while a cluster of apolipoproteins showed increased expression at week 14. Conclusions Differences in disease activity and/or blood innate/adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axial spondyloarthritis, where a model including clinical and blood gene expression variables shows high predictive power. Our results suggest novel molecular biomarkers of response to adalimumab at baseline. Trial registration Axial spondyloarthritis patients were selected from participants of the Bioefficacy study - Biomarkers Identification of Anti-TNFα Agent’s Efficacy in Ankylosing Spondylitis Patients Using a Transcriptome Analysis and Mass Spectrometry ( clinical trials.gov identifier NCT02492217 )

The MyoSpA Study Protocol

declArAtions ETHICS APPROVAL AND CONSENT TO PARTICIPATE The current study was submitted and approved by the ethical committee of University of Lisbon and Centro Hospitalar de Lisboa Ocidental, Hospital de Egas Moniz, EPE (Reference Number: 20170700050). The study will be conducted in accordance with the International Conference on Harmonization Good Clinical Practice (GCP) and the Declaration of Helsinki. Furthermore, voluntary written informed participants’ consent will be obtained from all subjects before the start of the study procedures. FUNDING This study was supported by iNOVA4Health (consortia to create a multidisdiplinary/translational network at the NOVA University, Lisbon, Portugal) and Portuguese Society of Rheumatology grants.BACKGROUND: Axial Spondyloarthritis (axSpA) is a chronic, inflammatory rheumatic disease that affects the axial skeleton, causing pain, stiffness, and fatigue. Genetics and environmental factors such as microbiota and microtrauma are known causes of disease susceptibility and progression. Murine models of axSpA found a decisive role for biomechanical stress as an inducer of enthesitis and new bone formation. Here, we hypothesize that muscle properties in axSpA patients are compromised and influenced by genetic background. OBJECTIVES: To improve our current knowledge of axSpA physiopathology, we aim to characterize axial and peripheral muscle properties and identify genetic and protein biomarker that might explain such properties. METHODS: A cross-sectional study will be conducted on 48 participants aged 18-50 years old, involving patients with axSpA (according to ASAS classification criteria, symptoms duration < 10 years) and healthy controls matched by gender, age, and levels of physical activity. We will collect epidemiological and clinical data and perform a detailed, whole body and segmental, myofascial characterization (focusing on multifidus, brachioradialis and the gastrocnemius lateralis) concerning: a) Physical Properties (stiffness, tone and elasticity), assessed by MyotonPRO®; b) Strength, by a dynamometer; c) Mass, by bioimpedance; d) Performance through gait speed and 60-second sit-to-stand test; e) Histological and cellular/ molecular characterization through ultrasound-guided biopsies of multifidus muscle; f) Magnetic Resonance Imaging (MRI) characterization of paravertebral muscles. Furthermore, we will perform an integrated transcriptomics and proteomics analysis of peripheral blood samples. DISCUSSION: The innovative and multidisciplinary approaches of this project rely on the elucidation of myofascial physical properties in axSpA and also on the establishment of a biological signature that relates to specific muscle properties. This hitherto unstudied link between gene/protein signatures and muscle properties may enhance our understanding of axSpA physiopathology and reveal new and useful diagnostic and therapeutic targets.publishersversionpublishe