Cytokines as Potential Biomarkers of Disease Clinical Course in Muscular Dystrophies View PDF

Muscular dystrophies (MDs) represent a complex, varied, and important subset of neuromuscular disorders ranging from severe and fatal congenital muscular dystrophies with onset in infancy to mild forms of limb and girdle weakness with onset in adolescence or adulthood [1]. Cardiac involvement is a frequent feature in this group of diseases, presenting as both systolic dysfunction, evolving to dilated cardiomyopathy (DCM) and heart failure (HF), and conduction system abnormalities [2].

There is an emerging interest and need for nonclinical biomarkers in assessing the clinical course and diagnosing MDs, as well as in monitoring drug therapy [3-5]. However, the problem is not so much finding potential candidates as their validation and translation into clinical practice. This difficulty is even more evident in MDs due to their phenotypic variability, but above all because of their rarity, which prevents assembling a large cohort of patients.

Cytokines are mediators of inflammation released by a variety of cell types and regulate a wide variety of metabolic, inflammatory, and regenerative processes [6,7]. The abnormal increase in the level of proinflammatory molecules such as TNF-α and IL-1 β has been suggested as being among the major mechanisms triggering muscle wasting [6,7]. Increasing evidence indicates that the inflammatory processes are highly integrated into muscle wasting in DMD and that circulating inflammatory cytokines maybe 1000 times higher in DMD human patients than healthy controls [6-8]. It is also known that oxidative stress and chronic inflammation are involved in the physiopathology of MDs. Disruption of the dystroglycan complex leads to high susceptibility to injury, with repeated, eccentric contractions as well as inflammation, resulting in significant damage and necrosis. Chronic damage and repair cycling leads to fibrosis and weakness [9].

Several trials demonstrated that the process of inflammation might be responsible for the initiation of DCM and progression to HF [10- 11]. In HF there is an activation of the immune system, production, and release of autoantibodies, pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and complement system recruitment [10-11]. High levels of circulating cytokines correlate with the severity of HF measured with the use of the New York Heart Association (NYHA) classification, and prognosis of the disease [10-13]. They also cause alterations in the heart function due to the increase of cardiomyocyte apoptosis, cardiac hypertrophy, and matrix metalloproteinase activation [10]. Cytokines levels in serum of 24 Muscular Dystrophy patients, DMD (n=7), DM1 (n=10), and LGMD2A (n=7) were evaluated by Comim CM, et al. (2019) They found an increase of IL-1β in DMD and DM1 patients, while TNF-α was increased only in DMD patients, and IL-10 decreased in all the analyzed groups. They suggested that in muscular dystrophies there could be a pro-inflammatory state due to the disease itself and an anti-inflammatory counterpart trying to balance the generated inflammation and perhaps restrain the progressive characteristic of the disease [14].

Cappelletti C, et al. (2020) [15] investigated the serum levels of 51 pro-and anti-inflammatory molecules, including cytokines, chemokines, and growth factors, in 85 patients with different types of MDs and 35 healthy controls. They showed that a) the levels of interleukin-17 (IL-17), granulocyte colony-stimulating factor (G-CSF), and transforming growth factor-beta (TGF-β2) significantly discriminated patients affected by muscular Laminopathies (Musc- LMNA) from controls; b) interleukin-1β (IL-1β), interleukin-4 (IL-4) and interleukin-8 (IL-8) were differentially expressed in Musc-LMNA patients compared to those with non-muscular laminopathies; c) IL- 17 was significantly higher in Musc-LMNA patients who presented cardiac involvement.

In this preliminary communication, we analyzed the cytokine profile in patients with X-linked Emery Dreifuss Muscular Dystrophy (EDMD1), Duchenne and Becker muscular dystrophies, (DMD/ BMD), and Myotonic Dystrophy type 1 (DM1) to determine whether an abnormal profile of circulating cytokines could be identified, helping to distinguish the different MDs, and, within the same disease, identify early onset of cardiac involvement.

a. Patients and controls

The study included 17 MD patients, among which 8 EDMD1, 3 DMD, 3 BMD, and 3 DM1. Four EDMD1 patients, 3 DMD, 3 BMD patients, and 1 DM1 had cardiomyopathy. Twenty-two age-matched healthy individuals were in the control group (Table 1).

Table 1: Demographic data of subjects included in the study.

b. Serum sample collection

The peripheral blood of patients, as well as the healthy controls, was drawn between 8:00 and 11:00 a.m. on the day of inclusion into the study and collected in Greiner Bio-One VACUETTE™ Z Serum Sep Clot Activator Tubes (Thermo Fisher Scientific, Waltham, MA, USA), centrifuged at 3000 rpm for 10 min at room temperature. The serum, transferred in cryogenic vials, was immediately stored in liquid nitrogen pending assays. The sera samples were provided by the Naples Human Mutation Gene Biobank (NHMGB), the genetic biobank of Cardiomyology and Medical Genetics of the Luigi Vanvitelli Campania University, which is a member of EuroBioBank and Telethon Network of Genetic Biobanks (TNGB). All patients and healthy controls were not affected by ongoing infections nor immunosuppressed at the time of blood samples and did not perform any relevant physical activity before the blood collection, being demonstrated that some cytokines (e.g., IL-6, TNF-α) are released by immune and muscle cells in response to exercise [16-17].

c. Cytokine, chemokine and growth factor quantification

A Bio-Plex ProTM Human Cytokine 27-plex Immunoassay 96- well kit (Bio-Rad Laboratory, Hercules, CA, USA) was used to measure the serum concentration of pro-and anti-inflammatory cytokines, chemokines, and growth factors. The kit includes: gamma interferon (IFN-γ), interleukin-1, (IL-1), IL-1 receptor antagonist (IL-1ra), interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, tumor necrosis factor-alpha (TNF-α), interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP- 1), MIP-1, RANTES, platelet-derived growth factor-BB (PDGFBB), basic fibroblast growth factor (bFGF), eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), and vascular endothelial growth factor (VEGF). The three isoforms of TGF-β were detected in the same serum samples using a Bio-Plex ProTM TGF-β3-plex assay (Bio-Rad Laboratory). The serum of each patient and healthy control was diluted 1:4 and tested in duplicate; sera from patients and healthy controls was always mixed in all plates to reduce the variability. Values are expressed as a concentration (pg/mL). The concentration of analyte bound to each bead was proportional to the median fluorescence intensity (MFI) of the reporter signal and was corrected by the standards provided in the kit (Bio-Rad) [15].

d. Statistical analysis

Data are shown in terms of means with standard deviations (SDs) or medians with value ranges for continuous data. Mann-Whitney unpaired test was used to assess differences in cytokine levels between muscular disorders and controls, whereas Kruskal-Wallis test with Dunn’s multiple comparison post-test was used in the comparison among MD subgroups. Differences were considered significant at p < 0.05. Statistical analyses were performed using Prism version 8.0 for Macintosh (GraphPad Software, La Jolla, CA, USA).

Out of 17 patients included in the study, 3 were females and 14 males. The mean age at blood collection was 32.5 ± 14.24 years. The control group included 19 females and 3 males, with a mean age of 30.73 ± 7.99 (Table 1). In the whole group of MDs patients, a different pattern of the cytokine profile was observed compared to controls. In particular, the serum levels of PDGF-2b, IL-2 IL-17, GM-CSF, MCP- 1a, TGF-b2, and TGF-b3, collectively referred to as Group A cytokines, were increased, while those of IL-5, IL-7, IL-8, IL12-p70, IL-13, MIP-1a, and VEGF, collectively referred to as Group B cytokines, were reduced, (Table 2). Furthermore, when considering separately EDMD1, DMD/ BMD, and DM1, and focusing on patients displaying cardiomyopathy, values of Cytokine group A showed a progressive increase whilst those of Cytokine group B showed a decrease, ranging from EDMD1 to DMD/ BMD to DM1 patients (Table 3). However, differences statistically significant were observed only for PDGF-2b (p=0.0051 in the DMD/ BMD), and for TGFβ3 (p=0.0167 in EDMD1, and p=0.054 in DMD/BMD groups). A trend to significance was found for IL-13 (p=0.0528 in DMD/BMD and p=0.0548 in DM1). In the group B cytokines, the mean values of IL-5, IL-12(P70), IL-13, and MIP-1a showed a similar trend, with a sharper reduction in cardiomyopathic patients. However, the level of significance was reached only for IL-13 (p =0.0314) in DMD/BMD group and MIP-1a (p=0.05) in EDMD1.

Table 2: Serum concentration of pro-and anti-inflammatory cytokines, chemokines and growth factors in patients and controls.

Table 3: Serum concentration of pro-and anti-inflammatory cytokines, chemokines and growth factors stratified according to the type of Muscular Dystrophy and myocardial involvement.

The ability to have serum cytokine profiling as monitors of disease progression or predictors of heart involvement in MD is challenging, as cytokine profiles could replace more invasive markers in clinical trials [5]. Cardiomyopathy progressing to dilated cardiomyopathy and heart failure is an almost constant feature of EDMD1, DMD/BMD, and DM1. Several trials demonstrated that inflammation might be responsible for the initiation of DCM and progression to HF [10-13], through activation of the immune system, production, and release of autoantibodies, pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), and complement system recruitment [10-13]. High levels of circulating cytokines cause alterations in the heart function through the increase of cardiomyocyte apoptosis, cardiac hypertrophy, and matrix metalloproteinase activation [10]. In this preliminary study investigating the cytokine profile in specific MDs with and without cardiomyopathy, the serum dosage of 17 inflammatory cytokines showed a significant increase in PDGF-2b, IL-17, and TGF-β3 (Group A), and a decrease in IL-7, IL-13, and MIP-1a (Group B). As an inflammatory cascade mediates the development of heart lesions with overexpression of inflammatory cytokines including IL-17, its expression may have a possible role in inflammatory processes resulting in cardiomyopathy. Wang et al have recently shown that the continued elevation of plasma IL-4 and IL-17 in patients with viral myocarditis (VCM) was associated with a high incidence of dilated cardiomyopathy at 3 months, and these 2 cytokines were independent predictors for the progression from VCM to DCM [18]. Within Cytokine group B, the most remarkable reduction regarding the values of IL-7, IL-13, and MIP-1a was observed in patients with DMD/BMD followed by those with EDMD1. This observation is not surprising if considering their function [19]. IL-7 is a non-redundant cytokine that plays a key role in T-cell development and function. It is important for early T-cell development as well as for T-cell homeostasis. MIP-1a is responsible for recruiting pro-inflammatory cells and plays a crucial role in the T-cell trans-endothelial migration. IL-13 is pro-fibrotic and responsible for Th2 responses in humans; it is released by T cells, mast cells, and eosinophils and can induce collagen expression by fibroblasts. IL-13 has been shown to reduce cardiac injury and protect heart function in viral myocarditis via enhanced M2 macrophage polarization [20].

The ability to early identify the onset of cardiomyopathy, the main cause of death in MDs patients, based on their serum cytokine levels, would be of great value in clinical practice. According to the preliminary results of this study, PDGF-2b, IL-17, and TGFβ on one side, and IL-13 and MIP-1a on the other side, might be considered as potential biomarkers to discriminate among MDs patients, those with and without cardiac involvement. The systematic monitoring of these cytokines since the first stages of the muscle disease could be useful in an early identification of the onset of cardiomyopathy allowing for earlier and appropriate treatments.

The small sample size was the main limitation of our study. We are aware that further large-scale studies are required to confirm our results.

All authors of the manuscript are members of the Italian Network of Laminopathies (NIL). We thank all patients and families, the Naples Human Mutation Genetic Biobank (NHMGB) of Cardiomyology and Medical Genetics, member of EuroBioBank and of the Telethon Network of Genetic Biobanks (TNGB), the BioLaM biobank (Rizzoli Orthopedic Institute, Bologna).

This research was funded by AIProSaB (grants 02/2013 and 03/2016); AIDMED onlus (grant 17/2015); Associazione Alessandra Proietti onlus (IGM prot. 0002041).

Data are available on request.

C.C. performed experiments, acquisition, analysis and interpretation of data; L.M. participated to the analysis and interpretation of data and critically revised the manuscript. N.C. selected the patients and collected blood samples; GL made samples available and critically revised the manuscript; LP designed the work, selected patients and collected blood samples, performed analysis and interpretation of data, wrote and supervised the manuscript.

The authors declare no conflict of interest.

All authors have read and agreed to the published version of the manuscript.

No. 001850-2016 (Bologna).

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