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Article Validation of Biomarkers in Duchenne Muscular Dystrophy

Michael Ogundele 1,2, Jesslyn S. Zhang 1, Mansi V. Goswami 1, Marissa L. Barbieri 1, Utkarsh J. Dang 3, James S. Novak 4, Eric P. Hoffman 1, Kanneboyina Nagaraju 1, CINRG-DNHS Investigators † and Yetrib Hathout 1,*

1 Department of Pharmaceutical Science, School of Pharmacy and Pharmaceutical Sciences, Binghamton University—SUNY, Johnson City, NY 13790, USA; [email protected] (M.O.); [email protected] (J.S.Z.); [email protected] (M.V.G.); [email protected] (M.L.B.); [email protected] (E.P.H.); [email protected] (K.N.) 2 Department of Biomedical Engineering, Binghamton University—SUNY, 4400 Vestal Pkwy E, Binghamton, NY 13902, USA 3 Department of Health Sciences, Carleton University, Ottawa, ON K1S 5B6, Canada; [email protected] 4 Center for Genetic Medicine Research, Children’s Research Institute, Children’s National Hospital, Washington, DC 20010, USA; [email protected] * Correspondence: [email protected] † Names and affiliations of the CINRG-DNHS Investigators are provided in the Acknowledgments.

Abstract: Duchenne muscular dystrophy (DMD) is a progressive muscle disease involving complex skeletal muscle pathogenesis. The pathogenesis is triggered by sarcolemma instability due to the 2+  lack of dystrophin protein expression, leading to Ca influx, muscle fiber apoptosis, inflammation,  muscle necrosis, and fibrosis. Our lab recently used two high-throughput multiplexing techniques ® Citation: Ogundele, M.; Zhang, J.S.; (e.g., SomaScan aptamer assay and tandem mass tag-(TMT) approach) and identified a series of Goswami, M.V.; Barbieri, M.L.; serum protein biomarkers tied to different pathobiochemical pathways. In this study, we focused on Dang, U.J.; Novak, J.S.; Hoffman, E.P.; validating the circulating levels of three proinflammatory (CCL2, CXCL10, and CCL18) Nagaraju, K.; CINRG-DNHS that are believed to be involved in an early stage of muscle pathogenesis. We used highly specific Investigators; Hathout, Y. Validation and reproducible MSD ELISA assays and examined the association of these chemokines with DMD of Chemokine Biomarkers in pathogenesis, age, disease severity, and response to glucocorticoid treatment. As expected, we Duchenne Muscular Dystrophy. Life confirmed that these three chemokines were significantly elevated in serum and muscle samples of 2021, 11, 827. https://doi.org/ DMD patients relative to age-matched healthy controls (p-value < 0.05, CCL18 was not significantly 10.3390/life11080827 altered in muscle samples). These three chemokines were not significantly elevated in Becker muscular dystrophy (BMD) patients, a milder form of dystrophinopathy, when compared in a one- Academic Editor: Gopal J. Babu way ANOVA to a control group but remained significantly elevated in the age-matched DMD group p Received: 22 July 2021 ( < 0.05). CCL2 and CCL18 but not CXCL10 declined with age in DMD patients, whereas all three Accepted: 11 August 2021 chemokines remained unchanged with age in BMD and controls. Only CCL2 showed significant Published: 13 August 2021 association with time to climb four steps in the DMD group (r = 0.48, p = 0.038) and neared significant association with patients’ reported outcome in the BMD group (r = 0.39, p = 0.058). Furthermore, Publisher’s Note: MDPI stays neutral CCL2 was found to be elevated in a serum of the mdx mouse model of DMD, relative to wild-type with regard to jurisdictional claims in mouse model. This study suggests that CCL2 might be a suitable candidate biomarker for follow-up published maps and institutional affil- studies to demonstrate its physiological significance and clinical utility in DMD. iations. Keywords: Duchenne muscular dystrophy; Becker muscular dystrophy; disease severity; inflamma- tory biomarkers; chemokines; validation studies

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and Duchenne muscular dystrophy (DMD) is an X-linked genetic disease with complex conditions of the Creative Commons and progressive muscle pathogenesis involving a cascade of altered biological and bio- Attribution (CC BY) license (https:// chemical events leading to muscle wasting and connective tissue replacement (fibrosis). creativecommons.org/licenses/by/ The pathogenesis is triggered by sarcolemma instability and Ca2+ influx due to the lack of 4.0/).

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dystrophin expression, an essential protein that plays a role in stabilizing the sarcolemma during muscle fiber contraction and relaxation via interaction with dystrophin-associated protein complex [1,2]. Our lab recently generated a large dataset of serum protein biomark- ers for DMD using highly multiplexing technologies such as SomaScan® aptamer-based assay (Somalogic, Boulder, CO, USA) [3] and a tandem mass tag (TMT) mass spectrometry- based method [4]. Different groups of biomarkers associated with different pathological pathways of the skeletal muscle were thus identified. These include muscle-centric pro- tein biomarkers reflecting sarcolemma fragility and muscle injury and non-muscle-centric protein biomarkers such as proinflammatory and extracellular matrix remodeling proteins most likely associated with disease pathogenesis and progression. This study focuses on validating and testing three proinflammatory chemokines, CCL2, CCL18, and CXCL10, previously identified by SomaScan® analysis, as being elevated in serum samples of glucocorticoid naïve DMD patients compared to age-matched healthy controls [3]. We hypothesize that chemokines play a role in the early stage of skeletal muscle pathogenesis in DMD, and their circulating levels reflect disease activity. Chemokines are a group of small secreted proteins involved in chemotaxis and recruit- ment of immune cells to inflammation sites [5]. They are subdivided based on their primary structures, number, and position of cysteine residues into two major groups: the alpha CXC group, where the two cysteine residues located in the N-terminal end are separated by one , and the beta CC group, where the two N-terminal cysteine residues are adjacent to each other. Two other minor groups have also been described, the gamma C group and CX3C-d group, but only two and one chemokines discovered under these minor categories to date [6]. All chemokines exert their biological activity by interacting with their respective G-protein coupled receptors and integrins [7]. Proinflammatory chemokines are believed to be involved in the recruitment of different types of immune cells to the site of muscle injury in DMD and other muscle inflammatory diseases [5,8]. However, the role of these recruited immune cells in muscle pathogenesis in DMD remains poorly understood. Some studies have shown that the proinflammatory M1 phenotype activated in the early stage of the disease are responsible for phagocytosis and muscle fiber damage; this is subsequently replaced by the M2-phenotype macrophages (anti-inflammatory) responsible for muscle regeneration and fibrosis in the later stage of the disease [9]. The recruitment of M1 and M2 macrophages is orchestrated by chemokines. CCL19, CCL21, CCL24, CCL25, CXCL8, CXCL10, and CXCL2 influence the recruitment of the M1 phenotype macrophages, while CCL7 influences the chemotaxis of both M1 and M2 macrophages [10]. In this study, we used a highly specific and reproducible MSD ELISA assay to examine the levels of the three chemokines (CXCL10, CCL2, and CCL18) in serum and muscle samples collected from DMD patients, Becker muscular dystrophy (BMD) patients, and age-matched healthy controls. This approach was also used to investigate the difference in chemokine levels in sera samples of mdx and wild-type mice. The assay was then used to assess the response of these chemokines to glucocorticoid (GC) treatment, their association with age, disease severity, and clinical outcomes. DMD and BMD are two clinically distinct dystrophinopathies. DMD is the most severe form of the disease due to the complete loss of dystrophin expression, while BMD is a milder form due to the partial expression of truncated but functional dystrophin [11–13]. Therefore, comparing the levels of circulating chemokines in DMD and BMD will help us understand their association with disease severity and eventually disease progression.

2. Materials and Methods 2.1. Study Participants and Sample Collection All specimens used in this study were collected under study protocols approved by the Institutional Review Boards (IRB) at all participating sites. Serum samples from DMD patients and age-matched healthy controls were from a subset of volunteers enrolled in the Cooperative International Neuromuscular Research Group (CINRG) [14]. Participating Life 2021, 11, 827 3 of 13

sites under the CINRG study protocol include the Office of Research IRB administrations at the University of California Davis in Davis, CA, the University of Pittsburgh in Pittsburgh, PA, the Children’s National Hospital in Washington, DC, and the Conjoint Health Research Ethics Board at the University of Calgary in Calgary, Alberta. Informed written consent was obtained from the parents or legal guardians before sample collection. Serum samples from BMD patients were collected remotely by a phlebotomist under a study protocol approved by the Human Subject Research Review Committee at Binghamton University, NY, and shipped to our laboratory for biomarker studies. In the BMD study, following the approval of parental consent/assent, participants were asked to provide a copy of their DNA diagnostic bloodwork to confirm a diagnosis of BMD disease. Additional serum samples from 4-year-old healthy controls were purchased from a third-party company (BIOIVT, Hicksville, NY, USA). Muscle tissue samples from BMD, DMD, and age-matched healthy controls were a gift from Dr. Hoffman’s laboratory previously collected and bio-banked under a study protocol approved by the Regulatory Affairs and Research Compliance administration at the Children’s National Hospital in Washington, DC. All participants consented to their sample being collected for research purposes. Detailed demographics of the patients are listed in Supplemental Table S1. Mouse serum samples were a gift from Dr. Novak (Children’s National Hospital in Washington, DC) and Dr. Nagaraju’s laboratory (Department of Pharmaceutical Sciences (Binghamton University in Binghamton, NY) and were originally collected from 6 to 8-week-old male C57Bl/10J (wild-type) and C57Bl/10J-mdx-23 (mdx-23) mice, respectively. All serum samples were processed with a standardized operating protocol and stored at −80 ◦C as workable aliquots in polypropylene cryogenic Nalgene vials (Thermo Fisher Scientific, Waltham, MA USA) to avoid repetitive freeze–thaw cycles.

2.2. Meso Scale Discovery ELISA Assay of Chemokines ELISA reagents and plates for measuring the three chemokines CXCL10, CCL2, and CCL18 concentrations were purchased from Meso Scale Diagnostics (Rockville, MD, USA). Analysis of CCL2 and CXCL10 was performed using a U-plex multiplex assay platform for both human and mouse samples, while CCL18 concentrations were measured using an R- plex assay platform. The plates were read using the MSD MESO QuickPlex SQ120 imager, and the values were reported in pg/mL for each target analyte. To test the reproducibility and linearity of the multiplex CXCL10 & CCL2 assay, we compared the results of this assay with the singleplex assay against CCL2 and CXCL10 using longitudinal serum samples of GC naïve DMD patients (n = 20). In order to check the reproducibility and stability of the assay, the same samples were analyzed twice on independent days (two weeks apart) by two independent laboratory technicians on the same serum samples from healthy controls (n = 10), BMD (n = 12) and DMD patients (n = 12).

2.3. Protein Extraction from Skeletal Muscle Tissue Muscle tissue samples were lysed in RIPA buffer containing protease inhibitors. RIPA buffer was added to tissue slices at a ratio of 3:1, v/v. Samples were vigorously vortexed, spun, and kept on ice for 15 min. This action was repeated once, and the samples were kept on ice for 5 min. Samples were then centrifuged at 14,000 rpm for 10 min. Supernatants were collected, and protein concentration was determined using the PierceTM BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA USA). Samples were then aliquoted in workable volumes and stored at −80 ◦C until analysis. The levels of the three chemokines CXCL10, CCL2, and CCL18, were measured in muscle extract using the same MSD plates and reagents as described above. Data were normalized to total muscle protein in each sample.

2.4. Data Processing and Statistical Analysis The protein concentrations were log-transformed (base 2) and the normality evaluated by the Shapiro–Wilk normality test. A cross-sectional analysis was performed using Student’s t-test to compare the levels of the three chemokines (CXCL10, CCL2, and CCL18) Life 2021, 11, 827 4 of 13

in serum and muscle samples of GC naïve DMD patients vs. age-matched healthy controls. Using the same method, we monitored the differences of these chemokines in serum samples of mdx and wild-type mice. Then, we conducted a one-way ANOVA to compare the level of the proteins between DMD, control, and BMD in both serum and muscle samples. Tukey’s test for multiple comparisons was used to compare the three groups in a pairwise fashion. Student’s paired t-test was conducted on DMD patients pre- and post-GC treatment (n = 11) to test for response to GC. Most patients were treated with deflazacort in this study. A Pearson correlation coefficient was used to investigate the association of chemokine serum levels with age using cross-sectional samples from DMD, BMD, and healthy controls. Similarly, a correlation was conducted to determine the association of the chemokines with clinical outcome measures and disease severity in DMD and BMD patients, respectively. A p-value < 0.05 was considered statistically significant for the data. All statistical analyses and figures were prepared using GraphPad Prism version 7.0 (GraphPad Software, Inc., San Diego, CA, USA).

3. Results 3.1. Reproducibility and Stability of the MSD ELISA Assays for Chemokines in Serum Samples MSD ELISA assays are well-standardized and use validated calibrators and antibodies that undergo rigorous testing and QCs. These QCs include sensitivity, precision, repro- ducibility, stability of calibrators, with a rigorous cross-reactivity or nonspecific binding cut-off of <0.5% [15]. In this study, we used the following assay kits from MSD: the sin- gleplex U-plex (CXCL10, CCL2), singleplex R-plex assay kits for CCL18, and the U-plex multiplex assay kit (CXCL10 and CCL2 combined). We separately evaluated the singleplex and the multiplex kits for consistency and stability of the assay. A Pearson correlation coefficient was used to check the correlation between experimental runs. To assess the repeatability of the assay kit, we conducted two independent experiments with independent operators on two separate days using the same serum samples from DMD patients (n =12), BMD patients (n = 12), and healthy controls (n = 10). The time interval between each experimental run was two weeks, and there was no repetitive freeze– thaw cycle of the serum samples to avoid changes due to sample handling. For each sample, we used a starting serum volume of 10 µL that was diluted six times, and then 25 µL were taken in duplicate from each diluted sample to perform the assay for CXCL10. As shown in Figure1a, there was a strong correlation between the two runs on independent days by two different operators in all three sample groups (<15% CV, average correlation 0.98) with a p-value < 0.0001; this indicates that the method is reliable and consistent for the serum tested. There was no substantial change in CXCL10 trends between each experimental run, and both operators obtained similar results. Similar repeatability between the two operators was obtained for the other two chemokines CCL2 and CCL18, further confirming the robustness of the assay kit. We then conducted an independent run to verify if multiplexing affects measurements of a given target. A U-plex 2-plex assay was created for CCL2 and CXCL10. Generally, the R-plex assays are run on separate plates; hence, CCL18 was analyzed separately. Both CCL2 and CXCL10 were multiplexed on a single 96-well plate tested with longitudinal serum samples collected from untreated DMD patients. The results of this experiment were then compared with a singleplex assay of both CCL2 and CXCL10 ran on independent days by two different operators on the same serum samples. The volume and dilution used for this assay are the same as the singleplex method above and were also ran in duplicates. The result obtained showed a strong correlation between the multiplex and the single- plex assay kits for CCL2 and CXCL10 (r = 0.94, p-value < 0.0001); this implies that whether the target was analyzed alone or multiplexed with a second target, similar results were obtained. These results suggest that multiplexing the two chemokines will not affect the outcome of the data (Figure1b). Moving forward, we multiplexed CXCL10 and CCL2 on Life 2021, 11, x FOR PEER REVIEW 5 of 14 Life 2021, 11, x FOR PEER REVIEW 5 of 14

Both CCL2 and CXCL10 were multiplexed on a single 96-well plate tested with longitudi- Both CCL2 and CXCL10 were multiplexed on a single 96-well plate tested with longitudi- nal serum samples collected from untreated DMD patients. The results of this experiment nal serum samples collected from untreated DMD patients. The results of this experiment were then compared with a singleplex assay of both CCL2 and CXCL10 ran on independ- were then compared with a singleplex assay of both CCL2 and CXCL10 ran on independ- ent days by two different operators on the same serum samples. The volume and dilution ent days by two different operators on the same serum samples. The volume and dilution used for this assay are the same as the singleplex method above and were also ran in used for this assay are the same as the singleplex method above and were also ran in duplicates. duplicates. The result obtained showed a strong correlation between the multiplex and the sin- The result obtained showed a strong correlation between the multiplex and the sin- gleplex assay kits for CCL2 and CXCL10 (r = 0.94, p-value < 0.0001); this implies that Life 2021, 11, 827 gleplex assay kits for CCL2 and CXCL10 (r = 0.94, p-value < 0.0001); this implies 5 that of 13 whether the target was analyzed alone or multiplexed with a second target, similar results whether the target was analyzed alone or multiplexed with a second target, similar results were obtained. These results suggest that multiplexing the two chemokines will not affect were obtained. These results suggest that multiplexing the two chemokines will not affect the outcome of the data (Figure 1b). Moving forward, we multiplexed CXCL10 and CCL2 the outcome of the data (Figure 1b). Moving forward, we multiplexed CXCL10 and CCL2 the same plate.on This the action same wasplate. performed This action in was all the performed following in experiments all the following to evaluate experiments the to evaluate on the same plate. This action was performed in all the following experiments to evaluate level of these chemokinesthe level of inthese subsequent chemokines samples. in subsequent samples. the level of these chemokines in subsequent samples.

Figure 1. CorrelationFigure 1. Correlation showing the showing reproducibility the reproducibility and stability and of stability the singleplex of the singleplex and multiplex and multiplex assay kits assay from MSD (p < Figure 1. Correlation0.0001). showing (a) kitsSerum the from reproducibility samples MSD (fromp < 0.0001 control and). stability ( a(n) Serum= 10), of DMD, the samples singleplex and from BMD controland (n =multiplex 12) (n were= 10), assaytested DMD, kits for and thefrom BMD stability MSD (n = ( p 12)and < were reproducibility 0.0001). (a) Serum samples from control (n = 10), DMD, and BMD (n = 12) were tested for the stability and reproducibility of the singleplextested kit for using the stability CXCL10 and as reference reproducibility protein, of ( theb) Data singleplex obtained kit from using the CXCL10 single run as reference of CCL2 protein,and CXCL10 were of the singleplex kit using CXCL10 as reference protein, (b) Data obtained from the single run of CCL2 and CXCL10 were plotted against(b) Data data obtainedobtained fromby the the multiplex single run run of of CCL2 CCL2 and and CXCL10 CXCL10. were plotted against data obtained by plotted against data obtained by the multiplex run of CCL2 and CXCL10. the multiplex run of CCL2 and CXCL10. 3.2. Data Validation Using Serum Samples from DMD Patients and Cross-Species Comparison 3.2. Data Validation Using Serum Samples from DMD Patients and Cross-Species Comparison 3.2. Data Validation UsingWe previously Serum Samples reported from that DMD CCL2, Patients CXCL10, and Cross-Species and CCL18 Comparisonwere elevated in DMD (n = We previously reported that CCL2, CXCL10, and CCL18 were elevated in DMD (n = We previously18) vs. reported control (n that = 12) CCL2, using CXCL10, SomaScan and® [3]. CCL18 We confirmed were elevated these results in DMD in a subset of the 18) vs. control (n = 12) using SomaScan® [3]. We confirmed these results in a subset of the (n = 18) vs. controlsame (n DMD= 12) patients using SomaScan (n = 9) using® [3]. ELISA We confirmed and plotted these the results correlation in a subset between of the two tech- same DMD patients (n = 9) using ELISA and plotted the correlation between the two tech- the same DMDniques. patients As ( ndepicted= 9) using in Figure ELISA 2, and we plotted found a the significant correlation correlation between between the two the two meth- niques. As depicted in Figure 2, we found a significant correlation between the two meth- techniques. Asods depicted for CCL2 in Figureand CXCL10.2, we found However, a significant there was correlation a statistically between insignificant the two correlation for ods for CCL2 and CXCL10. However, there was a statistically insignificant correlation for methods for CCL2CCL18. and This CXCL10. discrepancy However, between there was SomaScan a statistically® and MSD insignificant ELISA for correlation CCL18 could be due to CCL18. This discrepancy between SomaScan® and® MSD ELISA for CCL18 could be due to for CCL18. Thisthe discrepancy small sample between size SomaScanused for theand comparative MSD ELISA study. for CCL18 Unfortunately, could be due we did not have the small sample size used for the comparative study. Unfortunately, we did not have to the small sampleenough size of usedthe same for the samples comparative used in study. our previous Unfortunately, study to we increase did not the have sample size here. enoughenough of of the the same same samples samples used used in in our our previous previous study study to to increase increase the the sample sample size size here. here.

® Figure 2. Correlation plots® of SomaScan data vs. ELISA assay data obtained on same DMD serum Figure 2. Correlation plots of SomaScan® data vs. ELISA assay data obtained on same DMD serum Figure 2. Correlationsamples plots (n of = SomaScan9) for CXCL10 data (left vs.), ELISA CCL2 assay(middle data) and obtained CCL18 on (right same). DMD serum samplessamples ( (nn == 9) 9) for for CXCL10 CXCL10 (left (left),), CCL2 CCL2 (middle (middle) )and and CCL18 CCL18 ( (rightright).).

The standardized MSD-ELISA assay above was then tested on a larger sample size of GC naïve DMD patients (n = 26) and age-matched healthy controls (n = 25) (age range

of 4–30 years old). Each sample was run in duplicates, and the CV was generally <15%. The data obtained for each of the tested chemokines is shown under Table1 with their respective p-value and fold change in the DMD group relative to healthy controls. As expected, all three chemokines were significantly elevated in serum samples from DMD patients relative to age-matched healthy controls with a p-value < 0.001 for our data. Life 2021, 11, x FOR PEER REVIEW 6 of 14

The standardized MSD-ELISA assay above was then tested on a larger sample size of GC naïve DMD patients (n = 26) and age-matched healthy controls (n = 25) (age range of 4–30 years old). Each sample was run in duplicates, and the CV was generally <15%. The data obtained for each of the tested chemokines is shown under Table 1 with their respective p-value and fold change in the DMD group relative to healthy controls. As ex- Life 2021, 11, 827 pected, all three chemokines were significantly elevated in serum samples from DMD6 pa- of 13 tients relative to age-matched healthy controls with a p-value < 0.001 for our data.

Table 1. ELISA assay data obtained for the three tested chemokines on serum samples of DMD patientsTable 1. andELISA age-matched assay data healthy obtained controls. for the three tested chemokines on serum samples of DMD patients and age-matched healthy controls. Protein Name Cnt (pg/mL) DMD (pg/mL) p-Value DMD/Cnt ProteinCXCL10 Name 580Cnt (pg/mL)± 103 DMD 942 ± (pg/mL) 51 <0.0001p-Value DMD/Cnt 1.62 CCL2CXCL10 333 580 ±± 21103 518 942 ±± 2751 <0.0001 <0.0001 1.55 1.62 CCL18CCL2 57,047 333 ±± 527921 87,746 518 ±± 893627 0.0004 <0.0001 1.54 1.55 Data areCCL18 presented as mean 57,047 ± standard± 5279 error, 87,746 p-values± 8936 are for differences 0.0004 in the mean levels 1.54 be- tweenData areDMD presented (n = 26) as and mean healthy± standard controls error, (np =-values 25) and are were for differences generated in using the mean Student’s levels t between-test. DMD (n = 26) and healthy controls (n = 25) and were generated using Student’s t-test.

ToTo verify verify if if the the increased increased levels levels of of these these three three chemokines chemokines are are effectively effectively associated associated withwith dystrophin dystrophin deficiency, deficiency, wewe conductedconducted ELISAELISA assays assays to to measure measure CCL2 CCL2 and and CXCL10 CXCL10 on onserum serum samples samples collected collected from from mdx-23 mdx-23 mice mice ( n(n= =10) 10) andand wild-typewild-type mice (n = 14). 14). CCL18 CCL18 waswas omitted omitted in in this this experiment experiment because because of of the the unavailability unavailability of of a aCCL18 CCL18 MSD MSD kit kit for for mice. mice. AsAs shown shown in in Figure Figure 3,3, only only CCL2 CCL2 was was found found to to be be significantly significantly elevated elevated in in serum serum samples samples ofof the the mdx-23 mdx-23 mice mice compared compared to to wild-type wild-type BL10 BL10 mice mice (Fold (Fold change change 2.19, 2.19, p-pvalue-value < < 0.05). 0.05). CXCL10CXCL10 serum serum levels levels were were not not significantly significantly altered altered between between the the two two groups groups studied. studied.

FigureFigure 3. 3. BoxBox plot plot showing showing the level of CXCL10CXCL10 andand CCL2CCL2 in in serum serum samples samples of of mdx-23 mdx-23 mice mice (n (=n 10)= 10)vs. vs. wild-type wild-type BL10 BL10 mice mice (n = ( 14).n = Left14). panelLeft panel CXCL10 CXCL10 remained remained unchanged unchanged between between the two the groups two groups(p-value (p-value = 0.6732). = 0.6732). Right Right panel panel CCL2 CCL2 was significantly was significantly elevated elevated in serum in serum samples samples of mdx of mdx vs. BL10 vs. BL10mice mice (p-value (p-value = 0.0046). = 0.0046).

3.3.3.3. CCL2 CCL2 and and CXCL10 CXCL10 Are Are Elevated Elevated in in Skeletal Skeletal Muscle Muscle Samples Samples of of DMD DMD Patients Patients Compared Compared to to Healthy Controls Healthy Controls Levels of the three chemokines studied herein were further examined in skeletal Levels of the three chemokines studied herein were further examined in skeletal mus- muscle biopsies collected from GC naïve DMD and age-matched healthy control (age cle biopsies collected from GC naïve DMD and age-matched healthy control (age 2–8 years 2–8 years old). We hypothesize that chemokines drive DMD pathogenesis and are expected old). We hypothesize that chemokines drive DMD pathogenesis and are expected to alter to alter in their levels in the skeletal muscle, the site of injury, and pathogenesis. Due to in their levels in the skeletal muscle, the site of injury, and pathogenesis. Due to the limi- the limitation of muscle biopsies in pediatrics, we only tested a small number of samples tation of muscle biopsies in pediatrics, we only tested a small number of samples (n = 4 (n = 4 per group). As shown in Figure4, CXCL10 and CCL2 were found to be significantly per group). As shown in Figure 4, CXCL10 and CCL2 were found to be significantly ele- elevated by a factor of 2.88- and 2.78-fold, respectively, in muscle extracts of DMD patients vated by a factor of 2.88- and 2.78-fold, respectively, in muscle extracts of DMD patients relative to healthy controls (p-value < 0.05). CCL18 was also elevated in DMD muscle rela- relative to healthy controls (p-value < 0.05). CCL18 was also elevated in DMD muscle rel- tive to healthy control muscle but did not reach statistical significance (Fold change = 2.21, ativep-value to healthy = 0.26), control probably muscle due tobut the did small not reach sample statistical size. This significance data agrees (Fold with change= data reported 2.21, pfor-value CCL2 = 0.26), in the probably skeletal due muscle to the of small the mdx sample mouse size. model This data [16] andagrees may with indicate data reported a role of forthis CCL2 chemokine in the skeletal in recruiting muscle immune of the cellsmdx tomouse the site model of muscle [16] and injury. may indicate a role of this chemokine in recruiting immune cells to the site of muscle injury.

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Figure 4. Cross-sectional analysis of chemokine biomarkers in skeletal muscle samples of DMD (n = 4) and age-matched healthy controls (n = 4). CXCL10 and CCL2 were significantly elevated in DMD vs. control (p = 0.026, p = 0.0174). CCL18, although elevated in DMD vs. control, was not sig- Figurenificant 4. Cross-sectionalFigure (p-value 4. Cross-sectional = analysis 0.26). Note: of chemokine analysis Due to of muscle biomarkers chemokine sample inbiomarkers skeletal limitation muscle in skeletal from samples the muscle pediatric of DMD samples (population,n = of 4) DMD and age-matched ( nonly healthythree controls =samples 4) (andn = 4). age-matchedwere CXCL10 tested and healthyfor CCL2 CCL18 werecontrols in significantly healthy (n = 4). controls. CXCL10 elevated and in DMDCCL2 vs. were control significantly (p = 0.026, elevatedp = 0.0174). in CCL18, although elevatedDMD invs. DMD control vs. ( control,p = 0.026, was p = not 0.0174). significant CCL18, (p-value although = 0.26). elevated Note: in Due DMD to musclevs. control, sample was limitation not sig- from the nificant (p-value = 0.26). Note: Due to muscle sample limitation from the pediatric population, only pediatric3.4. population, Association only of three Circulating samples wereChemokines tested for with CCL18 Age in healthy controls. three samples were tested for CCL18 in healthy controls. Age is an important3.4. Association confounding of Circulating variable Chemokines in DMD, with Ageas most circulating protein bi- omarkers,3.4. Association especially of Circulatingmuscle injury Chemokines biomarkers, with Age have been shown to decline with age be- Age is an important confounding variable in DMD, as most circulating protein cause of theAge loss is an biomarkers,of important muscle massconfounding especially [17,18]. muscle variable As shown injury in DMD, biomarkers, in Figure as most 5, have bothcirculating been CCL2 shown protein and to CCL18 declinebi- with age declinedomarkers, with ageespeciallybecause in DMD muscle of patients the lossinjury of(age musclebiomarkers, range: mass 4–29 have [17 years,18 been]. As old, shown shown n = to26, in decline Figurep-value 5with, < both 0.05) age CCL2 be-while and CCL18 CXCL10cause remained of the lossdeclined unchanged of muscle with age massover in DMD[17,18].time with patients As shownage (age (n = range:in 25, Figure p 4–29-value 5, yearsboth = 0.3952) old,CCL2n = andin 26, agreement pCCL18-value < 0.05) while with declinedour earlier with studyCXCL10 age in [18]. DMD remained However, patients unchanged (agethe three range: over chemokines 4–29 time years with old, remained age n = (n 26,= 25,p -valueunchangedp-value < 0.05) = 0.3952) withwhile age in agreement in bothCXCL10 the BMD remained withgroup ourunchanged (age earlier range: study over 6–68 [time18]. years However,with old,age ( thenn = three25)25, pand-value chemokines in =healthy 0.3952) remained volunteersin agreement unchanged (age with age with our earlier study [18]. However, the three chemokines remained unchanged with age range: 4–32 yearsin old, both n = the 25). BMD group (age range: 6–68 years old, n = 25) and in healthy volunteers (age in both the BMDrange: group 4–32 (age years range: old, 6–68n = 25). years old, n = 25) and in healthy volunteers (age range: 4–32 years old, n = 25).

Figure 5. Association of circulating chemokines with age. Left panel: healthy controls (age range 4– FigureFigure 5. Association 5. Association of circulating of circulating chemokines chemokines with age. Leftwith panel: age. healthyLeft panel: controls healthy (age controls range 4–32 (age years range old, 4–n = 25), middle panel:32 DMDyears (ageold, n range = 25), 4–29 middle years panel: old, DMDn = 26), (age and range right 4–29 panel years is BMD old, n (n == 26), 25) and for CCL2,right panel CXCL10, is BMD and CCL18, 32 years(n = old, 25) nfor = CCL2,25), middle CXCL10, panel: and DMD CCL18, (age respectively. range 4–29 There years was old, no n change = 26), and in CXCL10 right panel in all is the BMD respectively. There was no change in CXCL10 in all the samples tested. CCL2 and CCL18 declined over time in DMD (n = 25)samples for CCL2, tested. CXCL10, CCL2 and and CCL18 CCL18, declined respectively. over time in There DMD waspatients no changebut remained in CXCL10 unchanged in all in the patients but remained unchanged in healthy controls and BMD patients. sampleshealthy tested. controls CCL2 and and BMD CCL18 patients. declined over time in DMD patients but remained unchanged in healthy controls and BMD patients. 3.5. Circulating and Skeletal Muscle Chemokines Are Elevated in DMD Patients Compared to 3.5. CirculatingAge-Matched and Skeletal BMDMuscle Patients Chemokines Are Elevated in DMD Patients Compared to 3.5. CirculatingAge-Matched and BMD Skeletal Patients Muscle Chemokines Are Elevated in DMD Patients Compared to The rationale behind this experiment relies on the fact that BMD is less severe than Age-MatchedThe BMDrationaleDMD; Patients behind hence, this the experiment levels of the relies chemokines on the fact may that differ BMD between is less severe these than two diseases and TheDMD; rationale hence, correlatethe behind levels with ofthis the disease experiment chemokines severity. reliesmay Of thediffer on three the between testedfact that these chemokines, BMD two diseasesis onlyless severe CCL2 and cor- was than significantly DMD;relate hence, with the disease elevatedlevels severity. of inthe serum chemokines Of the samples three maytested of DMD differ chemokines, patients between comparedonly these CCL2 twoto was diseases age-matched significantly and BMDcor- patients elevated in serum samples of DMD patients compared to age-matched BMD patients (fold relate with disease(fold severity. change Of 2.12, thep -valuethree tested = 0.024) chemokines, and age-matched only CCL2 healthy was controls significantly (fold change 2.08, elevated in serum samples of DMD patients compared to age-matched BMD patients (fold

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Life 2021, 11, x FOR PEER REVIEW 8 of 14

Life 2021, 11, 827 8 of 13 change 2.12, p-value = 0.024) and age-matched healthy controls (fold change 2.08, p-value = change0.03). Although 2.12, p-value CXCL10 = 0.024) was and elevated age-matched in the serumhealthy of controls DMD patients (fold change compared 2.08, p with-value BMD= 0.03). patients Althoughp by-value 2.03-fold, CXCL10 = 0.03 ).this was Although difference elevated CXCL10 didin the not serum wasreach elevated ofstatistical DMD in patients difference the serum compared ( ofp-value DMD with = patients com- 0.0882).BMD patients CCL18pared wasby 2.03-fold, with not statisticallyBMD this patients difference altered by 2.03-fold, did in itsnot circulating thisreach difference statistical levels did difference between not reach the (p statistical-value three = difference groups0.0882). with CCL18 a (p-value-value was of = not 0.08820.885 statistically (Figure). CCL18 6). altered was not in statistically its circulating altered levels in its between circulating the levels three between the groups withthree a p-value groups of 0.885 with a(Figurep-value 6). of 0.885 (Figure6).

Figure 6. Box plots showing the serum levels of chemokine biomarkers in DMD, BMD, and control. Figure 6. BoxLeft plotsFigure panel: showing 6. BoxCXCL10 plots the serum serumshowing levelslevels the ofserumin chemokineDMD levels (n = of biomarkers10) chemokine vs. BMD in biomarkers( DMD,n = 12) BMD, vs. in healthy DMD, and control. BMD,control Leftand (n control.= panel: 12). CXCL10 serum levelsMiddle inLeft DMD panel: panel: (n = CXCL10 10)CCL2 vs. serum BMD serum (levelsn =levels 12) in vs. inDMD healthyDMD vs. ( BMDn control = 10) vs. vs. ( ncontrol= BMD 12). Middle((nn == 12). 12) panel: Rightvs. healthy CCL2panel: serumcontrolCCL18 levels serum(n = 12). in DMD vs. levels in DMD vs. BMD vs. control (n = 12). * denotes p < 0.05, ns denotes not significant. A one-way BMD vs. controlMiddle (n = 12).panel: Right CCL2 panel: serum CCL18 levels serum in DMD levels vs. inBMD DMD vs. vs.control BMD (n vs. = 12). control Right (n panel:= 12). CCL18 * denotes serump < 0.05, ns ANOVAlevels in was DMD used vs. for BMD statistical vs. control analysis. (n = 12). * denotes p < 0.05, ns denotes not significant. A one-way denotes not significant. A one-way ANOVA was used for statistical analysis. ANOVA was used for statistical analysis. Consistent withConsistent the serum with samples, the serum CCL18 samples, levels CCL18were not levels significantly were not altered significantly in altered in skeletalConsistent muscleskeletal samples with musclethe between serum samples DMD samples, betweenand CCL18BMD DMD (Figure levels and 7).were BMDInterestingly, not (Figure significantly7 CCL2). Interestingly, wasaltered sig- in CCL2 was nificantlyskeletal elevatedmusclesignificantly samples in muscle between elevated samples DMD in of muscle DMD and BMDpatients samples (Figure compared of DMD 7). Interestingly, patients with BMD compared patients CCL2 was with(fold sig- BMD patients changenificantly 2.87, elevated p-value(fold change =in 0.02) muscle and 2.87, samples age-matchedp-value of =DMD 0.02) healthy patients and controls age-matched compared (fold withchange healthy BMD 2.76, controls patients p-value (fold (fold = change 2.76, 0.03).change CXCL10 2.87,p pwas-value-value also = 0.02)significantly 0.03). and CXCL10 age-matched elevated was alsoin healthy DMD significantly patientscontrols relative(fold elevated change to inBMD DMD2.76, patients, p-value patients = relative to and0.03). healthy CXCL10 controls,BMD was patients, also 3.28- significantly and and 3.33-fold, healthy elevated controls,respectively, in DMD 3.28- with patients and a 3.33-fold, significant relative respectively, top-value BMD patients,< 0.05. with a significant Theseand healthyresults suggestpcontrols,-value

Figure 7. Box plots showing chemokine biomarker levels in skeletal muscle of DMD vs. BMD vs. healthy control. Left panel: CXCL10 in muscle samples of DMD (n = 4) vs. BMD (n =4) vs. healthy controls (n = 4). Middle panel: CCL2 in Figure 7. Box plots showing chemokine biomarker levels in skeletal muscle of DMD vs. BMD vs. muscle samples of DMD (n = 4) vs. BMD vs. healthy controls (n = 4). Right panel: CCL18 in muscle samples of DMD healthyFigure control. 7. Box plotsLeft panel: showing CXCL10 chemokine in muscle biomarker samples levels of DMD in skeletal (n = 4) vs.muscle BMD of ( nDMD = 4) vs.vs. healthy BMD vs. (n = 4) vs. BMDcontrolshealthy vs. healthy (n control. = 4). controlsMiddle Left panel:panel: (n = 3). CXCL10CCL2 * denotes in inmuscle pmuscle< 0.05, samples samples ns denotes of DMDof DMD not ( significant.n =( n4) = vs. 4) BMDvs. A BMD one-way vs. healthy(n = ANOVA4) vs. controls healthy was used for statistical analysis.(n controls= 4). Right (n =panel: 4). Middle CCL18 panel: in muscle CCL2 samples in muscle of samplesDMD (n of= 4) DMD vs. BMD (n = 4) vs. vs. healthy BMD vs. controls healthy (n controls= 3). * denotes(n = 4). p Right < 0.05, panel: ns denotes CCL18 not in musclesignificant. samples A one-way of DMD ANOVA (n = 4) vs. was BMD used vs. for healthy statistical controls analysis. (n = 3). * denotes p < 0.05,3.6. ns Response denotes ofnot Circulating significant. Chemokines A one-way ANOVA to Glucocorticoid was used Treatment for statistical analysis. 3.6. Response of CirculatingIn this study, Chemokines we sought to Glucocorticoid to examine theTreatment response of these three circulating chemokines 3.6.In Response this study,to of glucocorticoidsCirculating we sought Chemokines to (GCs) examine treatment. to Glucocorticoidthe response Previously, ofTreatment these we found three nocirculating change in chemo- the levels of CXCL10, ® kines toIn glucocorticoids this study,CCL2, we and (GCs)sought CCL18 treatment. to in examine pre- and Previously, the post-GC response we treated foundof these patients no threechange using circulating in the the SomaScan levels chemo- of measuring CXCL10,kines to CCL2,glucocorticoidstechnique and CCL18 [3 (GCs)]. Toin pre- confirmtreatment. and thispost-GC Previously, finding, treated the we samepatientsfound set no using of change pre- the and in SomaScan the post-GC levels® of treated DMD ® measuringCXCL10, techniqueCCL2,patients and [3]. (CCL18n To= 11,confirm in age pre- range this and finding, 4–10 post-GC years the treatedsame old) set used patients of pre- for the andusing SomaScan post-GC the SomaScan treatedstudy ® were tested DMDmeasuring patients techniquefor (n = longitudinal 11, age [3]. range To confirm changes 4–10 years this in the finding,old) circulating used the for same the levels SomaScan set of of CXCL10,pre-® and study CCL2,post-GC were and tested treated CCL18 using the forDMD longitudinal patientsstandardized (changesn = 11, age in MSDtherange circulating ELISA 4–10 years assay levels old) herein. ofused CXCL10, Together, for the CCL2, SomaScan the data and obtained ®CCL18 study were forusing all tested threethe chemokines standardizedfor longitudinal MSDshow changes ELISA that they assayin the did herein.circulating not respond Together, levels to GC theof evenCXCL10, data after obtained CCL2, two years for and all ofCCL18 three treatment usingchemo- (theTable 2 ). These ® kinesstandardized show thatresults MSD they ELISA agreeddid not assay withrespond herein. the to SomaScan GC Together, even afterassay the twodata and years obtained confirm of treatment for that all the three (Table three chemo- 2). chemokines are kines show thatunresponsive they did not to GC respond treatment. to GC even after two years of treatment (Table 2).

Life 2021, 11, x FOR PEER REVIEW 9 of 14

These results agreed with the SomaScan® assay and confirm that the three chemokines are Life 2021, 11, 827 unresponsive to GC treatment. 9 of 13

Table 2. GC does not affect the level of chemokines pre- and post-treatment.

TableProtein 2. GC Name does not affect DMD the Pre-Treat level of chemokines (pg/mL) pre-DMD and Post-Treat post-treatment. (pg/mL) p Value CXCL10 702 ± 118 533 ± 82 0.097 ProteinCCL2 Name DMD Pre-Treat 449 ± (pg/mL) 75 DMD Post-Treat 426 ± (pg/mL)64 p 0.8765Value CXCL10CCL18 702 64,271± 118 ± 5523 533 64,856± 82 ± 4690 0.097 0.8793 Data areCCL2 presented as mean ± 449 standard± 75 error, p-values are for 426 differences± 64 in the mean levels 0.8765 be- tweenCCL18 DMD before and after 64,271 treatment± 5523 with GC use. Data 64,856 were± generated4690 using paired 0.8793 t-test. Data are presented as mean ± standard error, p-values are for differences in the mean levels between DMD before 3.7.and Association after treatment of with Circulating GC use. Data Chemokines were generated with usingClinical paired Outcomet-test. Measures in DMD Patients and3.7. Reported Association Patient of Circulating Outcomes Chemokinesin BMD Patients with Clinical Outcome Measures in DMD Patients and ReportedTo examine Patient the Outcomesassociation in BMDof the Patients three studied chemokines with clinical outcome measuresTo examine (6 min walk the association test, run/walk of the velocity, three studiedstand velocity, chemokines and climb with clinicalvelocity) outcome we fo- cusedmeasures on GC (6 min naïve walk DMD test, patients run/walk (4-years-old) velocity, stand to avoid velocity, the and confounding climb velocity) variable we focused of GC useon GCand naïveage. There DMD was patients no correlation (4-years-old) with to clinical avoid the outcomes confounding except variable for CCL2, of which GC use neg- and ativelyage. There correlated was no with correlation climb velocity with clinical and was outcomes not adjusted except for for multiple CCL2, which testing negatively (Figure 8a).correlated with climb velocity and was not adjusted for multiple testing (Figure8a).

FigureFigure 8. 8. CorrelationCorrelation plots plots of of serum serum levels levels for for the the three three tested tested chemokines chemokines with with clinical clinical outcome outcome measures:measures: (a (a) )correlation correlation with with velocity velocity climbing climbing four four steps steps in in DMD DMD patients patients ( (nn == 18). 18). Left Left panel: panel: CXCL10CXCL10 no no correlation; correlation; middle middle panel: panel: CCL2 CCL2 negatively negatively correlated correlated with with climb climb velocity velocity (r = ( r−0.49,= −0.49, p = 0.034),p = 0.034), and andright right panel: panel: CCL18 CCL18 no correlation. no correlation. (b) Correlation (b) Correlation with withNeuroQOL NeuroQOL scores scores in BMD in BMD pa- tientspatients (n = (n 25).= 25). Left Left panel: panel: CXCL10 CXCL10 nono correlation; correlation; middle middle panel: panel: CCL2 CCL2 neared neared significant significant negative negative correlationcorrelation with with NeuroQOL NeuroQOL ( (rr == −0.4,−0.4, pp == 0.058), 0.058), and and right right panel: panel: CCL18 CCL18 no no correlation. Pearson Pearson correlation coefficient was used. correlation coefficient was used.

Similarly,Similarly, we we checked checked the the association association of of the the three three chemokines chemokines with with patient-reported patient-reported outcomesoutcomes in in BMD. BMD. Using Using a a subset subset of of BMD BMD patients patients ( (nn == 25, 25, age age range range 6 6 to to 68), 68), we we evaluated evaluated thethe association association of of circulating circulating CXCL10, CXCL10, CCL2, CCL2, and and CCL18, CCL18, with with the the Neuro-QoL Neuro-QoLTM (Quality(Quality ofof Life Life in in Neurological Neurological Disorders)-based Disorders)-based clinical clinical severity severity score score used used to to group group the the patients patients basedbased on on disease disease progression, progression, symptoms, andand ambulationambulation status.status. TheThe scorescore ranged ranged from from 0 0(asymptomatic/very (asymptomatic/very mild) mild) to to 64 64 (most (most severe). severe). Of all Of the all three the three chemokines chemokines studied, studied, CCL2 CCL2was the was only the chemokine only chemokine nearing nearing significance significance with Pearson with Pearson correlation correlation with a pwith-value a p of- value0.058 of (Figure 0.0588 (Figureb). 8b).

4.4. Discussion Discussion BloodBlood accessible biomarkersbiomarkers are are becoming becoming highly highly attractive attractive to useto asuse tools as tools for assessing for as- sessingdisease diseaseprogression progression and response and toresponse therapies, to especially therapies, in especially pediatric diseases in pediatric such diseases as DMD, where other outcome measures remain challenging and labor-intensive for young boys [19]. Although previous high throughput serum proteome profiling to study biomarkers in DMD patients was proven useful for large-scale biomarker discovery [3,4,17,20], a validation step is still required to confirm and test the identified biomarkers. Defining robust biomarker candidates requires a well-defined context of use and an assay that is specific, sensitive, Life 2021, 11, 827 10 of 13

accurate, and reproducible [21]. In this study, we focused on validating three candidate chemokines (CXCL10, CCL2, and CCL18) in DMD and test their association with age, disease severity, GC use, and clinical outcomes. These chemokines were selected because of their relevance in muscle pathogenesis [22–24] and the availability of a customized ELISA assay from MSD. The MSD ELISA assays used to measure these three chemokines were found to be specific and stable over time. Furthermore, the multiplexing of two chemokines, CXCL10 and CCL2, under one assay did not affect the accuracy of the data output; this helps reduce analysis time and sample usage. CCL18 was not multiplexed with CCL2 and CXCL10 because R-plex assays often run as a standalone. The intra- and inter-assay experiments had a CV < 15% in agreement with the FDA’s Bioanalytical Method Validation Guidance for Industry [25]. Using the standardized and validated MSD ELISA assay above, we confirmed that CXCL10, CCL2, and CCL18 were significantly elevated in the GC naïve DMD group relative to the healthy control group. Interestingly, CCL2, but not CXCL10, was significantly elevated in serum samples of the mdx mouse model for DMD relative to wild-type mice, further suggesting that increased levels of this circulating CCL2 may be associated with muscle pathogenesis due to dystrophin deficiency. This data agrees with previous studies showing that CCL2 was effectively elevated in the serum of the mdx mouse model [26]. CCL18 was not tested in the mdx mouse model, as there was no validated ELISA assay available for this murine chemokine. Age is a major confounding variable when confronting biomarker and pathogenesis studies in DMD. We, and others, have shown that many circulating protein biomarkers and especially muscle-centric proteins, decline with age in DMD because of the loss of muscle mass [18,27]. In this study, we further show that CCL2 and CCL18 but not CXCL10 also declined with age in DMD patients. These three chemokines were not affected by age in BMD and healthy controls. The differential effect of age on CCL2 and CCL18 compared with CXCL10 in DMD could suggest a different role for these chemokines in DMD pathogenesis and progression. CCL2 and CCL18 may be involved in chemotaxis and recruit immune cells to the site of muscle injury. Their decline with age in DMD patients could be simply due to the loss of muscle mass and fewer cycles of muscle injury and inflammation. Although the levels of these studied chemokines seem to be associated with inflam- mation and disease severity (e.g., DMD vs. BMD), there was no significant correlation with timed function tests in DMD nor patient-reported outcomes (NeuroQOL scores) in BMD for CXCL10 and CCL18. The exception was CCL2, which showed a significant negative correlation with the four steps climbing velocity in GC naïve DMD and neared a significant negative correlation with NeuroQOL in the BMD group, exhibiting a heterogeneous degree of severity; this means that patients with elevated levels of circulating CCL2 fare better in the climb test velocity and NeuroQOL than patients with low levels of circulating CCL2. This data, although intriguing, suggests that elevated CCL2 may have a beneficial effect. This hypothesis is supported by an earlier study conducted in mice showing that CCL2 is required for repairing injured muscle [28]. The role of CCL2, also known as chemoattractant protein-1 (MCP-1), in the inflammatory processes of muscle injury and repair was reviewed in more detail, and its role in both degeneration and regeneration were discussed [8]. To further link these chemokines to muscle pathogenesis and disease severity, we examined their levels in serum and skeletal muscle samples collected from DMD patients with a severe form of the disease and BMD patients with a milder form of the disease. The rationale behind this experiment is that inflammation leading to muscle pathogenesis is greater in DMD patients than BMD patients, which might be driven by chemokine levels in both serum and muscle samples. Indeed, CCL2, CXCL10, and to some extent CCL18 were found to be significantly elevated in both serum and muscle samples of DMD patients relative to BMD patients and healthy controls. These chemokines were unchanged in serum and skeletal muscle samples of BMD patients relative to age-matched healthy Life 2021, 11, 827 11 of 13

controls. CCL2 exhibited a significant level (~three-fold greater) in skeletal muscle extract of DMD patients compared with BMD and healthy controls; this is further supported by previous studies conducted in animal models showing increased levels of CCL2 in the mdx mouse model in both serum and muscle samples [7,16,26]. It was suggested that CCL2 plays a central role in the recruitment of immune cells at the site of muscle injury, and it could potentially induce the cytotoxic effect of M1 macrophages [7,8,23]. They accomplish this by binding to specific transmembrane receptors, which initiates signals that attract macrophages to the site of damage [8]. A study conducted on the tibialis anterior muscle and diaphragm showed that the mRNA levels of CCL2 and its receptor CCR2 were significantly elevated in the mdx mice model relative to the wild type mice. These expression levels of mRNAs increased with disease progression [23]. Another study conducted on muscle samples of young DMD patients (age 2 years) demonstrated an increase in expression of CCL2 mRNA in this presymptomatic phase [29]; this strengthens the idea that this chemokine is being released at an early stage of the disease and could influence the chemotaxis of infiltrating macrophages in DMD. Although CCL2 is likely involved in chronic inflammation in DMD patients, it has also been reported to play a role in muscle regeneration [7]. Hence, factors such as muscle fiber degeneration, dysregulated regenerative processes, chronic inflammation, and abrogated cell-to-cell communications may affect the levels of circulating CCL2 over time [30]. Therefore, careful studies to define the role of CCL2 in the context of this complex pathogenesis are needed to develop potential therapeutic strategies targeting CCL2 and its receptor [23]. These previous studies on animal models with our data obtained on DMD and BDM patients support our hypothesis that these chemokines, especially CCL2, are likely involved in muscle pathogenesis. However, additional experiments using different muscle diseases and mouse knockout experiments with different inflammation severity are needed to verify this hypothesis. Interestingly, none of the chemokines responded to GC treatment, confirming our previous SomaScan® data [3]. This observation contrasts with juvenile dermatomyositis patients (JDM), another pediatric inflammatory disease, in which CCL2 and CXCL10 are reported to normalize with treatment to levels similar to healthy controls [31]. This could be due to extensive combination therapies that JDM patients are often treated with. A significant limitation of this study is the limited number of samples available, especially muscle biopsies. In addition, serum samples and the muscle biopsies reported herein are not from the same subjects, which would have preferably linked the chemokines result from the two specimens to a given patient.

5. Conclusions In summary, we validated three circulating chemokines, CCL2, CCL18, and CXCL10, in DMD serum samples using a reproducible and stable MSD ELISA assay, thereby con- firming our previous high-throughput SomaScan® data. CCL2 was especially elevated in the serum of the mdx mouse model and muscle tissue of DMD patients, further supporting its implication in the disease. This data suggests CCL2 as a strong candidate for follow-up studies to define its physiological significance and potential for targeted therapy.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/life11080827/s1, Table S1: Demographics and ambulation status for DMD and BMD patients whose serum was used for biomarker analysis. Author Contributions: Conceptualization, Y.H.; methodology, Y.H., U.J.D. and M.O.; validation, M.O. and J.S.Z.; formal analysis, M.O.; resources, M.V.G., M.L.B., J.S.N., E.P.H. and K.N.; writing—original draft preparation, M.O.; writing—review and editing, Y.H., U.J.D. and J.S.N.; funding acquisition, Y.H. The CINRG DNHS study was operated by the CINRG DNHS investigators who collected the biosamples. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by DoD grant W81XWH-16-1-0557, NIH grant P50HD090254. Life 2021, 11, 827 12 of 13

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of Binghamton University for DMD study (protocol code # 4049-16, approval date 03/16/2017) and for the BMD study (protocol code # 0419, approval date 02/14/2018). The animal serum study was approved by the Institutional Animal Care and Use Committee (IACUC) of the Children’s National Hospital (CNH) (IACUC Protocol # 00030662, Approval date 1/24/18). Informed Consent Statement: Informed consent was obtained from all subjects involved in the biomarker research study. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Tchilabalo Alayi for his help in data interpreta- tion. The authors would also like to thank the patients and their families for their participation in the study. In addition, the authors would like to thank study team members from our participating CINRG sites, which include the University of California Davis: C. McDonald, E’. Henricson, M. Cregan, L. Johnson, J. Han, N. Joyce, A. Nicorici, and D. Reddy; Sundaram Medical Foundation and Apollo Children’s Hospital, Alberta Children’s Hospital, Calgary: J. Mah, A. Chiu, T. Haig, M. Harris, M. Kornelsen, N. Rincon, K. Sanchez, and L. Walker; Queen Silvia Children’s Hospital: M. Tulinius, A. Alhander, A. Ekstrom, A. Gustafsson, A. Kroksmark, U. Sterky, and L. Wahlgren; Children’s National Health System: R. Leshner, N. Brody, B. Drogo, M. Leach, C. Tesi-Rocha, M. Birkmeier, B. Tadese, A. Toles, and M. Thangarajh; Royal Children’s Hospital: A. Kornberg, K. Carroll, K. DeValle, R. Kennedy, V. Rodriguez, and D. Villano; Hadassah Hebrew University Hospital: Y. Nevo, R. Adani, A. Bar Leve, L. Chen-Joseph, M. Daana, V. Panteleyev-Yitshak, E. Simchovitz, and D. Yaffe; Instituto de Neurosciencias de la Fundación Favaloro: L. Andreone, F. Bonaudo, J. Corderi, L. Levi, L. Mesa, and P. Marco; Children’s Hospital of Pittsburgh of UPMC and the University of Pittsburgh: P. Clemens, H. Abdel-Hamid, R. Bendixen, C. Bise, A. Craig, K. Karnavas, C. Matthews, G. Niizawa, A. Smith, and J. Weimer; Washington University: J. Anger, T. Christenson, J. Florence, R. Gadeken, P. Golumbak, B. Malkus, A. Pestronk, R. Renna, J. Schierbecker, C. Seiner, and C. Wulf; Children’s Hospital of Rich- mond at VCU: J. Teasley, S. Blair, B. Grillo, and E. Monasterio; University of Tennessee: T. Bertorini, M. Barrett-Adair, C. Benzel, K. Carter, J. Clift, B. Gatlin, R. Henegar, J. Holloway, M. Igarashi, F. Kiphut, A. Parker, A. Phillips, and R. Young; Children’s Hospital of Westmead: K. North, K. Cornett, N. Gabriel, M. Harman, C. Miller, K. Rose, and S. Wicks; University of Alberta: H. Kolski, L. Chen, and C. Kennedy; Centro Clinico Nemo: M. Beneggi, L. Capone, A. Molteni, and V. Morettini; Texas Children’s Hospital: T. Lotze, A. Gupta, A. Knight, B. Lott, R. McNeil, G. Orozco, and R. Schlosser; University of Minnesota: G. Chambers, J. Day, J. Dalton, A. Erickson, M. Margolis, J. Marsh, and C. Naughton; Mayo Clinic: K. Coleman-Wood, A. Hoffman, W. Korn-Petersen, and N. Kuntz; University of Puerto Rico: B. Deliz, S. Espada, P. Fuste, C. Luciano, and J. Torres, and the CINRG Coordinating Center: Lauren Morgenroth, M. Ahmed, A. Arrieta, N. Bartley, T. Brown-Caines, C. Carty, T. Duong, J. Feng, F. Hu, L. Hunegs, Z. Sund, W. Tang, and A. Zimmerman. The CINRG-DNHS was funded by the U.S. Department of Education/NIDRR (#H133B031118, #H133B090001); U.S. Department of Defense (#W81XWH-12-1-0417); National Institutes of Health/NIAMS (#R01AR061875); Parent Project Muscular Dystrophy. Conflicts of Interest: The authors declare no conflict of interest.

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