Duchenne Muscular Dystrophy (DMD), also referred to as dystrophinopathy, is a devastating X-linked disease caused by mutations in the dystrophin gene which affects 1 in 5,000 to 1 in 6,000 live male births (Duan et al., 2021). Dystrophin acts as a bridge between the cytoskeleton and the extracellular matrix in skeletal and cardiac muscle fibers. The absence of dystrophin destabilizes cell membranes and causes sarcolemmal fragility, cell necrosis and inflammatory responses. The regenerative capacity of the muscle fibers is impaired in a chronic inflammatory environment and necrotic muscle cells are eventually replaced by fat and fibrotic tissue. Most patients with DMD are wheelchair-bound in their early teens, require ventilatory support in their late teens, and eventually die in their early twenties from cardiac/respiratory failure (Rosenberg et al., 2015). Consequently, there is an urgent need to improve therapeutic strategies for patients with DMD. However, current treatments mainly rely on steroid-based therapy, non-sense readthrough, and exon skipping, which have limited efficacy, short duration effects, and various adverse effects (Shieh, 2018). Some promising treatments, such as genome editing and stem cell treatment, are limited by the immune response of the human body (Maffioletti et al., 2014; Duan et al., 2021; Hakim et al., 2021). Therapies to attenuate the inflammatory response in the muscle microenvironment could be an important and effective approach to prevent degeneration and adjunctive therapy to other emerging treatments aiming to repair the muscle (Mendell et al., 2010; Cordova et al., 2018). Therefore, it is vital to further characterize the immune microenvironment and identify novel therapies for DMD.

Previous studies have shown the infiltration of immune cells plays a crucial role in the progression of DMD. Initial fiber necrosis caused by sarcolemmal instability contributes to a severe inflammatory response, including the infiltration of immune cells and the increased secretion of proinflammatory cytokines, thus leading to a 400–500% increase in damage to the membranes of muscle cells (Porter et al., 2002; Tidball et al., 2018). For example, infiltrating M1 macrophages can promote the lysis of muscle cells via the expression and secretion of various pro-inflammatory cytokines. M2 macrophages are thought to be essential partners in muscle regeneration but may also promote myocardial and skeletal muscle fibrosis (Deconinck and Dan, 2007; Perandini et al., 2018). CD8+ T cells can directly destroy muscle fibers via perforin-mediated cytotoxicity while CD4+ T cells can enhance the cytocidal effects of CD8+ T cells and macrophage activity by secreting cytokines (Spencer et al., 2001). However, how immune cells affect the pathological progression of DMD needs further study. Evaluating the infiltration of immune cells and identifying the differences between the components of infiltrating immune cells are critical to fully elucidate the biological mechanisms underlying DMD and identify new immunotherapeutic targets.

Advances in genome sequencing technologies and the microarray analysis of disease-related datasets have made it possible for us to quantify immune cell infiltration in a diverse set of different diseases. Over recent years, several studies have provided an overview of the immune response as a key molecular pathway in DMD and identified key genes by using Gene Expression Omnibus (GEO) datasets (Wang et al., 2021; Xu et al., 2021). However, the specific relationships between immune cells and the key genes which influence inflammatory responses have yet to be studied systematically; the differences in the expression of key genes between patients with DMD and normal individuals remain unclear.

In the present study, bioinformatic analyses were applied to identify auxiliary biomarkers of DMD and the result was validated by real-time quantitative reverse transcription PCR (RT-qPCR). To investigate the infiltration of immune cells in DMD, we performed the single-sample gene set enrichment analysis (ssGSEA) and found the effects of immune-related genes on the main immune-related pathways by using gene set variation analysis (GSVA). Our findings emphasize the association between key genes and the immune microenvironment at the molecular level and provide new immunotherapeutic targets for patients with DMD.

Duchenne muscular dystrophy is a genetic disease characterized by progressive muscle wasting and persistent inflammation. Although mechanical damage and cell membrane defects are known to be basic pathological changes in DMD, the infiltration of immune cells also has a significant impact on the occurrence and progression of DMD (Tidball et al., 2018). In particular, the immune environment of the muscle tissue in DMD patients is known to restrict the therapeutic effects of some promising treatments, such as gene editing and stem cell transplantation (Duan et al., 2021; Tulangekar and Sztal, 2021). Therefore, exploring potential diagnostic biomarkers and characterizing the infiltration pattern of immune cells in DMD is of great significance to enhance the diagnosis and prognosis of DMD (Mendell et al., 2010).

In the present study, we sought to identify auxiliary diagnostic markers related to DMD immunity and analyze their relationship with immune cell infiltration in DMD. First, we identified 445 DEGs between DMD and normal muscle tissues. Functional enrichment analysis demonstrated that the DEGs were closely related to immune response and inflammatory signaling pathways, thus demonstrating DMD has a strong immune activation process which may lead to the inflammatory process in muscle tissue, thereby inducing muscular necrosis and atrophy. We also identified four immune-related DEGs (C3, SPP1, TMSB10, and TYROBP) as candidate genes by LASSO regression; ROC curves showed the performance of the LASSO model was superior. Finally, we found that the infiltration of immune cells into the muscle microenvironment might be critical in the occurrence and development of DMD.

Complement C3 is a central protein of the complement system which plays a core role in the activation of the complement system (Dalakas et al., 2020). A previous study reported the deposition of C3 and C9 in both necrotic and non-necrotic fibers of dystrophic muscles following pathological examination of muscle biopsies (Spuler and Engel, 1998). Moreover, Hodgetts and Grounds (2001) reported the depletion of C3 increased initial donor male myoblast survival in murine models. Another study confirmed that dysferlin-deficient mice with C3 knockout exhibited a mitigating effect on muscle pathology. Unexpectedly, the ablation of C3 did not affect muscle pathology in the murine mdx model of DMD (Han et al., 2010), which may reflect the lower degree of muscle pathology in mdx mice than that in DMD patients or may indicate species-specific differences in complement activity. Therefore, further studies are needed to validate the feasibility of C3 as a biomarker of DMD and its role as a potential therapeutic target in DMD. Secreted phosphoprotein 1 (Spp1; osteopontin or micropontonin), which enhances extracellular matrix synthesis and turnover, is significantly elevated in DMD and mdx limbs (Haslett et al., 2002; Porter et al., 2002). A study showed that Spp1 contributed to the aggravation of inflammatory-dependent pathology in the mdx model by skewing the polarization of macrophages and promoting fibrosis. The genetic deletion of Spp1 in mdx mice presented with milder pathology, a greater contractile force in the muscle, and less fibrosis compared with control mdx littermates (Vetrone et al., 2009). Studies have also demonstrated that single nucleotide polymorphisms (SNPs) of SPP1 loci may be associated with disease severity in DMD patients (Bello et al., 2012; Hoffman et al., 2013). Furthermore, as a genetic modifier, Spp1 has been shown to have a direct role in the regulation of myofiber damage and may serve as a novel therapeutic option for dystrophic muscle (Quattrocelli et al., 2017). Collectively, these studies implicated the profound effect of Spp1 protein and its gene upregulation in DMD. Thymosin beta 10 (TMSB10), a protein localized to the cytoskeleton, is thought to enable actin monomer binding activity and participates in the regulation of cell migration. Although many studies have supported that TMSB10 promotes macrophage M2 conversion and serves as a biomarker in cases with malignant tumors (Bouchal et al., 2015; Zeng et al., 2020; Yan et al., 2021), few studies have investigated its specific role in DMD cases. The TYROBP gene, also known as DAP12, serves as a downstream adaptor for many immune receptors such as trigger receptor expression myeloid 1 (TREM1) (Bouchon et al., 2000), trigger receptor expression myeloid 2 (TREM2) (Colonna, 2003), and complement receptor 3 (CR3) (Linnartz and Neumann, 2013). As a key component in the activation of signal transduction, TYROBP is mainly expressed in macrophages and NK cells (Lanier et al., 1998; Bostanci et al., 2011). The Toll-like receptors (TLRs), which are highly expressed in macrophages and skeletal muscle cells, TLR2/4 and TLR7/8/9 in particular, are known to play a prominent role in the sustained chronic inflammation exhibited by the muscle tissue in DMD (De Paepe, 2020). It has also been reported TLR4 can amplify the role of the NF-κB pathway via TREM1/TYROBP after binding to antigen (Campanholle et al., 2013; Pelham and Agrawal, 2014; Liu et al., 2019). Abnormal activation of the NF-κB signaling pathway contributes to the persistence of inflammation and skeletal muscle wasting during the course of DMD (Li et al., 2008). Therefore, we speculate TYROBP may be involved in the progression of DMD by mediating key inflammatory pathways. Recent studies have also identified TYROBP as a key gene in DMD co-expression networks based on other DMD datasets; these findings are consistent with those reported herein (Wang et al., 2021; Xu et al., 2021). Thus, C3, SPP1, TMSB10, and TYROBP are likely to affect the progression of the inflammatory response in DMD and serve as auxiliary markers, although further clinical research is needed to validate the diagnostic significance of these candidates.

Next, we investigated differences in the proportion of immune infiltration cell types between DMD and normal muscle tissue by ssGSEA. Analysis revealed the increase of infiltration in 23 out of 28 different types of immune cells in DMD, including macrophages, activated dendritic cells, activated CD4+ T cells, activated CD8+ T cells, regulatory T cells (Tregs), and CD56bright natural killer cells. The levels of infiltration for active B cells, CD56dim natural killer cells and type 17 T helper cells were decreased in DMD. These differences might be associated with DMD occurrence and progression, which were in good agreement with those from previous studies (Tripodi et al., 2021) and further confirmed the importance of the immune response in the pathogenesis of DMD. It is generally known that macrophages are an essential component of human innate immunity, and these cells can also exert potential influence on the pathogenesis of DMD. Studies have shown the activation of two functional subsets of macrophages contributes to the physiopathology of DMD: the classically activated M1 (pro-inflammatory M1) and alternatively, activated M2 (anti-inflammatory M2) macrophages. The predominant molecular mechanisms underlying the infiltration of M1 macrophages include enhanced oxidative stress, the production of pro-inflammatory cytokines (TNF-α, IL-6, and IFN-γ) and the expression of fibrotic genes. M2 macrophages can secrete anti-inflammatory cytokines (such as IL-10, IL-4) and promote regeneration (Deconinck and Dan, 2007). However, activated M2 macrophages have also been implicated in the pathogenic pro-fibrogenic response (Perandini et al., 2018). The interaction of persistently activated macrophages with the cytokines they produce leads to progressive necrosis in the muscle fibers, the accumulation of extracellular matrix, and the eventual replacement of muscle fibers by regenerated adipose and fibrous tissue throughout the course of the disease (Tidball and Villalta, 2010). Serving as professional antigen presenting cells (APCs), dendritic cells (DCs) are generally thought to represent a bridge which connects the innate and the adaptive immunity. It has been demonstrated that DCs can modulate the differentiation of T cells toward Th1, Th2, Th17 or regulatory T (Treg) cells and thus play a role in the dystrophic muscle environment (Lorant et al., 2018). In addition, DCs may regulate the pathways mediated by TLRs and recognize several cytokines and pro-inflammatory mediators which are highly expressed in patients with DMD (Madaro and Bouche, 2014). However, few studies have addressed the potential contribution of DCs to the progression of DMD pathology via TLR7-mediated signals or the modulation of TGF-β expression (Lemos et al., 2015). Adaptive immunity, predominantly involving T cells, has also been shown to play an important role in the occurrence of DMD (Spencer et al., 2001); the infiltration of B cells is much rarer (Peccate et al., 2016). Our present data supports this viewpoint. The infiltration of CD8+ T cells into DMD muscle is known to cause direct damage to the muscle fibers via perforin-mediated cytotoxicity. Furthermore, CD4+ T cells can differentiate further into Th1 cells and secrete a series of cytokines, including IL-6, TGF-β, and IFN-γ to enhance the cytotoxic effects of CD8+ T cells and the activity of macrophages, thus leading to further enhancement of the inflammatory response (Spencer et al., 2001). We also found that another class of T cells, Tregs, was increased in DMD muscle tissue. Previous studies have shown Tregs can mitigate both inflammatory responses and fibrotic processes in DMD muscle and promote myofiber regeneration through the activation of satellite cells (Villalta et al., 2014). Therefore, factors that promote and inhibit inflammatory responses co-exist in DMD muscle tissue, which reflects the complexity of the muscle immune environment. Our results indicate that Type17 T helper cells (Th17) are reduced in DMD. As another differentiated subtype of CD4+ T cells, Th17 cells mainly produce the cytokine interleukin-17 (IL-17) (Miossec and Kolls, 2012). However, few studies have focused on the specific relationship between the infiltration of Th17 cells and DMD. The role of Th17 in DMD progression requires further investigation. We observed reduced levels of infiltration of CD56dim natural killer cells in DMD muscle tissue, along with increased CD56bright natural killer cells. CD56dim NK cell subsets predominantly exert cytotoxic functions, while CD56bright NK cell subsets exert their effects by producing pro-inflammatory factors (Cooper et al., 2001). These findings suggested NK cells might enhance the inflammatory response by secreting pro-inflammatory cytokines in DMD patients. However, further research needs to address the specific mechanisms controlling the infiltration of immune cells in DMD.

Over recent decades, numerous studies have revealed the sustained infiltration of inflammatory cells in patients with DMD plays an important role in disease pathogenesis. Immune cells and immune-related genes may prove to be promising therapeutic targets (Iftikhar et al., 2021). Importantly, the clinical outcomes and therapeutic response of DMD are closely related to the infiltration of innate and adaptive immune cells (Lu et al., 2003; Park et al., 2008; Doudna, 2020; Hakim et al., 2021). A therapeutic strategy based on the characteristics of the immune response represents an attractive approach to estimating the clinical course and outcomes of DMD. By performing ssGSEA to analyze microarray data from DMD, researchers could know the specific relationships between different immune cell subpopulations, which enhanced our understanding of the immune environment in the muscles of patients with DMD.

Studying the correlation between immune cells and candidate genes, we found C3, Spp1, TMSB10, and TYROBP were strongly correlated with infiltrating immune cells. In addition, GSVA analysis indicated a close relationship between these genes and immune pathways. The mRNA expression levels of C3, Spp1, TMSB10, and TYROBP in DMD patients were significantly higher than that in healthy control. These results indicate that the four newly identified markers may serve as new immunotherapeutic targets for DMD. However, the specific mechanisms underlying the interaction between immune-related genes and immune cells require further investigation.

Although the current study included a relatively large sample size by integrating GEO datasets to characterize the immune microenvironment and successfully discovered four genes with potential diagnostic value for DMD, some limitations need to be considered. First, as the present study explored the infiltration of immune cells by ssGSEA, it is essential to verify our results by flow cytometry. Second, the interactions between immune-related genes, immune cell infiltration and potential immune pathways require an in-depth investigation. Third, the expression levels of C3, SPP1, TMSB10 and TYROBP in the muscular tissues of DMD were validated by RT-qPCR in the study. Nevertheless, due to the relatively small number of muscle tissues available for study, the patients were not stratified by pathological stage. In addition, more reference genes are suggested to improve the accuracy of RT-qPCR data (Vandesompele et al., 2002). Further studies are warranted to validate the prognostic function of these genes. Large research studies, incorporating clinical information and functional evaluation are needed to test and validate these important findings.

In the present study, we analyzed the overall landscape of immune cell infiltration in DMD and identified C3, SPP1, TMSB10, and TYROBP as auxiliary diagnostic biomarkers for DMD. Next, we found the expressions of these biomarkers were strongly correlated with infiltrating immune cells in DMD. Finally, we validated that the mRNA expression level of these genes was significantly higher in DMD compared with healthy controls by RT-qPCR. Overall, the infiltration of immune cells into the muscle microenvironment might exert a critical impact on the occurrence and development of DMD. Our findings may provide new immunotherapeutic targets and serve as an adjunct to other treatments to reduce the immune-related side effects of these treatments and improve efficacy.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

The studies involving human participants were reviewed and approved by Research Ethics Committee of the Second Hospital of Hebei Medical University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

XH, JH, and XS: study concept and design. XH, NW, and RG: data collection and processing. HW and SM: clinical sample collection. XH, JL, and PF: experiment conduction. XH: original draft writing. XS, JH, and GJ: manuscript review and editing. XS: funding acquisition. All authors read and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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