Proteomic profiling identifies SPP1 associated with rapidly progressive interstitial lung disease in anti-MDA5-positive dermatomyositis

Anti-MDA5 antibody-positive dermatomyositis (MDA5⁺ DM), a distinct subtype of idiopathic inflammatory myopathies (IIM), is significantly associated with interstitial lung disease (ILD), particularly the rapidly progressive interstitial lung disease (RP-ILD). MDA5⁺ DM with RP-ILD is often resistant to traditional treatment, and despite aggressive interventions, the mortality rate is still as high as 50-70% [1, 2]. Hence, early identification of high-risk RP-ILD patients is a vital aspect of improving prognosis.

MDA5⁺ DM is a heterogeneous disease, with substantial variability in clinical presentation, treatment response, and patient outcomes. About 30 ~ 50% of patients exhibited the life-threatening RP-ILD [1, 3], conversely, while about 60% of patients MDA5⁺ DM displayed mild symptoms with good prognosis. Therefore, accurate prediction of patients at high risk of RP-ILD may aid in decision-making on therapeutic strategies and assist in preventing overtreatment of “low-risk” patients.

During the last decade, efforts have been made to identify prognostic markers in MDA5⁺ DM patients. Male, old age, short disease duration, skin ulceration, and forced vital capacity are suggested as risk factors for RP-ILD [2, 4]. Some serum markers like the co-existence of anti-MDA5 antibody and anti-RO-52 antibody, elevated serum C-reactive protein (CRP), ferritin level, and Krebs von den Lungen-6 (KL-6) levels have been linked to poor outcome in RP-ILD patients [1, 2, 5]. However, due to the exact mechanisms underlying RP-ILD development have not yet been determined, most prognostic risk research mainly focused on the epidemiological and clinical characteristics of MDA5⁺ DM. Currently, no reliable biomarkers for predicting RP-ILD or morality in MDA5⁺ DM are available.

We hypothesized that although the specific pathogenesis and mechanism of RP-ILD are largely unknown, the rapidly progressive lung injury can induce distinctive molecular changes. Proteins with these changes can be detected in the peripheral blood of MDA5⁺ DM patients, potentially serving as surrogate markers for disease progression. High-throughput proteomics offers a promising approach to identify these biomarkers for RP-ILD. In the current study, we performed proteomic analysis to explore potential pathways and biomarkers that linked to RP-ILD development in MDA5⁺ DM patients.

RP-ILD is a life-threatening complication of MDA5⁺ DM, and there is currently a lack of reliable biomarkers to predict RP-ILD. Using LC–MS/MS analysis, we analyzed the protein profile in the plasma of MDA5⁺ DM and made two major novel findings. First, we identified 79 proteins that changed in the plasma of RP-ILD patients as compared with non-RP-ILD patients. These molecular changes mainly involve acute phase proteins, complement, and coagulation activation pathways, suggesting a potential role in the progression of RP-ILD. Second, our data supports plasma SPP1 as a potential biomarker for RP-ILD prediction. Taken together, our findings offer fresh insights for further elucidating the pathogenesis in MDA5⁺ DM with RP-ILD and provide valuable clues for developing promising therapeutic and prognostic biomarkers.

Our proteomic data showed a substantial activation in the biological process of inflammatory response in RP-ILD patients. MDA5⁺ DM patients tend to have high sera inflammation status, with markedly increased serum ferritin and high type I interferon [25]. These factors are associated with the severity and poor prognosis. We previously reported that MDA5⁺ DM patients with high-RP-ILD risk were characterized by elevated CRP, lactate dehydrogenase (LDH), and ferritin at baseline compared with low or medium RP-ILD risk patients, supporting our current findings that a persistent and severe inflammatory response promotes lung injury.

We revealed a variety of acute phase proteins (APPs) elevated in RP-ILD patients, including SSA1, SAA2, SERPINA3, and lipopolysaccharide-binding protein (LBP). This observation is consistent with other studies indicating that high CRP levels are associated with RP-ILD [2, 26, 27]. APP levels rise rapidly in response to inflammation or tissue damage. Proteins such as CRP and SAA1 can activate the complement system and promote the production of cytokines and chemokines [28, 29], contributing to the “cytokine storm” and acute lung injury. Our data suggest that active control of inflammation is vital for halting RP-ILD progression in MDA5⁺ DM.

The complement system plays a critical role in innate and adaptive immune responses, but its excessive activation may result in tissue injury. The role of the complement system has been well-defined in lung disease derived from coronavirus infection including COVID-19, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS) [30‐32]. Excessive complement activation could induce endothelial cell injury, blood clotting, and systemic microangiopathy, eventually, resulting in multi-systemic organ failure in patients with COVID-19 [33]. Complement activation products, such as C3a and C5a, can recruit inflammatory cells to the lungs and promote the release of pro-inflammatory cytokines, contributing to lung inflammation and injury [34]. Complement inhibitors are currently not used in MDA5⁺ DM. Therefore, further exploration of the pathophysiologic importance of complement in RP-ILD is needed and could suggest a novel specific intervention for RP-ILD.

Lung is the primary site of terminal platelet production and accounts for approximately 50% of total platelet production [35]. Platelets may in turn contribute to lung injury [36, 37]. Five proteins (GP5, PF4, PF4V1, TREML1, ITGB3) that were associated with platelet aggregation and adhesion were found to be downregulated in our data. Moreover, the suppressed platelet degranulation was observed in the plasma of MDA5⁺ DM patients with RP-ILD. In addition to regulating thrombosis, activated platelets could directly interact with immune cells, thereby promoting an inflammatory phenotype [38]. In acute respiratory distress syndrome (ARDS) animal model or clinical data from the study of bronchoalveolar lavage fluid in ARDS patients, platelets are proven to contribute to the development of acute lung injury [39]. Data from animal models [40, 41] and human studies [42, 43] have suggested antiplatelet therapies with aspirin could reduce incidence and mortality in ARDS. Collectively, our evidence suggests that the inhibition of complement activation and inflammatory responses, as well as antiplatelet therapies, might be helpful in the treatment of MDA5⁺ DM patients with RP-ILD.

With the SARS-CoV-2 pandemic outbreak worldwide, increasing evidence showed the striking similarities between COVID-19 and MDA5⁺ DM with RP-ILD, including chest computed tomography feature, high serum cytokine levels, severe acute respiratory symptoms, and high mortality, suggesting these two conditions share common pathophysiological mechanisms. Interestingly, recent studies have revealed that complement activation, complement-induced inflammatory response, and blood clotting were particularly evident in severe COVID-19 patients in response to viral infection [33, 44, 45]. How certain MDA5⁺ DM patients develop RP-ILD remains largely unclear. Viral infection has long been considered a suspected trigger of disease progression in MDA5⁺ DM patients. Our data imply that COVID-19 and MDA5⁺ DM may share a common underlying molecular mechanism for lung injury.

We validated 6 proteins and found SAA1, SPP1, and KNG1 are associated with the risk of RP-ILD. Among them, SPP1 showed the best predictive value. SPP1, also known as osteopontin, is involved in a variety of physiological and pathological processes including wound healing, bone turnover, tumorigenesis, inflammation, ischemia, and immune responses [46, 47]. SPP1 is expressed by a variety of inflammatory cells in culture, including T cells, macrophages, and NK cells [48‐50]. During inflammation, SPP1 could induce cell adhesion and migration, mediate proinflammatory lymphocytes activation and cytokine production, and inhibit the apoptosis of inflammatory cells [51, 52]. Single-cell transcriptome analysis found that SPP1 was significantly higher in bronchoalveolar lavage in severe cases of COVID-19 compared to control and mild cases [53]. Levels of SPP1 were also found to correlate with highly aggressive lung adenocarcinoma [54].

In addition to participating in inflammatory processes, increasing evidence suggests that the upregulation of SPP1 is associated with fibrosis. Anna et al. conducted a study where they performed single-cell RNA sequencing on 11 explanted lungs from patients with systemic sclerosis-associated ILD, and they identified the presence of SPP1⁺ profibrotic macrophages [55]. Furthermore, studies have demonstrated that the downregulation of SPP1 effectively reduces pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis mouse model [56]. Given that both inflammation and fibrosis are crucial pathological processes in the development of ILD, it is not surprising that SPP1 is linked to ILD risk in MDA5⁺ DM patients. The exact mechanism requires further validation through experiments.

Previous studies have found that SPP1/osteopontin was upregulated in MDA5⁺ DM [57, 58]. Based on the risk stratification, we found that SPP1 was specifically increased in the high-risk RP-ILD subgroup. The typical clinical features of the high-risk RP-ILD subgroup are the elevated levels of sera inflammation markers (ESR, CRP, ALT, AST, and LDH), as compared with the low- or medium-risk subgroup. These results demonstrate that SPP1 could serve as a novel biomarker for monitoring RP-ILD progression, and has the potential for developing new therapies for RP-ILD treatment.

In our study, we also observed SAA1 were upregulated in MDA5⁺DM-RPILD. As an acute-phase protein, SAA1 plays a role in regulating inflammation and immunity. Previous studies have reported its upregulation in fibrotic sarcoidosis [59] and COVID-19-related ARDS [60]. However, further research is needed to elucidate the specific mechanisms by which SAA1 contributes to interstitial lung lesions.

Several limitations remain in this study. First, the sample size was limited, and the role of identified biomarkers or pathways should be validated in a larger sample prospective cohort. Second, dynamic observations will be necessary to evaluate the effect of the treatments on the expression of the differential proteins. Third, the molecular functions of the differential proteins should be examined in further study to understand their role in the pathogenesis of MDA5⁺ DM.

Our study provides novel insights into the pathogenesis of RP-ILD development in MDA5⁺ DM and highlights the significance of plasma SPP1 as both a novel prognostic biomarker and a therapeutic target for MDA5⁺ DM.