Idiopathic inflammatory myopathy (IIM), also referred to as myositis, is a heterogeneous autoimmune disease with distinctive characteristics of chronic inflammation of the muscles, progressive muscle weakness, and increased muscle enzymes. Dermatomyositis (DM) is one of the most common clinical subtypes of IIM (1, 2). Anti-melanoma differentiation-associated gene 5-positive DM (anti-MDA5⁺ DM) refers to a rare and unique subtype of IIM. The clinical features of anti-MDA5⁺ DM usually include a characteristic DM rash, inflammatory muscle involvement, interstitial lung disease (ILD), and rapidly progressive ILD (RP-ILD) (3, 4). Numerous studies worldwide have revealed obvious regional and ethnic differences in the incidence of anti-MDA5⁺ DM, and it is mainly distributed in East Asia, especially among the Japanese and Chinese populations (5).The incidence of anti-MDA5⁺ DM ranges from 10% to 20% in Japan, from 17.6% to 22.6% in China, and from 7% to 13% in the United States (6–9). The cumulative 100-month survival rate of patients with anti-MDA5⁺ DM is 66%, and fatal outcomes occur very frequently within the first 6 months of diagnosis (10).

ILD is the most common and severe pulmonary manifestation of IIM patients, and the incidence of ILD in patients with IIM ranges from 5% to 80% (11). Patients with anti-MDA5⁺ DM are prone to the development of ILD with a probability of 50% to 100%. A previous cohort study showed that the 6-month mortality rate of patients with anti-MDA5⁺ DM was relatively high, ranging from 33% to 66% (12). Our previous study confirmed that the simultaneous presence of anti-MDA5⁺ and RP-ILD is a risk factor for a poor prognosis in patients with DM (13). Another study also showed that among patients with anti-MDA5⁺ DM, the mortality rate was significantly higher among those with than without RP-ILD, most patients died within 6 months of developing symptoms, and the 6-month survival rate was only 41% (14). The primary cause of death was respiratory failure caused by RP-ILD (10). Despite aggressive treatment with immunosuppressants and corticosteroids, patients with concurrent anti-MDA5⁺ DM and RP-ILD have a high 6-month mortality rate of 50% to 70% after the development of symptoms (15, 16). RP-ILD is an important subtype of anti-MDA5⁺ DM with ILD, and its prevalence is higher in East Asian populations (4). It progresses rapidly and has no effective treatment, making it an important cause of death in patients with anti-MDA5⁺ DM. Few studies to date have focused on the predictive effect of the clinical features of anti-MDA5⁺ DM with RP-ILD. Therefore, the present study was performed to identify the serological markers for anti-MDA5⁺ DM complicated with RP-ILD to assist in the achievement of a definitive diagnosis, accurate assessment of the patient’s condition, and improvement of the prognosis for patients with anti-MDA5⁺ DM.

In recent years, numerous studies have been conducted to identify markers that can predict the diagnosis and prognosis of secondary ILD with anti-MDA5⁺ DM in the preclinical stage. However, no ideal results have been obtained. Evidence has shown that the LDH concentration and PNI are related to the diagnosis and prognosis of various inflammatory diseases. Gómez et al. (23) found that in patients with influenza A-associated pneumonia, the LDH concentration was higher in patients who did than did not require mechanical ventilation, suggesting that the LDH concentration is related to the severity of pneumonia. Liu et al. (24) confirmed that an increased LDH concentration was related to the increased mortality of patients with community-acquired pneumonia. Ding et al. (25) found that patients’ preoperative LDH concentration was a predictive value for postoperative pneumonia, and the incidence of postoperative pneumonia was significantly higher in patients with a preoperative LDH concentration of >250 U/L. In addition, the serum LDH concentration is a commonly used inflammatory index in clinical practice. Moreover, varying degrees of the systemic inflammatory response are associated with the active period of connective tissue disease; thus, the LDH concentration can also be used to evaluate the activity of such diseases (26). Previous studies have proven that the PNI is associated with CTD. Some researchers have proven that the PNI is associated with the disease activity of systemic lupus erythematosus (27, 28). According to some studies, a low PNI could increase the risk of rheumatoid arthritis complicated with severe infection (29), and also be used for the diagnosis and evaluation of adult-onset Still’s disease (30). However, few studies have confirmed the relationship between the PNI, LDH concentration, and anti-MDA5⁺ DM with RP-ILD. Therefore, the present study was performed as a preliminary investigation of this relationship.

LDH is a stable cytoplasmic enzyme that is present in all cells. Cell membrane permeability increases with cell damage or death, resulting in the release of LDH (31). Macrophages such as liver Kupffer cells can be activated, leading to the injury of liver and then the elevated levels of ALT and AST (3). In addition, in our study, AST not only predicted poor prognosis in MDA5 positive DM patients, but also predicted the occurrence of RP-ILD with predictive values of 58.35 U/L (sensitivity, 67.9%; specificity, 69.8%; 95% CI = 0.536 – 0.795; P = 0.019) and 71.27 U/L (sensitivity, 47.6%; specificity, 82.7%; 95% CI = 0.506 – 0.794; P = 0.046), respectively ( Supplementary Figures 1 , 2 ). Therefore, extracellular LDH may be used as an indicator of cell damage or death of various causes (32). In our study, the LDH concentration in patients who had anti-MDA5⁺ DM with RP-ILD was higher than that in the control group. The LDH concentration was also found to predict RP-ILD, which is similar to the results reported by So (5) and Lian et al. (33). These findings suggest that the LDH concentration may be a predictive marker of the severity of ILD. In the present study, the alanine aminotransferase, aspartate aminotransferase, and LDH concentrations were significantly higher in patients with than without RP-ILD. The specific mechanism of LDH involvement in pulmonary fibrosis is still unknown, but studies have shown that LDH is a marker enzyme of macrophages, and its activity can be used as an indicator of macrophage activation (31). Recent studies have confirmed that activated macrophages were involved in the occurrence and development of pulmonary fibrosis in various ways, such as by causing neutrophils activation and triggering the formation of neutrophil extracellular traps, which in turn contribute to the development of ILD in patients with IIM (34–36). Seto (36) found that anti-MDA5 promotes the formation of neutrophil extracellular traps, which induce epithelial cell injury and the release of inflammatory cytokines. It is also speculated that LDH may bind to macrophage receptors, activate macrophages, and produce a variety of proinflammatory cytokines, including interleukins 2, 6, 8, and 12 and TNF-α. These inflammatory cytokines can stimulate the activation of neutrophils, leading to the exacerbation of pulmonary fibrosis (37, 38).

Serum LDH is a classic marker reflecting the progression of ILD (39). The concentration of LDH could indicate the disease activity and severity of idiopathic pulmonary fibrosis. Moreover, a high LDH concentration is always associated with severe pulmonary fibrosis and lung injury (40). In previous study, an LDH concentration of >300 U/L was an independent predictor of RP-ILD (5). In the present study, however, the cutoff value of LDH for prediction of RP-ILD was 365.62 U/L, and LDH was an independent predictor of RP-ILD. Factors such as the sample size and analysis methods may be the reasons for this difference. Therefore, more studies are needed to further determine the optimal LDH threshold for predicting RP-ILD in patients with anti-MDA5⁺ DM. Furthermore, the WBC count in the RP-ILD group was significantly higher than that in the non-RP-ILD group, and it had a predictive effect on RP-ILD. Infection has been considered one of the causes of acute exacerbation of idiopathic pulmonary fibrosis, and it is also a nonspecific inflammatory marker of infection (41). Our study showed that the combination of the LDH concentration and WBC count had a better predictive effect on RP-ILD than either the LDH concentration or WBC count alone.

The mortality rate of patients with anti-MDA5⁺ DM in our study (39.4%) was similar to that of Japanese patients (36%–41%) but higher than that of European patients (27.3%) (42). The incidence of RP-ILD is high in patients with anti-MDA5⁺ DM, and RP-ILD is always associated with a lower survival rate (43). Chen et al. (9) reported that 78.9% of patients with anti-MDA5⁺ DM developed RP-ILD. And another study showed that 85.7% of patients with MDA5⁺ DM died of RP-ILD (44). Among patients with anti-MDA5⁺ DM, the survival rate was significantly lower in those with RP-ILD than without RP-ILD, and their risk of death was increased by 9.7 times (5). The survival rate of patients with RP-ILD in our study was significantly lower than that of patients without RP-ILD, and RP-ILD was the most important predictor of an adverse prognosis (P = 0.002),this is similar to the results reported by Li (45). Moreover, patients with anti-MDA5⁺ had the highest mortality rate (85.71%) within the first year of onset, so early and effective treatment is essential for patients with concurrent RP-ILD and anti-MDA5⁺. Previous studies have shown that the LDH concentration is an indicator of a poor prognosis for patients with anti-MDA5⁺ DM (46), and the present study showed that the LDH concentration in the non-survival group was significantly higher than that in the survival group. LDH was an independent prognostic indicator in patients with anti-MDA5⁺ DM, consistent with the study by Niu et al. (12). In our study, we found an association between the LDH concentration and RP-ILD, indicating that a high LDH concentration could contribute to increased mortality in patients with anti-MDA5⁺ DM.

PNI was initially proposed by Onodera et al. (47) and used to predict the postoperative prognosis of gastric cancer. In addition to the prognosis of gastric cancer, the PNI is also associated with short-term postoperative complications and long-term adverse prognoses of lymphoma, lung malignant tumor, colorectal cancer, and cardiovascular disease (48–51). In our study, PNI in the survival group was significantly higher than that in the non-survival group, and it was associated with a good prognosis of patients. Additionally, PNI in the RP-ILD group was significantly lower than that in the non-RP-ILD group. Wang et al. (52) reported that the serum albumin level and total peripheral blood lymphocyte count were closely related to the inflammatory response. Studies have shown that RNA helicase encoded by MDA5 is involved in the innate immune defense mechanism during viral infection; thus, it is considered that viral infection may play an important role in the pathogenesis of anti-MDA5⁺ DM with RP-ILD, leading to the consumption of peripheral blood lymphocytes (53). In addition, studies have shown that among patients with anti-MDA5⁺ DM, the peripheral CD4⁺ and CD8⁺ T-cell counts in those with RP-ILD are significantly lower than in those with chronic ILD, indicating that lymphocytes play an important role in the disease progression (54). Moreover, previous researchers have speculated that the activation of inflammation also lead to a decrease in the lymphocyte count, and the decreased peripheral blood lymphocyte count may be caused by migration of these cells to the lung to participate in the local immune response (55). Advanced malnutrition may lead to deficiencies of essential vitamins and amino acids, which will further inhibit cellular or humoral immunity; this will in turn lead to significant reductions in the number and function of B cells and T cells, resulting in a decrease in the lymphocyte count (56). More studies are needed to elucidate the precise role of lymphocytes in anti-MDA5⁺ DM patients.

Albumin can inhibit endothelial cell apoptosis, prevent the generation of oxygen radicals, and reduce platelet aggregation; thus, it is a potential protective factor for human health (57). When systemic inflammation occurs, numerous inflammatory cytokines could be produced, which may inhibit the synthesis of albumin in the liver and thus reduce the albumin content; moreover, inflammation can also promote catabolism of albumin. Therefore, the exacerbation of MDA5⁺ DM-associated ILD may be related to a decrease in albumin, weakening of albumin protection, and activation of fibroblasts (28, 58). PNI is a comprehensive indicator of the body’s immune function and nutritional status. As one of its significant advantages, it is readily available and can be efficiently calculated from routinely measured serum albumin and lymphocytes. In this study, the PNI had a more significant clinical effect in evaluating the prognosis of anti-MDA5⁺ DM than did either the lymphocyte count or albumin concentration alone.

In this study, the detection rate of anti-Ro52 antibody in patients who had anti-MDA5⁺ DM was 75%, but the antibody level was not related to the prognosis. The predictive value of anti-Ro52 antibody combined with anti-MDA5 on RP-ILD and the prognosis of patients with DM require further confirmation by clinic studies. The prognosis of anti-MDA5⁺ DM with RP-ILD is not ideal, and most deaths occur in the first 6 months (59). Therefore, for patients with anti-MDA5⁺, treatment should be started before the development of respiratory symptoms or lung function damage. Immunosuppressive therapy should be administered in a timely manner, especially when serum markers such as LDH are elevated (39). These measures many help to significantly improve the survival of patients (60).

This study had several limitations. First, this was a retrospective study in which incomplete data collection may have led to systematic errors. Second, the sample size of this study was relatively small; future prospective, multicenter, population-based cohort studies with larger samples are needed. Third, all patients in this study were Chinese Han population, and the predictive effect on other races needs to be further validated. Fourth, although we confirmed the LDH cutoff values for prediction of RP-ILD, further studies are needed to demonstrate the dynamics of the LDH concentration during the progression of RP-ILD.

The LDH and PNI were independent prognostic factors in patients with MDA5⁺ DM, with LDH being associated with increased mortality and PNI with decreased mortality. This study also showed that among patients with MDA5⁺ DM, the WBC count and LDH concentration were significantly higher in RP-ILD patients than without RP-ILD. The WBC count and LDH concentration were independent and important risk factors for RP-ILD, and they also had some predictive value; Patients with MDA5⁺ DM can benefit from measurement of the LDH concentration and PNI, which are inexpensive and simple parameters that can be used for diagnosis as well as prediction of the extent of lung involvement and prognosis.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding author/s.

The studies involving humans were approved by Ethics Committee of Shandong Provincial Hospital, Shandong First Medical University. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

ML conducted the data collection and completed the data analysis, interpretation and writing of the manuscript. XZ conducted the data collection and completed the data analysis. BL and YZ and XL conducted the data collection. ZM and QY conceived the research plan and participated in the revision of the final manuscript. All authors were involved in reading and reviewing the manuscript. All authors contributed to the article and approved the submitted version.