Systemic sclerosis (Scleroderma; SSc) is a chronic, autoimmune disease characterized by vascular damage and activation of the immune system which triggers the excessive production and deposition of extracellular matrix (ECM) proteins. This process is driven by heightened inflammation, autoimmunity, and small vessel vasculopathy promoting abnormal connective tissue remodeling and scarring. These changes lead to progressive fibrosis affecting the skin and internal organs including the heart, kidneys, and lungs. Currently, the highest mortality rates among SSc patients are those with cardiopulmonary manifestations, accounting for ~50% of SSc-related deaths (1–3). Major pulmonary complications are systemic sclerosis-associated pulmonary artery hypertension (SSc-PAH), and systemic sclerosis-associated pulmonary fibrosis (SSc-PF) (4–6). SSc-PAH and SSc-PF are distinct disease endotypes, involving common and unique pathologic mechanisms that are not yet fully understood, leading to distinct clinical associations, predictive factors, and treatment regimens (7, 8).

SSc-PAH results from the extensive remodeling of the small pulmonary arteries, resulting in a narrowed lumen and increased pulmonary vascular resistance. The pathogenesis has been attributed to an imbalance of vasoactive mediators, pro-inflammatory cytokines, dysregulated thrombosis and disruption of several pathways, including cytokines involved in the Transforming Growth Factor-beta (TGF-β) signaling pathway. This results in altered endothelial and smooth muscle cell proliferation within the pulmonary arterioles (9, 10). There is also an indication that SSc-PAH patients exhibit distinctive, unfavorable metabolic profiles, which are attributable to oxidative stress and mitochondrial dysfunction (11, 12).

SSc-PF is an interstitial lung disease characterized by inflammation, fibrosis, and deposition of ECM within the lung parenchyma leading to diminished gas exchange and decreased vital lung capacity (5). Endothelial cell injury with subsequent vascular damage and alveolar epithelial cell injury are key initial insults that precede fibrosis. Upon injury, pro-inflammatory and pro-fibrotic mediators are released, that activate interstitial fibroblasts (13). Over time fibroblasts acquire features of smooth muscle cells and become myofibroblasts, resulting in dysregulated accumulation of collagen and other ECM components and ultimately leading to fibrosis (14). Cytokines and growth factors, such as TGF-β, interleukin-6 (IL-6) and connective tissue growth factor (CTGF), have all been implicated in the pathogenesis of the disease (15–17). Targeted anti-inflammatory and anti-fibrotic therapies such as tocilizumab, mycophenolate mofetil and nintedanib have been shown to improve patient prognosis, however these lack disease specificity (18, 19).

In the early stages of disease, the pathogenesis of SSc-PAH and SSc-PF appear to involve common mechanisms including vascular dysfunction and inflammatory processes, creating a disparity between mediators and autoimmunity (20–22). These are likely to reflect some shared factors whose overlapping roles are not completely understood, yet lead to distinct features as the diseases progress. SSc-PF can also contribute to pulmonary hypertension in SSc, due to chronic hypoxia and vessel damage from fibrosis in advanced interstitial lung disease (23). This complex overlapping of disease pathogenesis makes risk stratification, early intervention and management challenging. Thus, robust methods to predict the onset and nature of pulmonary complications associated with SSc would help in guiding early diagnosis and intervention. Moreover, specific discriminatory markers between SSc-PAH and SSc-PF could allow for the identification of new disease mechanisms and therapeutics targeting the underlying causes.

In recent years, omics-approaches including transcriptomics, proteomics and metabolomics have made significant advances in understanding disease pathogenesis and in detecting new biomarkers and therapeutic targets (24–26). A transcriptomic study of SSc-PF patients detected abnormalities in levels of IL-6, insulin growth factor binding protein-2 (IGFBP-2), insulin growth factor like-2 (IGFL-2) and toll-like receptor-8 (TLR-8) (27). In SSc-PAH, proteomics has identified changes in collagen IV, endostatin, IGFBP-2, IGFBP-7, matrix metallopeptidase-2 (MMP-2), neuropilin-1, N-terminal pro-brain natriuretic peptide and RAGE (28). More recently, using bioactive metabolomics analysis, metabolites of fatty acid oxidation, eicosanoid metabolism, and sex hormones were reported to distinguish between SSc-PAH and IPAH (29). However, to date, there have been no studies using a multi-omics approach to identify markers of SSc-PAH and SSc-PF within SSc cohorts. Therefore, the aim of this study was to use proteomics and metabolomics, to uncover systemic proteome factors and metabolites with the potential to differentiate between SSc-PAH and SSc-PF patients.

Key cohort demographics and clinical characteristics are shown in Table 1.

Pulmonary complications associated with SSc (SSc-PAH and SSc-PF) are the leading cause of disease-related death. Both conditions have complex etiologies which are not fully understood. Available therapeutics aim to alleviate symptoms, but do not resolve the underlying inflammatory and metabolic dysfunction. Identification of discriminatory biomarkers and novel therapeutic targets would improve patient prognosis and outcomes. This is the first study to produce a combined cytokine-metabolite panel which may potentially distinguish between SSc-PAH and SSc-PF.

MCP-1, MCP-3, and MCP-4 were identified as discriminatory markers for SSc-PF, as their levels were significantly elevated in this group compared to others. These chemokines are members of the monocyte chemoattractant protein family, and are strong chemotactic factors, recruiting immune cells to sites of infection, inflammation, and stress. MCP-1 has been associated with initiation of inflammation in SSc. High levels of its receptor, CCR2, has been detected on CD4⁺ T cells within the fibrotic lung of bleomycin-treated mice (40, 41). Elevated serum MCP-1 has also been shown to correlate with the development of pulmonary fibrosis in SSc patients (42). Similarly, serum MCP-3 levels in SSc patients levels have been associated with greater skin fibrosis and reduced FVC, suggesting a central role in fibrogenesis (43). MCP-4, while not as extensively studied as other members of the MCP family, has been reported to be expressed in cells of the bronchoalveolar lavage and may also contribute to fibrotic disease processes. However, members of the MCP family have also been reported to be elevated in other inflammatory pulmonary conditions, such as idiopathic pulmonary fibrosis (IPF). To date, proteomic studies in IPF have not consistently identified MCPs as established biomarkers. A direct comparison between IPF and SSc associated pulmonary fibrosis would therefore be required to determine whether MCPs represent a robust and disease-specific biomarker (44, 45).

The discriminatory value of MCP-1 between SSc-PAH and SSc-PF was validated in this study by ELISA. IL-33, IL-6, sIL-6R, sgp130 and CTGF, while not found to distinguish between the pulmonary groups, were also investigated. IL-33 levels were found to be reduced in SSc-PAH compared to HC. In trans-signaling, IL-6 bound to its soluble receptor, sIL-6R, and the signal transducer gp130 to promote inflammation, while soluble gp130 blocks this interaction and dampens the response. In this study, IL-6 and sIL-6R were found to be elevated in both SSc-PAH and SSc-PF. However, this may occur through distinct pathogenic pathways in the disease subsets, as suggested by the efficacy of anti-IL-6R therapy with tocilizumab in SSc-PF, but not in SSc-PAH (46, 47). Interestingly, in the SSc-NLD group, there was a significant positive correlation between IL-6 and sgp130. This is potentially a protective mechanism, halting progression and development of pulmonary complications. CTGF has also been implicated in the pathogenesis of SSc. Representational difference analysis (RDA) demonstrated increased CTGF gene transcription in fibroblasts isolated from SSc patients (48). In addition, selective deletion of CTGF in mouse models of PAH and PF significantly reduced pulmonary interstitial scarring and vascular remodeling (49). While no significant differences in CTGF levels were observed across patient groups in this study, there was a strong positive correlation between CTGF and MCP-1 in the SSc-PF group, suggesting that CTGF signaling may contribute to this disease pathway.

Network analysis identified SSc-PF as the group with the highest node degree, indicating that cytokines within this subset exhibited a greater number of interactions with one another. This finding aligns with previous studies suggesting that SSc-PF represents a more inflammation-driven phenotype. Collectively, the cytokines involved and their interconnectedness point to a state of inflammatory dysregulation within this group. Of the seven FDA-approved drugs targeting the cytokines within these networks, four (tocilizumab, sarilumab, siltuximab and satralizumab) target the IL-6 signaling pathway and have been trialed in SSc with varying degrees of success. Anakinra is an anti-IL-1 inhibitor. While increasing evidence suggests that IL-1-mediated inflammation may play a role in SSc-associated cardiac failure, to date, there are no reports of its use in SSc-related disease (50). Ustekinumab targets the p40 subunit, shared by IL-12 and IL-23, and has demonstrated efficacy in immune mediated conditions such as psoriasis. However, there is currently no evidence supporting its use in SSc (51). Spesolimab is a monoclonal antibody targeting the IL-36 receptor and has shown clinical benefit in the treatment of generalized pustular psoriasis; however, its role in SSc has not been investigated (52). Repurposing these drugs may potentially offer a wider range of therapeutic options for patients.

A 10-metabolite signature was uncovered for SSc-PAH. Dimethylarginines (SDMA + ADMA together), Hydroxyasparagine, QUIN, AYA, T6A, VLA, DMTPA, N-acetylserine, N-isoputreanine and Orotidine were significantly elevated in this group compared to all others. These metabolites are important for energy metabolism, mitochondrial oxidation, amino acid catabolism, and inflammation. Dimethylarginine (ADMA) is known to regulate vascular tone, response to injury, and is a known inhibitor of nitrous oxide (NO) synthase. Decreased levels of NO is a key driver of chronic inflammation and cellular disruption, as it leads to endothelial dysfunction. ADMA levels have been shown to be elevated in the plasma of IPAH and chronic thromboembolic pulmonary hypertension (CETPH) patients compared to healthy controls, and this correlated with low endothelial NOS expression (53, 54). Clinically, serum ADMA has been shown to positively correlate with mPAP and pulmonary vascular resistance, and so could serve as a biomarker for monitoring the effectiveness of treatment (55). However, ADMA was not measured separately in this study.

Hydroxyasparagine is important in post-translational modification of calcium-binding epidermal growth factor (cbEGF)-like domains. These domains are present in a wide variety of extracellular proteins such as fibrillin. Fibrillin has been shown to induce the trans differentiation of resident fibroblasts into myofibroblasts, which produce ECM proteins and pro-inflammatory cytokines that exacerbate inflammation (56). This is a key inflammatory pathway involved in regulating chemotaxis of inflammatory cells, which can lead to inflammatory cascades within the vasculature (57).

High levels of QUIN, a metabolite formed through the kynurenine pathway (KP) of tryptophan metabolism, has been associated with lower survival rates in treatment naïve PAH patients. Interestingly, in human lung primary cells, treatment with IL-6/sIL-6R complex lead to similar KP metabolic profiles as in PAH, suggesting that the metabolic dysfunction within the KP is driven by IL-6 trans signaling (58).

N-acetyl-isoputreanine is a by-product in the catabolism and metabolism of spermidine. Spermidine levels have been shown to be elevated in IPAH patients, and may promote proliferation and migration of PASMCs, encouraging vascular remodeling in rat pulmonary hypertensive models (59). Vanillactate (VLA), N-acetylalanine, T6A, DMTPA, N-acetyl-serine and orotidine were also shown to be elevated in the plasma of SSc-PAH patients compared to the other three groups. However, none of these have been well-characterized in PAH, and so further research into the mechanisms of these metabolites is necessary. However, these metabolites may still act as biomarkers if the results are reproduced. The robustness of the identified metabolites were evaluated using Random Forest. The analysis demonstrated that the identification of metabolite markers in the SSc-PAH group was significantly robust compared to other groups.

Nicotinate and nicotinamide metabolism was the most significantly affected metabolic pathway in SSc-PAH. Nicotinate and nicotinamide are precursors of the co-enzyme’s nicotinamide-adenine dinucleotide (NAD+) and NADP +. CD38, which hydrolyses NAD and nicotinamide, is overexpressed in skin of patients with diffuse SSc compared to healthy individuals. Depletion of NAD+ by CD38 impairs the ability for PARP to repair DNA lesions which encourage pro-inflammatory and pro-fibrotic phenotypes (60). Enrichment of the nicotinate and nicotinamide pathways may therefore imply an imbalance of the resolution of inflammation and fibrosis.

In this study, most of the protein-metabolite, protein-protein and metabolite-metabolite correlations were found to be comparable across groups. However, several notable differences were observed. In HC and SSc-NLD groups, IL-33 showed strong negative correlations with most metabolites, consistent with its role in maintaining metabolic homeostasis through ST2-mediated regulation of key metabolic checkpoints (61). These associations weakened in SSc-PF, indicating diminished IL-33–mediated control of immune–metabolic balance, and were completely lost in SSc-PAH, suggesting disruption of this pathway. Reduced systemic IL-33 levels have been reported in idiopathic PAH, suggesting a loss of its protective function. IL-33 has been implicated in cardiopulmonary protection by reducing inflammation and limiting vascular remodeling, mediated primarily via the ST2L receptor. Notably, IL-33 exhibits context dependent activity and can either activate, promoting inflammation, or reduce resolving the inflammatory response. In this context, our findings of reduced IL-33 levels in PAH may reflect a loss of protective IL-33 signaling (62–64). Moreover, IL-33 and IL-12 were not correlated in HC, but positively associated in SSc-NLD, likely reflecting early immune activation where IL-33 release enhances IL-12 production (65). In contrast, the pulmonary groups exhibited a negative correlation, suggesting immune polarization, where chronic IL-33 signaling suppresses IL-12 activity, driving a Th2-dominant, fibrotic environment (66).

In conclusion, this proof-of-concept study proposes a combined cytokine–metabolite signature which may potentially distinguish between SSc-PAH and SSc-PF. While the biomarker panel identified in this study largely aligns with previous findings, it may also hold novel diagnostic, prognostic, and therapeutic potential. However, its clinical applicability requires validation in a larger, independent cohort.

Key limitations of this proof-of-concept study include the small sample size and the absence of detailed information on comorbidities and treatment regimens. Although the cohorts were ethnicity and age matched, there were sex differences between the groups, reflecting the higher prevalence of SSc in women. As a result, potential sex related differences may not have been fully captured in the analysis. Finally, differences in medication in the groups may play a role in the metabolite signatures of each of these groups and the possibility that these metabolite profiles may be, at least in part, a medication-associated phenomenon cannot be discounted.