To our knowledge, this is the first study reporting an IgAV-associated skin mRNA signature in adults. Importantly, samples were collected from immunosuppressive treatment-naïve IgAV patients thus showing unmodulated pathological processes. PCA of skin transcriptome could distinguish between IgAV patients and HC. The IgAV patient closest to HC in the space spanned by the two most important PCs had a mild disease with nonnecrotic purpura and also low levels of inflammatory parameters (CRP, SAA and ESR), which might be the reason for the gene signature similar to controls. Differences observed in DEG in patients’ skin between sl-IgAV and IgAVN may indicate the activation of additional pathogenic processes in patients with systemic disease. The higher number of DEGs in IgAVN compared to sl-IgAV patients are not consequence of more severe skin involvement, as necrotic purpura was observed in only one patient from each group, while bullous purpura was also present in one sl-IgAV patient (Table S1).
Serum analytes measured in our study were selected in hypothesis free approach based on transcriptomic changes in the affected skin. Acute-phase response was one of the most highly enriched BP in IgAV skin, with the increased levels of acute-phase reactants, pro-inflammatory cytokines and chemokines. A recent study performing bidirectional Mendelian randomization analysis showed a causal effect of CRP, procalcitonin (PCT), and circulating inflammatory regulators on IgAV (19). LBP was identified among the top five DEGs in IgAV skin and increased LBP was found also in patients’ sera, where the levels correlated with SAA and CRP. Moreover machine learning approach identified LBP as the variable with the highest predictive value for IgAV. LBP was the only analyte in our study that was significantly increased in IgAVN compared to sl-IgAV patients, thus LBP could potentially be useful as a predictor/biomarker of renal involvement. Serum LBP level was also significantly increased in patients with IgA nephropathy, a separate disease that shares pathogenesis with IgAV (20). During acute-phase response LBP binds to bacterial lipopolysaccharide (LPS) to elicit immune responses by presenting the LPS to cell surface pattern recognition receptors CD14 and Toll-like receptor (TLR) 4 (21). Intriguingly, TLR4 expression was also increased in transcriptome of IgAVN patients. Blockade of the LPS-LBP interaction was a strategy to limit neutrophil infiltration in a mouse sepsis model (22). As positive correlation was observed between LBP and blood neutrophil count, and LBP was higher in patients with neutrophil infiltrates compared to those with mixed immune cells infiltrates, inhibition of LBP-TLR4 pathway might represent a potential therapeutic option also in IgAV (23).
Positive regulation of cytokine production and leukocyte mediated immunity are among enriched GO BP processes in IgAV skin. Due to their roles in leukocyte activation and migration, cytokines IL-15, IL-18, CCL3, CXCL5, CCL19 and CXCL10 were measured (24, 25). IL-15, IL-18, CCL3 and CCL19 were increased in all IgAV patients compared to HC. Positive correlation was observed between serum IgA and IgA deposits in the skin with CCL19 and CXCL10. These two chemokines are type I Interferons (IFN)-regulated, associated with type I IFN signature in systemic lupus erythematosus (SLE) (26, 27). CCL19 serum concentration was significantly increased in IgAV patients with necrotic compared to nonnecrotic purpura, suggesting that chemotactic activity might contribute to a more severe skin involvement.
Modifications in the skin's ECM, identified through enriched KEGG pathways and GO CC, can be consequence of inflammation and tissue damage. Both measured ECM proteins MMP1 and osteopontin were elevated in IgAV sera compared to HC, with osteopontin among the analytes with the highest predictive value for IgAV.
Among the most highly enriched KEGG pathways identified in IgAV skin were Regulation of lipolysis in adipocytes and PPAR signalling pathway. Deregulations in lipid metabolism and adipokines are reported also in other skin and autoimmune diseases (28) as inflammatory response could lead to lipid and adipokine dysregulations (29). Recent study reported alterations of serum lipids also in paediatric IgAV (30). We found DEG of many adipokines, namely adiponectin, leptin, FABP4, SERPIN A12 and ANGPTL4 in IgAV skin and increased serum levels of leptin, FABP4 and ANGPTL4 in IgAV vs. HC. ANGPTL4 has shown a positive correlation with SAA and CRP and inverse correlation with serum albumin. In patients with more severe skin involvement, histologically presented as fibrinoid necrosis or with necrotic purpura we found higher ANGPTL4 levels than in nonnecrotic purpura. As ANGPTL4 is implicated in vascular inflammation and endothelial dysfunction further investigations of ANGPTL4 function in adult IgAV are warranted. Although serum adiponectin levels were not changed in all IgAV patients vs HC, significantly lower levels were observed in IgAV_GI vs sl-IgAV and IgAVN. Any imbalance of adiponectin from optimal serum levels might have detrimental effects, as indicated across various SARDs, including elevated levels in rheumatoid arthritis (RA) and SLE, and decreased levels in systemic sclerosis (SSc) (31).
The performance of the RF and ST-EasyEnsemble algorithms in predicting IgAV using the measured analytes showed promising results, although it was difficult to predict visceral organ involvement. Nevertheless, we identified potential molecular markers of GI involvement, such as decreased adiponectin and CXCL10. Limitations of our study are the small number of patients included in the RNA sequencing and the potential influence of comorbidities on the measured analytes levels.
In conclusion, processes related to acute phase/inflammation, lipid metabolism and ECM are activated in IgAV adult skin lesions and are even more pronounced in patients with renal involvement. In our study, the most prominent biomarkers for predicting IgAV were LBP, ANGPT4 and osteopontin, involved in major deregulated processes, and found to be associated with clinical characteristics. Furthermore, our study revealed new potential serum biomarkers of adult IgAV-related GI and renal involvement, namely adiponectin, CXCL10 and LBP.