Project description:Background: A 58-year-old male with anti-synthetase syndrome-associated interstitial lung disease (ASS-ILD) underwent bilateral lung transplantation (LTx) but experienced ILD progression and myositis worsening 38 weeks post-transplant, despite glucocorticoids, tacrolimus, and mycophenolic acid treatment. High-dose glucocorticoids led to adverse effects and ILD exacerbation upon tapering. Objective: To identify persistent inflammatory pathways despite anti-rejection therapy. Methods: Two sets of single-cell RNA-seq (scRNA-seq) analyses were conducted on peripheral blood mononuclear cells (PBMCs) from our patient who experienced recurrent ASS-ILD following bilateral LTx: one before regimen adjustment and one after. The goal these analyses is to investigate systemic inflammatory profiles. Results: In comparison to five ASS-ILD patients without LTx, our patient following bilateral LTx (before regimen adjustment) displayed a higher proportion of CD14⁺ and CD16⁺ monocytes, alongside reduced T, B, and NK cells. Notably, interferon- and JAK-STAT–associated pathways were significantly enriched, as evidenced by marked up-regulation of key genes (e.g. JAK1, JAK2, STAT1, STAT2, STAT3, IL4R, IL6R) in the patient’s monocytes. Elevated serum ferritin levels and the enrichment of macrophage and neutrophil activation pathways further underscored hyperactivation of the mononuclear phagocyte system. Due to poor tolerance of high-dose steroids, limited efficacy of tacrolimus and MPA, and the need to manage post-transplant rejection while controlling ASS, the patient’s regimen was modified. Considering the activation of the mononuclear phagocyte system in the patient, tacrolimus was replaced with cyclosporine A, which is more widely used in macrophage activation syndrome (target trough concentration 120-150 ng/ml). Given the significant activation of the interferon and JAK-STAT pathways in the patient’s activated monocytes, we replaced EC-MPS with the Janus kinase inhibitor (JAKi) tofacitinib at 5 mg twice daily. Short-term high-dose glucocorticoids (methylprednisolone 120 mg/d for 3 days) were also administered before rapid tapering. After regimen adjustment, scRNA-seq was conducted again, which indicated a reduction in CD14⁺ and CD16⁺ monocytes, with T, B, and NK cells rebounding. Neutrophils, characterized by high S100A8, S100A9, and DEFA3 expression, became more prominent. Interferon-related gene expression decreased in monocytes and neutrophils, indicating successful suppression of the interferon pathway. Conclusions: We presented a complicated case of a patient with early recurrence of underlying ASS-ILD after bilateral lung transplantation. We used two sets of scRNA-seq analyses to investigate the systemic inflammatory responses.

Project description:Rationale: The diagnosis of idiopathic pulmonary fibrosis (IPF) requires exclusion of an underlying autoimmune disease, as present in interstitial lung diseases associated with connective tissue diseases (CTD-ILD). The prevalence of autoantibodies in IPF patients is currently unknown. Objectives: An unbiased assay for de novo discovery of autoantigens can help characterizing autoreactivities in IPF patients beyond clinically established autoimmune panels. Methods: We developed the proteomic Differential Antigen Capture (DAC) assay, capturing patient antibodies from plasma, followed by affinity purification coupled to mass spectrometry (AP-MS). The DAC assay quantifies the binding capacity of patient antibodies to proteins in a pooled native extract from lung explants (ILD explants n=41; donor controls n=12). Plasma antibodies from patients with IPF (n=35), CTD-ILD (n=24) and age-matched controls (n=32) were analyzed and validated in an independent cohort (IPF: n=40; CTD-ILD: n=20). Plasma antibody binding profiles were associated with clinical meta-data including diagnosis, lung function and transplant free survival. Measurements and Main Results: We identified 586 putative autoantigens in both study cohorts with a broad heterogeneity among disease entities and cohorts. On average, in IPF a mean±SD of 16±40 autoantigens and in CTD-ILD a mean±SD of 9±15 autoantigens were identified per patient. We identified 18 IPF-specific autoantigens validated in the second cohort. Interestingly, there was also a high number of shared autoantigens in IPF and CTD-ILD patients, with 17 being present in IPF and CTD-ILD of both cohorts. Presence of antibodies to Thrombospondin 1 (THBS1) and tubulin beta-1 chain (TUBB1) was associated with a significantly reduced survival in patients with IPF (p=0.002 and p=0.019, respectively). This signature was often associated with autoreactivity against talin-1 (TLN1), latent-transforming growth factor beta-binding protein 1 (LTBP1), epididymis secretory protein Li 112 (HEL-S-112), zyxin (ZYX), the LIM and senescent cell antigen-like-containing domain protein 1 (LIMS1) and caldesmon (CALD1). Conclusions: Unbiased proteomic profiling reveals that the overall prevalence of autoantibodies is similar in IPF and CTD-ILD patients and identifies novel IPF specific autoantigens associated with patient survival.

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Spatial transcriptomic profiling of the lung tissues from a patient with recurrent anti-synthetase syndrome associated interstitial lung disease after bilateral lung transplantation and one untreated patient

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