CO:01:2 | BAG3 in systemic sclerosis: possible therapeutic target and biomarker for pulmonary fibrosis

Claudia Iannone^1|2, Margot De Marco³, Antonina Minniti¹, Paola Manzo³, Angela D'Ardia³, Antonia Falco³, Liberato Marzullo³, Angela Rosato³, Maria Caterina Turco³, Roberto Caporali^1|2, Nicoletta Del Papa^1|2. | ¹ASST G. Pini-CTO. UOC Clinica Reumatologica, Milano; ²Department of Clinical Sciences and Community Health, University of Milano; ³Department of Medicine, Surgery and Dentistry Schola Medica Salernitana, Baronissi (SA), Italy

Background: BAG3 (Bcl2-associated athanogene 3) regulates apoptosis and autophagy while inducing fibroblast-to-myofibroblast transformation. In systemic sclerosis (SSc), myofibroblasts drive skin and organ fibrosis, with interstitial lung disease (ILD) affecting ~50% of patients as the leading mortality cause. BAG3 serum levels correlate with diffuse cutaneous SSc and nintedanib treatment response. This study investigated BAG3's role in SSc pathophysiology using complementary experimental approaches.

Methods: Immunohistochemistry assessed BAG3 expression in lung tissue from SSc patients (N=3) and healthy controls (N=5). For in vivo studies, bleomycin-induced pulmonary fibrosis was established in mice (N=23), followed by vehicle (N=11) or anti-BAG3 mAb treatment (20 mg/kg twice weekly, N=12) from day 7-28. Label-free shotgun proteomics using SP3 protocol analyzed lung samples, with Proteome Discoverer 2.4 software processing protein expression data.

Results: BAG3 dysregulation was demonstrated across multiple experimental platforms. Immunohistochemistry analysis (Figure 1A) showed significant BAG3 overexpression in SSc lung specimens versus controls, with quantitative analysis confirming elevated BAG3 mean intensity (p=0.0002) (Figure 1B). In the bleomycin-induced pulmonary fibrosis model (Figure 1C), anti-BAG3 mAb treatment markedly reduced fibrotic areas compared to PBS controls (Figure 1D, p=0.02). Comprehensive nanoLC-Orbitrap-MS/MS proteomic analysis revealed distinct molecular signatures between groups. Heat map clustering (Figure 1E) and principal component analysis (Figure 1F-G) demonstrated separation of experimental groups, with anti-BAG3 treated animals displaying proteomic profiles distinct from bleomycin-induced pathology. Functional pathway analysis (Figure 1H) revealed BAG3 inhibition significantly modulated key biological processes, downregulating harmful pathways including apoptosis (-8.095), fibrosis (-9.756), necrosis (-9.101), and respiratory inflammation (-2.806), while activating protective mechanisms including cell survival (14.579) and viability (14.244).

Conclusions: This study establishes BAG3 as central to SSc-associated fibrosis. Significant BAG3 overexpression in SSc lung tissue provides compelling evidence for its pathogenic role. Anti-BAG3 monoclonal antibody therapeutic efficacy, coupled with comprehensive proteomic evidence showing restored protective pathways, strongly supports BAG3 as a promising therapeutic target. The dual modulation suggests multifaceted mechanisms addressing multiple SSc pathogenesis aspects. These findings identify BAG3 as both therapeutic target and biomarker for SSc-ILD treatment response monitoring, warranting investigation in larger cohorts and clinical translation studies.