Combined anti-fibrotic and anti-inflammatory properties of JAK-inhibitors on macrophages in vitro and in vivo: Perspectives for scleroderma-associated interstitial lung disease
Abstract
Janus kinase (JAK) inhibitors, also known as Jakinibs, are a class of small-molecule drugs that target specific isoforms of JAKs—namely JAK1, JAK2, JAK3, and tyrosine kinase 2 (Tyk2). These inhibitors have demonstrated anti-inflammatory effects, partly through modulation of pro-inflammatory M1 macrophage activation. However, their broader impact on the full spectrum of macrophage activation states remains unclear, particularly in diseases characterized by the simultaneous activation of both pro-inflammatory M1 and profibrotic M2 macrophages. This is especially relevant in autoimmune pulmonary fibrotic conditions such as scleroderma-associated interstitial lung disease (ILD), where both M1 and M2 macrophages play central pathogenic roles.
In this study, we evaluated and compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors—ruxolitinib (JAK2/1 inhibitor), tofacitinib (JAK3/2 inhibitor), and itacitinib (JAK1 inhibitor)—across five distinct activation states of primary human monocyte-derived macrophages (MDMs). All three inhibitors significantly reduced inflammatory responses in M1 macrophages stimulated with IFNγ and LPS or IFNγ alone, as evidenced by the downregulation of polarization markers (CD86, MHCII, TLR4) and decreased secretion of key pro-inflammatory cytokines (CXCL10, IL-6, and TNFα).
Additionally, these JAK inhibitors attenuated the M2a phenotype induced by IL-4 and IL-13, as shown by reduced expression of the M2a surface marker CD206 and lowered CCL18 secretion. The inhibitors also diminished expression of markers such as CXCL13, MARCO, and SOCS3 in M2c macrophages, activated either with IL-10 alone or in combination with dexamethasone (M2c + dex). Across all macrophage polarization states, JAK inhibitors with activity against JAK2 consistently showed the strongest effects at both 1 μM and 0.1 μM concentrations.
Based on these in vitro findings, we further investigated the in vivo effects of JAK2/1 inhibition using ruxolitinib in a mouse model of HOCl-induced ILD, which replicates features of scleroderma-associated ILD. Ruxolitinib treatment significantly suppressed the expression of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1, Chi3L3), alongside improvements in both skin and lung pathology.
Together, these findings support the potential of JAK2/1 inhibitors to modulate macrophage-driven inflammation and fibrosis, suggesting their therapeutic relevance in autoimmune and inflammatory lung diseases that Itacitinib currently lack effective disease-modifying treatments.