In this analysis of data from 300 patients with IPF, we identified several circulating proteins that strongly associated with a composite outcome of respiratory death or lung transplant, after adjusting for clinical variables known to be associated with mortality in this population [8]. Many of these proteins have functions in inflammation, immune activation/regulation, cell–cell adhesion, or pathways reported to play a role in fibrogenesis (e.g. TGF-β signaling, bone morphogenetic protein signaling, Janus kinase signaling).
While some of our findings are consistent with previous data, such as the association between elevated levels of chemokine CXCL13 and reduced survival [9], our analyses identified several additional candidate proteins as biomarkers of mortality risk, including proteins not measured in previous studies. These results extend previous analyses of data from the IPF-PRO Registry that identified several proteins that associated with clinical measures of IPF severity (% predicted FVC, % predicted DLco, composite physiologic index) at enrollment [3]. In the current analyses, each of the proteins that was associated with all three disease severity measures in this prior work (SPON1, ICAM5, roundabout homolog-2 [ROBO2], polymeric immunoglobulin receptor [PIGR]) was selected by the multivariable model that considered both proteins and clinical factors. While none of these proteins has been well characterized in lung fibrosis, it has been shown that ROBO2 is overexpressed in a mouse model of toxin-induced liver fibrosis, and that the interaction between ROBO2 and its ligand promotes fibrogenic activity within stellate cells [10]. Notably, inclusion of the proteins along with the clinical measures enhanced the discriminatory ability of the model compared with a model that included only clinical factors. This suggests that proteins may confer information that is independent from that captured by measures commonly performed in the clinic.
Among the top protein predictors of the composite of respiratory death or lung transplant were AHSG and PIAS4. Higher AHSG levels and lower PIAS4 levels were associated with reduced risk. These proteins have opposing roles in regulating TGF-β signalling, a pathway known to be important in IPF. Thus it is plausible that they may contribute to the development or progression of IPF. In experimental models, AHSG is an antagonist of TGF-β, with animals genetically lacking in AHSG expression showing increased SMAD2 phosphorylation [11, 12]. Furthermore, TGF-β-mediated suppression of immune cell function was exaggerated in AHSG-deplete animals, as shown by inhibition of macrophage activation [12]. In an experimental model of liver fibrosis, PIAS4 silencing blocked recruitment of SMAD3, decreasing pro-fibrotic gene expression and ameliorating hepatic fibrosis [13]. In the context of these experimental data, our findings compel mechanistic and clinical studies to define the contribution of these proteins to the pathogenesis of IPF and clarify their potential as biomarkers of IPF progression.
Strengths of our analysis include the multi-center nature of the cohort and the adjustment for clinical variables known to influence survival in patients with IPF. Our analyses also have limitations. First, the cohort was a population of mainly white patients enrolled at expert centers in the US, thus our findings may not be applicable to all patients with IPF. Second, while a broad array of proteins were analyzed, some potentially important proteins may have been missed as they were not included on the platform. An aptamer-based approach to protein detection does not always produce results that are reproducible using ELISA and analyses using ELISA are planned.
In conclusion, we identified several novel candidate circulating protein biomarkers for predicting respiratory death or lung transplant in patients with IPF. These data underscore the opportunity to develop biomarker-inclusive algorithms that provide meaningful risk stratification for patients with IPF.