Study population, patient characteristics, and clinical data
We prospectively recruited patients with SSc (SSc-no ILD, n=63; SSc-no ILD, n=39) from our ambulatory care policlinic at CHU Liege and compared them to healthy subjects (HS) (n=39) (Fig. 1). Demographic, functional and treatment characteristics of the subjects are given in Table 1. The average age of patients compared to HS was similar. FEV1 was moderately lowered in the SSc-no ILD and SSc-ILD groups compared to HS (p<0.05 and p<0.05, respectively). SSc-ILDs present lower levels of FEV1, FVC, TLC and DLCO compared to SSc-no ILD patients (p<0.05; p<0.001; p<0.0001 and p<0.001, respectively). The blood cell count analysis showed that patients with SSc-no ILD and SSc-ILD exhibited higher levels of neutrophils (p<0.0001 and p<0.0001, respectively) and reduced levels of lymphocytes (p<0.001 and p<0.001, respectively) compared to HS. There was not difference between SSc-no ILD and SSc-ILD patients. Of note 30% of patients were receiving maintenance treatment with immunosuppressive drugs and 27% were receiving oral corticosteroids.
Serum biomarkers at baseline
First, we compared the levels of different serum biomarkers associated with lung fibrosis (IGF-1, IGFBP-1, IGFBP-2, IGFBP-3, TGF-β, IL-8, TNF-α, YKL-40, MMP-7, MMP-9 and CRP) between HS and SSc groups. There is a significant increase in IGFBP-1 (8 to 12.9 ng/ml, p<0.05), IGFBP-2 (83 to 117 ng/ml, p<0.001), IL-8 (3.6 to 9.3 pg/ml, p<0.001), MMP-9 (412 to 967 ng/ml, p<0.001) and CRP (0.7 to 2.1 mg/l, p<0.001) levels in SSc patients compared to HS (Fig. 2b, c, e, f). Of note, IGF-1 and IGFBP-3 were significantly reduced SSc patients compared to HS (13 to 8.9 ng/ml, p<0.05; and 806 to 694 ng/ml, p<0.05, respectively) (Fig. 2a, d).
Then, we compared the levels of serum biomarkers between the two sub-group of SSc patients (SSc-ILD and SSc-no ILD) and HS (concentrations are listed in Table 2). There is a significant increase of the levels of IGFBP-2, IL-8 and MMP-9, as well as a decrease of IGFBP-3 in SSc-no ILD and SSc-ILD patients compared to HS, respectively. Of note, the level of IGFBP-1 was only increased in SSc-no ILD patients compared to HS (p<0.05). On the other side, the level of IGF-1 was reduced and CRP increased only in SSc-ILD patients compared to HS (p<0.05 and p<0.0001, respectively). Then, we focused our analysis on the difference between SSc-no ILD and SSc-ILD. Interestingly, we observed a significant reduction of the levels of IGFBP-1 and IGFBP-3 in SSc-ILD compared to SSc-no ILD (p<0.01 and p<0.05, respectively). We did not find any significant relation between biomarkers and therapies at baseline (immunosuppressive agent or systemic corticosteroids).
Correlation between serum biomarkers and pulmonary function tests at baseline
We performed correlation analysis to assess whether biomarkers were associated with pulmonary function tests at baseline. The correlation matrix is shown in table 3. IGFBP-2 was negatively correlated with alveolo-capillar function assessed by carbon monoxide transfer coefficient (KCO) (r=-0.29, p< 0.01) (Fig. 3). In addition, YKL-40 was also negatively correlated with Forced Vital Capacity (FVC) as well as the Diffusion Lung capacity for CO (DLCO) (r=-0.31, p<0.01 and r=-0.24, p<0.05 respectively) (Table 3).
Longitudinal analysis on serum biomarker variations and pulmonary function tests
To assess whether the variation over the time of the levels of serum biomarkers was associated with pulmonary function declines, we performed a longitudinal study in a subgroup of 28 patients analyzing biomarkers variations and pulmonary function tests over a period of 2 years (Fig. 1). Demographic and biological characteristics of SSc patients at baseline and after 2 years are given in Table 4. The 2-year longitudinal analysis of pulmonary function revealed that only KCO was significantly reduced (Baseline: 81 (±14) % and 2-years 73 (±12) %, p<0.001) (Fig. 4a). There was no significant variation of the levels of serum biomarkers between baseline and 2-year follow-up (supplementary Table S1). Next, we performed analysis to determine if pulmonary function decline was associated to the variation of serum biomarkers (Table 5). Interestingly, we found a positive correlation between the variation of IGFBP-2 and KCO at 2-year follow-up (r=0.6, p<0.001) (Fig. 4b). ROC curve analysis enabled us to identify that baseline IGFBP-2 < 105 ng/ml was associated with a better outcome assessed by KCO at 2 years follow-up in the overall SSc cohort (AUC 0.75 at 75% sensibility and 68% specificity) (Fig. 4c and Supplementary Fig. S1). Of note, we did not find any correlations between biomarkers evolution and immunosuppressive therapies. These results suggest a predictive value of IGFBP-2 on the progression of SSc disease.
Impact of IGFBP-2 on fibrotic and proliferative processes
In order to elucidate the impact of IGFBP-2 on the progression of SSc disease, we investigated its effect on SSc-related processes in vitro.
IGFBP-2 secretion is reduced in pro-fibrotic context
First, we investigated the secretion of IGFBP-2 by human lung fibroblasts (HLFs) at basal state (FCS free) and in pro-proliferative condition (FCS, 2%). Both quiescent and proliferating HLFs actively secrete IGFBP-2. Proliferating HLFs produce more IGFBP-2 (4062±141 pg/ml) than quiescent HLFs (958±445 pg/ml) (Fig. 5a). Then, we investigated the impact of pro-fibrotic factor TGF-β on the secretion of IGFBP-2. Under TGF-β stimulation, we observed a reduction of IGFBP-2 secretion in proliferating HLFs (2945±1074pg/ml) (p<0.05). TGF-β stimulation has no effect on the secretion of IGFBP-2 on quiescent HLFs.
IGFBP-2 reduces the proliferation of HLF fibroblasts
Then, we studied the effect of IGFBP-2 on HLF proliferation by BrdU assay. In presence of IGFBP-2 (100ng/ml), we observed a significant decrease in BrdU incorporation at 48h, which reveals a reduction of HLF proliferation (p<0.05) (Fig. 5b). As expected, the positive control (TGF-β) increases the proliferation of HLF fibroblasts.
IGFBP-2 reduces the fibrotic activity of HLF fibroblasts
The impact of IGFBP-2 on fibrotic activity of HLF fibroblasts was first assessed by measuring the level of α-SMA via immunocytochemistry (Fig. 5c) and FACS analysis (Fig. 5d), then completed by the quantification of pro-collagen I secretion (Fig. 5e). In presence of IGFBP-2, we observed by FACS a decrease of α-SMA levels in HLF fibroblasts (Fig. 5d). Furthermore, the secretion of pro-collagen I was also significantly reduced by 35% in presence of IGFBP-2 (p<0.05) (Fig. 5e). The immunocytochemistry analysis did not show any difference without or with IGFBP-2.
Taken together, these results highlighted the anti-proliferative and anti-fibrotic properties of IGFBP-2.