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. 2021 May 26;10(6):1321.
doi: 10.3390/cells10061321.

Aberrant B Cell Receptor Signaling in Naïve B Cells from Patients with Idiopathic Pulmonary Fibrosis

Stefan F H Neys  1 , Peter Heukels  1 , Jennifer A C van Hulst  1 , Jasper Rip  1 , Marlies S Wijsenbeek  1 , Rudi W Hendriks  1 , Odilia B J Corneth  1
Affiliations

Aberrant B Cell Receptor Signaling in Naïve B Cells from Patients with Idiopathic Pulmonary Fibrosis

Stefan F H Neys et al. Cells. .

Abstract

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal disease in which an impaired healing response to recurrent micro-injuries is thought to lead to fibrosis. Recent findings hint at a role for B cells and autoimmunity in IPF pathogenesis. We previously reported that circulating B cells from a fraction of patients, compared with healthy controls, express increased levels of the signaling molecule Bruton's tyrosine kinase (BTK). However, it remains unclear whether B cell receptor (BCR) signaling is altered in IPF. Here, we show that the response to BCR stimulation is enhanced in peripheral blood B cells from treatment-naïve IPF patients. We observed increased anti-immunoglobulin-induced phosphorylation of BTK and its substrate phospholipase Cγ2 (PLCγ2) in naïve but not in memory B cells of patients with IPF. In naïve B cells of IPF patients enhanced BCR signaling correlated with surface expression of transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) but not B cell activating factor receptor (BAFFR), both of which provide pro-survival signals. Interestingly, treatment of IPF patients with nintedanib, a tyrosine kinase inhibitor with anti-fibrotic and anti-inflammatory activity, induced substantial changes in BCR signaling. These findings support the involvement of B cells in IPF pathogenesis and suggest that targeting BCR signaling has potential value as a treatment option.

Keywords: B cell receptor (BCR) signaling; Bruton’s tyrosine kinase (BTK); autoimmunity; idiopathic pulmonary fibrosis (IPF); nintedanib.

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Conflict of interest statement

S.F.H.N., P.H., J.A.C.v.H., J.R., R.W.H. and O.B.J.C. declare no conflicts of interest. M.S.W. reports grants and consultancy fees from Boehringer Ingelheim and Hoffman la Roche and consultancy fees from Galapagos, Respivant, Novartis, BMS, Horizon and Safara, all outside this study. All grants and fees were paid to her institution.

Figures

Figure 1
Figure 1
Phosphoflow analysis of BCR downstream signaling molecules in peripheral blood B cells from HCs and IPF patients (cohort 1). (A–C) Phosphorylation levels, expressed as geometric mean fluorescence intensity (gMFI) under unstimulated (unst.) conditions and after BCR stimulation (anti-immunoglobulin; α-Ig) in total (CD3−CD19+IgM+/−), naïve (CD3−CD19+CD38loCD27−IgD+; 56.8% ± 15.4 in HCs and 69.0% ± 11.0 in IPF patients of total B cells), and memory (CD3−CD19+CD38loCD27+; 23.6% ± 5.8 in HCs and 21.7% ± 6.2 in IPF patients of total B cells) B cells from HCs and IPF patients for (A) pBTK Y223, (B) pSYK Y348, and (C) pPLCγ2 Y759 (left panel). Representative histograms are shown with the gray vertical line indicating the gate for selecting the population low/high in phosphorylation following α-Ig stimulation (middle panel). Proportions of (A) pBTK-low, (B) pSYK-low, and (C) pPLCγ2-low cells, expressed as percentage of total naïve or memory B cell fraction (right panel). Subjects are indicated by individual data points, and bars indicate mean values + SD. * p < 0.05, ** p < 0.01, *** p < 0.001 in ((A–C) (right panel)) by an unpaired two-tailed Student’s t-test or (in (C) (left panel)) by a Mann–Whitney U.
Figure 2
Figure 2
Correlation analysis of ex vivo measured B cell markers and in vitro measured phosphorylation of BCR signalosome molecules in naïve (CD3−CD19+CD38loCD27−IgD+) B cells from HCs and IPF patients (cohort 1). (A) Principal component analysis (PCA) of BCR signalosome molecules and B cell activation markers and (B) the individual contribution of each variable to PC1 and PC2 from IPF and HC naïve B cells. (C,D) Spearman’s rank correlation matrix for (C) HCs and (D) IPF patients for the indicated markers measured ex vivo and phosphorylated proteins of unstimulated (unst.) and BCR-stimulated (α-Ig) naïve B cells. (E,F) Spearman’s rank correlation analysis of (E) HCs and (F) IPF patients for the correlation of pPLCγ2 with pBTK, BAFFR, and TACI in unstimulated (unst.) and BCR-stimulated (α-Ig) naïve B cells. Subjects are indicated by individual data points. * p < 0.05 in (A) by an unpaired two-tailed Student’s t-test; ns, not significant.
Figure 3
Figure 3
Analysis of circulating BAFF and TGF-β levels in HC and IPF patients (subset of cohort 1). (A) TGF-β levels in plasma of HCs and IPF patients measured by ELISA. (B) BAFF levels in plasma of HCs and IPF patients measured by ELISA. (C) Pearson correlation analysis between BAFF levels in plasma and BAFFR surface expression on naïve (CD3−CD19+CD38loCD27−IgD+) and memory (CD3−CD19+CD38loCD27+) B cells in IPF patients. Subjects are indicated by individual data points, and bars indicate mean values + SD. * p < 0.05 in (B) by an unpaired two-tailed Student’s t-test.
Figure 4
Figure 4
Phosphoflow analysis of BCR downstream signaling in nintedanib-treated IPF patients’ peripheral blood B cells (cohort 2). (A–D) Phosphorylation levels in unstimulated (unst.) conditions and after α-Ig stimulation for (A) pBTK Y223, (B) pSYK Y348, (C) pPI3K p85 Y458, and (D) pPLCγ2 Y759 in naïve (CD3−CD19+CD38loCD27−IgD+) and memory (CD3−CD19+ CD38loCD27+) B cells from IPF patients before and after the start of nintedanib treatment. (E,F) Spearman’s rank correlation analysis of the phosphorylation levels for (E) pSYK (left) and pPLCγ2 (right) in naïve B cells following BCR stimulation, before versus after the start of nintedanib treatment. (F) Spearman’s rank correlation matrix for phosphorylation of BCR signalosome molecules before and after the start of nintedanib treatment. Subjects are indicated by individual data points. * p < 0.05 in (A) by a paired two-tailed Student’s t-test.
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