*3.4. Bcl10 Mediates LPS-Induced Pro-Fibrotic Cytokine Signaling in Bronchial Fibroblasts*

In order to assess the role of Bcl10 in pro-fibrotic signaling, the bronchial fibroblasts were stimulated with LPS and the level of pro-fibrotic cytokines, IL-6, IL-8 and TGFβ1, was analyzed. The fibroblasts were serum-starved for 24 h and then exposed to 10 μg/mL of LPS for different time-points. We then studied the effect of LPS stimulation on the gene expression of BCL10 using qRT-PCR. LPS stimulation was observed to significantly upregulate the expression of BCL10 in both NHBF and DHBF in comparison to their respective unexposed controls and this increase was found to be time-dependent (Figure 4A).

**Figure 3.** Subcellular localization of Bcl10 in bronchial fibroblasts and bronchial epithelial cells. NHBF and DHBF were cultured in DMEM complete medium, and NHBE and DHBE were cultured in Pneumacult complete medium. (**A**) Cellular clots of NHBF and DHBF, and (**B**) NHBE and DHBE were immunostained for Bcl10 and the white arrows indicate Bcl10-positive cells.

Since cross-linking of Toll-like receptor 4 (TLR4) with LPS induces the synthesis and secretion of pro-inflammatory and pro-fibrotic cytokines [27], we next examined the expression of IL-6, IL-8 and TGFβ1 in NHBF and DHBF upon LPS stimulation for 2 h. LPS strongly induced IL-6 gene expression in both NHBF and DHBF, whereas IL-8 and TGFβ1 was induced to a lower extent when compared to their unexposed control (Figure 4C). The induction of collagen expression by LPS in primary cultured mouse lung fibroblast [28] led us to next assess the expression of collagen markers, COL1A1 and COL5A1, and fibronectin in NHBF and DHBF. No significant alteration in the expression of these markers was observed in NHBF and DHBF upon LPS stimulation for 2 h (Figure 4D).

To further confirm the role of Bcl10 in LPS-induced pro-fibrotic cytokine signaling, we blocked the activity of IRAK1 and IRAK4, upstream adaptors that are responsible for recruiting Bcl10 to the TLR4 signaling complex and further signaling to NF-κB [29] using an IRAK1/4 inhibitor. In addition to restoring Bcl10 levels, the LPS-induced upregulation of IL-6, IL-8 and TGFβ1 was reversed upon exposure to the IRAK inhibitor (Figure 4B,C). The IRAK inhibitor suppressed the elevated gene expression of Bcl10 and IL-6 in both NHBF and DHBF. Interestingly, a reduction in COL5A1 expression and a reducing trend in COL1A1 and FN1 expression was noted in LPS-stimulated NHBF in the presence of IRAK1/4 inhibition (Figure 4D).

TLR4 can signal via the MyD88-dependent as well as the TRIF-dependent pathways, both of which, in turn, activate the canonical NF-κB pathway [30]. Bcl10-mediated signaling is a part of the MyD88-dependent pathway, which represents the early phase of activation of NF-κB. The TRIF-dependent pathway involves signaling through TBK1 and IRF complexes culminating in IFN-β expression and represents the late phase activation of NF-κB. Since TBK1 was reported to stimulate NF-κB and mediate the induction of inflammatory cytokine genes [31,32], we examined the expression of intermediates of the TRIF-dependent pathway upon stimulation with LPS and in the presence of IRAK1/4 inhibition. IRAK1/4 inhibition disrupts Bcl10 signaling, and therefore, Bcl10-mediated NF-κB signaling, but not TBK1mediated NF-κB signaling. Therefore, as expected, LPS induced the expression of TBK1, IRF3, IRF7 and IFN-β in both NHBF and DHBF, with a statistically significant increase noted in IRF3 (Figure 5). IRAK1/4 inhibition led to a further increase in IRF3 expression but the expression of TBK1, IRF7 and IFNβ was unaffected. The inhibition of LPS-induced expression of IL-6, IL-8 and TGFβ1 upon inhibiting Bcl10 signaling and not TRIF or TBK1 thus confirms that Bcl10-mediated signaling regulates pro-fibrotic cytokine expression in bronchial fibroblasts.

**Figure 4.** Bcl10 mediates LPS-induced pro-fibrotic cytokine signaling in bronchial fibroblasts. (**A**) NHBF and DHBF were serum-starved for 24 h, and thereafter, exposed to LPS (10 μg/mL) for the indicated time points for mRNA analysis. The effect of LPS treatment on the mRNA expression of BCL10 in NHBF and DHBF was analyzed by qRT-PCR and expressed as fold expression change relative to the respective untreated control at 0 h post normalization to housekeeping gene 18s rRNA. NHBF and DHBF were serum-starved for 24 h, and thereafter, pre-treated with IRAK 1/4 Inhibitor I (50 μM) for 1 h prior to LPS (10 μg/mL) stimulation for 2 h for mRNA analysis. The effect of LPS treatment and IRAK1/4 inhibition on the mRNA expression of (**B**) BCL10, (**C**) pro-fibrotic cytokines, IL-6, IL-8 and TGF-β1, and (**D**) ECM components, COL1A1, COL5A1 and FN1, in NHBF and DHBF was analyzed by qRT-PCR and expressed as fold expression change relative to the respective untreated control post normalization to housekeeping gene 18s rRNA. Data representative of at least 3 independent experiments. Data presented as mean ± SEM after normalization to untreated control. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, statistical significance assessed by one-way ANOVA with Tukey's multiple comparison tests.

**Figure 5.** TRIF-dependent pathway not involved in LPS-induced pro-fibrotic cytokine signaling in bronchial fibroblasts. NHBF and DHBF were serum-starved for 24 h, and thereafter, pre-treated with IRAK 1/4 Inhibitor I (50 μM) for 1 h prior to LPS (10 μg/mL) stimulation for 2 h for mRNA analysis. The effect of LPS treatment and IRAK1/4 inhibition on the mRNA expression of TBK1, IRF3, IRF7 and IFN-β in NHBF and DHBF was analyzed by qRT-PCR and expressed as fold expression change relative to the respective untreated control post normalization to housekeeping gene 18s rRNA. Data presented as mean ± SEM after normalization to untreated control. \* *p* < 0.05, \*\* *p* < 0.01, statistical significance assessed by one-way ANOVA with Tukey's multiple comparison tests.
