Modulation of NLRP3 Inflammasome through Formyl Peptide Receptor 1 (Fpr-1) Pathway as a New Therapeutic Target in Bronchiolitis Obliterans Syndrome

Abstract

Chronic rejection is the major leading cause of morbidity and mortality after lung transplantation. Bronchiolitis obliterans syndrome (BOS), a fibroproliferative disorder of the small airways, is the main manifestation of chronic lung allograft rejection. We investigated, using transgenic mice, the mechanisms through which the deficiency of IL-1β/IL-18, Casp-1, or Fpr-1 genes could be protective in an experimental model of BOS, induced in mice by allogeneic heterotopic tracheal transplantation. Fpr-1 KO mice showed a marked reduction in histological markers of BOS and of mast cell numbers compared to other groups. Molecular analyses indicated that the absence of the Fpr-1 gene was able to decrease NF-κB nuclear translocation and modulate NLRP3 inflammasome signaling and the mitogen-activated protein kinase (MAPK) pathway in a more significant way compared to other groups. Additionally, Fpr-1 gene deletion caused a reduction in resistance to the apoptosis, assessed by the TUNEL assay. Immunohistochemical analyses indicated changes in nitrotyrosine, PARP, VEGF, and TGF-β expression associated with the pathology, which were reduced in the absence of the Fpr1 gene more so than by the deletion of IL-1β/IL-18 and Casp-1. We underline the importance of the NLRP3 inflammasome and the pathogenic role of Fpr-1 in experimental models of BOS, which is the result of the modulation of immune cell recruitment together with the modulation of local cellular activation, suggesting this gene as a new target in the control of the pathologic features of BOS.

Keywords: bronchiolitis obliterans syndrome; inflammasome; inflammation.

Figure 1

Histopatology evaluation and mast cell density in IL-1β/IL-18 KO, Casp-1 KO, and Fpr-1 KO: Histological evaluation of tracheal transplantation: wild-type (WT) (A), IL-1β/IL-18 KO (B), Casp-1 KO (C), Fpr-1 KO (D). Masson trichrome staining of the graft: WT (E), IL-1β/IL-18 KO (F), Casp-1 KO (G), Fpr-1 KO (H). Evaluation of mast cell degranulation by toluidine blue: WT (I), IL-1β/IL-18 KO (J), Casp-1 KO (K), Fpr-1 KO (L). Histopathologic score (M). For histological analyses, n = 5 animals from each group were employed. A p-value less than 0.05 was considered significant. ## p < 0.01 versus the WT group.

Figure 2

Effects of the absence of IL-1β/IL-18, Casp-1, and Fpr-1 on the NRLP3 inflammasome pathway. Western blots and, respectively, the densitometric analysis of NRLP3 (A,A’) and ASC (B,B’). For Western blot analyses, n = 5 animals from each group were employed. A p-value less than 0.05 was considered significant. # p < 0.05 versus the WT group, ### p < 0.001 versus the WT group.

Figure 3

Effects of the absence of IL-1β/IL-18, Casp-1, and Fpr-1 on the NF-κB pathway and iNOS expression. Western blots and, respectively, the densitometric analysis of IkB-α (A,A’), NF-kB p65 (B,B’), and iNOS (C,C’). For Western blot analyses, n = 5 animals from each group were employed. A p-value less than 0.05 was considered significant. ## p < 0.01 versus the WT group, and ### p < 0.001 versus the WT group.

Figure 4

Effects of the absence of IL-1β/IL-18, Casp-1, and Fpr-1 on Nitrotyrosine formation and PARP activation. Immunohistochemistry evaluation of nitrotyrosine expression: WT (A), IL-1β/IL-18 KO (B), Casp-1 KO (C), Fpr-1 KO (D), densitometric analysis (E). Immunohistochemistry evaluation of PARP expression: WT (F), IL-1β/IL-18 KO (G), Casp-1 KO (H), Fpr-1 KO (I), densitometric analysis (J). Yellow arrows point the positive cells. For immunohistochemistry, n = 5 animals from each group were employed. A p-value less than 0.05 was considered significant. ### p < 0.001 versus the WT group.

Figure 5

Effects of the absence of IL-1β/IL-18, Casp-1, and Fpr-1 on apoptosis. TUNEL staining of tracheal transplantation: WT (A), IL-1β/IL-18 KO (B), Casp-1 KO (C), Fpr-1 KO (D), graphical quantification (E). For TUNEL staining, n = 5 animals from each group were employed. A 20× magnification is shown (50-µm scale bar). A p-value less than 0.05 was considered significant. ### p < 0.001 versus the WT group.

Figure 6

Effects of the absence of IL-1β/IL-18, Casp-1, and Fpr-1 on growth factor expression. Immunohistochemistry evaluation of VEGF expression: WT (A), IL-1β/IL-18 KO (B), Casp-1 KO (C), Fpr-1 KO (D), densitometric analysis (E). Immunohistochemistry evaluation of TGF-β expression: WT (F), IL-1β/IL-18 KO (G), Casp-1 KO (H), Fpr-1 KO (I), densitometric analysis (J). Yellow arrows point the positive cells. For immunohistochemistry, n = 5 animals from each group were employed. A p-value less than 0.05 was considered significant. ### p < 0.001 versus the WT group.

Figure 7

Effects of the absence of IL-1β/IL-18, Casp-1, and Fpr-1 on the mitogen-activated protein kinase (MAPK) pathway. Western blots and, respectively, the densitometric analysis of p-ERK 1/2 (A,A’) and p-p38 (B,B’). For Western blot analyses, n = 5 animals from each group were employed. A p-value less than 0.05 was considered significant. # p < 0.05 versus the WT group, ### p < 0.001 versus the WT group.