Myofibroblasts persist through immune privilege mechanisms to mediate oral submucous fibrosis: Uncovering the pathogenesis

Abstract

Immune privilege is the ability to tolerate foreign antigens without eliciting an inflammatory immune response. Several mechanisms explain a structure's immune privilege status, which is regulated by innate and adaptive immune responses. The role of myofibroblasts in perpetuating fibrosis by acquiring an immune privileged phenotype against the backdrop of oral submucous fibrosis (OSF) is evolving. Myofibroblasts persist through the Fas/FasL autocrine pathway and induce apoptosis in epithelial cells, explaining the juxtaposition of apoptotic cells in areas of fibrosis. However, increased matrix stiffness, in addition to activating TGF-β, reduces Fas surface expression in myofibroblasts, increasing their resistance to apoptosis. The reciprocal amplification loop between the immune checkpoint proteins programmed death-ligand 1 (PD-L1) and TGF-β involves the YAP-TAZ and SMAD2,3 pathways and dramatically enhances profibrotic signalling. Increased matrix stiffness also enhances cMYC expression, which subsequently amplifies PD-L1 levels on myofibroblasts. The increase in PD-L1 on the myofibroblast microengineers the phenotype of CD4⁺ T cells homing to fibrotic areas by acting on the programmed cell death protein 1 (PD-1) receptor on the T-cell surface, converting these cells from antifibrotic cells to profibrotic cells that produce IL-17A and TGF-β. This manuscript provides mechanistic insight into how myofibroblasts avoid apoptosis in OSFs by evading the immune system. Targeting an immune-privileged phenotype in myofibroblasts with FAS-FASL pathway-dependent characteristics is an ideal strategy for reversing OSF.

Keywords: Fas-FasL pathway; Immune checkpoint protein; Myofibroblast; Oral submucous fibrosis; PD1-PD-L1 pathway; Tregs.

Graphical abstract

Fig. 1

The mechanism leading to the immune privilege of myofibroblasts in OSF 1A. Areca nut-induced downregulation of CD8⁺ T-cell growth, differentiation, and maturation through H₂O₂ leads to the suppression of INF-γ, a reduction in IL-2 (this reduction leads to feedback suppression of CD8⁺ T cells), and an increase in AIF and Casp-3, which leads to an increase in CD8⁺ T-cell apoptosis. TGF-β further promotes the differentiation of CD4⁺ T cells into T-regs, which produce more TGF-β. TGF-β causes myofibroblastic differentiation and the production of Col-1A1 and Col-1A3. Upon crosslinking, this collagen activates the Integrin Subunit Alpha 2 (IGTA-2) receptor on T-regs to stimulate them to produce even more TGF-β. Areca nut chewing downregulates the immune system by suppressing CD1a⁺ Langerhans cells, reducing neutrophil phagocytic activity, reducing CD207⁺ dendritic cells, and increasing the number of immunosuppressive CD303⁺ DCs. The downregulation of CD8⁺ T cells and upregulation of CD4⁺ T cells lead to a high CD4⁺CD8⁺ T-cell ratio with fibrosis progression. The ECM-induced YAP-TAZ pathway in epithelial cells results in the upregulation of Tregs and MDSCs and the downregulation of CD8⁺ T cells, leading to the upregulation of fibrosis and malignancy. 1B. The circulating immunocomplex (CIC) found in OSF patients suppresses the NK cell-mediated elimination of senescent epithelial cells and senescent fibroblasts through the TRAIL-TRAIL-R interaction. This also results in reduced NK cell granule-dependent elimination of senescent epithelial cells and senescent fibroblasts. The upregulation of DcR2 in senescent epithelial cells also inhibits the NK-mediated elimination of senescent fibroblasts through the TRAIL-TRAIL-R interaction. This also results in epithelial and fibroblast accumulation and the promotion of fibrosis. The intracellular cascade initiated by DcR2 in senescent epithelial cells leads to the formation of the GRP78‒Caspase-7 complex, resulting in reduced cleaved caspase-7 and subsequent resistance to apoptosis. The combined SASP from senescent epithelial cells and senescent fibroblasts promotes the EMT and malignant transformation of DOKs. 1C. Prototypic model of immune cell-mediated elimination through the apoptosis of unwanted cells through the FasL-Fas interaction. 1D. FasL⁻ Myofibroblasts cannot drive Fas⁺ CD8⁺ cell apoptosis through the Fas/FADD/Cas-8/3/7 pathway but instead undergo apoptosis, which is the typical path of fibrosis resolution. 1E. As myofibroblasts acquire immune privilege, they start expressing FasL and induce the apoptosis of CD8⁺ T cells, allowing their persistence in fibrotic tissues. These FasL⁺ myofibroblasts induce apoptosis in the Fas⁺ epithelium through the Fas/FADD/Cas-8/3/7 pathway, explaining the juxtaposition of the atrophic epithelium with fibrosis in OSF.

Fig. 2

Further mechanisms of immune privilege of myofibroblasts in OSF 2A. Co-option of the Intrinsic Pathway of Apoptosis Apoptosis, triggered by intracellular death stimuli, induces mitochondrial outer membrane permeabilization (MOMP), leading to mitochondrial swelling, rupture, and the release of proapoptotic proteins. The effectors of MOMP include BCL-associated X protein (BAX) and BCL-2 homologous antagonist/killer (BAK), which are activated by BH3-interacting domain death agonist (BID) and Bcl-2-interacting mediator (BIM). Through activation of the Abelson murine leukemia (ABL) pathway, TGF-β blocks intrinsic apoptosis in myofibroblasts by upregulating BCL-2 and Bcl-xL. TGF-β can act downstream of the Casp-9 pathway to inhibit apoptosis through SMAD-3-induced XIAP. Myofibroblasts express an immune checkpoint protein called programmed death ligand 1 (PD-L1), which promotes TGF-β-dependent ECM deposition and cell migration. T cells express the cell surface receptor programmed cell death protein 1 (PD-1), which, in interaction with PD-L1 on myofibroblasts, prevents homing T lymphocytes from destroying other cells, such as cancerous cells and myofibroblasts. 2B. Co-option of the Extrinsic Pathway of Apoptosis Increased fibrosis, because of stiffness, leads to the activation of TGF-β and the downregulation of Fas surface expression. Fas surface expression was also reduced through the downregulation of miR29C. These mechanisms explain the persistence of myofibroblasts due to their immune privilege in fibrosing tissues. More severe fibrosis in users who added slaked lime to the betel quid mixture can be explained by the upregulation of cFlip. cFlip is also upregulated by TGF-β, IL-4, IL-6, IL-10, IL-1β, and ROS. The upregulation of cFlip through the formation of the multimolecular complex of RAF1, TRAF1,2, and RIP-1 promotes the proliferation of myofibroblasts through the ERK and NF-κB pathways and promotes myofibroblast survival through the JNK, Wnt, and AKT pathways. This signalling by cFLIP also converts the Fas apoptotic signal on myofibroblasts into autocrine proliferative signalling. The soluble form of FasL, the sFasL generated by the actions of MMP-3,7,9 and ADAM-10 on mFasL and sFas, upregulates TGF-β and induces apoptosis resistance in myofibroblasts. Through TGF-β₁, ET/Akt/GSK-3β/NFATc1 (nuclear), membrane decoy receptor 3 (DcR3) is upregulated. DcR3 binds to FasL and inhibits apoptosis. This antiapoptotic effect is amplified by the binding of myofibroblasts to the collagen matrix through DcR3 upregulation. 2C. The accumulation of the type I collagen matrix downregulates FoxO3a, which in turn promotes myofibroblast proliferation through Bim, p21, and P27 downregulation. FoxO3a reduction also leads to reduced Cav-1 levels, which, in turn, reduce Fas levels and cause Fas-induced apoptosis. High Fas⁺ levels on myofibroblasts interact with high mFasL⁺ levels on neighboring myofibroblasts to promote autocrine myofibroblast proliferation.