Uptake of cell debris and enhanced expression of inflammatory factors in response to dead cells in corneal fibroblast cells

Highlights

• Corneal fibroblast cells are macrophage-like fibroblasts.

• Corneal fibroblasts can engulf dead bacteria, processed cellular debris and entire dead cells.

• Dying and dead cells stimulate corneal fibroblasts to further induce inflammatory factors.

Abstract

Keratocytes synthesize stromal proteins and participate in wound healing through successive differentiation into corneal fibroblasts and myofibroblasts. Cultured keratocytes or corneal fibroblasts are also known as non-professional phagocytes and innate immune cells. However, whether the corneal fibroblasts phagocytize their dead cells and whether the associated innate immunity is enhanced remains unknown. We initially characterized immortalized corneal fibroblast cells with the expression of specific genes. The corneal fibroblasts strongly expressed extracellular matrix molecules (FN and COL1A1) and low or medium levels of macrophage markers (CD14, CD68, and CD36), inflammatory cytokines (IL1A, IL1B, and IL6), and chemokines (IL8 and CCL2), but not CD11b, suggesting that corneal fibroblasts are macrophage-like fibroblasts. We confirmed the phagocytic activity of the corneal fibroblasts with fluorescent dye labeled-dead E. coli and S. aureus bacteria using confocal microscopy and flow cytometry. To test corneal fibroblast phagocytosis of apoptotic and necrotic cells we co-cultured corneal fibroblasts with fluorescent dye labeled-apoptotic and -necrotic cells and analyzed their uptake using fluorescence and confocal microscopy. We observed that corneal fibroblasts can engulf digested or processed cellular debris and entire dead cells. Co-cultured dying and dead cells strongly enhanced the expression of cytokine (IL1A, IL1B, and IL6), chemokine (CCL2, CCL5, CCL20, IL8, and CXCL10), and MMP (MMP1, MMP3, and MMP9) genes through the NF-κB signaling pathway. Our findings suggest that dying and dead cells stimulate corneal fibroblasts to further induce inflammatory factors and that corneal fibroblasts contribute to the clearing of cell debris as non-professional phagocytes.

Introduction

Keratocytes are the main population of cells in the corneal stroma. They reside between regularly organized collagen bundles called lamellae and are responsible for producing extracellular matrix (ECM) molecules, such as collagens and proteoglycans, that are a main component of the corneal stroma (Hovakimyan et al., 2014). In normal corneal stroma, keratocytes are quiescent cells with a dendritic morphology (Hovakimyan et al., 2014). However, upon injury, keratocytes become activated. Activated keratocytes, called corneal fibroblasts as they resemble fibroblasts, enter into the cell cycle and migrate toward the injury site (Hovakimyan et al., 2014; Wilson et al., 2001; Zieske et al., 2001). They further convert into myofibroblast cells and participate in wound closure (Funderburgh et al., 2001; Mohan et al., 2003). To investigate keratocyte activation and its role in wound healing in vitro, cell culture methods have been developed that recapitulate the phenotypes of the cells in vivo. For instance, when primary keratocytes are cultured in serum-free or low serum-containing media, they exhibit characteristics similar to keratocytes in vivo (Beales et al., 1999; Funderburgh et al., 2003; Hovakimyan et al., 2014). However, when they are cultured in the presence of 10% fetal bovine serum, they proliferate and lose their dendritic morphology and marker genes of keratocytes including KERA and crystallins and they gain the phenotypes of the corneal fibroblast (Funderburgh et al., 2003; Hovakimyan et al., 2014; Jester et al., 2012; Yoshida et al., 2005). These corneal fibroblasts are further transformed into myofibroblasts by the addition of TGF-β (Hovakimyan et al., 2014; Jester et al., 2012). Even with much effort using in vivo and in vitro model systems, the role of corneal fibroblasts in stromal wound repair remains largely unknown.

In addition to synthesizing stromal proteins, keratocytes respond to immunological stimuli and function like macrophages in certain conditions. Cultured keratocytes overexpress inflammatory cytokines and chemokines (IL4, IL6, IL8, IL17, G-CSF, and CCL2) after LPS, IL1A, TNF-alpha, or CpG DNA stimulation (Ebihara et al., 2007; Fukuda et al., 2017; Hong et al., 2001). Subsequent monocytes or macrophage recruitment was demonstrated by injection of CCL2 into the corneal stroma (Hong et al., 2001), suggesting that keratocytes might amplify the immune response and be involved in recruiting immune cells to injured sites.

Although not professional phagocytes, keratocytes can phagocytize latex beads, fixed erythrocytes, and dead bacteria (S. aureus and E.coli) (Funderburgh et al., 2001; Lande et al., 1981; Mishima et al., 1987, 1988, 1992). Fibronectin and poly inosine-polycytidylic acid (poly(I:C)) enhance the phagocytic activity of rabbit keratocytes (Funderburgh et al., 2001; Mishima et al., 1987, 1988), while dexamethasone inhibits their phagocytic activity (Mishima et al., 1988). This phagocytic function is supported by the expression of marker genes associated with macrophages (Chakravarti et al., 2004). In a mouse model, compared with fibroblasts and myofibroblasts, keratocytes overexpressed genes associated with macrophages, such as Mmp3, Mmp12, Cd68, chemokine ligands (Ccl2, Ccl7, Ccl9, and Cxcl12), cathepsins, and complement component pathway genes, while fibroblasts and myofibroblasts overexpressed genes associated with wound healing (Chakravarti et al., 2004). Analysis of differentially expressed genes in microarray is based on relative expression levels, which often does not reflect absolute gene expression levels. These results do not indicate that fibroblasts do not express macrophage marker genes. Whether corneal fibroblasts also function as phagocytes remains to be explored.

Keratocytes adjacent to massive dead cells or cell debris become activated to fibroblasts after stromal injury (Fukuda et al., 2017; Hovakimyan et al., 2014). It might be possible that corneal fibroblasts phagocytize dead cells and cell debris, but their scavenger function and the innate immune response stimulated by cellular debris remain unexplored. Here, we report that in vitro corneal fibroblasts can uptake dead bacteria, apoptotic cells, necrotic cells and their debris, and that their phagocytosis occurs with enhanced innate immune response.