Basement membrane protein and matrix metalloproteinase deregulation in engineered human oral mucosa following infection with Candida albicans

Abstract

A variety of morphological changes in the basement membrane (BM) are known to occur in inflammatory diseases. Modifications of the BM can be associated with significant changes in protein content. Candida albicans (C. albicans) is normally a commensal organism and is a member of the natural flora of a large number of healthy individuals. However, under certain conditions, C. albicans can invade host tissues, causing inflammation and tissue damage. The aim of this study was to investigate the effect of C. albicans on the expression and production of structural (laminin-5 and type IV collagen) and inflammatory [matrix metalloproteinases (MMPs) and their inhibitors] proteins by human oral epithelial cells. Using engineered normal human oral mucosa infected with 10⁵ C. albicans/cm² for different periods of time, we were able to demonstrate that this yeast promotes significant laminin-5 and type IV collagen gene activation and protein secretion. These effects were accompanied by MMP-2 and MMP-9 gene activation. Interestingly, only the levels of active MMP-9 rose. The increase in MMP levels was paralleled by a decrease in the secretion of type 2 matrix metalloproteinase tissue inhibitors (TIMP-2). Our results demonstrated that C. albicans has a significant effect on tissue structure through BM protein and MMP modulation. This might help C. albicans overcome the mechanical and biological defenses of the tissue and allow it to disseminate, causing severe infections. If C. albicans uses MMPs (mainly MMP-9) to disseminate, inhibition of this protease could be of interest in treating a variety of inflammatory disorders, including oral candidiasis.

Introduction

A variety of yeast species normally live in the oral cavity. Their presence does not in itself imply a disease state since the prevalence of asymptomatic yeast carriers in healthy individuals ranges from 3% to 48% (Arendorf and Walker, 1980) and is even higher (45–65%) in healthy children (Odds, 1988). By far, the yeast species most commonly isolated from the oral cavity is Candida albicans (Samaranayake and MacFarlane, 1990). While a median carrier rate of 38.1% has been observed for C. albicans in community-dwelling outpatients (Odds, 1988), a rate reaching 78% has been reported in hospitalized elderly patients (Wilkieson et al., 1991) and is even higher in HIV-infected individuals (Hauman et al., 1993).

C. albicans is the main cause of nosocomial fungal infections leading to candidiasis (Calderone and Fonzi, 2001). It has been reported that immune protection against candidiasis could be site specific, emphasizing the complex nature of the disease (Fidel, 2002). During disease development, the microorganism invades tissues by yeast–hyphal transition (morphogenesis), although direct persorption of yeast cells by mucosal cells has also been observed (Calderone and Fonzi, 2001, Leigh et al., 2001). The other critical event in the disease process, which precedes morphogenesis, is adhesion of the yeast to host cells via cell surface adhesins, which have been shown to promote the virulence of C. albicans (Calderone and Fonzi, 2001). Invasion of mucosal epithelia follows the adhesion and morphogenesis steps (Calderone and Fonzi, 2001).

The oral mucosa is a highly specialized stratified epithelia that protects the body from physical and chemical damage, infection, dehydration, and heat loss through interactions with the mesenchymal tissue via BM proteins (French and Pollitt, 2004, Nomanbhoy et al., 2002, Rouabhia et al., 2002).

Basement membranes (BMs) are thin layers of specialized extracellular matrix that form the supporting structure on which epithelial cells grow. They provide mechanical support, divide tissues into compartments, and significantly influence cellular behavior (Sasaki et al., 2004). Type IV collagen and laminin are two major components of basement membranes. Type IV collagen forms a network that confers the distinct mechanical stability known to basement membrane (Sacca et al., 2002). Laminin binds to collagen IV and forms a second network by interacting with nidogen. Laminin-5 is specific to basement membranes underlying squamous epithelium and mucosa (Rousselle et al., 1995). Its principal role is to modulate stable epithelial cell attachment through interactions with α₃β₁ and α₆β₄ integrin (Colognato and Yurchenco, 2000). These interactions also play an important role in cell migration. BM protein modification is a prerequisite for tissue and extracellular matrix (ECM) degradation.

Matrix metalloproteinases (MMPs) are a large family of Zn²⁺- and Ca²⁺-dependent endopeptidases involved in tissue remodeling and chronic inflammation. They have broad, overlapping specificities and have the capacity to degrade all the components of the ECM (Nagase and Woessner, 1999). MMPs are produced by different cell types such as lymphocytes, granulocytes, fibroblasts, and epithelial cells (Owen et al., 2003, Warner et al., 2004). MMPs are secreted as proenzymes (Woessner, 1998), which are activated by proteolytic cleavage and regulated by a family of inhibitors (tissue inhibitors of matrix metalloproteinases; TIMPs) (Mannello and Gazzanelli, 2001). They are constitutively produced by a variety of cells (Johnatty et al., 1997, Nahm et al., 2004). MMP activities are thus dependent on the balance between MMP production and activation and local TIMP levels (Nagase and Woessner, 1999, Mannello and Gazzanelli, 2001). In rheumatoid arthritis, pulmonary emphysema, periodontal disease, and inflammatory bowel disease, MMPs are believed to be responsible for much of the associated tissue destruction (Birkedal-Hansen et al., 1993). In addition to their direct effects on ECM proteins, MMPs can exacerbate inflammation by activating or by releasing cytokines (Ito et al., 1996). Their ability to generate chemotactic fragments from ECM proteins may also contribute to the recruitment of inflammatory cells after tissues infection (Hunninghake et al., 1981).

Systemic Escherichia coli infections, acute Lyme neuroborreliosis, and pneumococcal meningitis can all lead to the secretion of significant amounts of MMP-9 (Kirchner et al., 2000, Leib et al., 2000, Paemen et al., 1997). In the case of pneumococcal meningitis, it has been suggested that MMP-9 contributes to the destruction of the blood–brain barrier and neuronal injury. In cell cultures, MMP production is induced by different bacterial products (Firth et al., 1997). Several proinflammatory cytokines produced in response to bacterial infections, including TNF-, IL-1, and granulocyte-macrophage colony-stimulating factor (GM-CSF), have also been shown to increase monocyte and macrophage MMP production in vitro (Zhang et al., 1998). A recent report showed that tuberculosis patients have increased levels of MMP-9 in bronchoalveolar lavage fluids and that heat-killed Mycobacterium tuberculosis as well as mycobacterial cell wall components increase MMP-9 mRNA production by a myelomonocytic cell line (Chang et al., 1996). However, little is known about MMP production during C. albicans infections and their contribution to immunity and pathology.

The interaction of Candida with mucosal cells is believed to be one of the critical initial events in the development of candidiasis (Calderone et al., 1994). However, the mechanism by which Candida, an innocuous oral commensal, may occasionally become pathogenic and induce oral lesions despite an intact immune surveillance system remains unclear. The physical host-Candida contact is mediated by epithelial cells (Nomanbhoy et al., 2002, Rouabhia et al., 2002). The switch from a commensal to a pathogenic form induces significant changes in tissue structure and BM protein synthesis and deposition, which in turn induce a breakdown of oral homeostasis. For example, laminin-5 plays a crucial role in epithelial cell-BM adhesion and may be modified when the tissue is under stress (French and Pollitt, 2004). Our goal is to further characterize some of the key events that occur in the BM of buccal mucosa following contact with Candida as well as the status of some inflammatory molecules involved in ECM degradation. We first examined the influence of C. albicans on type IV collagen and laminin-5 expression and production by engineered human oral mucosa (EHOM). We then studied the expression and secretion of MMP-2, MMP-9, TIMP-1, and TIMP-2 following C. albicans infections of EHOMs.