Study On The Anti-infective Immunity Of Candida Albicans In Keratinocytes

Objective To investigate the mechanism of anti-infective immunity on keratinocytes. Keratinocyte, which account for 95%, are the main structure in epidermis, the outer layer of skin. They were regarded as target cells from immune cells in inflammatory and immunological skin disorders, as well as physical barriers against invading pathogenic organisms during the past years. With the establishment and advance in the theory of skin immune system (SIS), it was proved that keratinocytes functioned as immune cells. Firstly, keratinocytes express surface markers of immune cells and help T cell participate in immune response. Secondly, they take part in inflammatory, immunological and pathological reaction in the skin by secreting cytokines, chemokines and antimicrobial peptides in response to stimulating promoters.Genus Candida is a commonly opportunistic pathogen which is ubiquitous in nature, normal oral cavity, digestive tract, vagina and skin surface. It is a commensal flora in normal condition. Whether skin infection is onset or not, it is mainly determined by virulence factors of genus Candida and immunity of host when contaminating with the pathogenic organism. Especially, the immunity in the host plays a predominant role in pathogenesis.C. albicans is the most important pathogenic species among the genus of Candida. Immune system can be divided into two branches: innate immunity and adaptive immunity. The former include phagocytosis and killing pathogen resulting from phagocyte and leukocyte, the latter indicate T lymphocyte and B lymphocyte-mediated immune response featuring in specificity and memory. The innate immune was regarded as a nonspecific response until the theory of pattern recognition established by Janeway made the concept reverse. The pattern recognition theory define the main signaling molecules from innate immune as pathogen-associated molecular patterns (PAMPs), and corresponding receptors are referred to as pattern recognition receptors (PRRs), which specifically recognize different PAMPs from surface of microorganisms. These pathogen-related products are often highly conserved structures that are critical for microbial survival, and relatively invariant within a given class of microorganisms, such as mannans in the yeast cell wall, lipopolysaccharide (LPS) of Gram-negative bacteria and peptidoglycan (PGN) of Gram-positive bacteria.Drosophila Toll (dToll) was originally identified as a kind of innate receptor protein required for the establishment of dorso-ventral polarity in the developing embryo. Not only dToll has an influence on development of Drosophila embryo, but also take part in immune response in adult. Drosophila was predisposed to fungal infection if the function of Toll was absent. Mammalian homologues of Drosophila Toll, designated Toll-like receptors were recently discovered. In the past few years, 10 different human TLRs have been identified, from TLR-1~TLR-10. TLRs are kinds of pattern recognition receptors closely related to innate immune. They recognizePAMPs and then transduce recognizing signal. For example, Toll-like receptor-2 ( TLR-2 ) recognizes mannan and peptidoglycan, TLR-3 recognizes double-stranded RNA (dsRNA), a molecular pattern associated with viral infection, TLR-4 is the receptor for Gram-negative lipopolysaccharide (LPS), TLR-5 is the Toll-like molecule that recognizes flagellin from both Gram-positive and Gram-negative bacteria. TLR-9 is involved in the recognition of specific unmethylated CpG-ODN sequences, which distinguishes bacterial DNA from mammalian DNA. It was discovered recently that TLR-7 and TLR-8 may participate in the discrimination of nucleic-like structure in microorganisms although their nature ligands remain unclear.To further understand the role of anti-infective immunity in keratinocytes and the mechanism of interaction between keratinocytes and C. albicans. TLR-2, which is involved in recognition of yeast cell wall components, and human bata-defensin-2 (HBD-2), which is an important member in innate immunity of skin, was detected in keratinocytes stimulated with C. albicans and its cell wall components. Translocation of nuclear factor-KB (NF-kB) in keratinocyte response to C. albicans was stained. Secretion of IL-8 and killing activity was tested in cells stimulated with C. albicans after or before TLR-2 function was neutralized by anti-TLR-2 mAb.Method (1) Primarily cultured keratinocytes, its immortalized cell lines HaCaT and standard strain of C. albicans (ATCC 10231), were prepared for the study.(2) To establish the culture system of keratinocytes in vitro, specimens from juvenile foreskins were used. After dissociating epidermal layer fromdermis of skin in Dispase, the cell was cultured with Serum-Free Keratinocyte Medium for Culture of Human Keratinocytes (KC-SFM). The cells were identified as keratinocytes by morphology and broad-spectrum cytokeratin (CK) stain by immunocytochemistry.(3) C. albicans was cultured in Sabouraud's broth. Supernatant, cell wall extract and cell plasma extract of yeasts were isolated by Lyticase, repeatedly freezing and thawing, sonication and super-speed centrifugation. The expressions of TLR-2 mRNA and HBD-2 mRNA were detected by reverse-transcription polymerase chain reaction (RT-PCR) in keratinocyte and its immortalized cell line (HaCaT) co-cultured with C. albicans and its components for 24h, and then semi-quantitive analysis was made.(4) To investigate the influence of C. albicans and its components on the activation of NF-kB in cultured keratinocytes, the expression and nuclear translocation of NF-kB were detected by laser confocal scanning microscope (LCSM) in cells co-cultured with C. albicans and cell wall components for 1.5h or 3h.(5) To investigate the influence of TLR-2 on IL-8 secretion and killing C. albicans activity, IL-8 was detected by ELISA, and the killing activity was detected by methylene blue staining. TLR-2 function was neutralized by anti-TLR-2mAb in keratinocyte after or before stimulation with C. albicans and its components.Results (1) During culture, no fibroblasts were contaminated. The isolated cells were keratinocytes, featured by the variform, polygonal and slabstone-like morphology and the positive stain by broad range cytokertin antibody. The cells maintained for 5-9 passages after cyropreservation andanabiosis.(2) HBD-2 mRNA and TLR-2 mRNA expressed constitutively in cultured keratinocytes and HaCaT cells. HBD-2 mRNA could be up-regulated by C. albicans and mannan, and moreover, TLR-2 mRNA keep invariability.(3) NF-kB in keratinocytes could be induced to translocation from cytoplasm to nucleus by C. albicans and mannan stimulation.(4) C. albicans and mannan resulted in the up-regulation of IL-8 secretion in keratinocytes. The presence of anti-TLR-2 antibodies could not inhabit the cellular responses to C. albicans and mannan. TLR-2 is involved in the killing of Candida by keratinocytes. Human anti-TLR-2 antibodies significantly inhibited the Candida-killing activity of keratinocytes.Conclusion TLR-2 mRNA express constitutively, and express inducibly in keratinocytes by C. albicans and its cell wall components, mainly referred as mannan. The recognition of pathogens by TLR-2 in keratinocytes leads to translocation and activation of NF-kB, and then up-regulated the express of HBD-2 and IL-8, which might be responsible for proliferation in epidermal cells, killing invading pathogens and recruiting inflammory cells to the sites of infections. Our findings stress the importance of the keratinocytes as a component of the innate immune response, and offer a novel idea to find new target sites for antifungal therapy.