Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes, thereby hindering a comprehensive understanding of their interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using a key oral pathobiont, Fusobacterium nucleatum, as the primary test bed, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. In addition, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. Finally, we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont, Porphyromonas gingivalis. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research.
Coculture Techniques/methods* ; Humans ; Fusobacterium nucleatum/physiology* ; Gingiva/microbiology* ; Keratinocytes/microbiology* ; Host Microbial Interactions ; Mouth/microbiology* ; Host-Pathogen Interactions ; Epithelial Cells/microbiology* ; Cells, Cultured ; Porphyromonas gingivalis

Atopic dermatitis (AD) is a prevalent inflammatory skin disorder in which patients experience recurrent eczematous lesions and intense itching. The colonization of Staphylococcus aureus (S. aureus) is correlated with the severity of the disease, but its role in AD development remains elusive. Using single-cell RNA sequencing, we uncovered that keratinocytes activate a distinct immune response characterized by induction of Il24 when exposed to methicillin-resistant S. aureus (MRSA). Further experiments using animal models showed that the administration of recombinant IL-24 protein worsened AD-like pathology. Genetic ablation of Il24 or the receptor Il20rb in keratinocytes alleviated allergic inflammation and atopic march. Mechanistically, IL-24 acted through its heterodimeric receptors on keratinocytes and augmented the production of IL-33, which in turn aggravated type 2 immunity and AD-like skin conditions. Overall, these findings establish IL-24 as a critical factor for onset and progression of AD and a compelling therapeutic target.
Dermatitis, Atopic/genetics* ; Interleukins/metabolism* ; Animals ; Methicillin-Resistant Staphylococcus aureus/immunology* ; Mice ; Keratinocytes/microbiology* ; Humans ; Interleukin-33/immunology* ; Inflammation/microbiology* ; Staphylococcal Infections/microbiology* ; Disease Models, Animal ; Hypersensitivity/microbiology* ; Mice, Inbred C57BL

BACKGROUNDTrichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dermatophytosis caused by T. rubrum. In this study, we intended to develop a new T. rubrum infection model in vitro, using the three-dimensional reconstructed epidermis - EpiSkin ®, and to pave the way for further investigation of the mechanisms involved in T. rubrum infection.

METHODSThe reconstructed human epidermis (RHE) was infected by inoculating low-dose (400 conidia) and high-dose (4000 conidia) T. rubrum conidia to optimize the infection dose. During the various periods after infection, the samples were processed for pathological examination and scanning electron microscopy (SEM) observation.

RESULTSThe histological analysis of RHE revealed a fully differentiated epidermis with a functional stratum corneum, which was analogous to the normal human epidermis. The results of hematoxylin and eosin staining and the periodic acid-Schiff staining showed that the infection dose of 400 conidia was in accord with the pathological characteristics of host dermatophytosis caused by T. rubrum. SEM observations further exhibited the process of T. rubrum infection in an intuitionistic way.

CONCLUSIONSWe established the T. rubrum infection model on RHE in vitro successfully. It is a promising model for further investigation of the mechanisms involved in T. rubrum infection.

Animals ; Disease Models, Animal ; Epidermis ; microbiology ; Humans ; Keratinocytes ; cytology ; Tissue Culture Techniques ; Trichophyton ; pathogenicity

Infection by microorganisms may cause fatally erroneous interpretations in the biologic researches based on cell culture. The contamination by microorganism in the cell culture is quite frequent (5% to 35%). However, current approaches to identify the presence of contamination have many limitations such as high cost of time and labor, and difficulty in interpreting the result. In this paper, we propose a model to predict cell infection, using a microarray technique which gives an overview of the whole genome profile. By analysis of 62 microarray expression profiles under various experimental conditions altering cell type, source of infection and collection time, we discovered 5 marker genes, NM_005298, NM_016408, NM_014588, S76389, and NM_001853. In addition, we discovered two of these genes, S76389, and NM_001853, are involved in a Mycolplasma-specific infection process. We also suggest models to predict the source of infection, cell type or time after infection. We implemented a web based prediction tool in microarray data, named Prediction of Microbial Infection (http://www.snubi.org/software/PMI).
Algorithms ; Cell Line ; Chondrocytes/cytology/metabolism/microbiology ; Databases, Genetic ; Gene Expression Profiling ; Host-Pathogen Interactions ; Humans ; Keratinocytes/cytology/metabolism/microbiology ; *Models, Genetic ; Mycoplasma/genetics/metabolism ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo investigate the co-culture of keratinocytes with Malassezia isolates which cause the pityriasis versicolor with different color and to analyze the changes of cytokines associated with melanogenesis.

METHODSThe effects of Malassezia species with different proportions on the growth rate of keratinocytes was assessed with 5 g/L methyl thiazolyl tetrazolium (MTT). Co-culture of keratinocytes and Malassezia species were performed with isolates from hyer- and hypo-pigmentation areas of pityriasis versicolor. The supernatants were collected at different time points, and the changes of basic fibroblast growth factor (b-FGF), endothelin-1 (ET-1), nerve growth factor-beta (NGF-beta), interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), stem cell factor (SCF) were recorded. Three control groups were established accordingly.

RESULTSWhen the ratio between keratinocytes and Malassezia species was lower than 1: 10, the growth rate of keratinocytes was not affected by Malassezia (P > 0.05). When the ratio was increased above 1:20, the growth rate of keratinocytes was significantly inhibited by Malassezia (P < 0.01). The secretions of IL-1alpha, IL-6, TNF-alpha, and ET-1 was significantly increased after the co-culture of keratinocytes and Malassezia (P < 0.01), while those of b-FGF, NGF-beta, and SCF had no significant changes (P > 0.05). Compared with the isolates from the hypo-pigmentation area, ET-1 induced by isolate from hyperpigmentation area significantly increased (P < 0.01).

CONCLUSIONWhen Malassezia isolates are co-cultured with keratinocytes, the secretions of cytokines associated with melanogenesis may differ from each other. ET-1 may play certain role in the hyper-pigmentation of pityriasis versicolor.

Cell Proliferation ; Cells, Cultured ; Cytokines ; biosynthesis ; Humans ; Keratinocytes ; cytology ; metabolism ; microbiology ; Malassezia ; isolation & purification ; physiology ; Melanins ; biosynthesis ; Tinea Versicolor ; microbiology

Staphylococcus aureus may perform an crucial function in atopic dermatitis (AD), via the secretion of superantigens, including staphylococcal enterotoxins (SE) A or B, and toxic shock syndrome toxin-1 (TSST-1). Dysregulated cytokine production by keratinocytes (KCs) upon exposure to staphylococcal superantigens (SsAgs) may be principally involved in the pathophysiology of AD. We hypothesized that lesional KCs from AD may react differently to SsAgs compared to nonlesional skin or normal skin from nonatopics. We conducted a comparison of HLA-DR or CD1a expression in lesional skin as opposed to that in nonlesional or normal skin by immunohistochemistry (IHC). We also compared, using ELISA, the levels of IL-1alpha, IL-1beta, and TNF-alpha secreted by cultured KCs from lesional, nonlesional, and normal skin, after the addition of SEA, SEB and TSST-1. IHC revealed that both HLA-DR and CD1a expression increased significantly in the epidermis of lesional skin versus nonlesional or normal skin in quite a similar manner. IL-1alpha, IL-1beta, and TNF-alpha secretion was also significantly elevated in the cultured KCs from lesional skin after the addition of SsAgs. Our results indicated that KCs from lesional skin appear to react differently to SsAgs and increased proinflammatory cytokine production in response to SsAgs may contribute to the pathogenesis of AD.
Tumor Necrosis Factor-alpha/biosynthesis/genetics ; *Superantigens/administration & dosage/immunology ; Staphylococcus aureus/*immunology/pathogenicity ; Male ; Keratinocytes/immunology/*microbiology ; Interleukin-1/biosynthesis/genetics ; Inflammation Mediators/metabolism ; Humans ; HLA-DR Antigens/metabolism ; Enterotoxins/administration & dosage/immunology ; Dermatitis, Atopic/etiology/immunology/*microbiology ; DNA, Complementary/genetics ; Case-Control Studies ; Base Sequence ; Bacterial Toxins/administration & dosage/immunology ; Antigens, CD1/metabolism ; Adult