Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

ABSTRACTTo protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either theCAMP,S100A8, andS100A9open reading frames,A8-IRES-A9(fusion sequence), orA8-nIRES-A9(fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion byListeriaandSalmonellafor up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.

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Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

Infection and Immunity ◽

10.1128/iai.00282-15 ◽

2015 ◽

Vol 83 (7) ◽

pp. 2614-2626 ◽

Cited By ~ 35

Author(s):

Rohitashw Kumar ◽

Darpan Saraswat ◽

Swetha Tati ◽

Mira Edgerton

Keyword(s):

Candida Albicans ◽

Epithelial Cells ◽

Oral Candidiasis ◽

Oral Microbiome ◽

Proteinase Activity ◽

Adhesion Properties ◽

Content Type ◽

Oral Epithelial Cells ◽

Oral Epithelial ◽

Cell Cell

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually withC. albicanscells overexpressing Sap6 (SAP6OE and a Δsap8strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6strain was attenuated. These hypervirulent strains had highly aggregative colony structurein vitroand higher secreted proteinase activity; however, the levels of proteinase activity ofC. albicansSaps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6OE and Δsap8cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increasedC. albicansadhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

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Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300099 ◽

2003 ◽

Vol 30 (2) ◽

pp. 99-108 ◽

Cited By ~ 16

Author(s):

G Aguilera ◽

S Volpi ◽

C Rabadan-Diehl

Keyword(s):

Transcriptional Activation ◽

Receptor Gene ◽

Mrna Translation ◽

Open Reading Frames ◽

Entry Site ◽

Internal Ribosome Entry ◽

Protein Levels ◽

Nuclear Extracts ◽

V1b Receptor ◽

Vasopressin Receptors

The number of V1b vasopressin receptors (V1bR) in the anterior pituitary plays an important role during adaptation of the hypothalamic-pituitary-adrenal axis to stress in rats. Regulation of V1bR expression involves transcriptional and translational mechanisms. One of the elements mediating transcriptional activation of the rat V1bR gene is a long stretch of GAGA repeats (GAGA box) in the promoter located near the transcription start point capable of binding a protein complex of 127 kDa present in pituitary nuclear extracts. There is a lack of correlation between changes in V1bR mRNA and the number of VP binding sites, suggesting that V1bR expression depends on the efficiency of V1b R mRNA translation into protein. Two mechanisms by which the 5' untranslated region (5'-UTR) of the rat V1bR mRNA can mediate either inhibition or activation of V1bR mRNA translation have been identified. First, upstream open reading frames (ORF) present in the 5'-UTR repress translation of the major ORF encoding the V1b receptor, and secondly, an internal ribosome entry site (IRES) activates V1bR translation. Stimulation of IRES activity through protein kinase C-mediated pathways results in V1bR mRNA translation increasing V1bR protein levels. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

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Correction: Activation of Notch-1 in oral epithelial cells by P. gingivalis triggers the expression of the antimicrobial protein PLA2-IIA

Mucosal Immunology ◽

10.1038/s41385-019-0152-6 ◽

2019 ◽

Vol 12 (4) ◽

pp. 1066-1066

Author(s):

Ahmad Al-Attar ◽

Yelena Alimova ◽

Sreenatha Kirakodu ◽

Anastasia Kozal ◽

Michael John Novak ◽

...

Keyword(s):

Epithelial Cells ◽

Antimicrobial Protein ◽

Notch 1 ◽

Oral Epithelial Cells ◽

Oral Epithelial

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Regulation of the Peptidoglycan Amidase PGLYRP2 in Epithelial Cells by Interleukin-36γ

Infection and Immunity ◽

10.1128/iai.00384-18 ◽

2018 ◽

Vol 86 (9) ◽

Cited By ~ 5

Author(s):

Glen M. Scholz ◽

Jacqueline E. Heath ◽

Jiamin Aw ◽

Eric C. Reynolds

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Bacterial Pathogen ◽

Oral Bacteria ◽

Oral Epithelium ◽

Content Type ◽

Oral Epithelial Cells ◽

Mitogen Activated Protein ◽

Mucosal Homeostasis ◽

Oral Epithelial

ABSTRACT Interleukin-36 (IL-36) cytokines are important regulators of mucosal homeostasis and inflammation. We have previously established that oral epithelial cells upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis. Here, we have established that IL-36γ can stimulate the gene expression of mechanistically distinct antimicrobial proteins, including the peptidoglycan amidase PGLYRP2, in oral epithelial cells (e.g., TIGK cells). PGLYRP2 gene expression was not stimulated by either IL-17 or IL-22, thus demonstrating selectivity in the regulation of PGLYRP2 by IL-36γ. The IL-36γ-inducible expression of PGLYRP2 was shown to be mediated by IRAK1- and p38 mitogen-activated protein (MAP) kinase-dependent signaling. Furthermore, our finding that IL-36γ-inducible PGLYRP2 expression was reduced in proliferating TIGK cells but increased in terminally differentiating cells suggests that control of PGLYRP2 expression is associated with the maturation of the oral epithelium. PGLYRP2 expression in TIGK cells can also be directly stimulated by oral bacteria. However, the extracellular gingipain proteases (Kgp and RgpA/B) produced by P. gingivalis, which are critical virulence factors, can antagonize PGLYRP2 expression. Thus, the expression of IL-36γ by oral epithelial cells in response to P. gingivalis might enable the subsequent autocrine stimulation of PGLYRP2 expression. In summary, our data identify how IL-36γ may promote oral mucosal homeostasis by regulating PGLYRP2 expression.

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Candida albicans Cell Wall Glycosylation May Be Indirectly Required for Activation of Epithelial Cell Proinflammatory Responses

Infection and Immunity ◽

10.1128/iai.05591-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 4902-4911 ◽

Cited By ~ 34

Author(s):

Celia Murciano ◽

David L. Moyes ◽

Manohursingh Runglall ◽

Ayesha Islam ◽

Celine Mille ◽

...

Keyword(s):

Cell Wall ◽

Epithelial Cells ◽

Proinflammatory Cytokine ◽

Cytokine Production ◽

Cell Damage ◽

Proinflammatory Cytokine Production ◽

Content Type ◽

Oral Epithelial Cells ◽

Hypha Formation ◽

Oral Epithelial

ABSTRACTOral epithelial cells discriminate between the yeast and hyphal forms ofCandida albicansvia the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether β-glucan andN- orO-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of β-glucan andN- andO-mannan mutants, we found thatN-mannosylation (via Δoch1and Δpmr1mutants) andO-mannosylation (via Δpmt1and Δmnt1Δmnt2mutants), but not phosphomannan (via a Δmnn4mutant) or β-1,2 mannosylation (via Δbmt1to Δbmt6mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, theN- andO-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the α-1,6-mannose backbone of theN-linked outer chain (corresponding to a Δmnn9mutant) may be required for epithelial adhesion, while the α-1,2-mannose component of phospholipomannan (corresponding to a Δmit1mutant) may contribute to epithelial cell damage. β-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary,N- andO-mannosylation defects affect the ability ofC. albicansto induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.

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Activation of Notch-1 in oral epithelial cells by P. gingivalis triggers the expression of the antimicrobial protein PLA2-IIA

Mucosal Immunology ◽

10.1038/s41385-018-0014-7 ◽

2018 ◽

Vol 11 (4) ◽

pp. 1047-1059 ◽

Cited By ~ 9

Author(s):

Ahmad Al-Attar ◽

Yelena Alimova ◽

Sreenatha Kirakodu ◽

Anastasia Kozal ◽

Michael John Novak ◽

...

Keyword(s):

Epithelial Cells ◽

Antimicrobial Protein ◽

Notch 1 ◽

Oral Epithelial Cells ◽

Oral Epithelial

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Interleukin-6 (IL-6) and IL-8 Are Induced in Human Oral Epithelial Cells in Response to Exposure to PeriodontopathicEikenella corrodens

Infection and Immunity ◽

10.1128/iai.67.1.384-394.1999 ◽

1999 ◽

Vol 67 (1) ◽

pp. 384-394 ◽

Cited By ~ 53

Author(s):

Hiromichi Yumoto ◽

Hideaki Nakae ◽

Keiko Fujinaka ◽

Shigeyuki Ebisu ◽

Takashi Matsuo

Keyword(s):

Epithelial Cells ◽

Bacterial Colonization ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Kb Cells ◽

Protein Levels ◽

Periodontal Tissues ◽

Oral Epithelial Cells ◽

Oral Epithelial ◽

Stimulation Of

ABSTRACT Periodontitis is the inflammatory response in periodontal tissues elicited by bacterial colonization in periodontal pockets. In this response, pocket epithelial cells are the first cells to come into contact with bacteria. To elucidate this mechanism, we determined the adherence of the periodontopathic bacterium Eikenella corrodens 1073, which has a GalNAc-sensitive lectin-like adhesin (EcLS), to a human oral epithelial carcinoma cell line (KB) and the induction of proinflammatory cytokine production in the cells following exposure to this bacterium in vitro. In the adherence assay, EcLS played a role as the adhesin of this bacterium in adherence to KB cells. In a reverse transcriptase PCR, significant interleukin-8 (IL-8) and IL-6 mRNA levels were induced in response to exposure to this bacterium. In an enzyme-linked immunosorbent assay after an 8-h bacterial exposure, the IL-8 and IL-6 protein levels were 13.5- and 8.3-fold higher than those in the nonexposed controls, respectively. These protein responses were time dependent. Interestingly, whenE. corrodens was separated from KB cells by cell culture inserts, a slight stimulation of the IL-6 and IL-8 mRNA and secreted protein levels was seen. These results imply that the direct contact ofE. corrodens 1073 with oral epithelial cells is not necessarily required for the stimulation of IL-6 and IL-8 secretion. We suggest that E. corrodens induces the epithelial cells to secrete proinflammatory cytokines which serve as an early signaling system to host immune and inflammatory cells in underlying connective tissues.

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Oral Epithelial Cells Distinguish between Candida Species with High or Low Pathogenic Potential through MicroRNA Regulation

mSystems ◽

10.1128/msystems.00163-21 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Márton Horváth ◽

Gábor Nagy ◽

Nóra Zsindely ◽

László Bodai ◽

Péter Horváth ◽

...

Keyword(s):

Immune Response ◽

Epithelial Cell ◽

Epithelial Cells ◽

Pathogenic Potential ◽

Content Type ◽

Oral Epithelial Cells ◽

Cell Responses ◽

Regulatory Processes ◽

Species Specific ◽

Oral Epithelial

ABSTRACT Oral epithelial cells monitor microbiome composition and initiate immune response upon dysbiosis, as in the case of Candida imbalances. Candida species, such as C. albicans and C. parapsilosis, are the most prevalent yeasts in the oral cavity. Comparison of healthy oral epithelial cell responses revealed that while C. albicans infection robustly activated inflammation cascades, C. parapsilosis primarily activated various inflammation-independent pathways. In posttranscriptional regulatory processes, several miRNAs were altered by both species. For C. parapsilosis, the dose of yeast cells directly correlated with changes in transcriptomic responses with higher fungal burdens inducing significantly different and broader changes. MicroRNAs (miRNAs) associated with carbohydrate metabolism-, hypoxia-, and vascular development-related responses dominated with C. parapsilosis infection, whereas C. albicans altered miRNAs linked to inflammatory responses. Subsequent analyses of hypoxia-inducible factor 1α (HIF1-α) and hepatic stellate cell (HSC) activation pathways predicted target genes through which miRNA-dependent regulation of yeast-specific functions may occur, which also supported the observed species-specific responses. Our findings suggest that C. parapsilosis is recognized as a commensal at low doses by the oral epithelium; however, increased fungal burden activates different pathways, some of which overlap with the inflammatory processes robustly induced by C. albicans. IMPORTANCE A relatively new topic within the field of immunology involves the role of miRNAs in innate as well as adaptive immune response regulation. In recent years, posttranscriptional regulation of host-pathogenic fungal interactions through miRNAs was also suggested. Our study reveals that the distinct nature of human oral epithelial cell responses toward C. parapsilosis and C. albicans is possibly due to species-specific fine-tuning of host miRNA regulatory processes. The findings of this study also shed new light on the nature of early host cell transcriptional responses to the presence of C. parapsilosis and highlight the species’ potential inflammation-independent host activation processes. These findings contribute to our better understanding of how miRNA deregulation at the oral immunological barrier, in noncanonical immune cells, may discriminate between fungal species, particularly Candida species with high or low pathogenic potential.

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