scholarly journals MicroRNAs Regulate Cytokine Responses in Gingival Epithelial Cells

2016 ◽  
Vol 84 (12) ◽  
pp. 3282-3289 ◽  
Author(s):  
Steven C. Y. Chen ◽  
Christos Constantinides ◽  
Moritz Kebschull ◽  
Panos N. Papapanou
Keyword(s):  
Epithelial Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Periodontal Pathogens ◽  
Loss Of Function ◽  
Microbial Infection ◽  
Western Blot Assay ◽  
Content Type ◽  
Protein Levels ◽  
Gingival Epithelial Cells ◽  
Protease Deficient

MicroRNAs (miRNAs) have been established as key regulators of various biological processes with possible involvement in the pathobiology of periodontal disease. Expanding our earlier observations of substantial differential expression of specific miRNAs between clinically healthy and periodontitis-affected gingival tissues, we used miRNA inhibitors (sponges) in loss-of-function experiments to investigate the involvement of specific miRNAs in the response of pocket epithelium-derived, telomerase-immortalized human gingival keratinocytes (TIGKs) to microbial infection. We constructed stable knockdown (KD) cell lines for five epithelium-expressed miRNAs (miR-126, miR-141, miR-155, miR-210, and miR-1246) and assessed their response to infection with periodontal pathogens using microarray analysis, quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot assay. miR-126 KD cells showed lower expression of interleukin 8 (IL-8) and CXCL1, both on the mRNA and protein levels, than did controls upon stimulation by heat-killed wild-typePorphyromonas gingivalis, liveP. gingivalisprotease-deficient mutant KDP128, and liveAggregatibacter actinomycetemcomitans. In contrast, infection of miR-155 KD and miR-210 KD cells with the same organisms resulted in higher IL-8 and CXCL1 mRNA and protein expression. These effects appeared to be regulated by NF-κB, as suggested by altered transcription and/or phosphorylation status of components of the NF-κB system. Reduced neutrophil-like HL-60 cell chemotactic activity was observed in response to infection of miR-126 KD cells, indicating that miR-126 plays an important role in immune responses. Our findings indicate that specific miRNAs regulate the expression of inflammatory cytokines in human gingival epithelial cells in response to microbial infection.

scholarly journals Uptake of Nanotitania by Gingival Epithelial Cells Promotes Inflammatory Response and Is Accelerated by Porphyromonas gingivalis Lipopolysaccharide: An In Vitro Study

2021 ◽  
Vol 22 (15) ◽  
pp. 8084
Author(s):  
Shiho Sugawara ◽  
Taichi Ishikawa ◽  
Shu Sato ◽  
Hidemichi Kihara ◽  
Masayuki Taira ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Cell Model ◽  
Quantitative Polymerase Chain Reaction ◽  
High Rate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Periodontal Pathogens ◽  
Gingival Epithelial Cells

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonas gingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

scholarly journals Shiga Toxin 2 Reduces Complement Inhibitor CD59 Expression on Human Renal Tubular Epithelial and Glomerular Endothelial Cells

2013 ◽  
Vol 81 (8) ◽  
pp. 2678-2685 ◽  
Author(s):  
Silvia Ehrlenbach ◽  
Alejandra Rosales ◽  
Wilfried Posch ◽  
Doris Wilflingseder ◽  
Martin Hermann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Shiga Toxin ◽  
Human Kidney ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Protein Levels ◽  
Glomerular Endothelial Cells ◽  
Shiga Toxin 2 ◽  
Renal Tubular

ABSTRACTInfections with enterohemorrhagicEscherichia coli(EHEC) are a primary cause of hemolytic-uremic syndrome (HUS). Recently, Shiga toxin 2 (Stx2), the major virulence factor of EHEC, was reported to interact with complement, implying that the latter is involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate the effect of Stx2 on the expression of membrane-bound complement regulators CD46, CD55, and CD59 on proximal tubular epithelial (HK-2) and glomerular endothelial (GEnC) cells derived from human kidney cells that are involved in HUS. Incubation with Stx2 did not influence the amount of CD46 or CD55 on the surface of HK-2 and GEnC cells, as determined by fluorescence-activated cell sorter analysis. In contrast, CD59 was significantly reduced by half on GEnC cells, but the reduction on HK-2 cells was less pronounced. With increasing amounts of Stx2, reduction of CD59 also reached significance in HK-2 cells. Enzyme-linked immunosorbent assay analyses showed that CD59 was not present in the supernatant of Stx2-treated cells, implying that CD59 reduction was not caused by cleavage from the cell surface. In fact, reverse transcription-quantitative PCR analyses showed downregulation of CD59 mRNA as the likely reason for CD59 cell surface reduction. In addition, a significant increase in terminal complement complex deposition on HK-2 cells was observed after treatment with Stx2, as a possible consequence of CD59 downregulation. In summary, Stx2 downregulates CD59 mRNA and protein levels on tubular epithelial and glomerular endothelial cells, and this downregulation likely contributes to complement activation and kidney destruction in EHEC-associated HUS.

scholarly journals Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

2013 ◽  
Vol 81 (11) ◽  
pp. 3975-3983 ◽  
Author(s):  
Xianqiong Zou ◽  
Brent S. Sorenson ◽  
Karen F. Ross ◽  
Mark C. Herzberg
Keyword(s):  
Epithelial Cells ◽  
Open Reading Frames ◽  
Antimicrobial Protein ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Content Type ◽  
Protein Levels ◽  
Oral Epithelial Cells ◽  
Mrna Transfection ◽  
Oral Epithelial

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.

scholarly journals Mobilization of TLR4 Into Lipid Rafts by Aggregatibacter Actinomycetemcomitans in Gingival Epithelial Cells

2016 ◽  
Vol 39 (5) ◽  
pp. 1777-1786 ◽  
Author(s):  
Haruka Imai ◽  
Tsuyoshi Fujita ◽  
Mikihito Kajiya ◽  
Kazuhisa Ouhara ◽  
Tetsuya Yoshimoto ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Map Kinase ◽  
Lipid Rafts ◽  
Inflammatory Cytokines ◽  
Aggregatibacter Actinomycetemcomitans ◽  
P38 Map Kinase ◽  
Mrna Levels ◽  
Periodontal Pathogens ◽  
Gingival Epithelial Cells ◽  
Tnf Α

Background: An investigation of the mechanisms underlying the production of inflammatory cytokines through the stimulation of microorganisms on gingival epithelial cells may provide insights into the pathogenesis of the initiation of periodontitis. Lipid rafts, microdomains in the cell membrane, include a large number of receptors, and are centrally involved in signal transduction. We herein examined the involvement of lipid rafts in the expression of interleukin (IL-6) and IL-8 in gingival epithelial cells stimulated by periodontal pathogens. Methods: OBA9, a human gingival cell line, was stimulated by Aggregatibacter actinomycetemcomitans or tumor necrosis factor (TNF)-α in the presence of methyl-β-cyclodextrin (MβCD). Results: A. actinomycetemcomitans or TNF-α increased IL-8 and IL-6 mRNA levels, and promoted the phosphorylation of ERK and p38 MAP kinase in OBA9. The pretreatment with MβCD abolished increases in IL-6 and IL-8 mRNA levels and the phosphorylation induced by A. actinomycetemcomitans, but did not suppress the response induced by TNF-α. The transfection of TLR4 inhibited A. actinomycetemcomitans-induced increases in IL-8 and IL-6 mRNA levels. Confocal microscopy revealed that MβCD inhibited the mobilization of TLR4 into lipid rafts. Conclusion: The mobilization of TLR4 into lipid rafts is involved in the expression of inflammatory cytokines and phosphorylation of MAP kinase in human gingival epithelial cells stimulated by A. actinomycetemcomitans.

scholarly journals Interleukin-8 and Intercellular Adhesion Molecule 1 Regulation in Oral Epithelial Cells by Selected Periodontal Bacteria: Multiple Effects of Porphyromonas gingivalis via Antagonistic Mechanisms

2001 ◽  
Vol 69 (3) ◽  
pp. 1364-1372 ◽  
Author(s):  
George T.-J. Huang ◽  
Daniel Kim ◽  
Jonathan K.-H. Lee ◽  
Howard K. Kuramitsu ◽  
Susan Kinder Haake
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Adhesion Molecule ◽  
Intercellular Adhesion ◽  
Interleukin 8 ◽  
Intercellular Adhesion Molecule ◽  
Mrna Levels ◽  
Periodontal Pathogens ◽  
Intercellular Adhesion Molecule 1 ◽  
Gingival Epithelial Cells

ABSTRACT Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalisor its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis andF. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.

scholarly journals Bronchial Epithelial Tet2 Maintains Epithelial Integrity during Acute Pseudomonas aeruginosa Pneumonia

2020 ◽  
Vol 89 (1) ◽  
pp. e00603-20
Author(s):  
Wanhai Qin ◽  
Xanthe Brands ◽  
Cornelis van't Veer ◽  
Alex F. de Vos ◽  
Brendon P. Scicluna ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cell ◽  
Mrna Levels ◽  
Wild Type ◽  
Bronchial Epithelial ◽  
Epithelial Integrity ◽  
Content Type ◽  
Protein Levels

ABSTRACTRespiratory epithelial cells are important for pulmonary innate immune responses during Pseudomonas aeruginosa infection. Tet methylcytosine dioxygenase 2 (Tet2) has been implicated in the regulation of host defense by myeloid and lymphoid cells, but whether Tet2 also contributes to epithelial responses during pneumonia is unknown. The aim of this study was to investigate the role of bronchial epithelial Tet2 in acute pneumonia caused by P. aeruginosa. To this end, we crossed mice with Tet2 flanked by two Lox-P sites (Tet2fl/fl mice) with mice expressing Cre recombinase under the bronchial epithelial cell-specific Cc10 promoter (Cc10Cre mice) to generate bronchial epithelial cell-specific Tet2-deficient (Tet2fl/fl Cc10Cre) mice. Six hours after infection with P. aeruginosa,Tet2fl/fl Cc10Cre and wild-type mice had similar bacterial loads in bronchoalveolar lavage fluid (BALF). At this time point, Tet2fl/fl Cc10Cre mice displayed reduced mRNA levels of the chemokines Cxcl1, Cxcl2, and Ccl20 in bronchial brushes. However, Cxcl1, Cxcl2, and Ccl20 protein levels and leukocyte recruitment in BALF were not different between groups. Tet2fl/fl Cc10Cre mice had increased protein levels in BALF after infection, indicating a disturbed epithelial barrier function, which was corroborated by reduced mRNA expression of tight junction protein 1 and occludin in bronchial brushes. Differences detected between Tet2fl/fl Cc10Cre and wild-type mice were no longer present at 24 h after infection. These results suggest that bronchial epithelial Tet2 contributes to maintaining epithelial integrity by enhancing intracellular connections between epithelial cells during the early phase of P. aeruginosa pneumonia.

scholarly journals Porphyromonas gingivalis Induction of MicroRNA-203 Expression Controls Suppressor of Cytokine Signaling 3 in Gingival Epithelial Cells

2011 ◽  
Vol 79 (7) ◽  
pp. 2632-2637 ◽  
Author(s):  
Catherine E. Moffatt ◽  
Richard J. Lamont
Keyword(s):  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Luciferase Assay ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Suppressor Of Cytokine Signaling ◽  
Content Type ◽  
Downstream Target ◽  
Qrt Pcr ◽  
Gingival Epithelial Cells

ABSTRACTPorphyromonas gingivalisis a pathogen in severe periodontal disease. Able to exploit an intracellular lifestyle within primary gingival epithelial cells (GECs), a reservoir ofP. gingivaliscan persist within the gingival epithelia. This process is facilitated by manipulation of the host cell signal transduction cascades which can impact cell cycle, cell death, and cytokine responses. Using microarrays, we investigated the ability ofP. gingivalis33277 to regulate microRNA (miRNA) expression in GECs. One of several miRNAs differentially regulated by GECs in the presence ofP. gingivaliswas miRNA-203 (miR-203), which was upregulated 4-fold compared to uninfected controls. Differential regulation of miR-203 was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Putative targets of miR-203, suppressor of cytokine signaling 3 (SOCS3) and SOCS6, were evaluated by qRT-PCR. SOCS3 and SOCS6 mRNA levels were reduced >5-fold and >2-fold, respectively, inP. gingivalis-infected GECs compared to controls. Silencing of miR-203 using a small interfering RNA construct reversed the inhibition of SOCS3 expression. A dual luciferase assay confirmed binding of miR-203 to the putative target binding site of the SOCS3 3′ untranslated region. Western blot analysis demonstrated that activation of signal transducer and activator of transcription 3 (Stat3), a downstream target of SOCS, was diminished following miR-203 silencing. This study shows that induction of miRNAs byP. gingivaliscan modulate important host signaling responses.

Electronic cigarette liquid substances propylene glycol and vegetable glycerin induce an inflammatory response in gingival epithelial cells

2020 ◽  
Vol 40 (1) ◽  
pp. 25-34
Author(s):  
A Beklen ◽  
D Uckan
Keyword(s):  
Epithelial Cells ◽  
Propylene Glycol ◽  
Western Blotting ◽  
Electronic Cigarettes ◽  
Periodontal Diseases ◽  
Biological Response ◽  
Electronic Cigarette ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gingival Epithelial Cells

Information on the effects of propylene glycol (PG) and vegetable glycerin (VG) and on cytotoxicity and subsequent activation of the biological mediators is limited in periodontal diseases. This study analyzes the effect of unflavored PG/VG alone or in combination with nicotine on gingival epithelial cells. The cells were exposed to different PG/VG (± nicotine) concentrations for 24 h and cytotoxicity was evaluated by calorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid assay. The expressions of interleukin (IL)-6, IL-8, and matrix metalloproteinases (MMPs)-9 were measured using an enzyme-linked immunosorbent assay and a western blotting. Stimulation with PG/VG mixtures reduced cell viability compared to nonexposed controls ( p < 0.05). Adding PG/VG increased the levels of IL-6, IL-8, and MMP-9, and the amount of PG had more biological impact compared to the VG amount. The nicotine augmented this effect compared to its nicotine-free counterparts. In western blotting result, MMP-9 was clearly activated in almost all samples. These findings suggest that the main constituents PG/VG are cytotoxic and able to induce biological response in gingival cells in vitro. Despite being advertised as less harmful than conventional cigarettes, electronic cigarette liquid pose certain risks on periodontal cells. Awareness about the effects of electronic cigarettes on periodontal diseases must be increased.

scholarly journals A Natural Vibrio parahaemolyticus ΔpirAVppirBVp+ Mutant Kills Shrimp but Produces neither PirVp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Kornsunee Phiwsaiya ◽  
Walaiporn Charoensapsri ◽  
Suwimon Taengphu ◽  
Ha T. Dong ◽  
Pakkakul Sangsuriya ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Vibrio Parahaemolyticus ◽  
Gene Sequence ◽  
Culture Broth ◽  
Upstream Region ◽  
Enzyme Linked Immunosorbent Assay ◽  
Content Type ◽  
Early Mortality Syndrome ◽  
Reverse Transcription Pcr ◽  
Acute Hepatopancreatic Necrosis Disease

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp . These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp . Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions. IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp . The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND. Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND. Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

scholarly journals Mucosal Immunization with a Staphylococcus aureus IsdA-Cholera Toxin A2/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice

2011 ◽  
Vol 18 (9) ◽  
pp. 1543-1551 ◽  
Author(s):  
Britni M. Arlian ◽  
Juliette K. Tinker
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Cholera Toxin ◽  
Cellular Proliferation ◽  
Mucosal Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mucosal Immunization ◽  
Content Type ◽  
Human Nasal Epithelial Cells

ABSTRACTStaphylococcus aureusis a leading cause of opportunistic infection worldwide and a significant public health threat. The iron-regulated surface determinant A (IsdA) adhesin is essential forS. aureuscolonization on human nasal epithelial cells and plays an important role in iron acquisition and resistance to human skin defenses. Here we investigated the murine immune response to intranasal administration of a cholera toxin A2/B (CTA2/B) chimera containing IsdA. Plasmids were constructed to express the IsdA-CTA2/B chimera and control proteins inEscherichia coli. Proper construction of the chimera was verified by SDS-PAGE, Western blotting, GM1 enzyme-linked immunosorbent assay (ELISA), and confocal microscopy. Groups of female BALB/c mice were mock immunized or immunized with IsdA-CTA2/B, IsdA mixed with CTA2/B, or IsdA alone, followed by one booster immunization at 10 days postpriming. Analysis of serum IgG and nasal, intestinal, and vaginal IgA suggested that mucosal immunization with IsdA-CTA2/B induces significant IsdA-specific humoral immunity. Functionalin vitroassays revealed that immune serum significantly blocks the adherence ofS. aureusto human epithelial cells. Splenocytes from mice immunized with IsdA-CTA2/B showed specific cellular proliferation and production of interleukin-4 (IL-4) afterin vitrostimulation. Immunization with IsdA-CTA2/B drove isotype switching to IgG1, indicative of a Th2-type response. Our results suggest that the immunogenicity of theS. aureusIsdA-CTA2/B chimera merits further investigation as a potential mucosal vaccine candidate.

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