RESUMEN
Dental plaque, a highly structured polymicrobial biofilm, persistently forms in the oral cavity and is a common problem affecting oral health. The role of oral defense factors in either collaborating or disrupting host-microbiome interactions remains insufficiently elucidated. This study aims to explore the role of LL-37, a critical antimicrobial peptide in the oral cavity, in dental plaque formation. Through immunostaining dental plaque specimens, we observed that LL-37 and DNA colocalized in the samples, appearing as condensed clusters. In vitro experiments revealed that LL-37 binds rapidly to oral bacterial DNA, forming high molecular weight, DNase-resistant complexes. This interaction results in LL-37 losing its inherent antibacterial activity. Further, upon the addition of LL-37, we observed a visible increase in the precipitation of bacterial DNA. We also discovered a significant correlation between the levels of the DNA-LL-37 complex and LL-37 within dental plaque specimens, demonstrating the ubiquity of the complex within the biofilm. By using immunostaining on dental plaque specimens, we could determine that the DNA-LL-37 complex was present as condensed clusters and small bacterial cell-like structures. This suggests that LL-37 immediately associates with the released bacterial DNA to form complexes that subsequently diffuse. We also demonstrated that the complexes exhibited similar Toll-like receptor 9-stimulating activities across different bacterial species, including Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus salivarius. However, these complexes prompted dissimilar activities, such as the production of IL-1ß in monocytic cells via both NLRP3 pathway-dependent and pathway-independent mechanisms. This study, therefore, reveals the adverse role of LL-37 in dental plaque, where it binds bacterial DNA to form complexes that may precipitate to behave like an extracellular matrix. Furthermore, the unveiled stimulating properties and species-dependent activities of the oral bacterial DNA-LL-37 complexes enrich our understanding of dental plaque pathogenicity and periodontal innate immune responses.
Asunto(s)
Placa Dental , Humanos , ADN Bacteriano , Placa Dental/microbiología , Porphyromonas gingivalis/genética , Fusobacterium nucleatum , ADNRESUMEN
The antimicrobial peptide LL-37 is known to have a potent LPS-neutralizing activity in monocytes and macrophages. Recently, LL-37 in gingival crevicular fluids is suggested to be the major protective factor preventing infection of periodontogenic pathogens. In this study, we tried to address the effect of LL-37 on proinflammatory responses of human gingival fibroblasts (HGFs) stimulated with Toll-like receptor (TLR)-stimulant microbial compounds. LL-37 potently suppressed LPS-induced gene expression of IL6, IL8 and CXCL10 and intracellular signaling events, degradation of IRAK-1 and IkappaBalpha and phosphorylation of p38 MAPK and IRF3, indicating that the LPS-neutralizing activity is also exerted in HGFs. LL-37 also suppressed the expression of IL6, IL8 and CXCL10 induced by the TLR3 ligand poly(I:C). LL-37 modestly attenuated the expression of IL6 and IL8 induced by the TLR2/TLR1 ligand Pam(3)CSK(4), but did not affect the expression induced by the TLR2/TLR6 ligand MALP-2. Interestingly, LL-37 rather upregulated the expression of IL6, IL8 and CXCL10 induced by another TLR2/TLR6 ligand FSL-1. Thus, the regulatory effect of LL-37 is differently exerted towards proinflammatory responses of HGFs induced by different microbial stimuli, which may lead to unbalanced proinflammatory responses of the gingival tissue to infection of oral microbes.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Quimiocina CXCL10/biosíntesis , Fibroblastos/efectos de los fármacos , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Infecciones Bacterianas/inmunología , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Fibroblastos/inmunología , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Encía/patología , Humanos , Inmunidad Innata , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ligandos , Enfermedades Periodontales/inmunología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , CatelicidinasRESUMEN
BACKGROUND: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. METHODS AND RESULTS: We performed experimental cutaneous wounding in mice with a deficiency of alpha(2)-antiplasmin (alpha(2)AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in alpha(2)AP-deficient (alpha(2)AP-/-) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in alpha(2)AP-/- mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in alpha(2)AP-/- mice than in WT mice. In addition, the amount of released-VEGF from the explanted fibroblasts of alpha(2)AP-/- mice increased dramatically more than in the WT mice. Finally, the intra-jugular administration of anti-VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of alpha(2)AP-/- mice. CONCLUSION: The lack of alpha(2)AP markedly causes an over-release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated-wound closure. CONCLUSIONS: This is the first report to describe the crucial role that alpha(2)AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of alpha(2)AP on cutaneous wound healing.
Asunto(s)
Neovascularización Fisiológica/fisiología , Piel/lesiones , Factor A de Crecimiento Endotelial Vascular/fisiología , Cicatrización de Heridas/fisiología , alfa 2-Antiplasmina/deficiencia , Animales , Ratones , Ratones Noqueados , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/genética , Heridas y Lesiones/patologíaRESUMEN
Chronic periodontitis is correlated with Porphyromonas gingivalis infection. In this study, we found that the expression of secretory leucocyte protease inhibitor (SLPI), an endogenous inhibitor for neutrophil-derived proteases, was reduced in gingival tissues with chronic periodontitis associated with P. gingivalis infection. The addition of vesicles of P. gingivalis decreased the amount of SLPI in the media of primary human gingival keratinocytes compared to untreated cultures. We therefore investigated how arginine-specific gingipains (Rgps) affect the functions of SLPI, because Rgps are the major virulence factors in the vesicles and cleave a wide range of in-host proteins. We found that Rgps digest SLPI in vitro, suppressing the release of SLPI. Rgps proteolysis of SLPI disrupted SLPI functions, which normally suppresses neutrophil elastase and neutralizes pro-inflammatory effects of bacterial cell wall compounds in cultured human gingival fibroblasts. The protease inhibitory action of SLPI was not exerted towards Rgps. These results suggest that Rgps reduce the protective effects of SLPI on neutrophil proteases and bacterial proinflammatory compounds, by which disease in gingival tissue may be accelerated at the sites with P. gingivalis infection.
Asunto(s)
Adhesinas Bacterianas/farmacología , Cisteína Endopeptidasas/farmacología , Encía/metabolismo , Líquido del Surco Gingival/metabolismo , Periodontitis/metabolismo , Porphyromonas gingivalis/fisiología , Proteínas/metabolismo , Adhesinas Bacterianas/análisis , Células Cultivadas , Enfermedad Crónica , Cisteína Endopeptidasas/análisis , Progresión de la Enfermedad , Electroforesis en Gel de Poliacrilamida , Cisteína-Endopeptidasas Gingipaínas , Encía/inmunología , Encía/microbiología , Líquido del Surco Gingival/microbiología , Humanos , Immunoblotting , Interleucina-8/análisis , Persona de Mediana Edad , Periodontitis/inmunología , Periodontitis/microbiología , Proteínas Inhibidoras de Proteinasas Secretoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Inhibidor Secretorio de Peptidasas Leucocitarias , Estadísticas no ParamétricasRESUMEN
The activities to induce TNF-alpha production by a monocytic cell line, THP-1, and ICAM-1 expression and IL-6 production by human gingival fibroblasts were detected in plural membrane lipoproteins of Mycoplasma salivarium. Although SDS-PAGE of the lipoproteins digested by proteinase K did not reveal any protein bands with molecular masses higher than approximately10 kDa, these activities were detected in the front of the gel. A lipoprotein with a molecular mass of 44 kDa (Lp44) was purified. Proteinase K did not affect the ICAM-1 expression-inducing activity of Lp44, but lipoprotein lipase abrogated the activity. These results suggested that the proteinase K-resistant and low molecular mass entity, possibly the N-terminal lipid moiety, played a key role in the expression of the activity. The N-terminal lipid moiety of Lp44 was purified from Lp44 digested with proteinase K by HPLC. Judging from the structure of microbial lipopeptides as well as the amino acid sequence and infrared spectrum of Lp44, the structure of the N-terminal lipid moiety of Lp44 was speculated to be S-(2, 3-bisacyloxypropyl)-cysteine-GDPKHPKSFTEWV-. Its analogue, S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF, was synthesized. The lipopeptide was similar to the N-terminal lipid moiety of Lp44 in the infrared spectrum and the ICAM-1 expression-inducing activity. Thus, this study suggested that the active entity of Lp44 was its N-terminal lipopeptide moiety, the structure of which was very similar to S-(2, 3-bispalmitoyloxypropyl)-cysteine-GDPKHPKSF.