RESUMEN
Human papillomavirus (HPV) 11/16 E6/E7 proteins have been recognized to be pivotal in viral pathogenesis. This study sought to uncover the potential mechanisms of how HPV11/16 E6/E7-transfected keratinocytes inhibit cytokine secretion in peripheral blood mononuclear cells (PBMC). Upon co-culturing HPV11/16 E6/E7-transfected keratinocytes with PBMC in a non-contact manner, we observed a marked decrease in various cytokines secreted by PBMC. To determine if this suppression was mediated by specific common secreted factors, we conducted transcriptomic sequencing on these transfected cells. This analysis identified 53 common differentially secreted genes in all four HPV-transfected cells. Bioinformatics analysis demonstrated these genes were predominantly involved in immune regulation. Results from quantitative PCR (qPCR) and an extensive literature review suggested the downregulation of 12 genes (ACE2, BMP3, BPIFB1, CLU, CST6, CTF1, HMGB2, MMP12, PDGFA, RNASE7, SULF2, TGM2), and upregulation of 7 genes (CCL17, CCL22, FBLN1, PLAU, S100A7, S100A8, S100A9), may be crucial in modulating tumor immunity and combating pathogenic infections, with genes S100A8 and S100A9, and IL-17 signaling pathway being particularly noteworthy. Thus, HPV11/16 E6/E7 proteins may inhibit cytokine secretion of immune cells by altering the expression of host-secreted genes. Further exploration of these genes may yield new insights into the complex dynamics of HPV infection.
Asunto(s)
Citocinas , Leucocitos Mononucleares , Proteínas Oncogénicas Virales , Humanos , Citocinas/metabolismo , Citocinas/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Leucocitos Mononucleares/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Proteínas Oncogénicas Virales/inmunología , Queratinocitos/virología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 11/genética , Papillomavirus Humano 11/inmunología , Perfilación de la Expresión Génica , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/genética , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas E7 de Papillomavirus/inmunología , Técnicas de Cocultivo , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genéticaRESUMEN
Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.
Asunto(s)
Leucocitos Mononucleares , Infecciones por Papillomavirus , Humanos , Citocinas , Escherichia coli/genética , Proteínas Recombinantes/genéticaRESUMEN
Talaromycosis, namely Talaromyces marneffei infection, is increasing gradually and has a high mortality rate even under antifungal therapy. Although autophagy acts differently on different pathogens, it is a promising therapeutic strategy. However, information on autophagy in macrophages and animals upon infection by T. marneffei is still limited. Therefore, several models were employed here to investigate the role of autophagy in host defense against T. marneffei, including RAW264.7 macrophages as in vitro models, different types of Caenorhabditis elegans and BALB/c mice as in vivo models. We applied the clinical T. marneffei isolate SUMS0152 in this study. T. marneffei-infected macrophages exhibit increased formation of autophagosomes. Further, macrophage autophagy promoted by rapamycin or Earle's balanced salt solution (EBSS) inhibited the viability of intracellular T. marneffei. In vivo, compared with uninfected Caenorhabditis elegans, the wild-type nematodes upregulated the expression of the autophagy-related gene lgg-1 and atg-18, and nematodes carrying GFP reporter were induced to form autophagosomes (GFP::LGG-1) after T. marneffei infection. Furthermore, the knockdown of lgg-1 significantly reduced the survival rate of T. marneffei-infected nematodes. Likewise, the autophagy activator rapamycin reduced the fungal burden and suppressed lung inflammation in a mouse model of infection. In conclusion, autophagy is essential for host defense against T. marneffei in vitro and in vivo. Therefore, autophagy may be an attractive target for developing new therapeutics to treat talaromycosis.
Asunto(s)
Caenorhabditis elegans , Talaromyces , Animales , Ratones , Autofagia , Sirolimus/farmacologíaRESUMEN
Acne vulgaris is a complex skin disease involving infection by Cutibacterium acnes, inflammation, and hyperkeratinization. We evaluated the activity of the retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and 16 other retinoid analogs as potential anti-C. acnes compounds and found that CD437 displayed the highest antimicrobial activity with an MIC against C. acnes (ATCC 6919 and HM-513) of 1 µg/mL. CD437 demonstrated an MBC of 2 µg/mL compared to up to 64 µg/mL for the retinoid adapalene and up to 16 µg/mL for tetracycline, which are commonly used clinically to treat acne. Membrane permeability assays demonstrated that exposure of C. acnes ATCC 6919 to CD437 damaged the integrity of C. acnes ATCC 6919 bacterial membranes, and this finding was confirmed with scanning electron microscopy. Additionally, CD437 downregulated the expression of C. acnes ATCC 6919 virulence factors, including the genes encoding Christie-Atkins-Munch-Petersen factor 1 (CAMP1), CAMP2, glycerol-ester hydrolase B (GehB), sialidase B, and neuraminidase. In a mouse skin infection model of C. acnes ATCC 6919, topical treatment with CD437 ameliorated skin lesions and reduced the bacterial burden in situ (P < 0.001). In human NHEK primary cells, CD437 reduced the transcriptional levels of the coding genes for inflammatory cytokines (interleukin-1α, ~10-fold; interleukin-6, ~20-fold; interleukin-8, ~30-fold; and tumor necrosis factor-alpha, ~6-fold) and downregulated the transcriptional levels of KRT10 (~10-fold), FLG (~4-fold), and TGM1 (~2-fold), indicating that CD437 can diminish inflammation and hyperkeratinization. In summary, CD437 deserves further attention for its dual function as a potential acne therapeutic that potentially acts on both the pathogen and the host.
Asunto(s)
Acné Vulgar , Retinoides , Ratones , Animales , Humanos , Retinoides/metabolismo , Retinoides/uso terapéutico , Acné Vulgar/tratamiento farmacológico , Acné Vulgar/microbiología , Citocinas/metabolismo , Antibacterianos/uso terapéutico , Inflamación , Propionibacterium acnesRESUMEN
Background: T helper (Th) cells are involved in the pathogenesis of pemphigus vulgaris (PV). However, the mechanism still needs more exploration. Aims: This study aimed to evaluate the molecular mechanism of the dysregulation of Th17 cells in the peripheral blood of patients with PV. Materials and Methods: Serum levels of IL-17 and anti-Dsg3 titres in patients with PV were analysed using ELISA. The mRNA expression of retinoic acid orphan receptor γt (RORγt) in CD4+ T cells was detected using reverse transcription-quantitative PCR (qPCR). The number of Th17 cells was examined using flow cytometry. Western blot analysis and immunofluorescent staining were also performed to investigate the expression levels of ERK/MAPK signalling proteins and Th17 lineage-associated proteins. Results: The proportion of Th17 cells and Th17 spectrum-associated proteins (p-STAT3, RORγt and IL-17) were upregulated in CD4+ cells in PV patients. The increased transcriptional levels of RORγt and IL-17 correlated positively with the severity of PV. Elevated phosphorylation of the ERK signalling factors was found in the collected CD4+ T cells in PV patients. The inhibition of the ERK signalling pathway significantly reduced the differentiation of Th17 cells in PV patients in vitro. Conclusion: Th17 cells are essential in the dysregulation of PV, and ERK signalling is involved in Th17-type immunity and promotes the development of PV. The study here provides us with a potential therapeutic target for PV.