RESUMO
Human genital Chlamydia infection is a major public health concern due to the serious reproductive system complications. Chlamydia binds several receptor tyrosine kinases (RTKs) on host cells, including the epidermal growth factor receptor (EGFR), and activates cellular signaling cascades for host invasion, cytoskeletal remodeling, optimal inclusion development, and induction of pathogenic epithelial-mesenchyme transition (EMT). Chlamydia also upregulates transforming growth factor beta (TGF-ß) expression, whose signaling pathway synergizes with the EGFR cascade, but its role in infectivity, inclusions, and EMT induction is unknown. We hypothesized that the EGFR and TGF-ß signaling pathways cooperate during chlamydial infection for optimal inclusion development and stable EMT induction. The results revealed that Chlamydia upregulated TGF-ß expression as early as 6 h postinfection of epithelial cells and stimulated both the EGFR and TGF-ß signaling pathways. Inhibition of either the EGFR or TGF-ßR1 signaling substantially reduced inclusion development; however, the combined inhibition of both EGFR and TGF-ßR1 signaling reduced inclusions by over 90% and prevented EMT induction. Importantly, EGFR inhibition suppressed TGF-ß expression, and an inhibitory thrombospondin-1 (Tsp1)-based peptide inhibited chlamydia-induced EMT, revealing a major source of active TGF-ß during infection. Finally, TGF-ßR signaling inhibition suppressed the expression of transforming acidic coiled-coil protein-3 (TACC3), which stabilizes EGFR signaling, suggesting reciprocal regulation between TGF-ß and EGFR signaling during chlamydial infection. Thus, RTK-mediated host invasion by chlamydia upregulated TGF-ß expression and signaling, which cooperated with other cellular signaling cascades and cytoskeletal remodeling to support optimal inclusion development and EMT induction. This finding may provide new targets for chlamydial disease biomarkers and prevention.
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Infecções por Chlamydia/fisiopatologia , Chlamydia/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Endocitose , Transição Epitelial-Mesenquimal , Corpos de Inclusão/microbiologia , Camundongos , Modelos BiológicosRESUMO
The authors would like to make the following corrections to their paper, published in the International Journal of Molecular Sciences [...].
RESUMO
The polymorphic membrane protein D (PmpD) is a highly conserved outer membrane protein which plays an important role in pathogenesis during Chlamydia psittaci infection. In this study, we evaluated the ability of the N-terminus of PmpD (PmpD-N) to modulate the functions of chicken macrophages and the signaling pathway(s) involved in PmpD-N-induced Toll-like receptors (TLRs), as well as interleukin (IL)-6 and IL-10 cytokine secretions. Thus, HD11 macrophages were treated with exogenous and intracellular PmpD-N of C. psittaci. The chlamydial growth was evaluated by enumeration of chlamydial loads in the infected macrophages. The phagocytic function of macrophages following PmpD-N treatment was detected by fluorescein-labeled Escherichia coli (E. coli). The concentration of nitric oxide (NO) secreted by HD11 macrophages was measured by the amount of NO2- in the culture supernatant using the Griess method. The cytokine secretions were assessed using multiplex cytokine ELISA kits. Expression levels of TLRs, myeloid differentiation factor 88 (MyD88), and nuclear factor kappa B (NF-κB) were analyzed by a Western blotting assay, as well as a luciferase assay, while NF-κB p65 nuclear translocation was assessed by confocal microscopy. The nuclear translocation of the transcription factor NF-κB was confirmed by evaluating its ability to combine with the corresponding promoter using the electrophoretic mobility shift assay (EMSA). After treatment with exogenous or endogenous PmpD-N, chlamydial loads and phagocytic functions were reduced significantly compared with those of the plasmid vector group, while NO secretions were reduced significantly compared with those of the lipopolysaccharide (LPS) treatment. Stimulation of HD11 cells with PmpD-N provoked the secretion of the Th2 cytokines, IL-6, and IL-10 and upregulated the expression of TLR2, TLR4, MyD88, and NF-κB. Furthermore, inhibition of TLR2, MyD88, and NF-κB in HD11 cells significantly decreased IL-6 and IL-10 cytokine levels, while NO production and phagocytosis increased significantly, strongly suggesting their involvement in PmpD-N-induced Th2 cytokine secretion and macrophage dysfunction. Our data indicate that C. psittaci PmpD-N inhibited macrophage functions by activating the Th2 immune response and the TLR2/MyD88/NF-κB signaling pathway.
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Proteínas Aviárias/imunologia , Proteínas de Bactérias/imunologia , Chlamydophila psittaci/imunologia , Macrófagos/imunologia , Proteínas de Membrana/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Células Th2/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Linhagem Celular , Galinhas , Macrófagos/microbiologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Psitacose/imunologia , Psitacose/microbiologia , Psitacose/veterináriaRESUMO
BACKGROUND: Genital C. trachomatis infection may cause pelvic inflammatory disease (PID) that can lead to tubal factor infertility (TFI). Understanding the pathogenesis of chlamydial complications including the pathophysiological processes within the female host genital tract is important in preventing adverse pathology. MicroRNAs regulate several pathophysiological processes of infectious and non-infectious etiologies. In this study, we tested the hypothesis that the miRNA profile of single and repeat genital chlamydial infections will be different and that these differences will be time dependent. Thus, we analyzed and compared differentially expressed mice genital tract miRNAs after single and repeat chlamydia infections using a C. muridarum mouse model. Mice were sacrificed and their genital tract tissues were collected at 1, 2, 4, and 8 weeks after a single and repeat chlamydia infections. Histopathology, and miRNA sequencing were performed. RESULTS: Histopathology presentation showed that the oviduct and uterus of reinfected mice were more inflamed, distended and dilated compared to mice infected once. The miRNAs expression profile was different in the reproductive tissues after a reinfection, with a greater number of miRNAs expressed after reinfection. Also, the number of miRNAs expressed each week after chlamydia infection and reinfection varied, with weeks eight and one having the highest number of differentially expressed miRNAs for chlamydia infection and reinfection respectively. Ten miRNAs; mmu-miR-378b, mmu-miR-204-5p, mmu-miR-151-5p, mmu-miR-142-3p, mmu-miR-128-3p, mmu-miR-335-3p, mmu-miR-195a-3p, mmu-miR-142-5p, mmu-miR-106a-5p and mmu-miR-92a-3p were common in both primary chlamydia infection and reinfection. Pathway analysis showed that, amongst other functions, the differentially regulated miRNAs control pathways involved in cellular and tissue development, disease conditions and toxicity. CONCLUSIONS: This study provides insights into the changes in miRNA expression over time after chlamydia infection and reinfection, as well as the pathways they regulate to determine pathological outcomes. The miRNAs networks generated in our study shows that there are differences in the focus molecules involved in significant biological functions in chlamydia infection and reinfection, implying that chlamydial pathogenesis occurs differently for each type of infection and that this could be important when determining treatments regime and disease outcome. The study underscores the crucial role of host factors in chlamydia pathogenesis.
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Infecções por Chlamydia/genética , Infecções por Chlamydia/microbiologia , Chlamydia , Genitália/microbiologia , MicroRNAs/genética , Transcriptoma , Animais , Biópsia , Linhagem Celular , Infecções por Chlamydia/patologia , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genitália/patologia , Humanos , Imuno-Histoquímica , CamundongosRESUMO
The reproductive system complications of genital chlamydial infection include fallopian tube fibrosis and tubal factor infertility. However, the molecular pathogenesis of these complications remains poorly understood. The induction of pathogenic epithelial-mesenchymal transition (EMT) through microRNA (miRNA) dysregulation was recently proposed as the pathogenic basis of chlamydial complications. Focusing on fibrogenesis, we investigated the hypothesis that chlamydia-induced fibrosis is caused by EMT-driven generation of myofibroblasts, the effector cells of fibrosis that produce excessive extracellular matrix (ECM) proteins. The results revealed that the targets of a major category of altered miRNAs during chlamydial infection are key components of the pathophysiological process of fibrogenesis; these target molecules include collagen types I, III, and IV, transforming growth factor ß (TGF-ß), TGF-ß receptor 1 (TGF-ßR1), connective tissue growth factor (CTGF), E-cadherin, SRY-box 7 (SOX7), and NFAT (nuclear factor of activated T cells) kinase dual-specificity tyrosine (Y) phosphorylation-regulated kinase 1a (Dyrk1a). Chlamydial induction of EMT resulted in the generation of α-smooth muscle actin (α-SMA)-positive myofibroblasts that produced ECM proteins, including collagen types I and III and fibronectin. Furthermore, the inhibition of EMT prevented the generation of myofibroblasts and production of ECM proteins during chlamydial infection. These findings may provide useful avenues for targeting EMT or specific components of the EMT pathways as a therapeutic intervention strategy to prevent chlamydia-related complications.
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Infecções por Chlamydia/complicações , Infecções por Chlamydia/patologia , Chlamydia/patogenicidade , Transição Epitelial-Mesenquimal/fisiologia , Fibrose/etiologia , Fibrose/patologia , Actinas/metabolismo , Animais , Caderinas/metabolismo , Linhagem Celular , Infecções por Chlamydia/microbiologia , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fibrose/microbiologia , Camundongos , MicroRNAs/metabolismo , Miofibroblastos/microbiologia , Miofibroblastos/patologia , Fatores de Transcrição NFATC/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: We have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10-/- DCs to identify differentially expressed proteins with immunomodulatory properties. RESULTS: The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10-/- DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response. CONCLUSIONS: The upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.
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Biomarcadores Tumorais/genética , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Proteínas de Ligação a DNA/genética , Células Dendríticas/fisiologia , Genitália/imunologia , Fosfopiruvato Hidratase/genética , Proteínas Supressoras de Tumor/genética , Animais , Apresentação de Antígeno , Biomarcadores Tumorais/metabolismo , Permeabilidade da Membrana Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/microbiologia , Feminino , Genitália/microbiologia , Imunidade Inata , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteômica , Ácido Pirúvico/metabolismo , RNA Interferente Pequeno/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para CimaRESUMO
BACKGROUND: We previously showed that the Vibrio cholerae ghost platform (VCG; empty V. cholerae cell envelopes) is an effective delivery system for vaccine antigens promoting the induction of substantial immunity in the absence of external adjuvants. However, the mechanism by which these cell envelopes enhance immunity and stimulate a predominantly Th1 cellular and humoral immune response has not been elucidated. We hypothesized that the immunostimulatory ability of VCG involves dendritic cell (DC) activation. OBJECTIVE: The aims of this study were: a) to investigate the ability of DCs [using mouse bone marrow-derived DCs (BMDCs) as a model system] to take up and internalize VCGs; b) to evaluate the immunomodulatory effect of internalized VCGs on DC activation and maturation and their functional capacity to present chlamydial antigen to naïve and infection-sensitized CD4+ T cells and; c) to evaluate the ability of VCGs to enhance the protective immunity of a chlamydial antigen. RESULTS: VCGs were efficiently internalized by DCs without affecting their viability and modulated DC-mediated immune responses. VCG-pulsed DCs showed increased secretion of proinflammatory cytokines and expression of co-stimulatory molecules associated with DC maturation in response to stimulation with UV-irradiated chlamydial elementary bodies (UV-EBs). Furthermore, this interaction resulted in effective chlamydial antigen presentation to infection-sensitized but not naïve CD4+ T cells and enhancement of protective immunity. CONCLUSIONS: The present study demonstrated that VCGs activate DCs leading to the surface expression of co-stimulatory molecules associated with DC activation and maturation and enhancement of protective immunity induced by a chlamydial antigen. The results indicate that the immunoenhancing activity of VCG for increased T-cell activation against antigens is mediated, at least in part, through DC triggering. Thus, VCGs could be harnessed as immunomodulators to target antigens to DCs for enhancement of protective immunity against microbial infections.
Assuntos
Apresentação de Antígeno , Antígenos de Bactérias , Chlamydia trachomatis , Células Dendríticas/imunologia , Células Th1/imunologia , Vibrio cholerae , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Chlamydia trachomatis/química , Chlamydia trachomatis/imunologia , Feminino , Células HeLa , Humanos , Ativação Linfocitária , Camundongos , Vibrio cholerae/química , Vibrio cholerae/imunologiaRESUMO
Tubal factor infertility (TFI) represents 36% of female infertility and genital infection by Chlamydia trachomatis (C. trachomatis) is a major cause. Although TFI is associated with host inflammatory responses to bacterial components, the molecular pathogenesis of Chlamydia-induced infertility remains poorly understood. We investigated the hypothesis that activation of specific cysteine proteases, the caspases, during C. trachomatis genital infection causes the disruption of key fertility-promoting molecules required for embryo development and implantation. We analyzed the effect of caspase inhibition on infertility and the integrity of Dicer, a caspase-sensitive, fertility-promoting ribonuclease III enzyme, and key micro-RNAs in the reproductive system. Genital infection with the inflammation- and caspase-inducing, wild-type C. trachomatis serovar L2 led to infertility, but the noninflammation-inducing, plasmid-free strain did not. We confirmed that caspase-mediated apoptotic tissue destruction may contribute to chlamydial pathogenesis. Caspase-1 or -3 deficiency, or local administration of the pan caspase inhibitor, Z-VAD-FMK into normal mice protected against Chlamydia-induced infertility. Finally, the oviducts of infected infertile mice showed evidence of caspase-mediated cleavage inactivation of Dicer and alteration in critical miRNAs that regulate growth, differentiation, and development, including mir-21. These results provide new insight into the molecular pathogenesis of TFI with significant implications for new strategies for treatment and prevention of chlamydial complications.
Assuntos
Caspase 1/metabolismo , Caspase 3/metabolismo , Chlamydia trachomatis/patogenicidade , Infertilidade Feminina/microbiologia , Infertilidade Feminina/prevenção & controle , Complicações Infecciosas na Gravidez/prevenção & controle , Animais , Apoptose , Caspase 1/genética , Caspase 3/genética , Infecções por Chlamydia/enzimologia , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Ativação Enzimática , Feminino , Células HeLa , Humanos , Infertilidade Feminina/enzimologia , Inflamação/microbiologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Complicações Infecciosas na Gravidez/enzimologia , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/patologiaRESUMO
Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial infection worldwide, potentially leading to severe pathologies including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility if left untreated. Current strategies, including screening and antibiotics, have limited effectiveness due to high rates of asymptomatic cases and logistical challenges. A multiepitope prophylactic vaccine could afford long-term protection against infection. Immunoinformatic analyses were employed to design a multiepitope Chlamydia vaccine antigen. B- and T-cell epitopes from five highly conserved and immunogenic Ct antigens were predicted and selected for the vaccine design. The final construct, adjuvanted with cholera toxin A1 subunit (CTA1), was further screened for immunogenicity. CTA1-MECA (multiepitope Chlamydia trachomatis antigen) was identified as antigenic and nonallergenic. A tertiary structure was predicted, refined, and validated as a good quality model. Molecular docking exhibited strong interactions between the vaccine and toll-like receptor 4 (TLR4). Additionally, immune responses consistent with protection including IFN-γ, IgG + IgM antibodies, and T- and B-cell responses were predicted following vaccination in an immune simulation. Expression of the construct in an Escherichia coli expression vector proved efficient. To further validate the vaccine efficacy, we assessed its immunogenicity in mice. Immunization with CTA1-MECA elicited high levels of Chlamydia-specific antibodies in mucosal and systemic compartments.
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Anticorpos Antibacterianos , Vacinas Bacterianas , Infecções por Chlamydia , Chlamydia trachomatis , Epitopos de Linfócito B , Epitopos de Linfócito T , Simulação de Acoplamento Molecular , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/genética , Infecções por Chlamydia/prevenção & controle , Infecções por Chlamydia/imunologia , Animais , Chlamydia trachomatis/imunologia , Epitopos de Linfócito T/imunologia , Camundongos , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Feminino , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Simulação por Computador , Epitopos/imunologia , Humanos , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Toxina da Cólera/imunologia , Toxina da Cólera/genética , Modelos Animais de DoençasRESUMO
We have previously shown that the time of Chlamydia infection was crucial in determining the chlamydial infectivity and pathogenesis. This study aims to determine whether the time of Chlamydia infection affects the genital tract microbiome. This study analyzed mice vaginal, uterine, and ovary/oviduct microbiome with and without Chlamydia infection. The mice were infected with Chlamydia at either 10:00 am (ZT3) or 10:00 pm (ZT15). The results showed that mice infected at ZT3 had higher Chlamydia infectivity than those infected at ZT15. There was more variation in the compositional complexity of the vaginal microbiome (alpha diversity) of mice infected at ZT3 than those mice infected at ZT15 throughout the infection within each treatment group, with both Shannon and Simpson diversity index values decreased over time. The analysis of samples collected four weeks post-infection showed that there were significant taxonomical differences (beta diversity) between different parts of the genital tract-vagina, uterus, and ovary/oviduct-and this difference was associated with the time of infection. Firmicutes and Proteobacteria were the most abundant phyla within the microbiome in all three genital tract regions for all the samples collected during this experiment. Additionally, Firmicutes was the dominant phylum in the uterine microbiome of ZT3 Chlamydia infected mice. The results show that the time of infection is associated with the microbial dynamics in the genital tract. And this association is more robust in the upper genital tract than in the vagina. This result implies that more emphasis should be placed on understanding the changes in the microbial dynamics of the upper genital tract over the course of infection.
Assuntos
Infecções por Chlamydia , Chlamydia , Feminino , Camundongos , Animais , Infecções por Chlamydia/microbiologia , Vagina/microbiologia , Útero/patologia , Camundongos Endogâmicos C57BLRESUMO
Chlamydia abortus (Cab) causes spontaneous abortion and neonatal mortality in infected ruminants and pregnant women. Most Cab infections are asymptomatic, although they can be treated with antibiotics, signifying that control of these infections may require alternative strategies, including the use of effective vaccines. However, the limitations imposed by live attenuated and inactivated vaccines further suggest that employment of subunit vaccines may need to be considered. The efficacy of a newly generated Vibrio cholerae ghost (rVCG)-based subunit vaccine harboring the N-terminal portion of the Cab Pmp18D protein (rVCG-Pmp18.3) in preventing Cab-induced abortion or neonatal mortality was evaluated in pregnant mice. Mice were intranasally (IN) immunized and boosted twice, 2 weeks apart with the vaccine, and immunized and unimmunized mice were caged with males 4 weeks postimmunization. The mice were then infected either IN or transcervically (TC) 10 days after pregnancy, and the fertility rate was determined 7 days postpartum. Eight days after delivery, the mice were sacrificed, and Cab infectivity in the lungs and spleens was evaluated by culturing tissue homogenates in tissue culture. Our results demonstrated that the vaccine induced immune effectors that mediated complete clearance of infection in the lungs and significantly reduced Cab infectivity in the spleen following IN immunization. Vaccine immunization also afforded protection against Cab-induced upper genital tract pathology (uterine dilation). Furthermore, while there was no incidence of abortion in both immunized and unimmunized mice, immunized mice were completely protected against neonatal mortality compared to unimmunized infected controls, which lost a significant percentage of their litter 7 days postpartum. Our results establish the capability of the rVCG-Pmp18.3 vaccine to prevent infection in the lungs (mucosal) and spleen (systemic) and protect mice from Cab-induced tubal pathologies and neonatal mortality, a hallmark of Cab infection in ruminants. To advance the commercial potential of this vaccine, future studies will optimize the antigen dose and the number of vaccine doses required for protection of ruminants.
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Infecções por Chlamydia , Chlamydia , Humanos , Gravidez , Feminino , Animais , Camundongos , Vacinas Bacterianas , Infecções por Chlamydia/prevenção & controle , Vacinas de Subunidades Antigênicas , RuminantesRESUMO
Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.
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Quitosana/farmacologia , Chlamydophila psittaci/imunologia , Nanopartículas/química , Psitacose/tratamento farmacológico , Administração Intranasal , Animais , Antígenos de Bactérias/farmacologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/farmacologia , Galinhas/microbiologia , Quitosana/química , Chlamydophila psittaci/patogenicidade , Humanos , Imunidade nas Mucosas/imunologia , Injeções Intramusculares , Interferon gama/genética , Camundongos , Vibrio cholerae/imunologia , Vibrio cholerae/patogenicidadeRESUMO
Genital Chlamydia trachomatis infection causes severe reproductive pathologies such as salpingitis and pelvic inflammatory disease that can lead to tubal factor infertility. MicroRNAs (miRNAs) are evolutionarily conserved regulators of mammalian gene expression in development, immunity and pathophysiologic processes during inflammation and infection, including Chlamydia infection. Among the miRNAs involved in regulating host responses and pathologic outcome of Chlamydia infection, we have shown that miR-378b was significantly differentially expressed during primary infection and reinfection. In this study, we tested the hypothesis that miR-378b is involved in the pathological outcome of Chlamydia infection. We developed miR-378b knockout mice (miR-378b-/-) using Crispr/Cas and infected them along with their wild-type (WT) control with Chlamydia to compare the infectivity and reproductive pathologies. The results showed that miR-378b-/- mice were unable to clear the infection compared to WT mice; also, miR-378b-/- mice exhibited a relatively higher Chlamydia burden throughout the duration of infection. However, gross pathology results showed that miR-378b-/- mice had significantly reduced uterine dilatations and pathologic lesions after two infections compared to WT mice. In addition, the pregnancy and fertility rates for infected miR-378b-/- mice showed protection from Chlamydia-induced infertility with fertility rate that was comparable to uninfected WT mice. These results are intriguing as they suggest that miR-378b is important in regulating host immune responses that control Chlamydial replication and drive the inflammation that causes complications such as infertility. The finding has important implications for biomarkers of Chlamydial complications and targets for prevention of disease.
RESUMO
Efficacious vaccines are needed to control genital chlamydial diseases in humans and the veterinary industry. We previously reported a C. abortus (Cab) vaccine comprising recombinant Vibrio cholerae ghosts (rVCG) expressing the conserved and immunogenic N-terminal region of the Cab polymorphic membrane protein D (rVCG-Pmp18.1) protein that protected mice against intravaginal challenge. In this study, we investigated the immunomodulatory effect of the hematopoietic progenitor activator cytokine, Fms-like tyrosine kinase 3-ligand (FL) when co-administered with the rVCG-Pmp18.1 vaccine as a strategy to enhance the protective efficacy and the potential mechanism of immunomodulation. Groups of female C57BL/6J mice were immunized and boosted twice intranasally (IN) with rVCG-PmpD18.1 with and without FL or purified rPmp18.1 or rVCG-gD2 (antigen control) or PBS (medium) per mouse. The results revealed that co-administration of the vaccine with FL enhanced antigen-specific cellular and humoral immune responses and protected against live Cab genital infection. Comparative analysis of immune cell phenotypes infiltrating mucosal and systemic immune inductive tissue sites following immunization revealed that co-administration of rVCG-Pmp18.1 with FL significantly enhanced the number of macrophages, dendritic and NK cells, γδ and NK T cells in the spleen (systemic) and iliac lymph nodes (ILN) draining the genital tract (mucosal) tissues compared to rVCG-Pmp18.1 alone. Furthermore, FL enhanced monocyte infiltration in the ILN, while CD19+ B cells and CD4+ T cells were enhanced in the spleen. These results indicate that the immunomodulatory effect of FL is associated with its ability to mobilize innate immune cells and subsequent activation of robust antigen-specific immune effectors in mucosal and systemic lymphoid tissues.
Assuntos
Adjuvantes de Vacinas/farmacocinética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/farmacologia , Infecções por Chlamydia , Proteínas de Membrana/imunologia , Animais , Chlamydia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Vibrio choleraeRESUMO
Vaccine-induced immune responses following immunization with promising Chlamydia vaccines protected experimental animals from Chlamydia-induced upper genital tract pathologies and infertility. In contrast, primary genital infection with live Chlamydia does not protect against these pathologies. We hypothesized that differential miRNA profiles induced in the upper genital tracts (UGT) of mice correlate with the disparate immunity vs. pathologic outcomes associated with vaccine immunization and chlamydial infection. Thus, miRNA expression profiles in the UGT of mice after Chlamydia infection (Live EB) and immunization with dendritic cell (DC)-based vaccine (DC vaccine) or VCG-based vaccine (VCG vaccine) were compared using the NanoString nCounter Mouse miRNA assay. Of the 602 miRNAs differentially expressed (DE) in the UGT of immunized and infected mice, we selected 58 with counts >100 and p-values < 0.05 for further analysis. Interestingly, vaccine immunization and Chlamydia infection induced the expression of distinct miRNA profiles with a higher proportion in vaccine-immunized compared to Chlamydia infected mice; DC vaccine (41), VCG vaccine (23), and Live EB (15). Hierarchical clustering analysis showed notable differences in the uniquely DE miRNAs for each experimental group, with DC vaccine showing the highest number (21 up-regulated, five down-regulated), VCG vaccine (two up-regulated, five down-regulated), and live EB (two up-regulated, four down-regulated). The DC vaccine-immunized group showed the highest number (21 up-regulated and five down-regulated compared to two up-regulated and four down-regulated in the live Chlamydia infected group). Pathway analysis showed that the DE miRNAs target genes that regulate several biological processes and functions associated with immune response and inflammation. These results suggest that the induction of differential miRNA expression plays a significant role in the disparate immunity outcomes associated with Chlamydia infection and vaccination.
Assuntos
Vacinas Bacterianas/administração & dosagem , Infecções por Chlamydia/prevenção & controle , Chlamydia muridarum/imunologia , Células Dendríticas/imunologia , Imunogenicidade da Vacina , MicroRNAs/genética , Transcriptoma , Transferência Adotiva , Animais , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/genética , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia muridarum/patogenicidade , Células Dendríticas/microbiologia , Células Dendríticas/transplante , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Camundongos Endogâmicos C57BL , MicroRNAs/imunologia , MicroRNAs/metabolismo , Vacinação , Vibrio cholerae/genética , Vibrio cholerae/imunologiaRESUMO
We investigated the hypothesis that the enhanced Ag-presenting function of IL-10-deficient dendritic cells (DCs) is related to specific immunoregulatory cytoskeletal molecules expressed when exposed to Ags. We analyzed the role of a prominent cytoskeletal protein, LEK1, in the immunoregulation of DC functions; specifically cytokine secretion, costimulatory molecule expression, and T cell activation against Chlamydia. Targeted knockdown of LEK1 expression using specific antisense oligonucleotides resulted in the rapid maturation of Chlamydia-exposed DCs as measured by FACS analysis of key activation markers (i.e., CD14, CD40, CD54, CD80, CD86, CD197, CD205, and MHC class II). The secretion of mostly Th1 cytokines and chemokines (IL-1a, IL-9, IL-12, MIP-1a, and GM-CSF but not IL-4 and IL-10) was also enhanced by blocking of LEK1. The function of LEK1 in DC regulation involves cytoskeletal changes, since the dynamics of expression of vimentin and actin, key proteins of the cellular cytoskeleton, were altered after exposure of LEK1 knockdown DCs to Chlamydia. Furthermore, targeted inhibition of LEK1 expression resulted in the enhancement of the immunostimulatory capacity of DCs for T cell activation against Chlamydia. Thus, LEK1 knockdown DCs activated immune T cells at least 10-fold over untreated DCs. These results suggest that the effect of IL-10 deficiency is mediated through LEK1-related events that lead to rapid maturation of DCs and acquisition of the capacity to activate an elevated T cell response. Targeted modulation of LEK1 expression provides a novel strategy for augmenting the immunostimulatory function of DCs for inducing an effective immunity against pathogens.
Assuntos
Chlamydia trachomatis/imunologia , Proteínas Cromossômicas não Histona/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/microbiologia , Animais , Biomarcadores/análise , Diferenciação Celular/imunologia , Células Cultivadas , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/biossíntese , Proteínas Cromossômicas não Histona/deficiência , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/fisiologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Feminino , Interleucina-10/antagonistas & inibidores , Interleucina-10/biossíntese , Interleucina-10/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Oligonucleotídeos Antissenso/farmacologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Shift work, performed by approximately 21 million Americans, is irregular or unusual work schedule hours occurring after 6:00 pm. Shift work has been shown to disrupt circadian rhythms and is associated with several adverse health outcomes and chronic diseases such as cancer, gastrointestinal and psychiatric diseases and disorders. It is unclear if shift work influences the complications associated with certain infectious agents, such as pelvic inflammatory disease, ectopic pregnancy and tubal factor infertility resulting from genital chlamydial infection. We used an Environmental circadian disruption (ECD) model mimicking circadian disruption occurring during shift work, where mice had a 6-h advance in the normal light/dark cycle (LD) every week for a month. Control group mice were housed under normal 12/12 LD cycle. Our hypothesis was that compared to controls, mice that had their circadian rhythms disrupted in this ECD model will have a higher Chlamydia load, more pathology and decreased fertility rate following Chlamydia infection. Results showed that, compared to controls, mice that had their circadian rhythms disrupted (ECD) had higher Chlamydia loads, more tissue alterations or lesions, and lower fertility rate associated with chlamydial infection. Also, infected ECD mice elicited higher proinflammatory cytokines compared to mice under normal 12/12 LD cycle. These results imply that there might be an association between shift work and the increased likelihood of developing more severe disease from Chlamydia infection.
Assuntos
Infecções por Chlamydia/etiologia , Ritmo Circadiano/fisiologia , Jornada de Trabalho em Turnos/efeitos adversos , Animais , Chlamydia/patogenicidade , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/patologia , Chlamydia muridarum/patogenicidade , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Doença Inflamatória Pélvica/etiologia , Fotoperíodo , Gravidez , Gravidez Ectópica/etiologiaRESUMO
We tested the hypothesis that the impact of the Fms-like tyrosine kinase 3-ligand (Flt3L; FL) on recombinant Vibrio cholerae ghost (rVCG) vaccine-induced chlamydial immunity is influenced by route of vaccine delivery. Female C57BL/6J mice were immunized rectally (IR) or intramuscularly (IM) with rVCG co-expressing the Chlamydia trachomatis PmpD and PorB proteins (rVCG- PmpD/PorB) with and without FL or glycoprotein D of HSV-2 (rVCG-gD2) as antigen control. Vaccine evaluation was based on measurement of T cell proliferation, Th1/Th2 cytokine, and humoral responses at systemic and mucosal compartments, and protection against intravaginal challenge infection. Results revealed that high levels of CD4+ T cell-mediated and humoral immune responses, were elicited in mice as a function of both IR and IM immunization. Unexpectedly, co-administration of vaccine with FL enhanced specific Th1-type cytokine levels and T cell proliferative responses following IR but not IM immunization. While administration of vaccine with FL enhanced the specific mucosal and systemic IgA antibody responses following both immunization routes, IgG2c responses were not enhanced following IR delivery. The vaccine-induced immune effectors protected mice against live heterologous C. muridarum infection irrespective of route of vaccine administration, with the regimen incorporating FL having a protective advantage. Further evaluation showed that protection afforded by the FL adjuvanted vaccine was facilitated by CD4+ T cells, as indicated by reduction in the intensity and duration of genital chlamydial shedding by naïve mice following adoptive transfer of immune CD4+ T cells. Taken together, the results indicate that comparable protective immunity, which is enhanced by co-delivery with FL, is elicited in the female genital tract against Chlamydia infection after mucosal and systemic administration, highlighting the ability of FL to function as an effective immunostimulator at both mucosal and systemic sites. The differential modulation of humoral and cellular immune responses, and protective immunity afforded by the FL adjuvanted vaccine following IR administration indicates that the immunomodulatory impact of FL on chlamydial-specific immunity is influenced by the route of vaccine administration. Thus, targeting of VCG-based vaccines to antigen presenting cells by co-delivery with FL is a feasible immunization approach for inducing effective chlamydial immunity in the female genital tract.
Assuntos
Vacinas Bacterianas , Chlamydia trachomatis/imunologia , Imunidade nas Mucosas , Tirosina Quinase 3 Semelhante a fms/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/farmacologia , Chlamydia trachomatis/genética , Feminino , Imunoglobulina A/imunologia , Linfogranuloma Venéreo/genética , Linfogranuloma Venéreo/imunologia , Linfogranuloma Venéreo/prevenção & controle , Camundongos , Porinas/genética , Porinas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Vibrio cholerae/genética , Vibrio cholerae/imunologia , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Genital chlamydia infection in women causes complications such as pelvic inflammatory disease and tubal factor infertility, but it is unclear why some women are more susceptible than others. Possible factors, such as time of day of chlamydia infection on chlamydial pathogenesis has not been determined. We hypothesised that infections during the day, will cause increased complications compared to infections at night. Mice placed under normal 12:12 light: dark (LD) cycle were infected intravaginally with Chlamydia muridarum either at zeitgeber time 3, ZT3 and ZT15. Infectivity was monitored by periodic vaginal swabs and chlamydiae isolation. Blood and vaginal washes were collected for host immunologic response assessments. The reproductive tracts of the mice were examined histopathologically, and fertility was determined by embryo enumeration after mating. Mice infected at ZT3 shed significantly more C. muridarum than mice infected at ZT15. This correlated with the increased genital tract pathology observed in mice infected at ZT3. Mice infected at ZT3 were less fertile than mice infected at ZT15. The results suggest that the time of day of infection influences chlamydial pathogenesis, it indicates a possible association between complications from chlamydia infection and host circadian clock, which may lead to a better understanding of chlamydial pathogenesis.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia muridarum/patogenicidade , Relógios Circadianos/imunologia , Doença Inflamatória Pélvica/imunologia , Vagina/microbiologia , Animais , Infecções por Chlamydia/sangue , Infecções por Chlamydia/complicações , Infecções por Chlamydia/microbiologia , Modelos Animais de Doenças , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Camundongos , Doença Inflamatória Pélvica/microbiologia , Fotoperíodo , Vagina/imunologia , Vagina/patologiaRESUMO
Trachoma is the world's leading cause of preventable disease and the third most common cause of blindness after cataract and glaucoma, affecting an estimated 84 million people and leaving 590 million at risk. As a crippling disease, trachoma causes an enormous loss of productivity and constitutes a major socioeconomic burden. Although antibiotics are effective in treating active cases of the illness, they do not prevent re-infection, which occurs with high frequency in susceptible populations. Also, once infection and pathology are established, treatment may be less effective. Another major public health challenge posed by trachoma is that a large number of infected individuals are asymptomatic and readily infect those with whom they interact. Thus, an inexpensive and easy to deliver vaccine for trachoma would be highly effective in reducing the devastation caused by this disease. Development of an effective vaccine for controlling and preventing trachoma will require an understanding of the complex immunological mechanisms that occur during infection, identifying those antigens that elicit a protective immune response and designing effective vaccine delivery systems. Significant progress has been made in the delineation of the immune correlates of protection that will form the basis of vaccine evaluation. Recent advances in chlamydial genomics and proteomics has identified a number of protective antigens or epitopes that when appropriately delivered will produce an efficacious vaccine. The challenge at this time is the development of effective methods for vaccine delivery. We have developed an effective bacterial ghost (BG) delivery system possessing intrinsic adjuvant properties and capable of simultaneously delivering multiple antigens to the immune system. Such a flexible delivery system can produce an effective vaccine that will prevent the development of trachomatous conjunctivitis and blindness. The safety and relatively cheap production cost of BG-based vaccines offer a technological and manufacturing advantage for a vaccine needed on a global scale.