Chlamydia trachomatis Infection Is Associated with E-Cadherin Promoter Methylation, Downregulation of E-Cadherin Expression, and Increased Expression of Fibronectin and α-SMA—Implications for Epithelial-Mesenchymal Transition

Chlamydia trachomatis (Ct) can induce scarring disease of the ocular mucosa, known as trachoma, the most common infectious cause of blindness worldwide. We hypothesized that epithelial-mesenchymal transition (EMT) contributes to the fibrotic process in trachomatous scarring. Infection of human conjunctival epithelial cells (HCjE) with Ct activated signaling pathways involved in EMT induction, which was correlated with decreased expression of E-cadherin, guardian of the epithelial phenotype. In addition, Ct infection was associated with increased expression of two mesenchymal cell markers: fibronectin and α-SMA. The DNA methylation statuses of selected regions of E-cadherin, fibronectin, and α-SMA genes revealed that Ct infection was accompanied with changes in DNA methylation of the E-cadherin promoter, while the expression of the two mesenchymal markers was not related with this epigenetic event. Our data suggest that Ct infection of conjunctival epithelial cells induces EMT-like changes that go along with modification of the methylation profile of the E-cadherin promoter and could, as one of the earliest events, contribute to processes triggering conjunctival scarring.Frontiers in Cellular and Infection Microbiology (2017), 7(JUN): 25

Modulation of functional characteristics of murine peritoneal macrophages by dehydrogenate polymer from coniferyl alcohol and alginate

The dehydrogenate polymer from coniferyl alcohol (DHP; a lignin model compound) in alginate hydrogel (ALG) has been shown to exert a strong antibacterial activity. To broadens a spectrum of potential DHP/ALG application, we aimed this study to evaluate the immunomodulatory activity of DHP/ALG. DHP and ALG were tested separately and in mixture (1:2 w/w) for their impact on in vitro production of cytokines (IL-6, IL-12, and IL-10) and reactive oxygen (ROS) and nitrogen (RNS) species by resident (RMs) and thioglycolate-elicited (TGMs) peritoneal macrophages of BALB/c mice. RMs and TGMs were stimulated (48h) with ALG and DHP in concentrations previously shown to be non-cytotoxic (up to 50 and 25 μg/ml, respectively). DHP/ALG promotes simultaneous production of inflammatory (IL-6, IL-12) and regulatory cytokines by RMs in a positive dose-dependent manner. Production of inflammatory cytokines was stimulated by ALG, while an increase in IL-10 production positively correlated to the concentration of DHP. ALG also stimulated the production of IL-12 by TGMs, which was mirrored in the outcome of ALG/DHP stimulation. The significant increase in the activity of myeloperoxidase (MPO) due to DHP and/or ALG stimulation was recorded in TGMs, while a slight increase in MPO activity in RMs was recorded only upon stimulation with the higher amount of ALG. ALG in a positive dose-dependent manner stimulated the production of ROS and RNS by both RMS and TGMs. In all cases, except ROS production by RMs, the impact of ALG stimulation was mirrored in the outcome of ALG/DHP stimulation. Our results suggest that DHP/ALG exerts an immunomodulatory activity that could complement already reported antimicrobial activity and warrants further investigation on the use of DHP/ALG in the treatment of infectious diseases

Chlamydia trachomatis infection and development of epithelial mesenchymal transition in conjunctiva : possible epigenetic mechanisms unveiled

Introduction: Trachoma is the most common cause of infectious blindness worldwide, initiated by repeated infection of the conjunctiva with Chlamydia trachomatis (Ct). The resulting chronic inflammation and formation of fibrotic tissue eventually lead to corneal damage. Based on the facts that epithelial to mesenchymal transition (EMT) plays an important role in the development of fibrosis and that EMT is epigenetically regulated process, the aims of this study were to reveal the capacity of Ct to induce EMT in vitro and to unveil potential underlying epigenetic mechanisms. Methods: Human conjunctival epithelial (HCjE) cells were infected with 107 IFU of Ct for 72 h. EMT-inducing signaling pathways, as well as mRNA and protein expression of EMT markers (E-cadherin, fibronectin and α-SMA) were evaluated by RT-qPCR, Immunoblotting and Immunocytochemistry. DNA methylation patterns of selected regions of E-cadherin, fibronectin and α-SMA genes were examined by Methylation-Specific PCR, High Resolution Melting analysis and Bisulfite Sequencing. Results: Infection with Ct was accompanied with the activation of EMT-inducing signaling pathways, downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers fibronectin and α-SMA. While DNA methylation status of E-cadherin gene promoter correlated with its expression, methylation status of fibronectin and α-SMA genes couldn’t be related to their expression levels. Conclusion: Ct infection of HCjE cells triggers EMT that goes along with changes in the methylation profile of the E-cadherin promoter. Sequence of events described herein could contribute to scarring process in trachoma and open up possibilities for development of new therapeutic strategies in trachoma treatment.Brajušković G, Đorđević A, editors. CoMBoS. 1st Congress of Molecular Biologists of Serbia; 2017 Sep 20-22; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 70

Chlamydia trachomatis infection and development of epithelial mesenchymal transition in conjunctiva : possible epigenetic mechanisms unveiled

Introduction: Trachoma is the most common cause of infectious blindness worldwide, initiated by repeated infection of the conjunctiva with Chlamydia trachomatis (Ct). The resulting chronic inflammation and formation of fibrotic tissue eventually lead to corneal damage. Based on the facts that epithelial to mesenchymal transition (EMT) plays an important role in the development of fibrosis and that EMT is epigenetically regulated process, the aims of this study were to reveal the capacity of Ct to induce EMT in vitro and to unveil potential underlying epigenetic mechanisms. Methods: Human conjunctival epithelial (HCjE) cells were infected with 107 IFU of Ct for 72 h. EMT-inducing signaling pathways, as well as mRNA and protein expression of EMT markers (E-cadherin, fibronectin and α-SMA) were evaluated by RT-qPCR, Immunoblotting and Immunocytochemistry. DNA methylation patterns of selected regions of E-cadherin, fibronectin and α-SMA genes were examined by Methylation-Specific PCR, High Resolution Melting analysis and Bisulfite Sequencing. Results: Infection with Ct was accompanied with the activation of EMT-inducing signaling pathways, downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers fibronectin and α-SMA. While DNA methylation status of E-cadherin gene promoter correlated with its expression, methylation status of fibronectin and α-SMA genes couldn’t be related to their expression levels. Conclusion: Ct infection of HCjE cells triggers EMT that goes along with changes in the methylation profile of the E-cadherin promoter. Sequence of events described herein could contribute to scarring process in trachoma and open up possibilities for development of new therapeutic strategies in trachoma treatment.Brajušković G, Đorđević A, editors. CoMBoS. 1st Congress of Molecular Biologists of Serbia; 2017 Sep 20-22; Belgrade, Serbia. Belgrade: University of Belgrade, Faculty of Biology; 2017. p. 70

A Probiotic Adjuvant Lactobacillus rhamnosus Enhances Specific Immune Responses after Ocular Mucosal Immunization with Chlamydial Polymorphic Membrane Protein C

Recent advances in the development of chlamydia vaccines, using live-attenuated or ultraviolet light-inactivated chlamydia, are paving the way for new possibilities to oppose the societal challenges posed by chlamydia-related diseases, such as blinding trachoma. An effective subunit vaccine would mitigate the risks associated with the use of a whole-cell vaccine. Our rationale for the design of an efficient subunit vaccine against Chlamydia trachomatis (Ct) is based on the membrane proteins involved in the initial Ct-host cell contact and on the route of immunization that mimics the natural infection process (i.e., via the ocular mucosa). The first aim of our study was to characterize the specific conjunctival and vaginal immune responses following eye drop immunization in BALB/c mice, using the N-terminal portion of the Ct serovar E polymorphic membrane protein C (N-PmpC) as the subunit vaccine antigen. Second, we aimed to examine the adjuvant properties of the probiotic Lactobacillus rhamnosus (LB) when formulated with N-PmpC. N-PmpC applied alone stimulated the production of N-PmpC-and Ct serovar B-specific antibodies in serum, tears and vaginal washes, whereas the combination with LB significantly enhanced these responses. The N-PmpC/LB combination initiated a T cell response characterized by an elevated percentage of CD25+ T cells and CD8+ effector T cells, enhanced CD4+ T-helper 1 skewing, and increased regulatory T cell responses. Together, these results show that eye drop vaccination with combined use of N-PmpC and a live probiotic LB stimulates specific cellular and humoral immune responses, not only locally in the conjunctiva but also in the vaginal mucosa, which could be a promising approach in Ct vaccine development

The effect of infectious dose on humoral and cellular immune responses in Chlamydophila caviae primary ocular infection

Following infection, the balance between protective immunity and immunopathology often depends on the initial infectious load. Several studies have investigated the effect of infectious dose; however, the mechanism by which infectious dose affects disease outcomes and the development of a protective immune response is not known. The aim of this study was to investigate how the infectious dose modulates the local and systemic humoral and the cellular immune responses during primary ocular chlamydial infection in the guinea pig animal model. Guinea pigs were infected by ocular instillation of a Chlamydophila caviae-containing eye solution in the conjunctival sac in three different doses: 1x10(2), 1x10(4), and 1x10(6) inclusion forming units (IFUs). Ocular pathology, chlamydial clearance, local and systemic C. caviae-specific humoral and cellular immune responses were assessed. All inocula of C. caviae significantly enhanced the local production of C. caviae-specific IgA in tears, but only guinea pigs infected with the higher doses showed significant changes in C. caviae-specific IgA levels in vaginal washes and serum. On complete resolution of infection, the low dose of C. caviae did not alter the ratio of CD4(+) and CD8(+) cells within guinea pigs' submandibular lymph node (SMLN) lymphocytes while the higher doses increased the percentages of CD4(+) and CD8(+) cells within the SMLN lymphocytes. A significant negative correlation between pathology intensity and the percentage of CD4(+) and CD8(+) cells within SMLN lymphocyte pool at selected time points post-infection was recorded for both 1x10(4), and 1x10(6) IFU infected guinea pigs. The relevance of the observed dose-dependent differences on the immune response should be further investigated in repeated ocular chlamydial infections

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background: In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen. Materials and methods: BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 x LD50) of tetanus toxin. Results: The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFN gamma and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p lt 0.05). Conclusion: Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world's leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1-893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/cmice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocularmucosa was well tolerated without signs of inflammation. N-PmpC- specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFN gamma immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Isolation of tetracycline-resistant Chlamydia suis from a pig herd affected by reproductive disorders and conjunctivitis

Due to various challenges in diagnosing chlamydiosis in pigs, antibiotic treatment is usually performed before any molecular or antibiotic susceptibility testing. This could increase the occurrence of tetracycline-resistant Chlamydia (C.) suis isolates in the affected pig population and potentiate the reoccurrence of clinical signs. Here, we present a case of an Austrian pig farm, where tetracycline resistant and sensitive C. suis isolates were isolated from four finishers with conjunctivitis. On herd-level, 10% of the finishers suffered from severe conjunctivitis and sows showed a high percentage of irregular return to estrus. Subsequent treatment of whole-herd using oxytetracycline led to a significant reduction of clinical signs. Retrospective antibiotic susceptibility testing revealed tetracycline resistance and decreased susceptibility to doxycycline in half of the ocular C. suis isolates, and all isolates were able to partially recover following a single-dose tetracycline treatment in vitro. These findings were later confirmed in vivo, when all former clinical signs recurred three months later. This case report raises awareness of tetracycline resistance in C. suis and emphasizes the importance of preventative selection of tetracycline resistant C. suis isolates