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Gonorrhea is the second most common sexually transmitted infection worldwide and is caused by Gram-negative, human-specific diplococcus Neisseria gonorrhoeae. It colonizes the mucosal surface of the female reproductive tract and the male urethra. A rapid increase in antibiotic resistance makes gonorrhea a serious threat to public health worldwide. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are not able to recapitulate all the features of infection. Therefore, a realistic in vitro cell culture model is urgently required for studying the gonorrhea infection. In this study, we established and characterized three independent 3D tissue models based on the porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. The histological, immunohistochemical, and ultra-structural analysis showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in the human host including the formation of epithelial monolayer, underlying connective tissue, mucus production, tight junction (TJ), and microvilli. In addition, functional analysis such as transepithelial electrical resistance (TEER) and barrier permeability indicated high barrier integrity of the cell layer. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results showed disruption of TJs and growing the interleukins production in response to the infection, which depends on the type of strain and cell. In addition, the 3D tissue models supported bacterial survival, which provided an appropriate in vitro model for long-term infection study. This could be mainly because of the high resilience of the 3D tissue models based on the SIS scaffold to the infection in terms of alteration in permeability, cell destruction, and bacterial transmigration.
During gonorrhea infection, a high level of neutrophils migrates to the site of infection. The studies also showed that N. gonorrhoeae can survive or even replicate inside the neutrophils. Therefore, studying the interaction between neutrophils and N. gonorrhoeae is substantially under scrutiny. For this purpose, we generated a 3D tissue model by triple co-culturing of human primary fibroblast cells, human colorectal carcinoma cells, and human umbilical vein endothelial cells. The tissue model was subsequently infected by N. gonorrhoeae. A perfusion-based bioreactor system was employed to recreate blood flow in the side of endothelial cells and consequently study human neutrophils transmigration to the site of infection. We observed neutrophils activation upon the infection. Furthermore, we demonstrated the uptake of N. gonorrhoeae by human neutrophils and reverse transmigration of neutrophils to the basal side carrying N. gonorrhoeae. In summary, the introduced 3D tissue models in this research represent a promising tool to investigate N. gonorrhoeae infections under close-to-natural conditions.
Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease associated with autoantibodies to type VII collagen, the major constituent of anchoring fibrils. Previous attempts to demonstrate the blister inducing potential of autoantibodies to this protein have failed. To address this question, we used an in vitro model involving cryosections of human skin incubated with patients’ autoantibodies and leukocytes from healthy donors. We show that sera from 14 out of 16 EBA patients, in contrast to sera from healthy controls, induced dermal-epidermal separation in the cryosections. The level of the experimentally induced split localizes to the lamina lucida of the dermal-epidermal junction. Recruitment and activation of neutrophils at the dermal-epidermal junction was necessary for split induction, whereas mononuclear cells were not required. Importantly, patients’ autoantibodies affinity-purified against a recombinant form of the non-collagenous 1 (NC1) domain of type VII collagen retained their blister-inducing capacity, while patients’ IgG that was depleted of reactivity to type VII collagen lost this ability. Monoclonal antibody LH7.2 to the NC1 domain of type VII collagen also induced subepidermal splits in the cryosections; F(ab’)2 fragments of autoantibodies to type VII collagen were not pathogenic. These findings demonstrate the capacity of autoantibodies to type VII collagen to trigger an Fcg-dependent inflammation leading to split formation in cryosections of human skin.