A Microphysiological Endocervix Model to Study Sexually Transmitted Infections and Host Immune Response

Introduction: Sexually transmitted infections (STIs) are common; in 2018 nearly 20% of people in the US were diagnosed with an STI, the majority in people 18-24 years old. In females, STIs, such as chlamydia and gonorrhea, can also increase the likelihood of infertility and preterm birth. However, direct mechanistic investigations of the pathogenesis and host immune response during infection are challenging as animal models often do not recapitulate human physiology needed for infection and common in vitro models lack complexity. As such, we have developed a microphysiological human cervical model that supports the culture of endocervical epithelia and immune cells that can be infected with pathogenic bacteria. This model enables tissue-relevant studies to elucidate how the innate immune system responds to infection as well as mechanistic studies of STI pathogenesis.

Materials and

Methods: We fabricated a modular two-component microfluidic system: (1) an “insert” that supported cell culture on a polycarbonate membrane and (2) a “cassette” to establish fluid flow through two insert channels. A2EN endocervical epithelial cells were cultured on one side of the insert while supporting BJ fibroblasts were cultured opposite side of the membrane, both for up to 7 days. Epithelial cells were kept at the air-liquid interface, and medium was circulated basally at a flow rate of 1-60 µl/min. Following model establishment, Neisseria gonorrhoeae (Gc) GFP-labeled piliated, OpaD non-variable strain FA1090 or Chlamydia trachomatis (Ct) strain L2/434/Bu VR-902B were added to the epithelialized channel to establish infection. After infection of the epithelial layer, polymorphonuclear leukocytes (PMNs) were added to fibroblast channel to permit the study of host-pathogen dynamics. Infection lifecycle and immune cell trafficking were analyzed via live-cell labeling visualized with fluorescence microscopy.

Results, Conclusions, and Discussions: Multicellular endocervical models were successfully created and cultured at the air-liquid interface (Figure 1A) as shown by immunofluorescent staining of junctional membrane markers and mucins in the endocervical monolayer. The A2EN cells formed an intact monolayer when co-cultured with supporting BJ fibroblasts in our simple membrane insert model. These data are consistent with studies using transwell inserts at the air-liquid interface. The cell-laden inserts were moved to the cassette and physiologically relevant shear stresses were added. A2EN cells more robustly produced tight junctional proteins and mucins when cultured under flow. To validate that Gc and Ct pathogenesis could be modeled using this system, each pathogen was inoculated on the apical surface of the A2EN cells and monitored via immunofluorescence imaging. Gc adhered to and replicated on A2EN cells within the model to form clonal micro colonies (Figure 1B). Ct could infect A2EN cells, form inclusions, and transition through its entire life cycle forming elementary bodies and reticulate bodies in the model (Figure 1C). We sought to determine if neutrophils could be recruited from the basal channel through the fibroblast-membrane-epithelial cell layer to the epithelial apical surface in this model to enable the study of the immune response to infection. Fluorescently labeled primary human PMNs were added to the fibroblast channel of a Gc infected device. PMNs could transmigrate through the fibroblast and epithelial layers into the apical channel and associate with Gc (Figure 1D). An increased level of myeloperoxidase (MPO) was detected in devices with PMN and Gc indicating PMNs are responding to Gc infection. These data indicate that PMNs can home to the site of infection and have a functional response in this model.
We developed a modular microphysiological model of the human endocervix that supports Gc and Ct infection. The tissue-level architecture incorporated epithelial, stromal, and immune cells, as well as pathogenic bacteria validating that sexually transmitted host-pathogen interactions can be studied in this model. Further, the low cost and ease of use of the design of this microphysiological model enables democratization and translation to the broader scientific community.

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