The development of a stratified keratinocyte model for chlamydia trachomatis pathogenesis studies.

Abstract

A number of different methods to generate stratified keratinocyte layers have been published. These involved the use of normal human epidermal keratinocytes (NHEKs/NEKS), which have a better ability to stratify compared to HaCaT keratinocytes, which usually require supplemented growth factors or stromal interactions with fibroblasts to do so.

This study aimed to generate a model of stratified keratinocytes, closely resembling in vivo skin, using HaCaT cells and to demonstrate the effect that C. trachomatis has on these layered keratinocytes, allowing us to gain insight on the pathophysiology of this organism. All cells and bacteria were propagated and titrated according to conventional protocols. HaCaT cells were subcultured upon confluence, seeded (1x106 cells/ml) onto collagen-coated PTFE Transwell membrane inserts and incubated at 33°C and 37°C for 24 days to allow differentiation and stratification. Once cells became confluent they were exposed to the air-liquid interface and fed with KGM Gold (Lonza) supplemented with 10% FBS and additional calcium. Thereafter, cells were fixed in 3.7% phosphate-buffered formaldehyde, embedded in a paraffin block, sectioned, stained and viewed. Hematoxylin and Eosin (H&E) staining was used to determine the resemblance to in vivo human skin. Immunofluorescence was used to detect keratin 10, keratin 14 and involucrin which are markers of keratinocyte differentiation. Stratified keratinocyte layers were infected with C. trachomatis and this was confirmed using the MicroTrak ® C. trachomatis Culture Confirmation Test Kit. Subsequent changes to the layers were also observed and recorded. It was shown that HaCaT cells grown at the air-liquid interface on collagen-coated PTFE Transwell membrane inserts were able to stratify at 33°C. However, more layers of keratinocytes were seen at 37°C after the same duration of incubation (24 days). Keratin 10, keratin 14 and involucrin were all detected in the layers grown at both temperatures, suggesting that the keratinocytes had committed to differentiation. However, the fluorescence seen at 33°C for keratin 10 and involucrin was more intense as compared to that seen at 37°. This suggests that although stratification was faster at 37°C, differentiation was quicker at 33°C. C. trachomatis was able to infect layered keratinocytes grown at both temperatures although not all layers formed at 33°C were infected. Degradation of keratinocyte layers after infection with C. trachomatis was more prominent in those grown at 37°C, which is in keeping with previous findings that the optimum growth temperature of the C. trachomatis LGV biovar is 37°C. This study provided a novel insight in suggesting the manner in which C. trachomatis is able to infect and migrate through in vivo skin, leaving room for further studies in which similar methods of generating stratified keratinocytes may be used to better understand the pathophysiology of various other organisms that affect keratinocytes.