Multiple studies have demonstrated inadequate long-term protection of azithromycin for treating trachomatous trichiasis [19–21]. Suboptimal efficacy of antibiotics was also evident amongst a chlamydia-associated reactive arthritis population where persistent chlamydial bodies were identified in fibroblasts and macrophages one month after doxycycline treatment . Further support for the poor antibiotic efficacy against chronic C. trachomatis infections was demonstrated in a population of women with post-infectious tubal infertility who remained infected despite antibiotic treatment . Urogenital chlamydial re-infections have been identified as probable treatment failure with azithromycin or doxycycline using ompA genotyping in approximately 8 and 13.7% of cases [33, 34]. Together the suboptimal efficacy of therapeutic antibiotics in the treatment of active and persistent chlamydial infections indicates the need for alternative treatments.
One potential alternative treatment utilizing 405 nm irradiation was evaluated in this study and demonstrated photo inactivation of C. trachomatis during active and persistent states. Small dosages starting at 5 J/cm2 had a significant growth inhibiting effect, with increasing energy densities positively correlating with growth inhibition. Therapeutic utility and clinical safety have been described using LED phototherapy at 405 nm against acne vulgaris  and gastric Helicobacter pylori infections, with the latter applied to the gastrointestinal mucosa via a light wand . In vitro anti-bacterial activity of 405 nm irradiation has been demonstrated against multiple medically relevant Gram-positive and Gram-negative extracellular pathogens like Staphylococcus aureus (including methicillin-resistant strains, MRSA), Streptococcus pyogenes, Pseudomonas aeruginosa, Clostridium perfringens, Campylobacter jejuni, Salmonella enteritidis and Escherichia coli[24, 37]. Overall Gram-negative bacteria appear more resistant to 405 nm irradiation than Gram-positive, with the exception of Enterococcus faecalis. The majority of Gram-positive bacteria appeared to require less than 11 J/cm2 for a log10 reduction in colony forming units, whereas Gram-negative bacteriocidal effects were apparent between 25 and 96 J/cm2. The portable LEDs used in this study were battery operated with 88 second dosing times, therefore making it difficult to obtain higher energy densities. Comparing the log10 reduction levels of other Gram negative bacteria with C. trachomatis is difficult due to its intracellular nature and considering it may exist as two-uncultivable life cycle forms: RBs and aberrant persistent forms. After irradiation with an energy density of 20 J/cm2 we demonstrated a nearly 70% reduction in chlamydial growth, reflecting levels similar to other Gram-negative bacteria. To our knowledge, this is the first data demonstrating chlamydial growth inhibition caused by 405 nm irradiation.
Photo inactivation by 405 nm irradiation is believed to be caused by excitation of androgenic porphyrins, resulting in oxygen free radical production and subsequent bacterial membrane disruption . Endogenously produced porphyrins, like coproporphyrin, uroporphyrins, and protoporphyrin IX, have been shown to be produced by both Gram positive and negative bacteria [25, 39] though, to our knowledge, porphyrin production by C. trachomatis has not yet been demonstrated. Considering the intracellular nature of C. trachomatis, a second photo inactivation mechanism might be associated with altered expression of eukaryotic proteins in response to 405 nm irradiation. Boncompain et al. demonstrated a transient upregulation of reactive oxygen species within C. trachomatis-infected HeLa cells for approximately six hours after infection, with subsequent basal levels ensuing nine hours post-infection. The regulation of reactive oxygen species appears to be mediated by C. trachomatis sequestration of the NADPH oxidase subunit, Rac1, to the inclusion membrane . Considering the significant growth inhibition effect when 405 nm was applied promptly two hours post-infection rather than 24 h, the irradiation might have altered chlamydial protein expression thus influencing its ability to sequester host Rac1, thereby increasing reactive oxygen species within the epithelial cells. An alteration in protein expression may have also delayed the formation and secretion of bacterial type III effector proteins, such as CPAF, that have previously been shown to be involved in binding and degrading eukaryotic proteins like cytokeratin 8, adhesion protein nectin-1, host transcription factor RFX5, and multiple host pro-apoptotic BH3 proteins [41–44]. Alternatively, the lack of 405 nm photo inactivation effect on chlamydial growth at 24 h post-infection might be due to the exponentially higher bacterial burdens within the inclusion body 24 h post-infection relative to two hours post-infection, potentially causing the differences after treatment to be less pronounced. It is also possible that multiple mechanisms co-exist and provide a cumulative anti-proliferative effect on C. trachomatis, though further studies are warranted.
The immunopathologic sequelae from conjunctival and genital chlamydial infections are likely mediated through the secretion of a group of pro-inflammatory cytokines. In trachoma, we demonstrated elevated levels of IL-6 during both acute and chronic grades of infection, with detectable chlamydial cases exhibiting more pronounced concentrations . The role of IL-6 in immunopathologenesis was also evident in women with ectopic pregnancies  and positively correlated with antibody titers against Chlamydophila pneumoniae amongst atherosclerotic patients . In an attempt to mimic chronic chlamydial infections, Macaca nemestrina fallopian tubes received repeated C. trachomatis infections, which resulted in fibrosis and elevated IL-6, IL-10, IL-2, and IFNγ levels . In TLR2 -/- KO mice infected with mouse pneumonitis (MoPn), decreased fibrosis and inflammation with in oviducts and mesosalpinx correlated with abated IL-6 concentrations . To determine the immunologic correlation of persistence in vitro with clinical presentation, we quantified IL-6 in penicillin-induced C. trachomatis persistent infections in HeLa cells. We demonstrated similar increases in IL-6 production in persistent infections compared to active infections in vitro. A previous study looked at persistent infections with C. pneumoniae in the presence of iron-depletion, IFNγ and penicillin, and demonstrated slightly diminished production of IL-6 after 24 h and 48 h . However, multiple experimental differences between these studies, including the use of different chlamydial species, might provide an explanation for the differences in results. For example, Peters et al. added penicillin 30 min after infection, followed by daily media change. This is in contrast to our study which added penicillin 24 h post-infection without a daily media change. Wang et al. provided more molecular details of this persistent state, demonstrating attenuated production of secreted chlamydial proteins from ampicillin-induced persistence of C. trachomatis infected HeLa cells , suggesting that secreted type III effector proteins like CPAF , Tarp , CT311 , and CT795  may be involved in regulating IL-6 levels. We are unaware of any other studies that examine inflammatory differences associated with penicillin-induced persistence. The elevation of IL-6 after penicillin-induced persistence supports the importance of this model in elucidating other inflammatory mediators that may be associated with chronic infections in vivo. Further research on molecular characterizations and their immunostimulatory properties is needed to understand this in vitro antibiotic-induced persistent model.
Considering the immunopathologic response to chronic chlamydial infections, we were interested in determining the role of 405 nm irradiation on cytokines previously associated with immunopathogenesis. In our study, irradiation with 405 nm had a dose-dependent effect on IL-6 production with 20 J/cm2 causing a 64% reduction. However, this effect was most likely associated with a decreased bacterial burden since previous studies demonstrated elevated IL-6 from UV-A (340-450 nm) exposed fibroblasts [53, 54] and minimal effects of UV-A (1 J/cm2) treated keratinocytes on IL-6 production . Interestingly, attenuation of IL-6 after 405 nm treatment was only evident if 405 nm irradiation was applied promptly after infection; the effect was lost if applied 24 h post-infection. We believe that at this later time point, multiple chlamydial proteins were already secreted by type III secretory pathways into the host cytoplasm and interacted with pattern recognition receptors (PRRs) resulting in IL-6 production.
Previously, we have identified CCL2 as a risk factor for trichiasis , and therefore analyzed the effect of 405 nm irradiation on C. trachomatis induced CCL2 production. To our knowledge, our findings are the first to demonstrate elevated levels of CCL2 after C. trachomatis infection in HeLa cells. In vivo analysis has shown elevated mRNA levels of CCL2 at two days post-infection with C. trachomatis mouse pneumonitis (MoPn) strain . Unlike IL-6, the use of 405 nm phototherapy on C. trachomatis infected HeLa cells did not have a significant effect on CCL2 production. More studies are needed to further understand the relationship between C. trachomatis infection and CCL2 production resulting in these inflammatory differences.