Immunopathogenesis of Progressive Scarring Trachoma: Results of a 4-Year Longitudinal Study in Tanzanian Children.

Trachoma is initiated during childhood following repeated conjunctival infection with Chlamydia trachomatis, which causes a chronic inflammatory response in some individuals that leads to scarring and in-turning of the eyelids in later life. There is currently no treatment to halt the progression of scarring trachoma due to an incomplete understanding of disease pathogenesis. A cohort study was performed in northern Tanzania in 616 children aged 6 to 10 years at enrollment. Every 3 months for 4 years, children were examined for clinical signs of trachoma, and conjunctival swabs were collected for C. trachomatis detection and to analyze the expression of 46 immunofibrogenic genes. Data were analyzed in relation to progressive scarring status between baseline and the final time point. Genes that were significantly associated with scarring progression included those encoding proinflammatory chemokines (CXCL5, CCL20, CXCL13, and CCL18), cytokines (IL23A, IL19, and IL1B), matrix modifiers (MMP12 and SPARCL1), immune regulators (IDO1, SOCS3, and IL10), and a proinflammatory antimicrobial peptide (S100A7). In response to C. trachomatis infection, IL23A and PDGF were significantly upregulated in scarring progressors relative to in nonprogressors. Our findings highlight the importance of innate proinflammatory signals from the epithelium and implicate interleukin 23A (IL-23A)-responsive cells in driving trachomatous scarring, with potential key mechanistic roles for PDGFB, MMP12, and SPARCL1 in orchestrating fibrosis

Immunohistochemical Analysis of Scarring Trachoma Indicates Infiltration by Natural Killer and Undefined CD45 Negative Cells.

INTRODUCTION: The phenotype and function of immune cells infiltrating the conjunctiva in scarring trachoma have yet to be fully characterized. We assessed tissue morphology and immunophenotype of cellular infiltrates found in trachomatous scarring compared to control participants. METHODOLOGY: Clinical assessments and conjunctival biopsy samples were obtained from 34 individuals with trachomatous scarring undergoing trichiasis surgery and 33 control subjects undergoing cataract or retinal detachment surgery. Biopsy samples were fixed in buffered formalin and embedded in paraffin wax. Hematoxylin and eosin (H&E) staining was performed for assessment of the inflammatory cell infiltrate. Immunohistochemical staining of single markers on individual sections was performed to identify cells expressing CD3 (T-cells), CD4 (helper T-cells), CD8 (suppressor/cytotoxic T-cells and Natural Killer, NK, cells), NCR1 (NK cells), CD20 (B-cells), CD45 (nucleated hematopoietic cells), CD56 (NK and T-cells), CD68 (macrophages/monocytes) and CD83 (mature dendritic cells). The degree of scarring was assessed histologically using cross-polarized light to visualize collagen fibres. PRINCIPLE FINDINGS: Scarring, regardless of clinical inflammation, was associated with increased inflammatory cell infiltrates on H&E and CD45 staining. Scarring was also associated with increased CD8+ and CD56+ cells, but not CD3+ cells, suggestive of a NK cell infiltrate. This was supported by the presence of NCR1+ cells. There was some increase in CD20+ cells, but no evidence for increased CD4+, CD68+ or CD83+ cells. Numerous CD45 negative cells were also seen in the population of infiltrating inflammatory cells in scarred conjunctiva. Disorganization of the normal collagen architecture was strongly associated with clinical scarring. CONCLUSIONS/SIGNIFICANCE: These data point to the infiltration of immune cells with a phenotype suggestive of NK cells in conjunctival trachomatous scarring. A large proportion of CD45 negative inflammatory cells were also present. Future work should seek to understand the stimuli leading to the recruitment of these cells and their role in progressive scarring

The conjunctival transcriptome in Ethiopians after trichiasis surgery: associations with the development of eyelid contour abnormalities and the effect of oral doxycycline treatment

Background: Surgery to correct trichiasis is a key component of the World Health Organisation trachoma control strategy, however unfavourable outcomes such as eyelid contour abnormalities (ECA) following surgery are relatively common. This study aimed to understand the transcriptional changes associated with the early development of ECA and the impact of doxycycline, which has anti-inflammatory and anti-fibrotic properties, upon these transcription patterns. Methods: One thousand Ethiopians undergoing trichiasis surgery were enrolled in a randomised controlled trial following informed consent. Equal groups of randomly assigned individuals were orally administered with 100mg/day of doxycycline (n=499) or placebo (n=501) for 28 days. Conjunctival swabs were collected immediately prior to surgery and at one- and six-months post-surgery. 3’ mRNA sequencing was performed on paired baseline and one-month samples from 48 individuals; 12 in each treatment/ECA outcome group. qPCR validation was then performed for 46 genes of interest in 145 individuals who developed ECA at one month and 145 matched controls, using samples from baseline, one and six months. Results: All treatment/outcome groups upregulated genes associated with wound healing pathways at one month relative to baseline, however no individual differences were detected between groups. The summed expression of a highly coexpressed cluster of pro-fibrotic genes was higher in patients that developed ECA in the placebo group relative to controls. qPCR validation revealed that all genes in this cluster and a number of other pro-inflammatory genes were strongly associated with ECA, however these associations were not modulated by trial arm. Conclusions: The development of post-operative ECA is associated with overexpression of pro-inflammatory and pro-fibrotic genes including growth factors, matrix metalloproteinases, collagens and extracellular matrix proteins. There was no evidence that doxycycline modulated the association between gene expression and ECA.</ns4:p

The conjunctival transcriptome in Ethiopians after trichiasis surgery: associations with the development of eyelid contour abnormalities and the effect of oral doxycycline treatment

Background: Surgery to correct trichiasis is a key component of the World Health Organisation trachoma control strategy, however unfavourable outcomes such as eyelid contour abnormalities (ECA) following surgery are relatively common. This study aimed to understand the transcriptional changes associated with the early development of ECA and the impact of doxycycline, which has anti-inflammatory and anti-fibrotic properties, upon these transcription patterns. Methods: One thousand Ethiopians undergoing trichiasis surgery were enrolled in a randomised controlled trial following informed consent. Equal groups of randomly assigned individuals were orally administered with 100mg/day of doxycycline (n=499) or placebo (n=501) for 28 days. Conjunctival swabs were collected immediately prior to surgery and at one- and six-months post-surgery. 3’ mRNA sequencing was performed on paired baseline and one-month samples from 48 individuals; 12 in each treatment/outcome group (Placebo-Good outcome, Placebo-Poor outcome, Doxycycline-Good outcome, Doxycycline-Poor outcome). qPCR validation was then performed for 46 genes of interest in 145 individuals who developed ECA at one month and 145 matched controls, using samples from baseline, one and six months. Results: All treatment/outcome groups upregulated genes associated with wound healing pathways at one month relative to baseline, however no individual differences were detected between groups. The summed expression of a highly coexpressed cluster of pro-fibrotic genes was higher in patients that developed ECA in the placebo group relative to controls. qPCR validation revealed that all genes in this cluster and a number of other pro-inflammatory genes were strongly associated with ECA, however these associations were not modulated by trial arm. Conclusions: The development of post-operative ECA is associated with overexpression of pro-inflammatory and pro-fibrotic genes including growth factors, matrix metalloproteinases, collagens and extracellular matrix proteins. There was no evidence that doxycycline modulated the association between gene expression and ECA.</ns4:p

Differential frequency of NKG2C/KLRC2 deletion in distinct African populations and susceptibility to Trachoma: a new method for imputation of KLRC2 genotypes from SNP genotyping data.

NKG2C is an activating receptor that is preferentially expressed on natural killer (NK) cells. The gene encoding NKG2C (killer cell lectin-like receptor C2, KLRC2) is present at different copy numbers in the genomes of different individuals. Deletion at the NKG2C locus was investigated in a case-control study of 1522 individuals indigenous to East- and West-Africa and the association with the ocular Chlamydia trachomatis infection and its sequelae was explored. The frequency of homozygous KLRC2 deletion was 13.7 % in Gambians and 4.7 % in Tanzanians. A significantly higher frequency of the deletion allele was found in West-Africans from the Gambia and Guinea-Bissau (36.2 % p = 2.105 × 10(-8), 26.8 % p = 0.050; respectively) in comparison to East-African Tanzanians where the frequency of the deletion is comparable to other human populations (20.9 %). We found no evidence for an association between the numbers of KLRC2 gene copies and the clinical manifestations of trachoma (follicular trachoma or conjunctival scarring). A new method for imputation of KLRC2 genotypes from single nucleotide polymorphism (SNP) data in 2621 individuals from the Gambia further confirmed these results. Our data suggest that NKG2C does not play a major role in trachomatous disease. We found that the deletion allele is present at different frequencies in different populations but the reason behind these differences is currently not understood. The new method offers the potential to use SNP arrays from genome wide association studies to study the frequency of KLRC2 deletion in other populations and its association with other diseases

The conjunctival microbiome before and after azithromycin mass drug administration for trachoma control in a cohort of Tanzanian children.

Background: Trachoma, caused by ocular infection with Chlamydia trachomatis, is a neglected tropical disease that can lead to blinding pathology. Current trachoma control programmes have successfully used mass drug administration (MDA) with azithromycin to clear C. trachomatis infection and reduce transmission, alongside promoting facial cleanliness for better personal hygiene and environmental improvement. In areas of low-trachoma endemicity, the relationship between C. trachomatis infection and trachomatous disease weakens, and non-chlamydial bacteria have been associated with disease signs. Methods: We enrolled a cohort of children aged 6-10 years from three adjacent trachoma endemic villages in Kilimanjaro and Arusha regions, Northern Tanzania. Children were divided into four clinical groups based on the presence or absence of ocular C. trachomatis infection and clinical signs of trachomatous papillary inflammation (TP). To determine the impact of treatment on the ocular microbiome in these clinical groups, we performed V4-16S rRNA sequencing of conjunctival DNA from children 3-9 months pre-MDA (n = 269) and 3 months post-MDA (n = 79). Results: Chlamydia trachomatis PCR-negative, no TP children had the highest pre-MDA ocular microbiome alpha diversity, which was reduced in C. trachomatis infected children and further decreased in those with TP. Pre-MDA, Haemophilus and Staphylococcus were associated with C. trachomatis infection with and without concurrent TP, while Helicobacter was increased in those with TP in the absence of current C. trachomatis infection. Post-MDA, none of the studied children had ocular C. trachomatis infection or TP. MDA increased ocular microbiome diversity in all clinical groups, the change was of greater magnitude in children with pre-MDA TP. MDA effectively reduced the prevalence of disease causing pathogenic non-chlamydial bacteria, and promoted restoration of a normal, healthy conjunctival microbiome. Conclusion: We identified Helicobacter as a non-chlamydial bacterium associated with the clinical signs of TP. Further investigation to determine its relevance in other low-endemicity communities is required. MDA was shown to be effective at clearing C. trachomatis infection and other non-chlamydial ocular pathogens, without any detrimental longitudinal effects on the ocular microbiome. These findings suggest that azithromycin MDA may be valuable in trachoma control even in populations where the relationship between clinical signs of trachoma and the prevalence of current ocular C. trachomatis infection has become dissociated

Progression of scarring trachoma in Tanzanian children: A four-year cohort study.

BACKGROUND: Trachoma is a progressive blinding disease initiated by infection of the conjunctiva with Chlamydia trachomatis. Repeated infections are thought to cause chronic inflammation, which drives scarring, leading to in-turning of the eyelids. The relationship between C. trachomatis, clinical inflammation and scarring development in children is not fully understood due to a paucity of longitudinal studies with infection data at frequent follow-up. METHODS AND FINDINGS: This longitudinal cohort study took place in northern Tanzania. Children aged 6-10 years at baseline were eligible for inclusion. Participants were visited every three months for four years. Clinical signs and conjunctival swabs for C. trachomatis detection by qPCR were collected at each time-point. Conjunctival photographs from baseline and final time-points were graded and compared side-by-side to determine scarring incidence and progression. Of the 666 children enrolled in the study, outcome data were obtained for 448. Scarring progression was detected in 103/448 (23%) children; 48 (11%) of which had incident scarring and 55 (12%) had progression of existing scarring. Scarring was strongly associated with increasing episodes of trachomatous papillary inflammation (TP). Weaker associations were found between episodes of C. trachomatis infection and follicular trachoma (TF) with scarring progression in unadjusted models, which were absent in multivariable analysis after adjusting for inflammation (multivariable results: C. trachomatis p = 0.44, TF p = 0.25, TP p = <0.0001, age p = 0.13, female sex p = 0.05). Individuals having TP at 30% or more of the time-points they were seen had an odds ratio of 7.5 (95%CI = 2.7-20.8) for scarring progression relative to individuals without any TP detected during the study period. CONCLUSIONS: These data suggest that the effect of infection on scarring progression is mediated through papillary inflammation, and that other factors contributing to the development of inflammation, in addition to C. trachomatis infection, may be important in driving conjunctival scarring progression in children. The addition of TP as a measure in trachoma control programs would provide an indication of the future risk of developing scarring sequelae

Mycobacterium tuberculosis Eis Regulates Autophagy, Inflammation, and Cell Death through Redox-dependent Signaling

The “enhanced intracellular survival” (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner

Structure of S. aureus HPPK and the Discovery of a New Substrate Site Inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, Kd, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC50 of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and 15N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including 15N-1H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway