Interleukin 17 Expression in Peripheral Blood Neutrophils From Fungal Keratitis Patients and Healthy Cohorts in Southern India

Interleukin 17A (IL-17) production by peripheral blood neutrophils was examined in patients with fungal keratitis and in uninfected individuals in southern India, which has high levels of airborne Aspergillus and Fusarium conidia. Il17a gene expression and intracellular IL-17 were detected in all groups, although levels were significantly elevated in neutrophils from patients with keratitis. There were no significant differences in plasma IL-17 and IL-23 between patients with keratitis and uninfected individuals; however, combined data from all groups showed a correlation between the percentage IL-17 producing neutrophils and plasma IL-23, and between plasma IL-17 and IL-6 and IL-23

Host response and bacterial virulence factor expression in Pseudomonas aeruginosa and Streptococcus pneumoniae corneal ulcers.

P. aeruginosa and S. pneumoniae are major bacterial causes of corneal ulcers in industrialized and in developing countries. The current study examined host innate immune responses at the site of infection, and also expression of bacterial virulence factors in clinical isolates from patients in south India. Corneal ulcer material was obtained from 49 patients with confirmed P. aeruginosa and 27 patients with S. pneumoniae, and gene expression of Toll Like Receptors (TLR), cytokines and inflammasome proteins was measured by quantitative PCR. Expression of P. aeruginosa type III secretion exotoxins and S. pneumoniae pneumolysin was detected by western blot analysis. We found that neutrophils comprised >90% cells in corneal ulcers, and that there was elevated expression of TLR2, TLR4, TLR5 and TLR9, the NLRP3 and NLRC4 inflammasomes and the ASC adaptor molecule. IL-1α IL-1β and IFN-γ expression was also elevated; however, there was no significant difference in expression of any of these genes between corneal ulcers from P. aeruginosa and S. pneumoniae infected patients. We also show that 41/49 (84%) of P. aeruginosa clinical isolates expressed ExoS and ExoT, whereas 5/49 (10%) of isolates expressed ExoS, ExoT and ExoU with only 2/49 isolates expressing ExoT and ExoU. In contrast, all 27 S. pneumoniae clinical isolates produced pneumolysin. Taken together, these findings demonstrate that ExoS/T expressing P. aeruginosa and pneumolysin expressing S. pneumoniae predominate in bacterial keratitis. While P. aeruginosa strains expressing both ExoU and ExoS are usually rare, these strains actually outnumbered strains expressing only ExoU in the current study. Further, as neutrophils are the predominant cell type in these corneal ulcers, they are the likely source of cytokines and of the increased TLR and inflammasome expression

Primer sequences.

<p>Primer sequences.</p

Protein expression of <i>S. pneumoniae</i> pneumolysin and <i>P. aeruginosa</i> exotoxins.

<p>A. Western blot of pneumolysin in the ATCC reference strain (Lane 1), and four representative clinical isolates (lanes 2–5). B. Western blot of culture supernatants from <i>Pseudomonas aerugenosa</i> reference strains PAO1, which expresses ExoS and ExoT, and PA103, which expresses ExoU and ExoT. C. ExoS, ExoT and ExoU expression in representative <i>Pseudomonas</i> clinical isolates. Lane 1 is similar to PAO1 in expressing ExoS and ExoT; Lane 2 is similar to PA103 in expressing ExoU and ExoT; Lane 3 is <i>P. otitidis</i>, which does not express Type III exotoxins; Lane-4 Clinical isolate express all three effector molecules; Lane-5 Exo U and Exo T expressing clinical isolate similar to PA103. D. percent and total exotoxin production by 49 clinical isolates.</p

Gene expression of Toll Like Receptors, inflammasome proteins and cytokines in corneal ulcers from patients with bacterial keratitis.

<p>RNA was extracted from corneal ulcers, reverse transcribed and processed for Q-PCR. Data points represent individual patients infected with <i>P. aeruginosa</i> (closed circles) or <i>S. pneumoniae</i> (open circles), and the values presented are the log of relative gene expression (log(RQ)) in relation to uninfected donor corneas calculated using the 2^−ΔΔct method described in Methods. A. Pro-inflammatory cytokines IL-1α, IL-1β and IFN-γ; B. Toll Like Receptors and C. Inflammasome proteins. There were no significant differences in gene expression between <i>P. aeruginosa</i> and <i>S. pneumoniae</i> (p>0.05).</p

Clinical characteristics.

<p>Data are number and percent (%) of patients, unless otherwise indicated. Patients (n = 48) had corneal ulcers and presented at the clinic within 1–2 weeks after infection; corneal scrapings from the ulcer were used in the present study. Ten donor corneas from individuals with no infection or inflammation were obtained from the International Rotary Aravind Eye Bank. NA, not applicable.</p>*<p>accumulation of neutrophils in the anterior chamber.</p

Cellular composition of corneal ulcers from patients with bacterial keratitis.

<p>Representative corneal ulcers of patients caused by <i>P. aeruginosa</i> (A), or by <i>S. pneumoniae</i> (B). C, D. Gram staining of corneal ulcer material showing Gram negative bacilli (C), and Gram positive diplococci and chains (D). Original magnification is x1000. E,F: Wrights Giemsa (Diff-Quik) stain of corneal ulcer material from <i>P. aeruginosa</i> (E), or <i>S. pneumoniae</i> (F) infected tissue Original magnification is x400. G. Percent neutrophils and mononuclear cells were determined by counting cells from ten <i>P. aeruginosa</i> and ten <i>S. pneumoniae</i> patients.</p