Expression of innate and adaptive immune mediators in human corneal tissue infected with Aspergillus or fusarium

Abstract

Background: Filamentous fungi of the genera Aspergillus and Fusarium are major causes of corneal ulcers in the United States and in the developing world and result in significant visual impairment and blindness.

Methods: RNA was extracted from 110 patients with corneal ulcers in southern India within 1 week of infection with either Fusarium solani or Aspergillus flavus, and gene expression was determined by quantitative polymerase chain reaction. Posttransplant corneas from later stage disease (>2 weeks after infection) were also examined.

Results: Expression of Dectin-1, Toll-like receptor 2 (TLR2), TLR4, TLR9, and NOD-like receptor protein (NLRP)3 messenger RNA was elevated >1000-fold compared with uninfected donor corneas, whereas Dectin-2 was constitutively expressed in uninfected corneas. Furthermore, interleukin 1β (IL-1β) expression was elevated >1000-fold, whereas IL-1α expression was not increased. Expression of IL-8, IL-17, and tumor necrosis factor α was also elevated. CD3(+)and CD4(+) T cells were detected in infected posttransplant corneas. Expression of IL-17 and interferon γ was elevated but not that of IL-4. There were no significant differences in the host response between Aspergillus- and Fusarium-infected corneas at any time point.

Conclusions: There is a common innate and adaptive immune response to these filamentous fungi, which includes the generation of T-helper 1 and T-helper 17 cells.

Figure 1.

Cellular composition of corneal ulcers from patients with fungal keratitis. Shown are representative corneal ulcers caused by Aspergillus flavus (A) or by Fusarium solani (B) with the arrow indicating accumulation of neutrophils in the anterior chamber (original magnification ×10). Ulcerative tissue was fixed and stained with Diff-Quik, and representative images show A. flavus hyphae (C) and the cellular infiltrate (D) in infected corneas (original magnification ×400). The inset in panel D shows the typical nuclear morphology of neutrophils. The percentages of neutrophils and mononuclear cells were determined by counting 100 cells from 10 patients with Aspergillus infection and 10 patients with Fusarium infection (E).

Figure 2.

Expression of pathogen recognition receptors in corneal ulcers from patients with fungal keratitis. RNA was extracted from corneal ulcerative tissue, reverse transcribed, and processed for quantitative polymerase chain reaction. Data points represent individual patients with Aspergillus or Fusarium keratitis, and the values are presented as the log of relative gene expression (log[RQ]) relative to uninfected donor corneas calculated using the 2^−ΔΔCt method described in the text. There were no significant differences in gene expression between Aspergillus-infected corneas (closed circles) and Fusarium-infected corneas (P > .05) (open circles). TLR, toll-like receptor.

Figure 3.

Expression of proinflammatory, regulatory, and chemotactic cytokines in corneal ulcers. Q-PCR analysis of corneal ulcerative tissue was performed as described in Methods. Data points represent individual patients infected with Aspergillus or Fusarium and values were calculated as described above. There were no significant differences in gene expression between Aspergillus- (closed circles) and Fusarium- (open circles) infected corneas (P > .05). IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; log(RQ); log of relative gene expression.

Figure 4.

β-glucan and cellular infiltration in infected posttransplant corneas. A, Five-micron sections of Aspergillus flavus–infected corneas stained with Gomori-Methanamine silver to identify fungal hyphae or immunostained with antibodies to β-glucan (Dectin-1 ligand) (original magnification ×100 in upper left panel, ×400 in upper right and lower left panels, and ×1000 in lower right panel). Stroma and anterior chamber (ac) are indicated. B, Percentage of cell types (neutrophils [neuts] and macrophages [macs]) in Fusarium- and Aspergillus-infected corneas; 5 slides were examined for each stain, and 100 cells were counted on each section. C, Representative corneal sections of Aspergillus- and Fusarium-infected corneas immunostained with antibodies to neutrophils (elastase), macrophages (CD68), CD3, and CD4. DAPI (blue) showed the presence of cell nuclei in the CD4-stained sections, and 3,3′-diaminobenzidine and hematoxylin were used to visualize cells in other sections (original magnification ×400). D, Results of quantitative polymerase chain reaction performed on corneal tissue as described in the legend to Figure 2. Data points represent corneas from individual patients infected with Aspergillus or Fusarium in relation to uninfected donor corneas as described in the text. There were no significant differences in gene expression between Aspergillus- and Fusarium-infected corneas (P > .05). IFN, interferon; IL, interleukin; log(RQ), log of relative gene expression.