CD3ε of a pan T cell marker involved in mouse Aspergillus fumigatus keratitis

Abstract

Aim: To explore whether CD3ε is involved in the adaptive immunity of Aspergillus fumigatus (A. fumigatus) keratitis in mice and the role of innate and adaptive immunity in it.

Methods: Mice models of A. fumigatus keratitis were established by intra-stromal injection and corneal epithelial scratching. Subconjunctival injections of natamycin, wedelolactone, LOX-1 inhibitor (poly I) or Dectin-1 inhibitor (laminarin) were used to treat mice with A. fumigatus keratitis. Mice were pretreated by intraperitoneal injection of anti-mouse CD3ε. We observed the corneal infection of mice under the slit lamp microscope and made a clinical score. The protein expression of CD3ε and interleukin-10 (IL-10) was determined by Western blotting.

Results: With the disease progresses, the degree of corneal opacity and edema augmented. In the intra-stromal injection models, CD3ε protein expression began to increase significantly on the 2^nd day. However, in the scraping epithelial method models, CD3ε only began to increase on the 3^rd day. After natamycin treatment, the degree of corneal inflammation in mice was significantly attenuated on the 3^rd day. After wedelolactone treatment, the severity of keratitis worsened. And the amount of CD3ε protein was also reduced, compared with the control group. By inhibiting LOX-1 and Dectin-1, there was no significant difference in CD3ε production compared with the control group. After inhibiting CD3ε, corneal ulcer area and clinical score increased, and IL-10 expression was downregulated.

Conclusion: As a pan T cell marker, CD3ε participate in the adaptive immunity of A. fumigatus keratitis in mice. In our mice models, the corneas will enter the adaptive immune stage faster. By regulating IL-10, CD3ε exerts anti-inflammatory and repairs effects in the adaptive immune stage.

Keywords: Aspergillus fumigatus keratitis; CD3ε; adaptive immunity; interleukin-10.

Figure 1. CD3ε increased earlier in mice corneas of A. fumigatus keratitis developed by intra-stromal injection than corneal epithelium scratch

A: Fungal keratitis models were established by intra-stromal injection of A. fumigatus spores, and mouse corneas were observed under a slit lamp at 12h, 1, 2, and 3d after infection. B: The mouse corneas (six per group) were taken at each time point for CD3ε Western blot. C: A. fumigatus keratitis models were established by scraping the epithelium, and mouse corneas were observed under a slit lamp at 1/2, 1, 2, and 3d after infection. D: The mouse corneas (six per group) were taken at each time point for CD3ε Western blot. dpi: Day post infection. (^cP≤0.001, ns: P>0.05).

Figure 2. CD3ε was down-regulated in natamycin treated A. fumigatus keratitis mice

A, B: Subconjunctival injection of natamycin was used to treat fungal keratitis in mice for 2d and 3d. The mouse corneal slit lamp pictures (A) and clinical scores (B) were compared. C: After euthanasia, corneas (six per group) were taken for Western blotting to detect the protein of CD3ε. dpi: Day post infection. ^aP≤0.05, ^bP≤0.01, ns: P>0.05.

Figure 3. CD3ε was down-regulated in wedelolactone treated A. fumigatus keratitis mice

A, B: Subconjunctival injection of wedelolactone was used to treat fungal keratitis in mice for 2d and 3d. The mouse corneal slit lamp pictures (A) and clinical scores (B) were compared. C: After euthanasia, corneas (six per group) were taken for Western blotting to detect the protein of CD3ε. Dpi: Day post infection. ^aP≤0.05, ^cP≤0.001, ns: P>0.05.

Figure 4. CD3ε up-regulation in response to A. fumigatus infection was independent of Dectin-1 and LOX-1

The LOX-1 inhibitor (poly I; A) and Dectin-1 inhibitor (laminarin; B) were pretreated with mice. Two days later, mouse corneas (six per group) were taken for Western blotting to detect CD3ε (^cP≤0.001, ns: P>0.05).

Figure 5. Disease response after CD3ε antibody treatment

Intraperitoneal injection of anti-mouse CD3ε (145-2C11) was used to pretreat fungal keratitis in mice. The mouse corneal slit lamp pictures (A) and clinical scores (B) were compared on day 2, 3 and 5 (^aP≤0.05, ns: P>0.05).

Figure 6. Blockade of CD3ε decreased expression of IL-10 in mouse A. fumigatus keratitis

After euthanasia, corneas (six per group) were taken for Western blotting to detect the protein of IL-10 (^bP≤0.01, ^cP≤0.001).