Fig 1.
Histopathological changes in ocular tissues infected with the TgCtwh6 strain.
Uninfected mice; infected mice at Day30. (A)Retina of a control eye from normal non-infected mice showing the normal architecture. All layers are present and unaltered. General aspect of TgCtwh6 infected retina showing the alteration of the retinal layer. Inflammatory cells were seen in the vitreous (*). Retinal vasculitis (black arrowhead) was evident and disorganization of the retinal architecture. Original magnification 400 ×, H&E stain. (B)Toxoplasma-ITS-1 PCR results, M: DNA Marker, n = 10 mice. (C)The mRNA expressions of IFN-γ and TNF-α in ocular tissues of naive mice and T. gondii-infected mice were measured by using a Real-time quantitative PCR. GAPDH was used as an internal control. The data were analyzed using Mann Whitney U test. mean ± SEM, *p < 0.05. (D)Representative experimental retinas stained for IFN-γ, TNF-α and IL-10 from control and OT murine model by immunohistochemistry as described in the Methods. 400 ×. Quantification of IFN-γ, TNF-α and IL-10 expression levels in retinas by the software Image J. Values represent the mean ± SEM, *P<0.05, **P<0.01.
Fig 2.
mRNA sequencing and regulatory gene analysis at Day 30 after TgCtwh6 infection.
(A) Volcano plots showing Log2(Fold Change) differences versus–Log10(False Discovery Rate) for indicated comparisons, using pooled data from each indicated sample type. Genes exhibiting Log2FC > 1 and FDR < 0.1 are colored. Red represents up-regulated significantly differentially expressed genes, blue represents down-regulated significantly differentially expressed genes and grey dots represent non-significant differentially expressed genes. The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and p value < 0.05 by R package edgeR or DESeq2, and then analysis GO enrichment (B) and KEGG enrichment (C) to the differentially expressed mRNAs.
Fig 3.
Representative flow cytometry plots for the analysis of NK cells and CD49a+ NK in the eyes and spleen of uninfected and Day 30-infected mice.
Percentage of NK (CD3- NK1.1+) cells in lymphocytes in the infected eyes (A) and spleen (B) from representative murine OT and control mice. (C) The percentage of CD49a+ NK (NK1.1+ CD3- CD49a+) cells was increased in infected eyes and spleen in the OT murine model. Pools of 10 mice were used and experiments were replicated three times. Values represent the mean ± SEM, *P<0.05, **P<0.01.
Fig 4.
T. gondii infection upregulates the expression of ICAM-1 and LFA-1 by NK cells in murine ocular tissue.
(A) The mRNA expressions of ICAM-1 and LFA-1 (ITGAL/ITGB2 gene) in ocular tissues of uninfected mice and TgCtwh6-infected mice were measured by using a Real-time quantitative PCR. GAPDH was used as an internal control. The results were representative of 3 independent experiments and the data were analyzed using Mann Whitney U test. mean ± SEM, *p < 0.05. (B) Western blot analysis showed that the protein expression of ICAM-1 and LFA-1 (CD11a/CD18) was upregulated in the eyes in comparison with the uninfected eye. GAPDH was used as an internal control. The results are from a pool of protein from two mice and are representative of results in three independent experiments. Expressions of LFA-1 on infiltrating CD3−NK1.1+ cells (C) and CD49a+ NK cells (D) in the eyes or spleen. Pools of 10 mice were used and experiments were replicated three times. The results were representative of 3 independent experiments and represented as mean ± SEM. The data were analyzed using Student’s t-test. mean ± SEM, *p < 0.05; **p < 0.01.
Fig 5.
The percentages of CD3- NK1.1+ cells and CD49a+ NK cells were assessed in the eyes and spleen at Day 30 after infection in the OT murine model treated with vehicle or lifitegrast.
(A-B) Representative flow cytometry plots for the analysis of NK cells (CD3- NK1.1+) and (C) CD49a+ NK cells. n = 5–11 mice; mean ± SEM; one-way ANOVA test; ns not significant, *p<0.05, **p<0.01. Data are representative of three independent experiments.
Fig 6.
Topical lifitegrast administration inhibited the expression of LFA-1 and ICAM-1 in OT murine model.
(A) Western blot analysis showed that ICAM-1 and LFA-1 were decreased in infected eyes after lifitegrast treatment. GAPDH was used as an internal control and the results are from a pool of protein from two mice. Flow cytometry analysis of LFA-1 expression in CD3- NK1.1+ cells (B) and CD49a+ NK cells (C) from lifitegrast-treated eyes or spleen of OT murine model. Pools of 10 mice and the experiment was repeated three times. The differences from the groups were compared using an ANOVA test. ns, not significant; *p<0.05; **p<0.01.
Fig 7.
Topical lifitegrast administration reduced ocular inflammation.
(A) Histology of the eye at Day 30 after infection in the OT murine model treated with vehicle or lifitegrast. Hematoxylin and eosin; 400 ×. (B) Representative experimental retinas stained for IFN-γ, TNF-α and IL-10 from vehicle or lifitegrast-treated murine OT. Immunohistochemistry showed that the production of pro-inflammatory cytokines (IFN-γ, TNF-α) but not anti-inflammatory cytokines (IL-10) was significantly inhibited by lifitegrast treatment. 400 ×. Quantification of IFN-γ, TNF-α and IL-10 expression levels in retinas by the software Image J. ns, not significant; *p<0.05. (C) Western blot analysis showed that IFN-γ and TNF-α were decreased, while IL-10 was increased in infected eyes after lifitegrast treatment. β-actin was used as an internal control. (D)Western blot analysis showed that caspase-3 were decreased in infected eyes after lifitegrast treatment. GAPDH was used as an internal control.