CD74 deficiency ameliorates Pseudomonas aeruginosa-induced ocular infection

Abstract

Eye trauma and contact lens wear are the main factors that predispose to the development of infectious keratitis. The existing therapies fail to control the inflammation-driven tissue damage that occurs during Pseudomonas aeruginosa infection. Antibiotic treatment reduces bacterial burdens, but better interventions are needed to alleviate tissue damage resulting from local inflammation. We have previously documented that inhibition of macrophage migration inhibitory factor (MIF) reduces the bacterial levels and the inflammatory damage during keratitis. Here, we report that mice deficient for CD74, the putative MIF receptor, developed milder Pseudomonas aeruginosa-induced disease, characterized by decreased proinflammatory mediators and reduced bacterial presence in the cornea. However, topical inhibition of MIF using antibodies applied to the cornea further promoted recovery from disease, suggesting that in addition to MIF-dependent signaling events, MIF-triggered CD74-independent signaling pathways regulate sensitization to P. aeruginosa-induced infection.

Figure 1. CD74 KO mice have milder P. aeruginosa-induced ocular keratitis.

(A) Pathology and levels of P. aeruginosa strain 6294, determined 48 h following infection with 1×10⁵ cfu onto eyes of CD74 KO and WT C57BL6 mice. Points indicate values for individual mice, bars the medians. Data are from a representative experiment out of two performed. (B) Histopathology images of corneal sections from C57BL6 and CD74 KO mice. (C) and (D) Levels and pathology of P. aeruginosa strain 6294, determined at 72 h following infection with 5×10⁶ cfu onto eyes of CD74 KO and WT C57BL6 mice. Points indicate values for individual mice, bars the medians. Data are from a representative experiment out of three performed.

Figure 2. CD74 KO mice exhibit reduced bacterial burdens in the cornea after infection with PAO1 and 6077 strains.

(A) and (B) Pathology and levels of P. aeruginosa strain PAO1, determined at 48 h (A) and 72 h (B) following infection with 5×10⁶ cfu onto eyes of CD74 KO and WT C57BL6 mice. Points indicate values for individual mice, bars the medians. Data are from a representative experiment out of two performed. (C) and (D) Pathology and levels of P. aeruginosa strain 6077, determined at 48 h (A) and at 72 h (B) following infection with 5×10⁵ cfu onto eyes of CD74 KO and WT C57BL6 mice. Points indicate values for individual mice, bars the medians. Data are from a representative experiment out of three performed.

Figure 3. CD74 regulates proinflammatory responses to P. aeruginosa.

Groups of 5 individual CD74 KO mice and 5 C57Bl6 mice were infected with 5 × 10⁶ cfu placed onto scratch-injured eyes. Mouse corneas were harvested at 48 h (A) and 72 h (B) after infection. The levels of mouse cytokines in corneal lysates were simultaneously measured using a MSD multiplex 7-spot electrochemiluminescence (ECL) assay. The levels of cytokines measured in the different groups were compared with the Mann-Whitney U test.

Figure 4. CD74 deficiency does not radically inhibit Th2 cytokines present in the cornea after P. aeruginosa infection.

Groups of 5 CD74 KO mice and 5 C57Bl6 mice were infected with 5 × 10⁶ cfu placed onto scratch-injured eyes. Mouse corneas were harvested at 48 h (A) and 72 h (B) after infection. The levels of mouse cytokines in corneal lysates were simultaneously measured using a MSD multiplex 7-spot electrochemiluminescence (ECL) assay. The levels of cytokines measured in the different groups were compared with the Mann-Whitney U test.

Figure 5. Treatment with anti-MIF antisera promotes further recovery from P. aeruginosa-induced corneal infection and pathology in CD74 KO mice.

(A) Groups of CD74 KO mice were treated with either rabbit control antisera or anti-MIF antisera every 8 h after the induction of infection for the duration of 2 days after which infected corneas were harvested, homogenized, and bacteria quantified. Points indicate values from individual mice, bars the medians. (B) Groups of C57BL6 and CD74 KO mice were treated with either gentamicin, non-immune rabbit antisera, or gentamicin and anti-MIF antisera every 8 hours after infection for 2 days after which infected corneas were harvested, and bacteria quantified. (C) Pathology scores plotted. Points indicate values from individual mice, bars the medians. Data analysis performed with 1way Anova with Dunn's Multiple Comparison Test (Prizm 4.0). Each experiment was repeated twice.