CLEC4E-Mediated Macrophage Activation Contributes to Host Defense in Acanthamoeba Keratitis

Acanthamoeba keratitis (AK) is a vision-threatening corneal infection characterized by intense inflammation and tissue damage. Although innate immunity is critical for host defense against Acanthamoeba, the pattern recognition receptors (PRRs) involved in pathogen sensing remain incompletely defined. This study investigated the role of the C-type lectin receptor CLEC4E in macrophage responses during AK.

Transcriptomic data from human AK corneas were analyzed to identify immune-related pathways and PRRs. RAW264.7 macrophages and bone marrow-derived macrophages were infected with Acanthamoeba trophozoites and subjected to RNA sequencing, quantitative real-time PCR, and Western blot analyses. CLEC4E function was examined using the agonist trehalose-6,6'-dibehenate and lentiviral short hairpin RNA-mediated knockdown. An experimental mouse model of AK was used to assess the effects of in vivo CLEC4E blockade.

A transcriptomic analysis of AK corneas revealed enhanced inflammatory signaling and increased expression of multiple PRRs, including CLEC4E. Acanthamoeba infection induced rapid and sustained CLEC4E upregulation in macrophages, accompanied by activation of nuclear factor κB signaling and increased expression of inflammatory mediators. Pharmacological activation of CLEC4E amplified inflammatory responses, whereas CLEC4E knockdown reduced cytokine production and nuclear factor κB activation. Correlation and functional analyses identified hypoxia-inducible factor 1-α (HIF-1α) as a mediator associated with CLEC4E-dependent inflammatory amplification. Inhibition of HIF-1α attenuated CLEC4E-enhanced inflammatory responses. In vivo, the neutralization of CLEC4E aggravated experimental AK, resulting in increased corneal opacity and clinical severity.

CLEC4E promotes macrophage activation and contributes to protective innate immune responses during Acanthamoeba infection. HIF-1α mediates CLEC4E-dependent inflammatory amplification, highlighting a potential link between CLEC4E signaling and macrophage inflammatory programs during AK.