ISG15 in Host Defense Against Candida albicans Infection in a Mouse Model of Fungal Keratitis

Abstract

PurposeISG15, a di-ubiquitin-like protein, is critical for controlling certain viral and bacterial infections. We sought to determine if ISG15 plays a role in corneal innate immunity against Candida albicans (C. albicans) using a C57BL/6 (B6) mouse model of human fungal keratitis.MethodsScarified corneas of adult B6 mice were pretreated with TLR5 ligand flagellin and then inoculated with C. albicans. The expression of ISG15 and other genes involved in ISG15 conjugation (ISGylation) was determined by real-time PCR. ISG15 expression and distribution in infected corneas were assessed by immunohistochemistry. ISGylation was examined by Western blotting. siRNA knockdown and recombinant ISG15 were used to elucidate the effects of ISG15 on controlling fungal keratitis by clinical scoring, fungal number plate counting, ELISA cytokine determination, and polymorphonuclear leukocytes (PMN) infiltration measurement.ResultsHeat-killed C. albicans induced expression of ISG15, and hBD2 was markedly enhanced by flagellin-pretreatment in cultured human primary corneal epithelial cells (CECs). In vivo, C. albicans infection induced the expression of ISG15, ISGylation-associated genes (UBE1L, UBCH8, and HERC5), and ISGylation in mouse CECs, all of which were enhanced by flagellin-pretreatment. siRNA knockdown of ISG15 increased keratitis severity, dampened flagellin-induced protection, and greatly suppressed the expressions of ISGylation enzymes, IFN-γ, but not CXCL2 in B6 mouse CECs. Recombinant ISG15, on the other hand, enhanced corneal innate immunity against C. albicans and suppressed infection-induced IL-1β, but not IL-Ra expression. ISG15 alone induced the expression of IL-1Ra, CXCL10, and CRAMP in mouse CECs. ISG15 was upregulated and secreted in cultured human CECs in response to challenge in a type 1 IFN-dependent manner.ConclusionsOur data, for the first time, demonstrate that ISG15 acts as an immunomodulator in the cornea and plays a critical role in controlling fungal keratitis.

ISG15 in antiviral immunity and beyond

“…Recently, the impact of ISG15 on fungal infections was explored in a mouse model of fungal keratitis. Candida albicans was shown to induce the expression of ISG15, ubiquitin-activating enzyme e1 homologue (uBe1l), ubiquitin/ ISG15-conjugating enzyme e2 l6 (uBCH8; also known as uBe2l6) and e3 ISG15-protein ligase (HeRC5) in corneal epithelial cells (CeCs), resulting in the upregulation of ISGylation 126 . Knockdown of Isg15 in CeCs that were exposed to C. albicans following ocular scarification increased the severity of keratitis.…”

Deregulated MicroRNA-21 Expression in Monocytes from HIV-Infected Patients Contributes to Elevated IP-10 Secretion in HIV Infection

“…By exploring the signaling pathway and reviewing previous reports, we proposed an ubiquitin-like protein, ISG15 as essential in regulating the IP-10 expression and it exhibited higher levels during monocyte–macrophage differentiation and HIV infection ( 30 , 48 ). Recent study revealed that ISG15 can induce the expression of IP-10 in Candida albicans infection ( 49 ). NF-κB is a well-known transcription factor for IP-10 gene activation ( 34 – 37 ).…”

Experimental Models for Fungal Keratitis: An Overview of Principles and Protocols

“…However, the extent of abrasion may vary between groups, with some generating 20 or more crosshatch scratches to the central cornea while others reporting just 3, 1-mm deep, scratches [34][35][36]. The inoculum is typically administered as a 5 µl droplet to the surface of the eye, with the inocula ranging between 10 5 and 10 6 total yeasts [27,[33][34][35][36][37][38][39][40][41]. Scratching has also been used by several groups studying Fusarium solani keratitis.…”

“…For example, cytokines can be detected at the protein level by ELISA, label-based protein assays, and Western blotting at the RNA level by PCR [32][33][34]36,40,46]. Myeloperoxidase (MPO) is highly abundant in neutrophil granulocytes and other immune cells, and its activity can be measured by commercially available kits; 1 unit of MPO activity corresponds to approximately 2.0 × 10 5 PMNs [36,40,59]. The release of metalloproteases, which mediate the destruction of the corneal extracellular matrix during infection, can easily be assayed by zymography [43].…”

“…Grocott-Gomori Methenamine-Silver (GMS) may also be used to stain fungal cell walls black, and this is typically used in conjunction with hematoxylin and eosin (H&E) staining of serial sections. Immunohistochemistry can more specifically label host and fungal cells through fluorophore-conjugated antibodies [33,34,37,39,40].…”