Wnt5a contributes to dectin-1 and LOX-1 induced host inflammatory response signature in Aspergillus fumigatus keratitis

Highlights

• Corneal Wnt5a production was dependent on dectin-1 and LOX-1 with contribution of Erk1/2 and JNK in A. fumigatus keratitis.

• Wnt5a production was dependent on dectin-1 and LOX-1 with contribution of Erk1/2 and JNK in response to A. fumigatus exposure.

• Wnt5a knockdown revealed decreased MPO levels, lower neutrophils recruitment, and a higher fungal load in A. fumigatus keratitis murine model.

• Wnt5a knockdown impaired pro-inflammatory cytokine IL-1β production in response to A. fumigatus exposure.

Abstract

Fungal keratitis causes devastating corneal ulcers which can result in significant visual impairment and even blindness. As a ligand that activates the non-canonical Wnt signaling pathways, Wnt5a triggers the production of important inflammatory chemokines and the chemotactic migration of neutrophils. In this study we aimed to characterize the role of Wnt5a production, in situ, in vivo and in vitro in response to fungal keratitis. Wnt5a expression in corneas of Aspergillus fumigatus (A. fumigatus) keratitis patients was determined by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. In vivo and in vitro experiments were then performed in mouse models and THP-1 macrophages cell cultures infected with A. fumigatus, respectively. C57BL/6 mice were pretreated with siRNAs or neutralizing antibodies for dectin-1, LOX-1 and Wnt5a, or inhibitors of erk1/2 and JNK. Changes in Wnt5a expression were assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blot and bioluminescence imaging system image acquisition. We confirmed that corneal Wnt5a expression increased with A. fumigatus keratitis in patients and a murine model. Wnt5a production was dependent on dectin-1 and LOX-1 expression with contributions by Erk1/2 and JNK pathways. Additionally, Wnt5a knockdown revealed decreased levels of MPO, lower neutrophil recruitment, and a higher fungal load in mouse models. Compared with controls, Wnt5a knockdown impaired pro-inflammatory cytokine IL-1β production in response to A. fumigatus exposure. Wnt5a also produces dectin-1 and LOX-1 induced inflammatory signature via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis. These findings demonstrate that Wnt5a is a critical component of the antifungal immune response.

Introduction

Fungal keratitis is an infectious disorder increasing in incidence worldwide [1]. Fungal keratitis, mostly caused by Aspergillus fumigatus (A. fumigatus) and Fusarium solani (F. solani), results due to ocular trauma secondary to irritation of the eye with vegetative matter. This condition affects general visual health predominantly in developing countries, such as China, India, and Mexico [2,3]. In industrialized countries, such as in the southeastern United States, the major risk factor for fungal keratitis is contact lens use [3,4]. Although new therapies have been used in clinical settings, fungal keratitis remains a challenge to ophthalmologists because of its difficult and thus delayed diagnosis as well as a lack of effective drugs and treatment methods [5].

Inflammation during fungal keratitis is initiated by various pattern recognition receptors (PRRs) which have been confirmed by recent studies, including toll-like receptor 2 (TLR2), dendritic cell-associated C-type lectin-1 (dectin-1) and lectin-type oxidized LDL receptor 1 (LOX-1) [3,[6], [7], [8]]. However, the intricate host-immune signaling pathways employed in response to pathogenic fungi invasion of the cornea are not well understood. Wnt signaling pathways, as a family of secreted glycoproteins, can be classified as canonical (β-catenin-dependent) or non-canonical (β-catenin-independent) pathways that play a critical role in the regulation of cell proliferation, differentiation, migration and inflammation [9,10]. In murine macrophages, pro-inflammatory cytokines IL-6, IL-12 and tumor necrosis factor α (TNF-α) are down-regulated, and anti-inflammatory cytokine IL-10 has been shown to be up-regulated by the Wnt canonical pathway upon infection with Francisella tularensis [11]. In contrast to the anti-inflammatory functions of Wnt canonical signaling pathway, there are reports indicating that the Wnt non-canonical pathway can regulate pro-inflammatory responses [12,13].

Wingless-type MMTV integration site family member 5A (Wnt5a) is a ligand that activates the non-canonical Wnt signaling pathway and plays a part in neutrophil recruitment, mediating inflammation in response to pathogenic microorganism infection [9,14,15]. Wnt5a β-catenin-independent signaling mediates planar cell polarity and Wnt–Ca²⁺ pathways by binding to different receptors, such as Frizzled 2, 5, 8 and receptor tyrosine kinase-like orphan receptor 2 (ROR2) [16]. Previous work has shown that Wnt5a is up-regulated upon mycobacterial infection or endotoxin stimulation (LPS) in human mononuclear cells and that Wnt5a knockdown of mature dendritic cells downregulates IL-12 [17,18]. Moreover, β-catenin-independent Wnt signaling was demonstrated to be mediated by Wnt5a promotion of pro-inflammatory cytokines expression, such as IL-1β, IL-6, IL-8, and macrophage inflammatory protein-1β (MIP-1β) in a JNK-dependent manner in human macrophages [19,20]. These results illustrate that Wnt5a plays a role in the induction of pro-inflammatory cytokine responses in inflammatory diseases. However, the production and role of Wnt5a in fungal keratitis remains unknown.

The present study demonstrates that Wnt5a protein expression is increased in the corneas of keratitis patients, human THP-1 macrophages and mice corneas infected with A. fumigatus. To better understand the production and role of Wnt5a in fungal keratitis, we focused on two PRRs, dectin-1 and LOX-1, which play important roles in antifungal immunity, as likely primary modulators increasing Wnt5a expression and its pro-inflammatory signaling function. We confirmed that Wnt5a is also a critical component of the antifungal immune response. Dectin-1 and LOX-1 were shown to induce an inflammatory signature dependent upon Wnt5a expression via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis.