Clustering of Pattern Recognition Receptors for Fungal Detection

The innate immune system is the first line of defense against invading pathogens. Innate cells recognize microbes via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs); initiate innate immune responses; and eventually trigger adaptive immunity. Association of heterologous PRRs synergistically enhances their signal intensity. Such PRR cluster formation is essential for fungal detection in generating antifungal immunity. In this review, PRR cluster formation to detect fungi and to initiate innate immune responses is discussed.


Introduction
The innate immune system is the first line of defense against invading pathogens. Innate cells recognize microbes via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs); initiate innate immune responses; and eventually trigger adaptive immunity. Association of heterologous PRRs synergistically enhances their signal intensity. Such PRR cluster formation is essential for fungal detection in generating antifungal immunity. In this review, PRR cluster formation to detect fungi and to initiate innate immune responses is discussed.

PRRs Involved in Fungal Detection
Upon fungal infection, host innate immune cells, such as macrophages, neutrophils, and dendritic cells (DCs), are the first cell types to detect the infection. During the early stages of infection, before T cells are activated, the host innate immune system initiates antifungal responses (i.e., phagocytosis; production of reactive oxygen species [ROS], cytokines, and chemokines) as sentinels. Antigen-presenting myeloid cells, such as DCs and macrophages, then activate T cells by presenting foreign antigens to trigger adaptive immune responses, along with secretion of cytokines and chemokines to instruct naïve T cells to polarize ''proper'' T cell subsets.

Synergy of PRRs
It is known that association of heterologous PRRs synergistically enhances their signal intensity. TLR2, dectin-1, and MMR play a critical role in the detection and clearance of Pneumocystis [1,10]. In particular, TLR2 and dectin-1 physically associate each other and synergize to augment antifungal responses [4,[10][11][12]. Co-localization of TLR2 and dectin-1 in the phagocytic cap engulfing zymosan on the macrophage membrane was also reported [10,13]. Zymosan does not activate NFkB when cells express either TLR2 or dectin-1 alone; however, co-expression of TLR2 and dectin-1 allows zymosan to activate NFkB [4], suggesting the importance of PRR synergy. Synergy of decin-1 and TLR2 was also demonstrated in human monocyte-derived macrophages via increased TNFa production with curdlan (dectin-1 ligand) and Pam3Cys (TLR2 ligand) co-stimulation [12]. Further, crosstalk between dectin-1 and TLR2 signaling pathways has been suggested from the failure to elicit collaborative receptor responses by the lack of a signaling molecule, Syk or MyD88, which are downstream of dectin-1 and TLR2, respectively [11]. In addition to TLR2, pathways of TLR4, TLR5, TLR7, and TLR9 appear to collaborate with the dectin-1/Syk signaling pathway based on the synergistic enhancement of TNF production [11,12]. Finally, it has been reported that DC-SIGN synergizes TLR signaling through RAF1, a serine/threonine kinase, which induces phosphorylation of the p65 subunit of NFkB, followed by prolonged and enhanced transcription of Il10 by NFkB activation [14]. A sizeable number of studies thus suggest the existence of synergistic collaboration, particularly between TLR and CLR signaling pathways.

PRR Clustering in Fungal Detection
In order to achieve synergy between heterologous PRR signaling pathways, it is considered crucial to have PRRs in physical proximity to one another. Clustering PRRs on a myeloid cell surface is required for the optimum detection of fungi [10,15]. TLR2 and dectin-1 are recruited to lipid rafts, plasma membrane microdomains rich in cholesterol and sphingolipids, after stimulation with their ligands; signals from the receptors are synergized to enhance the downstream responses [10,16]. Disruption of lipid rafts causes decreased cytokine expression, phagocytosis, Ca 2+ influx, and phosphorylation of both Syk and ERK (Extracellular signal Regulated Kinases) [16], suggesting the critical role of rafts in PRR signaling. Importance of receptor cluster formation in antifungal immunity was demonstrated by comparing soluble and particulate b-glucans immobilized on latex beads [15]. Treating bone marrow-derived macrophages with particulate b-glucans induced dectin-1 signaling but soluble b-glucans did not; the former was done through the mechanism of excluding regulatory tyrosine phosphatases CD45 and CD148 from synapse-like structures [15]. We speculate that the physical crosslink of PRRs by particulate ligands may also be critical for triggering strong receptor signaling [10]. The PRR cluster was termed ''phagocytic synapse'' [15], which provides a platform for fungal detection and phagocytosis (Figure 1). Such PRR clustering is reminiscent of the supramolecular activation cluster (SMAC) formed on T cell surfaces in the immunological synapse; although, PRR clusters do not appear to be as distinct and intricate as SMAC. The biology and precise mechanism of PRR clustering are clearly arenas for further research.
We reported that PRR cluster formation of TLR2 and dectin-1 is essential for antifungal innate immunity against Pneumocystis [10]. In the signaling pathway downstream of the TLR2/dectin-1 cluster, intracellular osteopontin (iOPN) [17] plays a critical role ( Figure 1). Upon detection of Pneumocystis, iOPN was essential for the clustering of TLR2, dectin-1, and MMRs in macrophages [10]. iOPN simultaneously associates with IRAK1 and Syk, which are signaling molecules downstream of TLR2 and dectin-1, respectively, while enhancing MAPK (Mitogen-Activated Protein Kinases) activation [10]. OPN-deficient macrophages have shown attenuated phagocytosis of Pneumocystis and ROS generation, as well as cytokines production, suggesting that iOPN is essential for antifungal effects through PRR cluster formation. In fact, OPNdeficient immunocompromised mice are extremely susceptible to opportunistic infection by Pneumocystis [10]. iOPN is known to co-localize with actin and induces cytoskeletal rearrangement [18,19]. It is also known that the actin cytoskeleton plays an important role in the dynamics of the clusters with transmembrane receptors [20]. Therefore, evaluating the role of iOPN in the PRR clustering in the context of cytoskeletal rearrangement upon fungal detection may be of interest.

Question to Be Asked
Depending on the fungal species to be detected, macrophages appear to form clusters comprised of a unique combination of PRRs to optimize detection of fungi and to create synergy among multiple PRRs. However, precisely how PRRs are recruited to the lipid rafts and how the synergistic signal transduction pathways crosstalk are not clear. Relative to viruses and bacteria, sizes of fungi are, in general, significantly larger. It is therefore logical for host cells to form PRR clusters in an effort to maximize the interaction between PRRs and the fungal surface. In this regard, we suggest that the mechanism of PRR cluster formation in host cells during fungal detection is an issue critical to our further understanding of detection of fungi by host cells.