Immunoglobulins at the interface of the gut mycobiota and anti-fungal immunity

Abstract

The dynamic and complex community of microbes that colonizes the intestines is composed of bacteria, fungi, and viruses. At the mucosal surfaces, immunoglobulins play a key role in protection against bacterial and fungal pathogens, and their toxins. Secretory immunoglobulin A (sIgA) is the most abundantly produced antibody at the mucosal surfaces, while Immunoglobulin G (IgG) isotypes play a critical role in systemic protection. IgA and IgG antibodies with reactivity to commensal fungi play an important role in shaping the mycobiota and host antifungal immunity. In this article, we review the latest evidence that establishes a connection between commensal fungi and B cell-mediated antifungal immunity as an additional layer of protection against fungal infections and inflammation.

1. Introduction

Known collectively as the “gut mycobiota”, the fungal component of the intestinal microbiota ranges between 1% and 2% of biomass [1], [2] or ~0.01% of DNA in human fecal material as assessed through metagenomic sequencing [3]. Recent evidence points to several fungal species associating with the gastrointestinal tract as either commensals or transients arriving through food sources or the outside environment [3], [4], [5]. Among these species and strains belonging to Candida, Saccharomyces, Pichia, Malassezia, Debaryomyces and several other genera have been more vigorously studied in the recent years to highlight the relationship of gut fungi to the gut immune system and various diseases including inflammatory bowel disease (IBD), diabetes, cancer, brain disorders, chronic kidney diseases, respiratory diseases, and liver diseases [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18].

Antibodies play a pivotal role in the interface between intestinal microbes and the host immune system due to their capacity to generate potent mucosal immune responses and maintain gut homeostasis. Recent investigation revealed that C. albicans and its hyphal morphotype are particularly potent inducers of antifungal sIgA, which promotes fungal commensalism in the gut. In this review, we will examine the body of work elucidating the mechanisms that regulate antifungal antibody (aFA) responses and a therapeutic potential of antibodies to fungal commensals and pathogens.

2. Systemic and mucosal antibody responses to bacteria and fungi

The diverse community of bacteria, fungi and viruses composing the gut microbiota necessitates a constant, broad, and finely tuned management of host-microbe interactions by the mucosal immune system throughout life. In this context, an expanding body of research has identified antibodies against commensal bacteria as a critical arm of the adaptive immune response, providing protection against toxins and invasive pathogens while also promoting commensal interactions. In the gut, vast quantities of secretory immunoglobulins (sIg) are secreted daily into the gut lumen to bind [19], [20], [21], neutralize [21], [22], [23], and chaperone microbial residents to lymphoid tissues for sampling by immune cells [24], [25], [26], [27], [28]. Although IgA – the most abundant mammalian sIg isotype produced in the gut mucosa – is considered to play an important role in maintaining homeostasis at mucosal barriers, more recent work revealed that IgG also recognizes a broad range of microbial antigens that are in some cases overlapping with IgA [29]. sIg plays a particularly critical protective role in early life, as maternally transferred sIg protects the immature neonatal intestinal environment by dampening the response to colonizing microbes (with observable long-term implications) [30], [31], [32], [33], [34], as well as playing a pivotal role in protection against enteric pathogenic bacteria such as Shigella flexneri and Salmonella typhimurium, [35], [36]. Recent studies utilizing novel techniques and improved resolution uncover more nuanced homeostatic functions of sIgA, acting as a buffer against offensive colonization [37], [38], [39], and triggering transcriptional modulation of sIgA -bound microbiota species [38], [40], [41], [42].

2.1. Role of Immunoglobulins to bacteria at the mucosal surfaces

Mounting evidence has demonstrated that intestinal microbiota participates in shaping B cells and humoral immunity in health and disease settings. Germ-free (GF) mice exhibit impaired lymphoid structures, dramatically reduced serum IgG and IgA titers, and antibody-producing plasma cells- features that can be reversed by microbial colonization [43], [44]. Another study also revealed that deletion of activation-induced cytidine deaminase (AID)-dependent antibodies, which fail to undergo class switch recombination (CSR) and somatic hypermutation (SHM) necessary for affinity maturation, allowed for expansion of anaerobic bacteria such as segmented filamentous bacteria (SFB), which could penetrate the mucus layer to reach the intestinal epithelium and induce isolated lymphoid follicle (ILF) hyperplasia [45], [46]. Interestingly, the Bacteroidetes, the major phylum of gut microbiota including Bacteroides faecis, Bacteroides caccae, and Bacteroides acidifaciens, was positively associated with sIgA production in Peyer’s patches and AID activity [47]. In addition, it was recently reported that the level of sIgA production in mice monocolonized with human gut isolates of Bacteroides ovatus depends on yet to be determined strain-specific features [48].

sIgA possess broad reactivity to a variety of microorganisms in the gut to prevent systemic dissemination of gut microbes. Generally, it is estimated that approximately 50% of luminal commensal microbiota in the small intestine is bound to sIgA. A fraction of microbes bound by highly-specific sIgA antibodies contain pathobionts that aggravate colon inflammation [49]. Analysis of fecal samples from elderly people revealed that age-related changes in the composition of gut microbiota correlates with a reduction of sIgA antibodies against several pathobionts [50]. Another study reported that pediatric patients with allergy have a low proportion of sIgA bound to fecal bacteria compared with healthy individuals [51]. While sIgA coating of pathobionts can limit their pathogenesis, it can also trigger inflammation in a context-dependent manner:

For example, opsonized bacteria engulfed by neutrophils, can recruit inflammatory cells into the colonic mucosa of ulcerative colitis patients, and promote inflammation via neutrophil extracellular trap (NET) formation [52], [53], [54]. Thus, antibody-coated pathobionts can cause tissue damage and inflammation by neutrophil activation in the context of ongoing intestinal disease.

2.2. Role of secretory IgA antibodies to fungi at the mucosal surfaces

Diverse fungal communities (the mycobiota) are associated with nearly all human barrier surfaces. Recent characterization of the mycobiota biogeography along the gastrointestinal tract revealed the presence of a subset of fungi associated with the intestinal mucosa of mice and humans [55], with several Candida and Saccharomyces species making the majority of these mucosa-associated fungi. Using a multistep flow cytometry-based technique for multi-kingdom antibody profiling (multiKAP) a recent study explored the antibody pool against gut mycobiota and determined a population of intestinal fungi that was bound by both luminal (sIgA) and systemic (IgG) antibodies [56]. The human gut commensal C. albicans was determined as the primary target (Fig. 1). In contrast to bacteria, C. albicans cells in the gut exist in both yeast and hyphal morphotypes that differ in cell size and function. However, how fungal species with the potential to cause disease can safely inhabit the intestinal mucosa without causing harm has been a mystery [57]. Using mouse models and several genetic approaches affecting the process of hyphal formation in C. albicans, two studies determined that sIgA antibodies bind preferentially to the larger hyphal morphotypes [58], [59].

Fig. 1. Function of secretory IgA and systemic IgG antibodies to fungi.

Several effects of antibody binding have been described in the context of antifungal immunity. From the oral cavity to the intestinal lumen, sIgA immobilizes and thus prevents fungi from encroaching on the epithelial layer, a process known as immunologic exclusion (A, top left). sIgA also promotes commensalism in C. albicans by targeting hyphae-associated (Als1 and Als3) and secreted (Candidalysin, SAP6) virulence factors to incur a fitness penalty on the hyphal morphotype (A, top) and protect host cells (A, top right). A significant fraction of the systemic antibody repertoire in circulation also recognizes fungi, from highly reactive IgG to natural IgM (A-B, bottom). These sIg have been demonstrated to protect against fungi that disseminate from the intestinal or the lung mucosa and cause systemic infection (A-B). Figures Created with BioRender.com.

Given the importance of hyphae for host cell invasion and the presence of multiple virulence factors associated with this morphotype, recent studies focused on hyphae-associated virulence factors as a potential target of these antibodies (Fig. 1). Adhesin proteins such as members of the Als family (particularly Als3 and Als1), and Hwp1 and Hyr1 [14], [60], [61] allow hyphae to bind to intestinal epithelial cells. Among these, Als3 is also critical for regulating iron homeostasis between the iron-rich intestinal lumen and to the iron-poor bloodstream upon invasion [61]. Als3, along with Ssa1, is involved in the hyphae-associated invasion strategy of inducing endocytosis through binding to N- or E-cadherin [14], [60], [61], [62]. Recent studies demonstrated that salivary sIgA prevent C. albicans adhesion and interferes with its iron acquisition through binding of Als3, further interfering with hyphae-associated activities [63], [64]. Thus, Als proteins play several roles that foster C. albicans invasion properties in different environments. Furthermore, sIgA in the oral cavity prevented fungal epithelial adhesion and invasion leading to decrease of the proinflammatory response [65]. What are the factors targeted by sIgA in this scenario remains to be determined.

Corresponding with the preferential sIgA coating of these hyphal-associated virulence factors, numerous studies point to associations between Candida commensalism and its intestinal fitness (Fig. 1). In the gut, transcription factor WOR1, induced upon gastrointestinal passage of C. albicans, results in unique “GUT” morphotypes that excel at intestinal colonization [66]. Competitive gut colonization of mice with a library of homozygous gene disruption C. albicans mutants resulted in successful gut colonization upon disruption of transcription factor Ume6, a master regulator of filamentation. Ume6, similar to the well-known regulator of morphogenesis Efg1 [13], promotes the expression of hyphae-associated virulence factors including the secreted aspartyl proteinase protease (Sap6) and adhesin Hyr1 [14]. Sap6 mutants exhibit enhanced colonization fitness, whereas Sap6-overexpression strains exhibit reduced competitiveness in the gut. Using a screening approach of homozygous deletion mutant libraries of C. albicans, a recent study determined that transcription factor Ahr1 is necessary for sIgA binding to C. albicans. Ahr1 is involved in adhesion and hyphal formation and regulates Als3, which the study determined to be the target of sIgA [59]. Expression of Als3 or two other hyphae-associated adhesins Als1 and Hwp1 on S. cerevisiae (yeast that induces little sIgA) was sufficient for sIgA binding. Similar sIgA binding was observed to C. glabrata that also expresses these adhesins. Repeated colonization of C. albicans into wild type (WT), but not into the adaptive immunity deficient Rag1^−/− mice, resulted in decreased expression of ALS1 and ALS3, demonstrating the fitness penalty conferred by the adaptive immunity [59]. It remains to be determined whether these effects on C. albicans are direct (B- and/or T-cells) or indirect (mediated by different microbiota in mice lacking adaptive immunity).

While adhesins are surface molecules with natural ability to bind to substrates allowing the attachment and penetration of pathogens into host cells, these proteins are not directly responsible for the damage caused by the pathogen itself. Indeed, it was demonstrated that during pathologies [12], C. albicans damage intestinal epithelial cells (IECs) and macrophages by secretion of pore-forming toxin candidalysin [67], which induces apoptotic cell death [68] and fuels intestinal inflammation by inducing macrophage cell damage and release of IL-1 [12]. Notably, strains of C. albicans, associated with different individuals harbor different potential to cause damage [12]. These findings underline a strain-specific nature of host–fungal interactions in the gut [12]. Candidalysin is produced by several Candida species that are present in the human gut [69], and it was identified along with Sap6 as a target of C. albicans colonization-induced sIgA that was notably decreased in Crohn’s Disease patients’ mucosal lavages [58]. Candida hyphal morphotypes and several hyphae-associated factors have a direct effect on host adaptive immunity by triggering B cell expansion and class switch recombination in Peyer’s Patches (PP) in response to intestinal C. albicans colonization [58], [59]. These findings reveal the nuanced effect of sIgA binding to C. albicans and demonstrate a role for sIgA in influencing the processes of yeast-to-hyphal transition in the intestines.

2.3. Systemic antifungal antibodies as a layer of protection against systemic fungemia

The systemic immunoglobulin response is dominated by IgG, making up ~70–75% of the total Ig in serum and tissues [70]. A continuously expanding body of research demonstrates that, in addition to the secretory anti-commensal sIgA response, a surprisingly large fraction of the systemic IgG repertoire circulating in the blood recognizes gut commensal epitopes. Multiple studies have documented the capacity of the systemic IgG to bind gut microbes under steady-state conditions and confer protection against bacteria escaping systemically from the gut [29], [30], [31], [32], [71], [72], [73]. Furthermore, extraintestinal B cells have been shown to recognize a significant fraction of the gut microbiota [74], [75].

Traditionally, the systemic antifungal antibody responses have been studied in the context of systemic Candida [76], [77] and Cryptococcus [78], [79] infections. Potent antifungal antibody responses, targeting fungal cell wall-associated epitopes, such as β-glucan and mannan [80], also arise during lung infections caused by fungal pathogens such as Pneumocystis and Aspergillus [81]. More recently, specificity or polyreactivity of antifungal antibodies present in the serum has been analyzed by flow cytometry [82] or a serological proteome analysis (SERPA) [83]. A high-throughput multi-kingdom antibody profiling (MultiKAP) was currently applied to explore the human antibody repertoires against gut commensal fungi (mycobiota), leading to several advances in the understanding of antibody mediated immunity to fungi [56].

The antifungal humoral response is necessary and sufficient for protection against systemic fungemia [56], [84], [85]. While B cell-deficient (μMT^−/−) mice, similar to their WT counterparts, are resistant to primary systemic infection with C.albicans, systemic antifungal IgG appear particularly important during secondary challenges with the same pathogen, which rely on the generation of B cell memory and is particularly lethal in the absence of B-cells [32], [56], [86]. Deficiencies in C. albicans phagocytosis, IL-10 production, and Treg priming, which could be remedied by a passive transfer of antifungal serum IgG, are thought to further contibute to susceptibility of μMT^−/− mice to fugnal infections [56], [85].

A recent study revealed the critical role of B-cell mediated immunity in protection against gut disseminated fungal disease [56]. The gut commensal C. albicans was the major inducer of antifungal IgG. C. albicans intestinal colonization of mice led to the induction of germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generation of systemic antibodies. Notably these circulating anti-fungal IgG antibodies conferred protection against disseminated C. albicans infection and cross-protection against C. auris. These findings revealed an important role of gut commensal fungi in shaping the human antibody repertoire and a role of antibody-mediated immunity in antifungal immunity and production.

In the lungs, Aspergillus fumigatus (Af) is one of the most frequent causes of invasive fungal disease in immunosuppressed individuals. Immunosuppressive drugs are well-recognized risk factors for developing invasive aspergillosis [87], lead to neutrophil impairment [88] and dramatic decrease in antifungal IgG antibodies in the serum [56]. Emergency of COVID-19–associated pulmonary aspergillosis (CAPA) was also reported, adding pulmonary viral infections to the risk factors for the development of fungal coinfections [89], [90]. A recent study reported that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–driven pneumonia depletes innate B lymphocytes and led to decrease of anti-Af IgG antibodies in the serum of severe COVID19 patients [91]. Neutrophils, provide essential defense by preventing conidial germination and invasive disease caused by Af [92] while systemic IgG antibodies further limit fungal disease . SARS-CoV-2 spike protein and corticosteroid therapy both induced B1a cell apoptosis [91]. Further experiments in a mouse model revealed that neutrophil-mediated host defense against Af was dependent on antifungal IgG. Altogether, these studies highlight the role of circulating antibodies and B cells in antifungal immunity and defense.

Reflecting the unique morphological flexibility of some fungal pathogens in the mammalian hosts, a wide range of effector functions have been connected to fungal antigen binding to systemic immunoglobulins during systemic infection. Clonally gut-related IgA⁺ B cells, which travel to the dural sinuses of the brain in response to commensal intestinal colonization, produce protective IgA to restrict systemic candidiasis to dural sinuses [93]. Supporting these findings, neutrophils and monocytes isolated from an invasive candidiasis patient with a missense mutation in CARD9 were deficient in C. albicans yeast killing, which could be restored by serum incubation to allow serum IgG opsonization [94]. In addition to the role of CARD9 in innate immunity against fungi, it was found that [56] antifungal IgG production also depends on the CARD9, revealing an important crosstalk between gut commensal fungi, innate and adaptive immunity for the generation of host-protective antifungal IgG.

3. Mechanistic factors governing anti-fungal antibody induction

The mosaic of diverse targets and reactivities of the secretory and systemic anti-commensal antibody repertoires is facilitated by multiple induction pathways (Fig. 2). In particular, we will discuss few components including B cell subsets, phagocyte subsets, and antibody isotypes.

Fig. 2. Multiple pathways for antibody repertoire development contribute to a variety of antifungal antibody isotypes and reactivities.

In the absence of presentation with gut microbial antigens or stimulation associated with gut microbial colonization, a subset of B cells will spontaneously mature and secrete natural antibodies (top). Gut fungal colonization-induced f antifungal antibodies occur through either T cell-independent (middle) or -dependent (bottom) mechanisms, although the exact pathways are yet to be elucidated. In the former, a combination of BCR engagement, PRR co-stimulation, and TNF-family ligands BAFF and APRIL induce CSR responses in the absence of T cell help. In the latter, BCR-engaged B cells undergo CSR and harmonize with T helper cells to form germinal centers that promote cycles of expansion/SHM and selection of high-affinity BCR clones, a process known as affinity maturation.

3.1. A diversity of B cell subsets that contribute to antifungal antibody reactivities

B cell diversity arises from two processes mediated by the activation-induced cytidine deaminase (AID): 1) CSR, the process of replacing the constant μ region of the heavy chain and with other constant regions to express a different antibody isotype without altering the variable region; and 2) SHM, the process of converting cytosines in the antigen-binding variable regions to uracil to induce DNA breaks that can be randomly filled in to generate diversity of Ig binding. The degree to which B cells will undergo either or both of these processes is dependent upon the secondary lymphoid tissue, the antigen sensed by pattern recognition receptors (PRRs) on innate immune cells, and co-stimulation by other immune cells.

A fraction of naïve mature B cells will produce antibodies spontaneously with minimal to no CSR or SHM. These “natural” antibodies, which can be observed in GF mice [95] in the absence of live gut microbiota or dietary antigen exposure [73], [96], exhibit light and heavy chain regions that are or are nearly identical to the “germline sequences” of B cells leaving the bone marrow. While natural IgA [74], [97], [98] and IgG [99] isotypes have been described, these “natural” antibodies are mostly associated with IgM isotypes. Typical epitopes of natural antibodies include polysaccharides common to the cell walls of a wide range of prokaryotes and eukaryotes [81], [98], making these antibodies highly polyreactive with low binding affinity. Notably, fungal cell wall polysaccharides such as beta-glucan and chitin are a common target of anti-fungal antibodies, and some of these antibodies are identified in GF mice in the absence of microbial colonization, suggesting the existence of a robust and protective “natural antifungal” antibody response [56], [78], [81], [95].

Polyreactivity and low binding affinity is a shared quality with antibodies produced by B cells that matured without T cell co-stimulation, thus collectively identified as the T cell-independent (TI) response. In the gut, this process is characteristically initiated by co-stimulation with both microbial antigens and myeloid cell-secreted TNF family ligands BAFF and APRIL [100], [101], [102]. Assessments of the breadth of TI anti-commensal sIgA coating and comparison of sIgA commensal coating frequencies in T cell-deficient mice, show similar results relative to immunocompetent mice, suggesting that TI anti-commensal antibodies can serve as redundant and compensatory protection upon T cell dysfunction, albeit with potentially different epitopes [103].

T cell-dependent (TD) antibody induction is the source of most immunoglobulins with high specificity to microbial antigens [74], [104]. The defining feature of TD antibody responses occur in germinal centers (GC), where SHM-derived B cell clones are selectively activated for clonal expansion and optimization depending on their ability to bind to presented antigens, a process known as affinity maturation. These dynamics have recently been reviewed [105]. In brief, GCs are polirized into a light zone (LZ) and a dark zone (DZ), and are the sites where B-cell clonal expansion, hypermutation, and selection occurs. GC-B cells are repeatedly cycling in between these zones. In the DZ, densely packed GC-B cells undergo extensive divisions and SHM, while upon migrating to the LZ GC-B cells test their B cell receptor (BCR) affinity against specially presented antigens [106]. The latter action is critically dependent upon proliferation signals from T follicular helper cells (Tfh), which promote B cell proliferation and expansion through their unique profile of co-stimulation and cytokine expression [107], [108], [109]. Additionally, multiple potential models have been suggested for the selection process upon LZ BCR engagement, including the culling of low-affinity GC-B cells through apoptosis as well as GC-B cell proliferation that correlates with the degree of BCR affinity [105]. In either case, the resulting B cells exhibit high specificity and affinity for their target antigen.

Evidence for C. albicans GC engagement could be found in the presence of its delivery to the GC-laden PP by certain subsets of intestinal dendritic cells [110]. Stimulation with fungal β-glucan induces the expression of activation markers such as CD40 and CD86, and is dependent upon spleen tyrosine kinase (Syk) signaling [111]. Such activation is critical for engaging naïve T cells and inducing their transition to Tfh cells. Investigations of the intestinal PP, show that C. albicans intestinal colonization promotes expansion of IgA⁺ GC-B cells , while the absence of GC formation in T cell-deficient Tcrb^−/− mice leads to a significant decrease in anti-C. albicans sIgA binding [58], [59] (Fig. 2). GC populations are also critical for the induction of antifungal systemic IgG, with a dramatic loss of antifungal systemic IgG observed in the serum of Tcrb^−/− mice [56].

3.2. Role of innate phagocytes in the generation of antifungal antibody responses

The persistent colonization of microbes at the intestinal barriers provide an abundant source of pathogen-associated molecular patterns (PAMP) and antigens that are sensed and processed by several phagocyte subsets in the intestines. Throughout the intestine, these cell types play varying roles in direct and indirect regulation of sIg production and response to opsonized microbial antigens. Two main populations of intestinal phagocytes, characterized by the expression of C-type lectin receptors (CLR), mediate the generation of antifungal antibodies in the murine gut: CX3CR1⁺ macrophages (Mfs) and type 2 conventional dendritic cells (cDC2) [56], [58], [112], [113]. A recent study has further described the presence of CLRs on a population of AIRE-expressing cells with features of both innate lymphoid (ILCs) and dendritic cells (DCs), and with unknown function in these processes [114].

CX3CR1⁺ intestinal Mfs are characterized by high expression of the integrin CD11b and chemokine receptor CX3CR1 [115], and are in a close proximity to intestinal epithelial cells (IEC) in the colon and the small intestine [116]. CX3CR1⁺ Mfs are also characterized by their relatively poor ability to migrate to other tissues such as the mesenteric lymph nodes (mLNs), in stark contrast to their cDC counterparts in the gut [115], [117]. Despite their relatively static nature, CX3CR1⁺ Mfs are critical contributors to both cellular and humoral protective immune responses, and have the ability to directly sample antigens coming from the lumen [118], [119]. Underlining this sensitivity, the perturbation of a steady-state gut microbiota by antibiotics or invasive pathogens can shift macrophages tolerogenic program towards a pro-inflammatory program [120], [121]. Importantly, these cells are critical mediators of antifungal Th17 responses to commensal fungi in the gut [113], [122].

Several conventional Zbtb46-dependent cDC subsets, are found at higher frequencies in the small intestine [123]. These cells lack canonical macrophage-specific markers and express CD11b and/or CD103 [124]. The latter two markers are used to further distinguish between CD103⁺CD11b⁻ cDC1s and CD103⁺CD11b⁺ cDC2s, whose differentiation is dependent on Batf3 and Irf4 respectively [125]. cDC2s sample luminal contents in the gut to induce gut-homing tolerogenic adaptive immune responses, primarily against gut microbial and dietary antigens in a TLR-dependent manner [126]. Lacking the ability to sample antigens across the epithelium, these cells instead access antigens through gap junctions-mediated interaction with macrophages [119], through transcytosis of antigens from goblet cells [127] or through the uptake of sIg-bound antigens [24], [128]. Unique to cDCs among intestinal MNPs is the ability to use the chemokine receptor CCR7 to enter lymphatic vessels [129] and migrate to draining lymph nodes. These cell types are critical for the priming of effector Th cells during homeostasis [130], [131], and for the induction of gut-homing receptors in T cells [132]. Failure to activate effective priming often results in a significant decrease of small intestine-homing effector T cells [130], [132], [133], [134], [135].

An increasing number of studies point to the critical involvement of the CX3CR1⁺ subset in the induction of antibody responses to commensal microbes. In vitro experiments using ovalbumin as a model antigen have demonstrated the ability of this MNP subset to induce IgA production when cocultured with B cells. Induction of low-affinity TI IgA antibodies by CX3CR1⁺ macrophages required BAFF and APRIL, but was independent of retinoic acid signaling [101]. Meanwhile, cDC2s that encounter antigens in the PPs also play a role in the generation of sIgA responses. The expression of CCL20 by follicle-associated epithelium (FAE) initially draws CCR6-expressing cDCs, along with CCR6-expressing B cells, to the PP where penetrant commensals and antigens can be sampled [136], [137].

Currently, the mechanisms of MNP-mediated modulation of antibody responses to fungi remain unclear. Experiments in mice demonstrate that both the CX3CR1⁺ macrophages and cDC2s contribute to the overall antifungal sIgA response in the intestine, albeit in compartmentalized fashion: while depleting either population leads to significant decreases in free luminal anti-C. albicans sIgA titers, cDC2 depletion abrogates PP IgA-CSR while CX3CR1⁺ macrophage depletion does the same for plasmablasts [58]. These findings are supported by previous studies demonstrating that human cDC2 subset found in gut associated lymphoid tissues (GALT) were capable of C. albicans phagocytosis in the PPs [110].

3.3. Innate immune receptors and their involvement in regulation of antifungal antibody responses in the gut

While a number of PRRs sense fungal cellular components, the primary and most studied contributors are several C-type lectin receptors (CLRs). The most extensively studies CLR is Dectin-1, which recognizes cell wall b-glucan and initiates signaling through Syk and caspase recruitment domain-containing protein 9 (Card9) [111], [138]. Signaling initiated through Dectin-1 has been tied to protective antifungal responses such as the production of reactive oxygen species, phagocytosis, the production of critical pro-inflammatory cytokines and chemokines, and priming Th1 and Th17 populations [2], [139], [140], [141]. Other CLRs notable for expression on macrophages and DCs include Dectin-2 and Mincle. The former binds mannan, induces IL-1β and IL-23 to promote protective Th17 responses [142], and exhibits a proclivity for binding hyphal filaments [142], [143]. The latter, which binds mannose and appears to play a critical role during systemic candidiasis involving the induction of TNF-α [144]. Notably, neither of these receptors have an intracellular ITAM-containing tail to activate Syk, instead forming complexes with Fc receptor (FcR) adaptor molecules that are necessary for mediating antifungal signaling through Syk [145], [146]. Despite lack of observed fungi-specific clinical phenotypes [147], TLR signaling – TLR2, TLR4, and TLR9 in particular – contribute to the induction of innate antifungal immunity in response to fungal cell components [148], [149], [150], [151], [152].

PRR signaling specifically, stimulation of Dectin-1, Dectin-2 CLRs, but not mincle, on macrophages results in secretory sIgA induction by co-cultured B cells [153]. Interestingly, Dectin-1 is expressed on FAE cells and contributes to transcytosis of recognized sIgA-bound complexes, suggesting that their contribution to the secretory antibody response may not be restricted to antigen presentation to B cells or Tfhs [26]. In contrast to sIgA, depletion studies suggest the systemic IgG responses to commensal mycobiota appear to be solely dependent on CX3CR1⁺ macrophages and solely induced in the spleen [56], raising important questions regarding site of fungal sensing and the site of induction.

3.4. Immunoglobulin isotypes

While the mechanisms guiding B cell activation and the subsequent affinity maturation in systemic lymphoid tissues match those of GALT, the antibodies produced by the resulting plasma cells exhibit drastically different effector functions upon binding. This is due mostly to the variety among the four IgG subclasses (IgG1–4) and their variable affinity for IgG Fc receptors [70]. While mice have a different set of IgG subclasses (IgG1, IgG2a/c, IgG2b, and IgG3), their subclasses IgG2b and IgG3 closely match the human IgG1 and IgG2, respectively. Profiles of IgG subclass binding reveal IgG1 and IgG3 reactivity against proteinaceous antigens [154], [155], while IgG2 exhibits near-exclusive reactivity against surface polysaccharides (although polysaccharide reactivity is not restricted to the IgG2 subclass) [156], [157]. While IgG4 also exhibits preferential reactivity to microbial proteins, this typically occurs after extended exposure ? in non-infectious conditions, and it is more commonly associated with reactivity to allergens such as pollen [158], [159].

While all but the IgG4 subclass can recruit complement, most other IgG effector functions require Fc-gamma receptor (FcgR) binding, systemic IgG effector functions are guided by their interaction with their cognate receptors, the FcgR superfamily. Reflecting the fact that only one FcgR family member (FcgRIIB) mediates anti-inflammatory signaling, IgG effector functions are overwhelmingly pro-inflammatory. Nevertheless, FcgRIIB plays a critical role in the calibration of pro-inflammatory IgG responses: a variety of FcgRs expressed on almost every immune cell, with a higher activating-to-inhibitory ratio (A:I) effectively reducing the IgG binding threshold for raising the appropriate downstream immune responses [160]. Notably, FcgR-mediated phagocytosis in macrophages increased the A:I ratio, demonstrating a positive feedback loop of IgG-mediated pro-inflammatory responses [161]. Among the IgG subclasses, IgG1 and particularly IgG3 are the most effective inducers of effector functions due to their longer and more flexible hinge region that facilitates more efficient FcgR binding and recruitment of complement [162]. However, a single AA substitution in IgG3 results in its intracellular out-competition for transcytosis by IgG1, significantly reducing the half-life of IgG3 antibodies [163]. Among these IgG subclasses, IgG2b and IgG3 dominate the IgG response to commensal fungi with IgG3 binding the largest fraction of the intestinal fungi [56].

4. Secretory and systemic antibody responses during fungal infections and IBD

Perturbation of gut microbiota composition or deficiencies in gut immunity result in a state of intestinal dysbiosis associated with IBD disorders, such as Cohn’s Disease (CD) and Ulcerative Colitis (UC), which affect over three million Americans [164]. Increasing focus is being devoted to understanding the role of antibody responses induced against gut bacteria, which depending on the context, can have a dual role by potentiating autoimmunity or dampening immune responses to prevent inflammation [32], [165], [166]. Interestingly, dysbiosis resulting from mucosal infections and inflammation during IBD provide examples of IgA and IgG antibodies reacting to the gut microbiota with opposite effects on intestinal health. The disruption of the intestinal barrier by invasive enteric infection can lead to changes in lymphoid tissue architecture that significantly alter the secretory IgA response. In the colon, this is characterized by the formation of additional tertiary lymphoid tissues (TLS), a category of ectopic lymphoid tissue that are induced into formation on-site by immunogenic intestinal microbes [167] or the development of inflammatory lesions in IBD patients. Mouse models of human salmonellosis reveal these TLSs, mediated by colon-resident CX3CR1^hi Mfs, are critical sites of Salmonella-specific IgA-CSR and generation of protective salmonella-specific sIgA (along with CD4⁺ T cell-mediated cellular immune responses), without which mice succumb to systemic infection within weeks [167]. Turning to chronic intestinal inflammation, sIgA binding to the gut microbiota is significantly increased in IBD patients relative to healthy individuals [49], [168]. Increases in sIgA gut microbiota coating can be transferred to healthy WT mice simply by co-housing them with littermates whose gut microbiota composition induces spontaneous colitis, demonstrating that colitogenic microbes induce robust protective sIgA responses [49]. IBD-associated sIgA also exhibits disrupted functionality, particularly with regards to the capacity in mediating Dectin-1 reverse transcytosis after binding gut microbes, the capacity of which is significantly decreased in UC patients but expanded in CD patients [169].

Meanwhile, an examination of human intestinal mucosal tissue samples revealed that the onset of inflammation in IBD patients was associated with the accumulation of IgG⁺ plasmablasts in the mucosa at significantly higher frequencies than healthy individuals [170]. Single-cell analyses revealed that these IBD-associated B cells exhibit immature phenotypes such as reduced diversity and lower SHM frequency [170], [171]. Notably, multiple studies have identified FcγR SNPs that significantly decrease the activating:inhibiting (A:I) ratio of MNPs resulting in greater protection during UC. These studies suggest a critical role for IgG binding to gut microbes and subsequent induction of pro-inflammatory signaling in the pathogenesis of IBD. Matched analyses of samples from UC patients and their healthy relatives sharing the same households support these observations [161]. Commensal-bound IgG complexes induced pro-inflammatory responses through the FcγR, including IL-1β production leading to neutrophil and Th17 infiltration.

Interestingly, the most prominent association between the gut mycobiota and systemic antibody responses are anti-Saccharomyces cerevisiae-antibodies (ASCA). These antibodies, which are reactive with S. cerevisiae cell wall mannan, are a robust clinical prognostic marker of Crohn’s Disease [172], [173] when titers are elevated in the blood [173], [174] and feces [175]. Although the mechanisms of ASCA induction are not precisely understood, both C. albicans and S. cerevisiae express ASCA epitopes, emphasizing the possibility that these clinically relevant antibodies could be cross-reactive with a number of fungal commensals in the intestine [176], [177]. Further complicating the ASCA induction picture, C. albicans ASCA epitope expression is dependent upon unique conditions, such as live systemic exposure (heat-killed C. albicans did not induce ASCA) [175], [176], once again highlighting the impact environmental conditions have on fungal immunogenicity. Despite these findings, the function of ASCA is currently unknown.

5. Prospects for an Antifungal Vaccination

Fungal pathogens present unique problems. Due in part to their ability to proliferate outside of their host and global travel, this fungus has spread to tens of countries since its first isolation in 1996 in South Korea. Additionally, its transmissibility appears to be positively associated by global warming [178], [179], suggesting that this threat will continue to grow. Like other Candida spp., C. auris persistently menaces hospitals worldwide by lying dormant on commonly used equipment such as catheter tubing [179], [180], [181]. However, unlike other Candida spp., it is multi-drug resistant, in some cases against all available anti-fungal drugs. Finally, within the host, the conditions inducing yeast-to-hyphae transition towards virulence are associated with immunocompromised state and/or dysbiosis, leaving little available recourse for treatment outside of passive immunization or vaccination before sickness.

While no fungal vaccines have progressed beyond clinical evaluation, a number of fungal antigens and strategies have been applied and have been extensively covered by several reviews in the past few years [7], [182]. Several rather broad targets include surface glycans, for which monoclonal antibodies have been generated and whose binding has been associated with promoting phagocytosis and killing while inhibiting filamentous growth [183], [184]. Several specific targets include hyphae-associated virulence factors that aid epithelial adhesion such as Als3 (NDV-3A) [185], Sap proteins, Hsp90, and Hyr1 include other promising targets [76], [186], [187]. The wide variety of targets reflects the primary challenge of developing these vaccines: fungi are present as a variety of morphologies as commensals [188] and during infection [189]. Additionally, systemic candidiasis can originate from any of several members of the Candida spp. including C. albicans, C. galabrata, C. auris, C. parapsilosis, each occurring in immunodeficient patients or those with a weakened immune system. A recent success of a phase 2 randomized, double-blind, placebo-controlled trial of a vaccine against Als3 (NDV-3A) for treatment of recurrent vulvovaginal candidiasis (RVVC) shows promise. NDV-3A administered to women with RVVC was safe, highly immunogenic and reduced the frequency of symptomatic episodes of vulvovaginal candidiasis for up to 12 months [185]. Thus, the most promising antifungal immunoglobulin therapy design would likely be directed against multiple fungal antigens, provide help to innate antifungal defense by activating neutrophils (although this will be challenging in neutropenic patients), or may simply rely on passive transfer of antibodies.

6. Conclusions

In this review, we discussed the existing body of literature related to the known functions of antifungal antibodies locally and at systemic levels. We also reviewed less-explored antibody induction sites, immune cell subsets, and signaling pathways involved in B cell-mediated antifungal immunity. Many challenges and roadblocks remain. For example, any extent to which the fungal and the bacterial microbiota affect each other’s metabolic activity, morphology, and fitness would have profound consequences on how antibody repertoires against specific mucosa-associated fungi will be generated. We also lack knowledge of the extent to which antifungal antibody repertoires are shaped by commensal bacteria (and vice versa) or other factors such as the diet.

While relatively unspecific and broadly cross-reactive, deeper understanding of TI antibody responses to fungi might shade light to mechanisms that limit fungal pathogenesis. Future studies will have to contend with the overlapping contributions of B cells and T cells to adaptive antifungal immunity. The evidence coming from current studies on adaptive immunity to fungi suggest that B cells , while not as critical for antifungal protection as T cell , are sufficient to boost antifungal response during systemic infection. Knowledge gaps remain regarding site-dependent mechanisms through which antifungal antibody responses are generated and regarding the specificities of such antibodies. In regard to mechanisms by which systemic antibody responses to commensal fungi are induced, it remains to be elucidated whether specific cell type(s) traffic fungal antigens from the gut or if those antigens and PAMPs passively disseminate to induce systemic IgG. Such investigations will further our knowledge and might lead to therapeutic implications.