The complex role of mast cells in fungal infections
Qingqing Jiao  Ying Luo  Jörg Scheffel  ZuoTao Zhao  Marcus Maurer
First published: 23 February 2019 https://doi.org/10.1111/exd.13907
Qingqing Jiao and Ying Luo contributed equally to this work.
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Abstract
In addition to their critical role in allergic disorders, mast cells (MCs) are well recognized for their protective effector functions during bacteria and parasite infections. This review describes recent advancements of our understanding of the complex role of MCs in fungal infections. Specifically, we outline key features of the contribution of MCs to infections with six fungal pathogens, namely Sporothrix, Paracoccidioides, Aspergillus, Malassezia, Candida and Dermatophytes. Evidence from studies of these pathogens suggests that MCs can function as positive regulators that detect and contain fungi at the site of infection. However, it appears that the inflammation induced by MCs following fungal infections may not always and only be beneficial to the host. MC responses during fungal infections may primarily benefit the pathogen by facilitating its spreading and contributing to a greater severity of fungal infections. This review also highlights key drivers of MCs activation and effector mechanisms that have been identified for the multidimensional function of MCs in fungal diseases and in allergic diseases combined with fungal infection.

1 INTRODUCTION
Fungi are a very diverse group of eukaryotic organisms that separate from plants and animals in their own kingdom. Today, fungal infections represent a serious public health problem around the world with more than 300 million people suffering from serious fungal infections and an estimated 1.6 million deaths annually.1

Fungi can cause a broad range of mycoses in humans: (a) Superficial fungal infections of the skin, nails and hair are commonly due to dermatophytes, for example Trichophyton rubrum or Trichophyton mentagrophytes, or yeasts, for example Candida or Malassezia furfur.2-5 (b) Subcutaneous fungal infections include sporotrichosis, chromoblastomycosis, mycetoma, phaeohyphomycosis, hyalohyphomycosis and lacaziosis.6, 7 (c) Paracoccidioidomycosis, coccidioidomycosis, histoplasmosis, mucormycosis and cryptococcosis are common systemic fungal infections.8 Subcutaneous and systemic mycoses occur mainly in immunosuppressed patients.7 Systemic fungal infections are linked to significant morbidity and mortality.8 In addition, fungal spores, for example, Aspergillus spores, are important allergenic agents and can cause persistent allergic inflammation of the airways.9

The immune mechanisms of defense against fungal infections are numerous, ranging from protective mechanisms that were present early in evolution (innate immunity) to sophisticated adaptive mechanisms that are induced specifically during infection and disease (adaptive immunity).10-12

Traditionally, mast cells (MCs) are seen as key effector cells in the elicitation of allergic signs and symptoms.13-15 However, there is an increasing number of studies that indicate that MCs are also essential players in innate immune responses.16-18 MCs are strategically located in tissues that are borders to the external environment such as the lung, the gut and the skin. Therefore, MCs are perfectly positioned to orchestrate protective immune responses against invading pathogens including fungi. Most studies of anti‐fungal immune responses are focused on the effects of epithelial cells, neutrophils, macrophages/monocytes and dendritic cells (DCs).19-23 As of now, only a few publications and reviews have addressed the role of MCs in the immune response to fungi infections.

In the innate immune system, MCs act as sentinels for invading pathogens, as initiators of inflammatory responses and as regulatory cells in the coordination of raising and limiting innate immune responses.18, 24, 25 Recent findings suggest that, during innate immune responses against invading pathogens, MCs function as positive or negative immunoregulatory cells depending on the situation. In this review, we focus on the recent advancements of our understanding of the complex role of MCs in fungal infections—as positive regulators that detect and contain fungi at the site of infection and as negative regulators that promote a wider dissemination and a greater severity of fungal infections. Specifically, we highlight key features of the contribution of MCs to infections with six fungal pathogens, namely Sporothrix, Paracoccidioides, Aspergillus, Malassezia, Candida and Dermatophytes.

2 WHAT IS THE ROLE AND RELEVANCE OF MCs IN SPOROTHRIX SCHENCKII INFECTION?
Sporothrix schenckii (S. schenckii) is an opportunistic dimorphic fungus with a global distribution.26, 27 S. schenckii is the cause of sporotrichosis, which usually affects the skin, less commonly the lungs, joints, bones, and brain of humans and other mammals.28 Veterinarians, animal technicians and caretakers, and owners of cats with sporotrichosis are regarded to be at increased risk for the acquisition of the disease.27 Sporotrichosis is transferred via the inoculation of S. schenckii (conidia or hyphae) from contaminated soil, plants or organic matter into sites of tissue injury such as wounds (traumatic inoculation).27 During interactions of S. schenckii with the host, the conidia‐phase of S. schenckii will transform into the yeast‐phase of S. schenckii in response to the host's temperature. Yeast is the dominant form of S. schenckii in sporotrichosis.29 In vitro studies showed that IgG‐seroreactive proteins from Sporothrix yeast cell extracts such as Gp 70 may be important for the pathogenesis and invasion of S. schenckii in human.30

In 2012, Romo‐Lozano et al. first reported that S. schenckii conidia can activate mouse peritoneal MCs to release early response cytokines such as TNF‐α and IL‐6 without degranulation.31, 32 By using an experimental murine model of systemic S. schenckii infection, which used intraperitoneal inoculation with S. schenckii conidia, they demonstrated that mice that had been depleted of peritoneal MCs presented with a decreased fungal load in organs and reduced severity of clinical manifestations. Their results suggest that mediators released by MCs may be implicated in promoting a wider dissemination of S. schenckii and a greater severity of the infection, indicating that MCs might act as negative immunoregulators in S. schenckii infection. However, this study used a functional MCs depletion model where MCs were depleted by injection of compound 48/80 into the peritoneal cavity. Moreover, their experimental model investigated systemic S. schenckii infection following intraperitoneal injection, whereas subcutaneous infection is the more common clinical manifestation of sporotrichosis. In summary, the in vivo evidence for a relevant role of MCs in S. schenckii infection is still limited. Based on the limited evidence available from mouse studies so far, MCs appear to promote S. schenkii, rather than protect from them. How MCs are involved in human sporotrichosis still needs to be clarified.

3 DO MCs PROMOTE PARACOCCIDIOIDES BRASILIENSIS INFECTION?
The fungus Paracoccidioides brasiliensis (Pb) causes paracoccidioidomycosis (PCM), an autochthonous systemic mycosis found primarily in Central and South America.33 PCM is characterized by mucous membrane ulcerations of the mouth and nose with spreading through the lymphatic system. The route of infection is assumed to be inhalation. The lungs, lymph nodes and mucous membrane of the mouth are the most frequently infected tissues.34 That MCs interact with Pb has been shown by Valim et al., who demonstrated that co‐culturing of Pb yeast cells with MC lines rat basophilic leukaemia (RBL)‐2H3 MCs induced morphological changes of MCs and their degranulation.35 Furthermore, endogenous PbPga1, which is an immunoregulatory protein on the surface of Pb yeast cells, is able to activate rodent MCs through the NF‐κB pathway, resulting in the release of the inflammatory interleukin IL‐6. IL‐6 is involved in the induction of inflammatory responses that can lead to tissue destruction.

Recently, a study of skin biopsies from patients with paracoccidioidomycosis suggested that MCs contribute to skin responses characterized by loose granuloma formation. This type of cutaneous response, which does not effectively contain or clear the infection, features markedly increased numbers of MCs and a Th2 cytokine profile.36 Interestingly, most MCs in these lesions were positive for IL‐10 suggesting that MCs are an important source of this immunosuppressive cytokine. Moreover, Batista et al. observed a downregulation of the inducible nitric oxide synthase (iNOS) in the tissues of oral paracoccidioidomycosis (OP). This results in reduced production of fungicidal nitric oxide (NO) in multinucleated giant cells and most mononuclear cells.37 MCs have been shown to be one of the major sources of NO in humans with fungus infections.38 These findings suggest that a low expression of iNOS in OP may represent a possible MC‐mediated mechanism that permits local fungal multiplication and maintenance of active oral lesions. Taken together, MCs appear to contribute to the morbidity of PCM, by several mechanisms. However, most of the above findings are from ex vivo or in vitro experiments, and proof that MCs play a harmful role in Pb infection needs to come from future in vivo studies.

4 MCs CONTRIBUTE TO THE MORBIDITY OF ASPERGILLUS FUMIGATUS INFECTION
Aspergillus fumigatus (A. fumigatus) is a ubiquitous, spore‐forming fungus that has been associated with multiple pulmonary disorders. Inhaled conidia of A. fumigatus adhere to pulmonary epithelial cells, causing opportunistic infection followed by inflammatory responses to A. fumigatus antigens.39

Most studies on the role of MCs in chronic lung disease due to A. fumigatus focus on IgE‐mediated allergic bronchopulmonary aspergillosis (ABPA).40-42 ABPA is associated with early allergic and late‐phase inflammatory responses of the lungs to A. fumigatus antigens that occur in a minority (less than 1%) of patients with asthma and, more often (7%‐10%), in patients with cystic fibrosis.43 ABPA is characterized by elevated total and A. fumigatus‐specific immunoglobulin E (IgE) levels accompanied by eosinophilia, pulmonary infiltrates, bronchiectasis and fibrosis leading to asthmatic attacks and wheezing in affected patients. A. fumigatus‐specific IgE has been shown to activate MCs by cross‐linking of the high‐affinity receptor for IgE(FcεRI) leading to the immediate release of preformed mediators from intracellular granules and the de novo synthesis of lipid mediators and cytokines, all of which contribute to the subsequent development of inflammatory responses and obstruction of the airways.44-46 Proteases released from MCs can promote the release of growth factors from airway epithelial cells driving strong and irreversible remodelling reactions in airways of patients with ABPA, resulting in bronchiectasis lesions and fibrosis.46 Treatment with omalizumab, a humanized mAb that binds to IgE, has been shown to be an effective therapy for patients with severe allergic asthma and ABPA as shown by attenuated asthma symptoms and improved pulmonary function parameters in patients with ABPA.47 Recently, clinical trials demonstrated that omalizumab treatment not only provided a clinically important reduction in serum IgE, exacerbation rates and steroid requirement but also showed attenuated asthma symptoms and improved pulmonary function parameters in patients with ABPA.47

Interestingly, however, the majority of patients with airway colonization by A. fumigatus do not develop ABPA, yet they still exhibit a progressive decline in lung function.48 IgE‐independent MC degranulation in response to A. fumigates may be involved in this response. Urb and co‐workers found that direct contact with mature A. fumigatus hyphae can induce the degranulation of bone marrow‐derived mast cells (BMCMCs) in the absence of IgE, whereas no significant degranulation is induced by either conidia or immature hyphae.49 Although the exact mechanism of this is still unknown, the fungal proteins StuA and MedA, which control the composition of the cell surface molecules of mature A. fumigatus hyphae, were shown to induce BMCMC degranulation in the absence of IgE.49 Therefore, the authors speculated that A. fumigatus may directly worsen pulmonary disorders, by inducing degranulation in MCs that are in contact to hyphae, which may aggravate the inflammation and obstruction of airway rather than eliminate the fungus.

Recent data shows that similar to ABPA in man, MCs may be implicated in recurrent airway obstruction (RAO), a type I hypersensitivity response to A. fumigatus in horses.49, 50 As in ABPA, specific IgE‐mediated cross‐linking of FcεRI results in MC degranulation and the release of histamine, serotonin, leukotrienes and eicosanoids such as thromboxane and 15‐hydroxyeicosatetraenoic acid. In addition, in experimental eosinophilic esophagitis induced by A. fumigatus, Niranjan et al. reported that MCs numbers are markedly increased and play a significant role in promoting oesophageal functional abnormalities, contributing to muscle cell hyperplasia and hypertrophy.51 Finally, a recent report suggests that MCs also contribute to A. fumigatus‐induced inflammation in acute invasive fungal rhinosinusitis (AIFR), an aggressive fungal infection with high morbidity and mortality rates in immunocompromised patients.52 Yan et al. created a rat model of AIFR based on A. fumigatus infection and investigated the role of MCs.53 Compared to control rats, the total number of MCs was unchanged, but MC degranulation was only found in the infected nasal cavities of AIFR rats. In summary, these findings suggest that MC responses to A. fumigatus appear to help the pathogen rather than the host. Further studies will be required to determine the underlying mechanisms of the contribution of MCs to A. fumigatus infection by valuable mice models.54

5 EXACERBATION OF MALASSEZIA‐INDUCED SKIN INFLAMMATION BY MCs IN PATIENTS WITH ATOPIC DERMATITIS
Malassezia comprises a family of opportunistic pathogenic fungi, which includes 19 species found on the skin of humans and animals. These Malassezia species have been associated with dermatological diseases such as seborrhoeic dermatitis, pityriasis versicolor, atopic dermatitis (AD) and folliculitis.55 AD is one of the most common chronic inflammatory diseases of the skin.56 Malassezia sympodialis (M. sympodialis) is frequently isolated from AD patients and healthy individuals.57 Recently, it was demonstrated that mouse and human MCs respond to extracts from M. sympodialis.58-60 For example, M. sympodialis can enhance IgE‐mediated MC degranulation by signalling through the TLR2/MyD88 pathway. Interestingly, Ribbing et al. further observed that MCs derived from peripheral blood CD34+ progenitor cells of Malassezia‐infected patients contain more preformed granule mediators and exhibit enhanced IL‐6 secretion in response to M. sympodialis, as compared to MCs from healthy controls.60 In addition, Malassezia also can elicit specific IgE and T‐cell reactivity in AD patients.61, 62 Hiragun and colleagues identified the fungal protein, MGL_1304, derived from Malassezia globosa (M. globosa) in purified sweat, as an allergen in AD patients.63-65 They confirmed its ability to induce IgE‐dependent degranulation in human basophils and RBL cells expressing the human FcεRI receptor. Their results suggest that MGL_1304 is a major allergen in human sweat and could cause type I allergy in patients with AD. These findings indicate that M. sympodialis can activate MCs and thereby exacerbate inflammatory responses in AD patients.

6 DO MCs CONTRIBUTE TO THE PATHOGENIC EFFECTS OF CANDIDA ALBICANS?
Candida albicans (C. albicans) is an opportunistic fungal pathogen, and they are normally grown as a harmless commensal on the skin and mucosal surfaces of healthy individuals.66 However, it can also cause superficial and systemic infections, especially in immunosuppression condition. C. albicans initially interacts with epithelial cells, resulting in fungal recognition and the formation of hyphae.67 The ability to undergo the morphological transition from yeast to hyphal growth is critical for its pathogenesis. Epithelial activation leads to the production of inflammatory mediators that recruit innate immune cells, which together work with epithelial cells to clear the fungal infection. The main immune cell in the defense against these candida infections is assigned to professional phagocytes, neutrophils and macrophages. In addition to the above cells, MCs also have been identified as the main cell types involved in the defense against this pathogen.68, 69

Yamaguchi et al. have shown that ovalbumin‐sensitized mice colonized with C. albicans show higher specific IgG and IgE titres than control mice, and colonized tissues promoted infiltration and degranulation of MCs.70 Their results suggest that gastrointestinal C. albicans colonization may promote sensitization against food antigens, at least partly due to MC‐mediated hyperpermeability in the gastrointestinal mucosa of mice. Furthermore, Takenaka and colleagues reported that increased levels of Candida‐specific IgE were linked to MC hyperplasia in lymph nodes in Kimura's disease.71 In addition, Segal and co‐workers observed that the injection of C. albicans together with Complete Freund's Adjuvant into the foot‐pads of guinea‐pigs provoked a state of hypersensitivity, characterized by direct degranulation of mouse peritoneal MCs when exposed in vitro to the antigen.72 Noverr and co‐workers reported that overgrowth of the yeast C. albicans following antibiotic treatment can promote the development of allergic airway disease characterized by increased levels of MCs.73 Taken together, these findings suggest that C. albicans may contribute to atopic diseases, at least in part by effects on MCs.

Interestingly, MCs may also protect from C. albicans infection. Recent evidence shows that rat peritoneal MCs can kill C. albicans in the extracellular environment, very likely through secreted granular mediators.74 That C. albicans can degranulate rat peritoneal MCs to release mediators has been shown as early as in 1974 when Nosál et al. reported that glycoprotein from C. albicans can induce histamine release in isolated rat MCs, which was confirmed by later in vivo studies showing C. albicans‐induced MCs degranulation in the gut of mice.75 More recently, Nieto‐Patlán et al. have shown that both yeasts and hyphae of C. albicans can induce BMCMCs degranulation, production of TNF‐α, IL‐6, IL‐10, CCL3, CCL4, and ROS as well as NO involving TLR2 and dectin‐1.76 Moreover, MC seems to be able to phagocytose C. albicans. According to Lopes JP et al., the protective response of primary human MCs in C. albicans infections has three stages: Initially, during the first 3 hours, MCs reduce C. albicans viability by degranulation. During the intermediate stage of the response, 3‐12 hours after infection, MC cytokine secretion and the recruitment of neutrophils by releasing chemoattractants such as CXCL1/CXCL2 help to contain the infections by phagocytosis, the release of proinflammatory mediators and by recruiting other leucocytes. Later responses, that is after 12 hours, are characterized by the release of anti‐inflammatory cytokines and the recruitment of effector cells of adaptive immunity.77 Taking together, these results suggest that MCs can help the infected host by killing C. albicans and by containing the infection by recruiting neutrophils and macrophages to the sites of infection. As for the role of MCs in C. albicans infection, there are still many questions to be answered, and further evidence from in vivo studies is needed to arrive at robust conclusions.78

7 WHAT IS THE RELEVANCE OF MCs IN DERMATOPHYTE INFECTIONS?
Dermatophytes, a group of keratinophilic fungi with approximately 40 species, are grouped into three genera: Trichophyton, Microsporum and Epidermophyton.79 Dermatophytes can invade human skin, hair, and nails and cause dermatophytosis (tinea).80 The role and relevance of MCs in dermatophyte infections are ill characterized. MCs have been observed to accumulate at sites of dermatophyte infections. For example, in Microsporum canis‐infected cats, the histopathological features of lesional skin include MC infiltration of the superficial dermis.81 Moskalewski et al. reported that the number of skin MCs is higher in mice infected with dermatophytes.82 In a Trichophyton quinckeanum dermatophytosis mouse model,83 MCs also accumulated in the dermis at the site of infection. This may or may not be relevant for the course of dermatophyte infections, and we will need further studies to clarify this.

8 WHAT CAN SPOROTHRIX, PARACOCCIDIOIDES, ASPERGILLUS, MALASSEZIA, CANDIDA AND DERMATOPHYTES TEACH US ABOUT THE EFFECTS OF MCs IN FUNGAL INFECTIONS?
It appears that the inflammation induced by MCs following fungal infections may not always and only be beneficial to the host, but may primarily benefit the fungal pathogen by facilitating spreading (Figure 1 and Table 1). Additional support for this comes from studies with the fungal cell wall product zymosan.84-86 Zymosan‐induced peritonitis was found to be suppressed in MC‐deficient mice (WBB6F1‐W/Wv), and this was restored by adoptively transferred BMCMCs.87 MCs also appear to be involved in the morphine‐mediated inhibition of zymosan‐induced peritonitis in mice.88, 89 Gelatinase B/MMP‐9 as an inflammatory marker enzyme in mouse zymosan peritonitis was constitutively present in unstimulated MCs,90 and zymosan‐activated peritoneal MCs reportedly contain and produce more MMP‐9 after zymosan administration.

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Figure 1
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Complex regulator role of MCs in immune responses against fungus. The red text is harmful, and the green text is beneficial in terms of functions of MCs on fungal infection
Table 1. Studies of MCs against fungus
Fungal species (family) Main colonized tissues Study host Study type IgE/non–IgE‐mediated response Reference
Sporothrix schenckii (Ophiostomataceae) Skin, lungs, joints, bones, brain Mouse In vivo Non–IgE‐mediated response 31, 32
Paracoccidioides brasiliensis (Ajellomycetaceae) Lymph nodes, mucous membrane of the mouth Mouse In vivo Non–IgE‐mediated response 35
Human In vitro Non–IgE‐mediated response 36, 37
Aspergillus fumigatus (Trichocomaceae) Lung Mouse In vivo IgE‐mediated response 40
Human In vivo IgE‐mediated response 41, 44, 47
Mouse In vivo Non–IgE‐mediated response 45
Mouse In vitro Non–IgE‐mediated response 49
Horse In vivo IgE‐mediated response 50
Mouse In vivo, In vitro Non–IgE‐mediated response 51
Malassezia (Malasseziaceae) Skin Human In vitro IgE‐mediated response 59-61
Candida albicans (Saccharomycetaceae) Skin, mucosal surfaces Mouse In vivo IgE‐mediated response 69, 72
Guinea‐pig In vitro IgE‐mediated response 71
Rat In vitro Non–IgE‐mediated response 73
Mouse In vivo Non–IgE‐mediated response 74
Mouse In vitro Non–IgE‐mediated response 75
Human In vitro Non–IgE‐mediated response 76
Dermatophyte Skin Mouse In vivo Unclear 87, 88
While the published findings from studies on MCs in fungal infections point to a role of MCs that is different from that in bacteria and parasite infection, they do not provide the underlying mechanisms. In Sporotrichosis, for example, MCs promote a wider dissemination of S. schenckii and a greater severity of the infection in mice, but it remains unclear how MCs can mediate these negative effects.

Also, most of the investigations performed so far, as described above, have been conducted with mouse models, and it has to be stressed that care should be taken in the translation of findings from the murine to the human system. MCs in mice and humans share many characteristic features, but also differ significantly, for example, in the expression of certain mediators such as proteases.91 In addition, the characterization of the role of MCs in fungal infections, as of now, has not made use of novel models and insights recently introduced to MC research, a limitation that will need to be addressed by future studies. Further evidence from ongoing and forthcoming research will need to provide a better picture of the mechanisms and the relevance of MC responses in fungal infections in humans. Finally, more focus has to come to the clinical relevance of characterizing the role of MCs in fungal infections. Several MC inhibitors are currently under development for the treatment of MC‐driven diseases. These inhibitors should be explored for their effects on fungal diseases. They may be especially helpful (and instructive) in the treatment of patients with allergic diseases and fungal infections. On the other hand, MC inhibitors may carry a risk of exacerbating some fungal infections, candidiasis for example, as MCs can reportedly contribute significantly to the clearance of C. albicans.

In conclusion, further studies are needed to characterize the complex function of MCs in fungal diseases and, especially, in allergic diseases exacerbated by fungal infections. In the long run, this will lead to better strategies for preventing and treating fungal infections.

ACKNOWLEDGEMENTS
This work was supported by grants from A Joint Sino‐German Research project (no. GZ901) and Jiangsu Provincial Medical Youth Talent (no. QNRC2016736).

CONFLICT OF INTEREST
The authors confirm that there are no conflicts of interest.

REFERENCES