Dendritic cell interactions with Histoplasma and Paracoccidioides

Abstract

Fungi are among the most common microbes encountered by humans. More than 100, 000 fungal species have been described in the environment to date, however only a few species cause disease in humans. Fungal infections are of particular importance to immunocompromised hosts in whom disease is often more severe, especially in those with impaired cell-mediated immunity such as individuals with HIV infection, hematologic malignancies, or those receiving TNF-α inhibitors. Nevertheless, environmental disturbances through natural processes or as a consequence of deforestation or construction can expose immunologically competent people to a large number of fungal spores resulting in asymptomatic acquisition to life-threatening disease. In recent decades, the significance of the innate immune system and more importantly the role of dendritic cells (DC) have been found to play a fundamental role in the resolution of fungal infections, such as in dimorphic fungi like Histoplasma and Paracoccidioides. In this review article the general role of DCs will be illustrated as the bridge between the innate and adaptive immune systems, as well as their specific interactions with these 2 dimorphic fungi.

Abbreviations

DC

dentritic cells(s)

TLR

toll-like receptor

PAMP

pathogen-associated molecular patterns

PRR

pattern recognition receptors

PCM

paracoccidioidomycosis

HKHC

heat-killed H.capsulatum

Introduction

The immune response to infecting microbes is vital to the survival of the host. The innate immune system has evolved to recognize molecular patterns common to many infecting pathogens by a variety of specialized cell receptors. This recognition initiates the innate response, orchestrates the induction of the adaptive response, and leads to a T cell response that helps to control or eradicate the infection. As the bridge between the innate and adaptive immune systems, DCs play a crucial role in this process. Their presence in pulmonary tissue is particularly important in mediation of the immune response to airborne pathogens including fungi. Over the past decades, there has been a growing body of knowledge regarding the interplay between DCs and fungal pathogens. In this review, we will present the current knowledge regarding the role of DCs in the innate and adaptive immune systems and their interactions with 2 important endemic dimorphic fungi studied in our laboratories, Histoplasma capsulatum and Paracoccidioides brasiliensis.

Innate and Adaptive Immune Responses

Historically, the innate and adaptive immune systems have been largely studied independently by immunologists and microbiologists. In more recent years their intricate and dependent relationship has been much more appreciated. The 2 systems are obligate and complementary parts of the response to infection and tissue injury.¹ Components of the innate immune system range from the epithelium's non-specific barrier function and the complement cascade, to a highly specific recognition of pathogens through the use of germline-encoded receptors.² Cellular components include DCs, monocytes, macrophages, granulocytes and natural killer cells. The innate immune system has developed the ability to recognize specific molecular patterns shared among several classes of pathogens termed pathogen-associated molecular patterns (PAMP). These include lipopolysaccharides, proteins, mannans, teichoic acids, denatured DNA and bacterial DNA.² The cellular components of innate immunity are able to recognize these patterns via cell surface receptors namely pattern-recognition receptors (PRR) and secreted molecules. The toll-like receptors (TLR) are one important type of these receptors. In the last several years, extensive research has established that TLRs are essential for the recognition of pathogenic microorganisms and subsequent activation of the innate immune response.³ Specifically, studies have demonstrated an involvement of TLRs in the recognition of fungal pathogens, such as Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Coccidioides posadasii.^4-7 In contrast to the innate immune system, the adaptive immune system uses modified antigen receptor genes to create new receptors for essentially any pathogen. However, it is still largely dependent on the innate response for instruction.⁸ Adaptive immune responses are slower but flexible, and help the host fight infections that have evolved mechanisms to evade innate responses.⁸

Dendritic Cells in the Immune Response

DCs are antigen-presenting cells that are constantly sampling antigens in their environment.^1,9,10 Their role is diverse, dynamic, and as unique at each stage in the immune response to an infection as the infection itself. DCs are the important link between innate and adaptive immunity and are vital determinants of the immune response to an infecting pathogen.¹¹ DC precursors originate in the bone marrow and develop into specialized cells that carry out different functions. At least 2 of these DC subsets reside in the skin – Langerhans cells and dermal DCs.¹² DCs are also present under most surface epithelia and in most solid organs such as the heart and kidneys.¹ In the lung tissue specifically, DCs are located within the airway epithelium, lung parenchyma, and submucosa; within alveolar septal walls and on the alveolar surface.^13,14 Their presence in pulmonary tissue is particularly important in mediation of the immune response to airborne pathogens such as, A. fumigatus, C. neoformans, M. tuberculosis and P. brasiliensis^15-19 as well as other fungal infections, which are typically acquired through the respiratory route. DCs have been subcategorized into 3 types; conventional myeloid, plasmacytoid, and lymphoid DCs. They all have immunostimulatory properties, function in tolerance, and express unique PRRs.²⁰

In peripheral blood, DCs are able to phagocytose pathogens or their components and present antigens to other cells, initiating the pathogen-specific adaptive immune response. Immature DCs are phagocytic but poor activators of an immune response and specialize in antigen uptake and processing,⁸ contrasting with their mature counterparts that almost exclusively serve as antigen-presenting cells.²¹ The induction of this immune response begins with the ingestion of a pathogen in infected tissue by immature DCs, which occurs by non-receptor mediated phagocytosis of entire microorganisms or through macropinocytosis. The ingestion of pathogens or their parts, results in DC activation and the production of inflammatory cytokines such as TNF-α, IFN-γ, IL-12. The newly mature DCs eventually migrate to regional lymph nodes where they interact with and activate naïve T lymphocytes via antigen presentation.^22-25 DCs are able to load antigenic peptides onto cells expressing MHC class I and MHC class II complexes, permitting presentation to both CD8 and CD4 T cells respectively.^26,27 The ability of DCs to process exogenous antigens with MHC I and MCH II molecule's for presentation to T cells is known as “cross presentation.”^27,28 Activated DCs undergo changes that enable them to activate pathogen-specific lymphocytes and secrete cytokines that influence both innate and adaptive responses. At this time, the mature, activated DCs forfeit their ability to capture antigens. DCs like other phagocytic cells are activated by specific receptors that transmit signals when they are bound by microorganisms or their components, and by cytokines produced during the inflammatory response.¹ They respond quickly to environmental changes and differentiate extensively to become immunogenic accessory cells. One of the major initiators of maturation are microbes, but examples of other strong inducers of the system include contact hypersensitivity and graft rejection.⁹ The mature DCs transfer important information about the invading pathogen and the subsequent innate response to T cells, commencing the adaptive response. This information includes the type of offending pathogen that incited their maturation²⁹ and in turn, influences whether T cells will respond, and where and how they will respond.⁸

Histoplasma capsulatum

H. capsulatum is a dimorphic fungus that exists in the soil as a mycelium and transforms into its yeast form once it invades tissue.³⁰ It has a worldwide distribution though highly endemic areas are present in the Mississippi and Ohio River valleys of the USA as well as certain areas within Central and South America. Out of an estimated 200,000 to 500,000 cases of infection per year, less than 5% of those infected exhibit acute clinical symptoms.³¹ H. capsulatum can cause a broad spectrum of disease, largely dependent on host immunity and its interaction with the pathogen as well as the amount of the initial inoculum. While the disease course is typically indolent in immunocompetent hosts, it is more likely to be severe in immunocompromised individuals such as patients with advanced HIV infection, hematologic malignancies or those receiving chemotherapy, corticosteroids or anti-TNF-α therapies.^31-33 A high inoculum can cause life-threatening disease in immunologically intact individuals.³⁴ Hence, the spectrum of disease manifestations range from asymptomatic to an acute respiratory illness with flu-like symptoms to life-threatening disseminated disease with high fever and respiratory compromise^30,31,35 The infection is acquired via inhalation of microconidia or small mycelial fragments into the terminal bronchioles and alveoli where the fungus transform into its yeast phase.^36-38 The stimuli that lead to this transformation have not been well-defined, but a shift in temperature is a major factor in this process.³⁹ This transformation is associated with diverse events including genetic, biochemical and physical changes.³⁰ The yeasts are then ingested by alveolar macrophages and DCs. Within the macrophages, the yeast replicates, destroying the macrophages and is subsequently taken up by surrounding neutrophils, inflammatory macrophages and DCs.⁴⁰ This leads to spread of the infection to lymph nodes and other organs during the acute stage of primary histoplasmosis. Consequently the resultant development of cell-mediated immunity against. H. capsulatum leads to inflammatory macrophage activation, and most frequently, resolution of the disease process.⁴¹ Resolution is associated with activation of cell-mediated immunity, particularly the T cell arm, however this does not eradicate the pathogen, rather the fungus becomes encased in granulomas that calcify.³⁶ The granulomas function to limit yeast replication, influenced by IFN-γ and TNF-α,³⁰ and disease reactivation may occur with immunosuppression.

One of the roles that DCs play in the defense against histoplasmosis is restricting the transformation of Histoplama conidia into yeast forms, thereby limiting dissemination of the fungus. This was demonstrated by Newman et al., using both human and murine lung DC models.³⁷ In these models, there was no evidence of active killing of H. capsulatum inside the DCs. Conversely, the authors found that within macrophages, there was no restriction of this transformation.³⁷ To further characterize the role of DCs in histoplasmosis, Gildea et al. demonstrated complete inhibition of Histoplasma yeast growth intracellulary within human DCs and the ability to kill and degrade the yeasts Contrastingly, within human macrophages there was rapid fungal growth.²¹ In DCs the binding of the Histoplasma yeast cells was facilitated by the fibronectin receptor, VLA-5, as opposed to CD18 receptors which mediated the binding in macrophages.⁴² H. capsulatum cyclophilin A is the ligand for VLA-5⁴³ whereas heat shock protein 60 is the ligand for CD18.⁴⁴ An additional observation in this study was that DCs were able to stimulate T cell proliferation when they contained viable H. capsulatum organisms and did so more efficiently than after ingesting heat-killed cells, suggesting that some immunogenic antigens are destroyed with heat.²¹

In a more recent article, Gildea et al. investigated the mechanism by which DCs kill H. capsulatum.⁴⁵ Using human DCs and macrophages, the authors demonstrated that the Histoplasma-infected DCs displayed marked phagosome-lysosome fusion and thus were able to restrict the growth of the yeast cells. Using suramin and incubating Histoplasma-infected human DCs at 18°C, both of which inhibit phagosome-lysosome fusion, resulted in decreased phagosome-lysosome fusion in DCs and decreased fungicidal activity.⁴⁵ This process was not affected by pH which is known to affect the activity in murine DCs. Taken together, these data propose that exposing the yeasts to lysosomal enzymes is the major mechanism by which human DCs exhibit fungistatic and fungicidal activity, overriding one of the strategies used by H. capsulatum yeasts to survive intracellularly within human macrophages. Nitric oxide and oxygen radicals did not appear to play a role in facilitating DCs to kill H. capsulatum as they do in macrophages.⁴⁵ The ability of DCs to kill intracellular Histoplasma and the inability of macrophages to do so may be due to the aforementioned difference in cell-surface receptors that are used to recognize the fungus, CD18 and VLA-5 receptors, respectively. This may lead to different signaling cascades resulting in phagosome-lysosome fusion and increased killing within DCs but not macrophages.⁴⁵

In the presence of specific cytokines, macrophages can alter their permissibility for the growth of H. capsulatum yeasts within them. Notably, a predominant Th1 response and its associated production of cytokines like IFN-γ and TNF-α promotes resolution of infection by activating macrophages which in turn increases NO production and degradation of yeasts.^46,47 In contrast, increased IL-4 production as a part of a Th2 pathway dampens the protective immune response against H. capsulatum.⁴⁸ CD4 T cells are one of the most potent producers of IL-4 and its presence causes DCs to stimulate further production of IL-4 by CD4 T cells.⁴⁹ Additionally, excess production of IL-4 impairs fungal clearance of H. capsulatum in mice deficient of chemokine receptor CCR-2 via its negative effect on DC recruitment.⁴⁸ CCR-2 is a necessary receptor for mobilization of monocytes from bone marrow to peripheral blood as demonstrated in bacterial infections.⁵⁰ By infecting mice lacking CCR-2 with heat killed yeast cells of H. capsulatum, Szymczak et al. found a reduced IL-4 level but no improvement in fungal clearance, possibly explained by an associated diminished Th1 response.⁵¹ Using antigen-free DCs, an increase in IL-4 level and an exacerbation of the infection was observed. Furthermore, when antigen-exposed DCs were used in CD4-depleted mice, there was increased fungal clearance. Seemingly, to suppress IL-4 production, a prerequisite increase in H. capsulatum-antigen exposed DC recruitment was necessary, suggesting that antigen presentation is a required step for IL-4 regulation to occur. The increase in fungal clearance in antigen-exposed CD4-depleted mice may be explained by the observed associated increase in the number of CD8 T cells in the lung.⁵¹ The CD8 T cells were not essential for protective immunity in immunocompetent mice, but their interaction with antigen-exposed DCs activates them and accelerates clearance of the fungus in MHC II-deficient mice, effects that have been implicated in vaccination models.^52,53 Hence, there are clearly opposing rolls of CD4 and CD8 T cells in histoplasmosis, underscoring the importance of antigen presentation of DCs and their resulting regulation of IL-4 and fungal clearance.⁵¹

A deficiency in either CD8 T cells or in MHC I has little effect on H. capsulatum clearance.^54,55 However in Histoplasma-infected mice that lack functional CD4 T cells, there appears to be a protective effect of CD8 T cells, mimicking the situation of advanced HIV infection where patients are nevertheless able to respond to fungal infections, albeit to varying degrees. Lin et al. used CD4-depleted mice, and found that the ingestion of H. capsulatum yeasts trigger macrophage apoptotic cell death, serving as Histoplasma antigen donors to cross-prime CD8 T cells,⁵² further illustrating the importance of CD8 T cells in the setting of CD4 depletion.

In another study that looked at the antigenic effects of Histoplasma and its ability to activate T cells in a vaccine model, Hseih et al., demonstrated that apoptotic peritoneal macrophages containing heat-killed H. capsulatum (HK Hc) activated both CD8 and CD4 T cells more efficiently than viable macrophages with ingested HK Hc, apoptotic macrophages alone, viable macrophages alone or HK Hc.⁵⁶ The fact that apoptotic peritoneal macrophages simply mixed with heat-killed Histoplasma before injection did not result in a strong CD8 or CD4 T cell response implies that in order for antigen presentation to efficiently occur, H. capsulatum antigens must be contained within apoptotic macrophages.⁵⁶ The study suggests that immunization with apoptotic peritoneal macrophages with ingested HK Hc activates both CD8 and CD4 T cells, providing protection against fungal infection. Hence, the function of apoptotic phagocytes as antigen donors and the resultant effective T cell response in combination with mechanism of cross-presentation can be applied to the development of fungal vaccines⁵⁶ as well as providing insight into disease pathogenesis.

The cell wall of H. capsulatum is one feature that the fungus has evolved in a manner that facilitates the evasion of the human immune system. The wall of most H. capsulatum strains are composed of α and β glucans. α-1,3-glucan specifically contributes to the pathogen's virulence, and β-1,3-glucan, functions in immune response modulation and has antigenic properties. The latter is the predominant component of the fungus in the mycelial stage, participates in leukocyte recruitment and stimulates the production of inflammatory mediators.⁵⁷ β-glucans interact with dectin-1, a C-type lectin. Dectin-1 is an important β-glucan receptor and one of the first PRRs identified that can mediate signaling both independently and synergistically with TLRs to initiate specific responses to microorganisms. Dectin-1 is found in neutrophils, natural killer cells, DCs and a subset of T cells.⁵⁷ Dectin-1 engagement is associated with enhanced phagocytosis,⁵⁸ reactive oxygen species production⁵⁹ and up-regulation of cytokine production.⁶⁰

α-1,3-glucan contributes to virulence in Histoplasma as avirulent strains produce significantly less α-1,3-glucan.⁶¹ Similarly, α-1,3-glucan is known to contribute to virulence in Paraccocidioides.⁶² Using strains of H. capsulatum that produce α-1,3-glucan and by manipulating expression of dectin-1 receptors on phagocytic cells, Rappleye et al. were able to develop a mechanism by which α-1,3-glucan interferes with recognition of H. capsulatum through specifically blocking host dectin-1 from binding to and β-1,3-glucan during the yeast phase, via direct obscuration of the binding site by α-1,3-glucan.⁶³ Interestingly, manipulation of α-1,3-glucan expression in certain H. capsulatum strains (chemotype I) did not decrease the virulence, suggesting that there are diverse pathogenic strategies used by different H. capsulatum strains, and that the virulence of only some strains (chemotype II) is largely dependent on α-1,3-glucan.⁶⁴

A less explored and more recently discovered type of T helper cell is the Th17 subset, which mainly produces IL-17 and is well described in mucosal immunity against extracellular bacteria.⁶⁵ Th17 cells also have an increasingly recognized role in the adaptive immune response to a variety of fungal pathogens.^66,67 Chamilos et al. found that exposure of the β-glucan component of the cell wall of a mutant strain of H. capsulatum decreased its pathogenicity and stimulated an increased production of IL-23 by DCs and a resultant Th17 antifungal response.⁶⁸ This was found using a mutant strain of H. capsulatum that is unable to generate α-glucan, thereby exposing β-glucan to DCs. Similarly a Th17 antifungal response was triggered in response to the opportunistic fungi Aspergillus and Rhizopus by exposing the β-glucan component of their cell walls to DCs during their growth stage. These authors also found that IL-23 production was much higher in these 2 fungi during the invasive stage but not the inactive stage. IL-23 production by DCs is known to be an important driver of the Th17 response.^69,70 Interestingly, like these opportunistic fungi, the mutant H. capsulatum yeast form (Δags1) resulted in an increased production of IL-23. After blocking dectin-1 receptors with laminarin, the interleukin response was not observed, suggesting that dectin-1 receptors were involved in the mechanism.⁶⁸ Hence, exposure of β-glucan in the wall of H. capsulatum is vital for the induction of the IL-23/Th17 response and this interleukin response may be a key regulator of protective immunity to opportunistic fungi but is not necessarily vital in pathogenic fungi.⁶⁸ In contrast, vaccine studies indicate that Th17 cells are vital in the induction of protection against H. capsulatum demonstrating significant differences in requirements for responses in the course of disease compared to for vaccine immunity.⁷¹

Galectin-3 (gal-3) is a member of the galectin family, and is expressed in macrophages, DCs, activated lymphocytes and epithelial cells.⁷² It is involved in diverse functions such as migration, adhesion, apoptosis, activation and phagocytosis.⁷³ Gal-3 has a known role in atopic dermatitis and asthma by promoting an anti-inflammatory Th2 response, while it diminishes the Th2 response in Paracoccidioides infection by negatively regulating IL-10 production.⁷⁴ Gal-3 affects Th1,⁷⁵ Th2,⁷⁴ and Th17 responses,⁷⁶ depending on the infecting pathogen. Presumably, this is dependent on the type of receptors involved and their interaction with the stimulant. Gal-3 has been shown to interact with dectin-1 and TLR-2.^77,78 Previous studies using knockout mice have demonstrated that TGF-β1 and IL-6 fuel Th17 differentiation, while IL-23 facilitates the differentiation and sustains the Th17 cells.^79-81 IL-17A acts on a variety of cell types to promote inflammation⁸² and helps eradicate extracellular pathogens.⁸³ Interestingly, blockade of IL-17A has been shown to aggravate pulmonary histoplasmosis,⁸⁴ suggesting that this interleukin promotes antifungal activity. Both Th1 and Th17 contribute to fungal clearance in mice infected with H. capsulatum. Using this concept, Wu et al. demonstrated that gal-3 deficient mice cleared Histoplasma more efficiently than wild type mice, and that the DCs from the mutant mice produced higher levels of IL-23, TGF-β1, and IL-1β compared to the control mice.⁸⁵ H. capsulatum also induced higher levels of IL-17A and greater percentages of Th17 cells, but lower levels of IFN-γ, IL-12 as well as a lower percentages of Th1 cells. The results of this study support the role of gal-3 in negatively regulating host IL-17 responses to Histoplasma by impeding IL-23/IL-17–axis cytokine production by DCs.⁸⁵

Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is caused by a complex group of fungi comprising 4 distinct phylogenetic lineages (PS2, PS3, S1 and Pb01-like). The first 3 lineages are speciated as Paracoccidioides brasiliensis whereas Pb01 is morphologically and genetically distinct and is designated as Paracoccidioides lutzii.⁸⁶ P. brasiliensis primarily infects lung tissue. It is endemic in Latin America and is most prevalent in Brazil, which accounts for 80% of total cases.⁸⁷ Colombia, Venezuela, Ecuador, and Argentina account for the majority of other endemic cases.^88,89 The infection is acquired through inhalation of the conidia produced by the fungus in its mycelial form.⁹⁰ The clinical presentation of the infection ranges from a localized and benign disease to a progressive and potentially fatal systemic infection.⁹¹ The disease severity is dependent on the interaction between several factors including immune competence and the virulence of the infecting P. brasiliensis strain.⁹¹ Other factors include the site of infection, age and the sex of the host.⁹² There is a strong male predominance (13:1) of symptomatic disease that may be explained in part by the inhibition of transformation from the mycelia to yeast form in the presence of estrogens, as shown in a study by Restrepo et al.⁸⁸ This phenomenon could lead to decreased fungal propagation and dissemination, allowing the immune system more time to develop an effective response to the infection.⁹³ PCM has 2 clinical presentations; a chronic or “adult” form, and an acute or subacute form, also termed the “juvenile” form. Severe disease is associated with deficiencies in cell-mediated immunity, high antibody levels, and a predominant production of anti-inflammatory type 2 cytokines.⁹¹ Several factors influence resistance or susceptibility to infection by P. brasiliensis including the amount of antigen, the number of antigen presenting cells, and the co-stimulatory microenvironment, which help determine the immune response pathway that will be initiated.^90,94 Many studies have shown the importance of the development of an appropriate CD4 Th subset for disease resolution and others have demonstrated different disease outcomes due to a dominant Th1 or Th2 response.^90,95 IL-12 produced by DCs seems to be the key cytokine that stimulates a preferential, inflammatory Th1 type immune response, which is an important defense in systemic fungal infections.⁹⁶

DCs are part of the first line of defense against P. brasiliensis infection and the migration of DCs to the lymph nodes is a key initial step toward the induction of a T cell response.^15,90 Silvana et al. observed an increased expression of CCR7, CD103 and MHC II on the surface of lung DCs after infection with P. brasiliensis, which enables migration to lymph nodes where T cell activation takes place and initiates a predominant T helper response.¹⁵ Numerous studies have established that pathogen recognition through PRRs activate DCs leading to an increase in DC expression of the chemokine receptors CCR7 and CD103.^97,98 Resistance to P. brasiliensis infection is associated with a predominant Th1 response, while susceptibility is related to Th2 activation.⁹⁹

Ferreira et al., using susceptible (B10.A), resistant (A/J) and TLR-2-knockout mice, demonstrated that after infection with P. brasiliensis, the susceptible mice had an increased expression of TLR-2 on DCs, leading to an increase in IL-10 production and expression of CD80 and CD54.¹⁰⁰ The data suggests that P. brasiliensis prompts regulatory DCs in susceptible mice to increase IL-10 secretion likely through activation of TLR-2 and dectin-1 receptors, leading to down-modulation of the host's microbicidal response. Furthermore, DCs from susceptible animals had a higher phagocytic index compared to resistant mice, but the phagocytosed yeasts were still viable. The presence of mannan inhibited phagocytosis in cells obtained from both types of mice while laminarin, a polysaccharide that blocks dectin-1 receptors, inhibited DCs only from susceptible mice. This result can partially explain the high phagocytic activity observed in the DCs from susceptible mice. The authors concluded that P. brasiliensis could be phagocytosed by the use of mannose and dectin-1 receptors,¹⁰⁰ as proposed in previous studies.^101-103

By studying the predominant DC and associated cytokine types, Pina et al. explored other mechanisms that could explain the resistance and susceptibility of mice to infection. In their study, Pina et al. found that when infected with P. brasiliensis, there was a predominance of myeloid bone marrow DCs in susceptible (B10.A) mice and plasmacytoid DCs in resistant (A/J) mice.²⁰ The susceptible mice produced high levels of TNF-α, IL-12, IL-1β, and IL-10, whereas DCs from resistant mice produced high concentrations of TGF-β and IL-6. The DCs of the resistant mice had increased naïve lymphocyte proliferation when activated by P. brasiliensis in comparison to susceptible mice. The resistant mice also induced a higher frequency of CD4⁺ CD25⁺ FoxP3⁺ Treg cells, indicating that DCs of resistant mice were not only stimulators of proliferation of lymphocytes but also of tolerogenic Treg cells. Furthermore, using pulmonary DCs infected with P. brasiliensis, the authors found that resistant mice had high levels of TNF-α and TGF-β, and susceptible mice displayed high levels of IL-12. Additionally, the resistant mice exhibited a mixed Th1/Th2/Th17 adaptive immune response, while the innate immune response in susceptible mice involved mainly IL-17 and IFN-γ.²⁰ Hence, an early pro-inflammatory immune response leads to susceptibility where a TGF-β-mediated anti-inflammatory response in combination with a counterbalancing pro-inflammatory reaction resulted in host resistance to P. brasiliensis. Specifically, the susceptibility is thought to be due to the excessive inflammatory activity of DCs and lymphocytes that help control the fungus early on in the infection, but which suppresses an effective T cell response, resulting in disease progression and mortality in infected mice. The resistant mice in this study however, showed concurrent tolerogenicity and immunity leading to a protective Th1/Th17 immunogenic response regulated by Th2 and Treg cells.²⁰ This is in contrast to previous studies that concluded that susceptibility and resistance was mainly due to the predominant T helper response, Th2 and Th1 responses respectively.¹⁰⁰

The cytokine IL-1β is one of the most important inflammatory mediators against opportunistic fungi, but its effects have not been extensively studied in primary fungal infections such as P. brasiliensis. Tavares et al. found that murine derived DCs released IL-1β in response to infection with the fungi, and released twice as much compared to macrophages.¹⁰⁴ The authors proposed that the fungus was specifically sensed by a nucleotide-binding oligomerization domain receptor (NLRP3) inflammasome, a cytoplasmic multi-protein complex that is dependent on a spleen tyrosine kinase, caspase-1, and NLRP3. When NLRP3 is activated, it recruits several peptides including pro-caspase-1 to form the NLRP3 inflammasome, activating caspase-1 and eventually leading to the cleaving of pro-IL-1β to IL-1β. The capacity of P. brasiliensis to activate the NLRP3 inflammasome are in agreement with previous studies using C. albicans, A. fumigatus and C. neoformans^105-107 and suggest that manipulating NLRP3 inflammasome activation may provide a new approach for the control of PCM.¹⁰⁴

Glycoprotein 43 (gp43) was first described in 1986, and it is the major diagnostic antigen of P. brasiliensis.¹⁰⁸ It stimulates an IFN-γ dependent Th1 response that protects against infection by yeast forms of P. brasiliensis.¹⁰⁹ Gp43 is recognized by host cells associated with both humoral and cellular responses^110,111 and DCs are crucial in this protective response. Ferreira et al. found that injection of gp43- pulsed DCs into lymph node cells from resistant mice resulted in the high production of IL-2 and IFN-γ by CD4 T cells, consistent with a Th1 response.¹¹¹ In contrast, the injection of gp43-pulsed macrophages into the lymph node cells led to the production of IFN-γ, IL-4 and IL-10, a Th0 response, and lymph node cells stimulated with gp43-primed B-cells induced a Th2 type pattern, producing IL-10 and IL-4.¹¹¹ Peptide 10 (P10) is derived from the P. brasiliensis gp43 and its potent ability to elicit protective T helper responses makes it a good candidate for development as a therapeutic vaccine against Paracoccidiodes.¹¹² Magalhães et al. reported that of P10-primed DCs injected either subcutaneously or intravenously into mice with known PCM results in the significant reduction of fungal burden as well as the induction of a Th1-predominant cytokine response with concomitant decreases in Th2-type cytokines. Similarly, P10-primed DCs produced a rapid, protective response in subsequently P. brasiliensis-challenged mice characterized by increased secretion of IFN-γ and IL-12 and a reduction in IL-10 and IL-4.¹¹² Supporting these results, Tavares et al. recently utilized microarrays to determine the expression of DC genes involved in immunity and found that genes encoding cytokines IL-12 and TNF-α, along with the chemokines CCL^-22, CCL^-27 and CXCL^-10, were up-regulated, suggesting that P. brasiliensis induces the production of cytokines and chemokines as well as other molecules that participate in the early response of DCs to this fungus.⁹⁶

Conclusions

DCs are an important bridge between the innate and adaptive immune systems and play a significant role in the immune response to dimorphic fungi such as H. capsulatum and P. brasiliensis. The interaction begins when DCs first encounter these microorganisms, typically in the lungs, and continues until the resolution of the infection. Starting with its phagocytic action, DCs contain these dimorphic fungi and through degradation, antigen presentation, and ultimately, activation of an effective T cell response, they are able to effectively orchestrate a protective host response. In histoplasmosis, DCs limit the transformation of mycelia into the yeast form, preventing dissemination, and can actively kill the fungus through phagosome-lysosome fusion and degradation of the yeast. In PCM, a preferential Th1 response at the beginning stages of infection followed by an anti-inflammatory and immune-tolerant response is fundamental for the resolution of infection. Gp43 represents a major P. brasiliensis antigen and is a significant stimulus for this response. The antigen has been used in studies that have validated the pursuit of gp43 and P10 for use in prophylactic and therapeutic vaccines. Similarly, analogous antigens have been used in attempt to create a potent vaccine against H. capsulatum.

This unique interaction between DCs and dimorphic fungi and the resulting immune response is an important determinant of the prognosis of infection in individuals. Moreover, there is exciting research utilizing DCs to aid in the development of vaccines against these endemic fungi that may prevent such potentially fatal infections in susceptible hosts.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.