Tissue-resident macrophages are derived from either blood monocytes or through local proliferation of yolk sac-derived phagocytes that are originally seeded during embryonic development (McGrath et al., 2015; Das Gupta et al., 2016). Depending on the organ they populate, macrophages receive different designations. Tissue-specific macrophages are distinguished by specific differentiation programs, cell morphologies, and specialized functions (Jurberg et al., 2018). Examples of such macrophages include microglia in the brain, alveolar macrophages in the lungs, Kupffer cells in the liver, osteoclasts in the bone, and chondroclasts in the cartilage. Self-renewal is the defining hallmark of most tissue-resident macrophages.

Macrophage development is dependent on the transcription factor PU.1 and macrophage colony-stimulating factor receptor (CSF1R) signaling (Kurotaki et al., 2017). Transcription factor PU.1 (encoded by the Spi1 gene) is a master switch that controls monocyte/macrophage development from hematopoietic stem cells (Roy, 2016). Nearly all macrophages, monocytes and mature B cells are absent in Spi1^–/– mice (Scott et al., 1994; McKercher et al., 1996). CSF1R signaling is required for the differentiation, survival and proliferation of macrophages and monocytes. Indeed, CSF1R-deficient mice show severely reduced numbers of most tissue-resident macrophages and monocytes (Dai et al., 2002). CSF1 and interleukin 34 (IL-34) are the two known ligands for CSFR1. CSF1 is abundant in many tissues including the lymph nodes, uterus, ovary, salivary gland and peripheral blood, whereas IL-34 is specifically expressed in the epidermis and brain (Wang and Colonna, 2014). Binding of these cytokines to CSF1R activates the PI3K–Akt and Ras–Raf–MEK–ERK signaling pathways, thereby controlling various downstream cellular processes including cytoskeletal remodeling, cell adhesion, survival, and proliferation (Pixley and Stanley, 2004; Mouchemore and Pixley, 2012). Blockade of CSF1R signaling suppresses the proliferation of tissue resident macrophages in vivo during inflammation (Davies et al., 2013). Thus, CSF1 and IL-34 are potent trophic factors for macrophages and monocytes (Figure 1).

Macrophages, as key innate immunity cells, express a plethora of receptors that allow them to sense the extracellular environment with exquisite sensitivity and specificity. Examples of these receptors include phagocytic receptors, cytokine receptors, receptors for other host-derived inflammatory mediators and several families of pattern recognition receptors (PRRs), such as the Toll-like receptors (TLRs) and Nod-like receptors (NLRs) (Das Gupta et al., 2016). Macrophages activated through PRRs produce potent pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-12, together with chemokines and toxic free radicals (Arango Duque and Descoteaux, 2014; Kawasaki and Kawai, 2014; Turner et al., 2014). Scavenger receptors bind a diverse range of ligands from bacteria to native proteins, allowing them to regulate both cell adhesion and the removal of noxious agents by phagocytosis (Prabhudas et al., 2014). Recognition and clearance of apoptotic cells is another important function of macrophages. Apoptotic cells are detected by macrophages through specific receptors that recognize externalized phosphatidylserine (PS) on the cell membrane of dying cells (Jurberg et al., 2018). Macrophages are also able to detect complement molecules by using cognate receptors and antibodies by using Fc receptors. These molecules opsonize pathogens and abnormal cells, thus stimulating their phagocytosis. The combined actions of these detection systems enable macrophages to initiate appropriate inflammatory programs upon perturbed homeostasis.

Macrophages are dynamic cells and can undergo transitions across a continuum of phenotypes and activation states. Depending on the microenvironmental milieu, macrophages are polarized into one of two subclasses, broadly classified as M1 or M2 (Mantovani et al., 2002; Mosser and Edwards, 2008; Murray et al., 2014). M1 macrophages, also known as “classically activated,” are induced by the canonical Th1 cytokines TNF-α and IFN-γ. In addition, granulocyte–macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS), and other TLR ligands also stimulate M1 polarization. In contrast, polarization of M2, or “alternatively activated macrophages (AAMs),” is induced by Th2 cytokines, IL-4 and IL-13 (Figure 1). The signal transducer and activator of transcription 1 (STAT1) and interferon-regulatory factor 5 (IRF5) play a major role in dictating M1 macrophage phenotypes, whereas, STAT3/STAT6, peroxisome proliferator-activated receptor-γ (PPARγ), and IRF4 direct M2 macrophage polarization (Lawrence and Natoli, 2011). M1 macrophages support Th1 responses and produce large amounts of reactive oxygen species (ROS) and nitric oxide (NO) to kill intracellular pathogens (Mantovani et al., 2002). Meanwhile, M2 macrophages are involved in tissue repair, vascularization, tumor promotion/invasion, and in responding to parasitic infections. AAMs are also characterized by their high expression of scavenger receptors in addition to high production of IL-10, VEGF, and MMPs (Mantovani et al., 2002). In cryptococcosis, STAT1-mediated M1 macrophage activation is critical to generate a host protective effector Th1 cell population (Leopold Wager et al., 2014; Leopold Wager et al., 2015).

Macrophages play diverse roles. Resident macrophages regulate tissue homeostasis by acting as sentinels and respond to changes in physiology as well as external challenges. Macrophages are uniquely equipped to sense and respond to tissue invasion by infectious microorganisms and tissue injury through various scavenger, pattern recognition and phagocytic receptors (Lavin et al., 2015). Macrophages also have homeostatic functions, such as the clearance of lipoproteins, debris and dead cells using sophisticated phagocytic mechanisms (Kohyama et al., 2009; Hussell and Bell, 2014). Unfortunately, these homeostatic and reparative functions are sometimes subverted due to continuous insult, resulting in causal association of macrophages with disease states, such as the inflammatory diseases of fibrosis, obesity, and cancer. Thus, macrophages are an incredibly diverse set of cells constantly shifting their functional state to new set points in response to changes in tissue physiology or environmental challenges.

C. neoformans is a fungal pathogen with a thick polysaccharide capsule. This capsule plays the dual role of “sword” by evading host immune responses and also acts as “shield” by protecting the fungi from the host’s anti-microbial oxidative burst attacks and phagocytosis. Interaction of C. neoformans with macrophages either results in (a) lysis of host cells due to capsular enlargement, (b) lysis of the fungi or (c) survival of both the host and the pathogen (Figure 2). In mammalian hosts, all possible outcomes result in either survival of host, active infection, or emergence of dormant/latent infection. The polysaccharide capsule is composed of three major components namely: glucuronoxylomannan (GXM), galactoxylomann (GalXM) and mannoproteins (MP). GXM influences the function of innate immune cells like macrophages, neutrophils, and dendritic cells by reducing the antigen presentation functions, altering costimulatory molecules, dysregulating secretion of proinflammatory and anti-inflammatory cytokines and by stimulating apoptosis via upregulation of the death receptor Fas/FasL. Overall, GXM has immunosuppressive effects on innate immune cells. Similarly, immunosuppressive effects of GalXM also inhibit adaptive immune responses by inducing T cell apoptosis and suppressing B cell activity (Decote-Ricardo et al., 2019) In addition to the immunosuppressive activity of the capsule, chemically diverse signals in the host, such as iron deprivation, serum, CO₂, can induce capsular enlargement to evade phagocytosis. Conversely, MPs stimulate a protective cell-mediated immune response against C. neoformans (Murphy, 1998; Levitz and Specht, 2006). MP1 and MP2 bind to dendritic cell mannose receptors to stimulate efficient antigen presentation, secrete pro-inflammatory cytokines (IL-12, IFN-γ, and TNF-α), and induce T cell proliferation (Pietrella et al., 2005). Furthermore, mannoprotein antigen MP98 and MP88 is known to stimulate T cell responses (Levitz et al., 2001; Huang et al., 2002; Dan et al., 2008). Another study characterized two novel MPs-MP 84 and MP115 which are recognized by serum antibodies during cryptococcosis (Biondo et al., 2005; Teixeira et al., 2014).These immunological features make MPs a potential vaccine candidate. Overall, the capsule exerts immunosuppressive effects on the host.

Laccase enzymes catalyze the synthesis of melanin. Therefore, laccase deficient fungal strains also show defective melanin production (Kwon-Chung et al., 1982, Rhodes et al., 1982). Melanin has antioxidant activity (Wang and Casadevall, 1994a; Wang and Casadevall, 1994b), mediates resistance to antifungal drugs (van Duin et al., 2002; Wozniak and Levitz, 2008) such as amphotericin B and caspofungin and provides rigidity to the cell wall. Melanin can also be recognized by host antibodies and drive a protective immune response (Rosas et al., 2001). C. neoformans has two laccase (Lac) isoforms, Lac1 and Lac2. Lac1 predominantly plays a role in infection and is expressed in the cell wall, whereas Lac2 is expressed in the cytoplasm (Polacheck et al., 1982; Missall et al., 2006). Furthermore, the investigators utilized a congenic pair of laccase positive and laccase deficient strains to determine the role of antifungal activity in murine AM. Using the recombinant cryptococcal laccase, they found oxidation of phagosomal iron with a resultant decrease in hydroxyl radical formation protects C. neoformans (Liu et al., 1999). Moreover, laccase also has immunomodulatory effects by catalyzing prostaglandin E2 production, which helps sustain infections (Erb-Downward and Huffnagle, 2007). Furthermore, laccase contributes to cryptococcal CNS dissemination via increased eosinophilia, M2 macrophage response, overall polarizing CD4+T cells towards a Th2 immune response (Qiu et al., 2012). Another study associated laccase activity with cryptococcal survival in human fluid (CSF) ex-vivo and poor fungal clearance (Sabiiti et al., 2014). Laccase also modulates nonlytic exocytosis, a function independent of melanin production. C. neoformans isolates that produce melanin faster frequently undergo nonlytic exocytosis resulting in increased dissemination of fungal cells (Frazão et al., 2020).Overall, the dual actions of melanin and laccase synergize for full virulence and promote dissemination of yeast cells.

C. neoformans cells show great heterogenicity in cell size during lung infection. C. neoformans possess a rare and complex ability to dramatically enlarge their size from a typical 5 μm cell body diameter up to 100 μm, thereby forming “titan cells”. Recent studies revealed that such morphologic alterations play an important role in fungal adhesion and penetration of biologic membranes for avoiding immune detection, and promoting fungal dissemination (Okagaki et al., 2010; Zaragoza et al., 2010; García-Barbazán et al., 2016). Thus, titan cells play a key role in pathogenesis due to their ability to survive and disseminate within the host.

Importantly, the main virulence factor of C. neoformans, capsule, has structural differences in titan cells, including being more dense, having an abnormally thick cell wall and having more crosslinked capsule (Zaragoza et al., 2010). The significantly enlarged cell size also corresponds to increased DNA content and undergoes endoreduplication to form polyploid cells containing 16, 34, 64, or more copies of the genome (Okagaki et al., 2010; Zaragoza et al., 2010; Kronstad et al., 2011). Despite polyploidy, titan cells produce haploid progeny, highlighting a distinct developmental transition likely required to disseminate into the CNS. Interestingly, like titan cells, their haploid progeny is also resistant to stress factors and antifungals (Zaragoza et al., 2010; Gerstein et al., 2015).

Recent descriptions of the in vitro conditions required to promote titanization have contributed to a better understanding of these cells. Titan cells can be induced in vitro by nutrient limitation-oxygen depletion (Trevijano-Contador et al., 2018), addition of polar lipids like PC (Chrisman et al., 2011), serum (Dambuza et al., 2018, Hommel et al., 2018), bacterial components like MTP and quorum sensing molecules (Albuquerque et al., 2014; Hommel et al., 2018; Dambuza et al., 2018). Other signals that contribute titanization include interaction of Ste3a pheromone receptor with alpha pheromone (Okagaki et al., 2010) and interaction of G protein-coupled receptor Gpr5 with a still unknown ligand (Okagaki et al., 2011).

Titan cells fail to undergo phagocytosis due to their large size, which also reduces the phagocytosis of normally sized cryptococcal cells (Crabtree et al., 2012; Okagaki and Nielsen, 2012) (Figure 2). Additionally, titan cells may prevent clearance by host immune cells, allowing them to maintain infection latency (Okagaki and Nielsen, 2012). During murine cryptococcal infection, titan cells promote disease through enhanced persistence in the host. Titan cells also promote the prevalence of lung eosinophils to stimulate a Th2 mediated immune response (Okagaki and Nielsen, 2012). Titan cells have been described to have interactions with amoeba (Chrisman et al., 2011) and wax moths (García-Rodas et al., 2011), suggesting these cells are formed under selective host pressures. Another interesting study demonstrated IgM inhibited titanization by inducing small capsules and downregulating the expression of stress and cell morphogenesis regulators (RIM101, HOG1), chitin synthetase (CHS1, CHS2, and CHS8) and cell wall carbohydrate synthetases (AGS1 and FKS1) (Trevijano-Contador et al., 2020). Bioengineered cytotoxic T cells expressing GXM-targeting chimeric antigen receptor (GXMR-CAR) recognized and controlled titanization (Kumaresan et al., 2014). Furthermore, second generation GXMR-CAR redirected T cells to recognize variable polysaccharide thicknesses of C. Gattii and C.neoformans, reduced titanization and provided protection against pulmonary cryptococcosis (da Silva et al., 2021). Taken together, targeting titanization will likely have a profound impact on disease progression and prevention.

C. neoformans has developed a fascinating mechanism, non-lytic exocytosis (or vomocytosis), to enhance its survival in hosts. This mechanism was first described by two groups independently, during imaging of macrophages infected with C. neoformans (Alvarez and Casadevall, 2006; Ma et al., 2006). Non-lytic exocytosis is the mechanism by which the live organisms are expelled out from a phagocyte, leaving both the host cell and the pathogen undamaged (Figure 2). Mammalian cells use non-lytic phagocytosis for a variety of functions, including phagocytosis, exocytosis of intracellular compartments required for phagosome formation, secretion of lysosomal enzymes and cytokines (Cruz-Acuña et al., 2019). During fungal infections, macrophages are the first responders. Macrophages recognize fungal cells based on their PAMPs and engulf them into phagosomes. Phagosomes then fuse with the lysosomes to form phagolysosomes where the invading fungi are killed (Figure 2). However, intracellular pathogens like C. neoformans and C. gattii have developed a novel mechanism to fuse their containment vacuoles with the cell membrane to evade lysosomal degradation (Alvarez and Casadevall, 2006; Ma et al., 2006; Bielska and May, 2016). Intracellular survival of C. neoformans inside the phagosome enables the ability to withstand oxidative burst, inflict damage to the phagolysosomal membrane, and impair critical host cell functions (De Leon-Rodriguez et al., 2018a). Interaction of C. neoformans and macrophages therefore would result either in 1) killing or restriction of C. neoformans growth by macrophages; 2) C. neoformans release following macrophage lysis; or 3) non-lytic exocytosis that results in the survival of both the macrophage and the fungus. Furthermore, non-lytic exocytosis could also be accompanied by a Trojan horse mechanism, whereby the fungus hijacks macrophages to disseminate into the brain to cause fatal cryptococcal meningitis (De Leon-Rodriguez et al., 2018b) (Figure 2). Within the lungs, C. neoformans can also become latent and localize within giant cells or macrophages in the form of granulomas. This latent infection can reactivate under conditions of weakened immunity, resulting in intracellular replication and dissemination (Fu et al., 2018) (Figure 2). Therefore, the coexistence of protective and deleterious roles of macrophages in the progression of cryptococcosis warrants further investigation.

Early observations showed that non-lytic exocytosis does not occur with heat-killed Cryptococcus or with latex beads, suggesting that host cells have a mechanism to detect phagosomal cargo viability (Alvarez and Casadevall, 2006; Ma et al., 2006). Cryptococcal virulence factors like phospholipase B, urease, and capsule influence non-lytic exocytosis (Cox et al., 2000; Cox et al., 2001). Cryptococcal strains with defective phospholipase B, urease production or acapsular strains show decreased non-lytic exocytosis (Alvarez and Casadevall, 2006). Furthermore, non-acidified phagosome also supports vomocytosis. The link between phagolysosomal pH and non-lytic exocytosis can be further explained by the inability of acapsular and urease lacking strains to buffer the acidic pH resulting lower vomocytosis (De Leon-Rodriguez et al., 2018a; Fu et al., 2018).

Host cytoskeleton rearrangement is an obvious target for regulating expulsion of pathogens. Addition of cytochalasin D, inhibitor of actin polymerization is known to enhance vomocytosis of C. neoformans (Johnston and May, 2010). Furthermore, Johnston et al. demonstrated a new mechanism by which the host cell attempts to retain internalized cargo and inhibits vomocytosis via assembly of transient actin cages surrounding the phagolysosome (Johnston and May, 2010). For proper non-lytic exocytosis, the internal vesicle must be positioned near the edge of the cell. This positioning allows the vesicle and plasma membrane to fuse together and allow expelling of the cargo (Stukes et al., 2016). This function of bringing the membranes together is carried out by Annexin A2. Loss of Annexin A2 reduces phagocytosis and vomocytosis, both in vitro and in vivo, resulting in enlarged capsule. These findings suggest a capsule either too small or too large may also abrogate non-lytic exocytosis (Stukes et al., 2016). Other host factors influencing non-lytic exocytosis include host membrane integrity (Tucker and Casadevall, 2002), composition (Nolan et al., 2017), and autophagy (Nicola et al., 2012).

Macrophages are highly plastic in nature and respond differently to different environmental cues. M2 or alternatively activated macrophages show an increased intracellular proliferation rate of cryptococcal cells and reduced non-lytic exocytosis. Conversely, M1 activated macrophages show reduced intracellular proliferation but increased events of non-lytic exocytosis (Seoane et al., 2020). A plausible explanation for this might be that alternatively activated macrophages have increased iron levels to support fungal growth (Weiss et al., 1997), whereas in classically activated macrophages, cryptococcal cells attempt to escape immune attack by non-lytic exocytosis. Thus, the polarization status of macrophages also affects non-lytic exocytosis. Lastly, Gilbert et al. demonstrated the role of host mitogen-activated kinase ERK5 in the regulation of non-lytic exocytosis (Gilbert et al., 2017). Suppressing ERK5 with a chemical inhibitor resulted in a significant increase of non-lytic exocytosis (Gilbert et al., 2017). Overall, different biological and immune cues influence the rate of non-lytic exocytosis. Understanding non-lytic exocytosis could help in the design of novel strategies to inhibit vomocytosis and prevent dissemination.

As a facultative intracellular pathogen, a key characteristic of C. neoformans is its ability to survive and replicate within the phagolysosome of macrophages (Huang et al., 2016). Multiple fungal proteins have been found to play a role in C. neoformans-macrophage interaction.

In cryptococcal infected macrophages, urease promotes yeast fitness by delaying intracellular replication, and reduces damage to the phagolysosomal membrane thus, enhancing fungal dissemination through non-lytic exocytosis (Fu et al., 2018). During infection, urease alkalizes and damages the surrounding cellular environment through the production of ammonia. In a murine model of cryptococcosis, urease aids the transcytosis of Blood Brain Barrier (BBB) (Olszewski et al., 2004) and also influences immune responses in the lungs (Osterholzer et al., 2009). A recent study also highlighted the role of urease in the growth and metabolism of C. neoformans. Urease positive strains show increased activity under nutrient-limited conditions at 37°C, whereas urease deficient strains showed higher melanin levels at 26°C, suggesting urease is closely linked to the functions of key metabolic pathways (Toplis et al., 2020). Moreover, phospholipase hydrolyzes phospholipids. Since Phospholipase B1 (Plb1) is active at 37°C and is stable in an acidic environment, Plb1 likely promotes C. neoformans proliferation and survival within macrophages. Indeed, Δplb1 mutants display growth and proliferation defects within macrophages, causing attenuated virulence by having reduced fungal burden in the lungs and reduced dissemination to the CNS (Santangelo et al., 2004; Maruvada et al., 2012). Overall, urease helps C. neoformans to both persistently enter and exit macrophages while phospholipase facilitates growth within macrophages.

Anti-phagocytic protein 1 (App1) is a cryptococcal protein that is secreted extracellularly and inhibits phagocytosis through a complement-mediated mechanism where App1 binds to complement receptor 3 and 2 (Stano et al., 2009). This function of App1 inhibits both binding and ingestion of yeast cells by macrophages (Luberto et al., 2003). App1 as a virulence factor is differentially expressed based on location. In pulmonary cryptococcosis, App1 is highly expressed in bronchioalveolar fluid (BAL) while weakly expressed in blood (Williams and Del Poeta, 2011). Similarly, expression of App1 is also upregulated in other body fluids like serum and cerebrospinal fluid (CSF) (Williams and Del Poeta, 2011). Overall, low glucose conditions stimulate increased App1 transcription and mRNA stability (Williams and Del Poeta, 2011).

Our recent studies revealed the potential function of fungal phospholipid membrane distribution in C. neoformans pathogenesis, including macrophage recognition during early pulmonary infection. These studies showed that expression of the regulatory subunit of lipid translocase (flippase) Cdc50 was highly induced when C. neoformans was co-cultured with the J774.16 macrophage cell line (Huang et al., 2016). Mutation of Cdc50 reduced phosphatidylserine (PS) inward translocation, leading to accumulation of exocytoplasmic PS (Huang et al., 2016). The cdc50Δ strain was engulfed and killed more efficiently than the wild-type strain, suggesting loss of fungal CDC50 leads to increased phagocytosis of C. neoformans. Furthermore, the cdc50Δ mutant was hypersensitive to macrophage-mediated killing, suggesting Cdc50 is essential for fungal survival in macrophages. Therefore, Cdc50 activity is likely required for counteracting the anti-microbial activities of host macrophages (Huang et al., 2016).

The hypersensitivity of the cdc50Δ mutant to macrophage killing may be due to not only its defect in membrane integrity, but also increased PS on the cell surface. Increased PS exposure on mammalian cell surfaces is known to act as a signal for macrophage recognition and phagocytosis. This is a classical feature of apoptotic cells for signaling their clearance by macrophages (Shor et al., 2016). It remains unclear how increased PS exposure on the fungal plasma membrane generates a signal that is recognized by host macrophages despite the presence of the capsule and thick cell wall. It is possible that macrophages may secrete factors that survey the environment and help recognize potential target cells for phagocytosis. Therefore, PS exposure on the cell surface may have multiple effects on C. neoformans-host interaction during infection, an intriguing future research direction.

Data from cryptococcus-macrophage interaction assays suggests fungal F-box protein (Fbp1) is required for fungal proliferation inside macrophages. The fbp1Δ mutant showed a defect in intracellular proliferation following phagocytosis, which likely contributes to its attenuated virulence. Fbp1 is part of the ubiquitin-proteasome system. The inositol phosphosphingolipid-phospholipase C1 (Isc1) substrate is required for fungal survival inside macrophage cells. This is consistent with the role of Fbp1 in regulating cryptococcus-macrophage interaction and fungal virulence. These findings suggest the Fbp1-mediated ubiquitin-proteasome pathway controls C. neoformans virulence by regulating fungal intracellular growth in macrophages. This findings also reveal a new determinant of fungal virulence that is mediated by post-translational regulation of inositol sphingolipid biosynthesis (Liu and Xue, 2014).