Hyphal‐associated protein expression is crucial for Candida albicans‐induced eicosanoid biosynthesis in immune cells

Candida albicans causes opportunistic infections ranging from mucosal mycoses to life‐threatening systemic infections in immunocompromised patients. During C. albicans infection, leukotrienes and prostaglandins are formed from arachidonic acid by 5‐lipoxygenase (5‐LOX) and cyclooxygenases, respectively to amplify inflammatory conditions, but also to initiate macrophage infiltration to achieve tissue homeostasis. Since less is known about the cellular mechanisms triggering such lipid mediator biosynthesis, we investigated the eicosanoid formation in monocyte‐derived M1 and M2 macrophages, neutrophils and HEK293 cells transfected with 5‐LOX and 5‐LOX‐activating protein (FLAP) in response to C. albicans yeast or hyphae. Leukotriene biosynthesis was exclusively induced by hyphae in neutrophils and macrophages, whereas prostaglandin E2 was also formed in response to yeast cells by M1 macrophages. Eicosanoid biosynthesis was significantly higher in M1 compared to M2 macrophages. In HEK_5‐LOX/FLAP cells only hyphae activated the essential 5‐LOX translocation to the nuclear membrane. Using yeast‐locked C. albicans mutants, we demonstrated that hyphal‐associated protein expression is critical in eicosanoid formation. For neutrophils and HEK_5‐LOX/FLAP cells, hyphal wall protein 1 was identified as the essential surface protein that stimulates leukotriene biosynthesis. In summary, our data suggest that hyphal‐associated proteins of C. albicans are central triggers of eicosanoid biosynthesis in human phagocytes.


Introduction
Acute inflammation is an important, life-saving, defense mechanism with the primary goal of eliminating noxious agents, such as pathogens, and restoring tissue homeostasis.Myeloid phagocytes such as macrophages and neutrophils form the first line of myeloid cell defense against pathogens [1,2].These cells are equipped with a specific set of effector mechanisms, including the release of pro-inflammatory cytokines, chemokines, and other immune mediators.The latter involves the formation of eicosanoids such as leukotrienes (LTs) and prostaglandins (PGs), which are formed from arachidonic acid (AA, C20:4, ω-6) by 5lipoxygenase (5-LOX) and cyclooxygenases (COXs), respectively [3].Cytosolic phospholipase A 2α (cPLA 2α ) releases AA from phospholipids upon inflammatory stimulation.Simultaneously, 5-LOX activated by Ca 2+ -binding and serine phosphorylation by p38-MAPK and ERK1/2-kinase translocates to the nuclear membrane, where it co-localizes with the membrane-bound 5-LOX-activating protein (FLAP), which is required for cellular LT formation [4].In another branch of the AA cascade, AA is a substrate for COX-1 and COX-2 yielding PGH 2 as an unstable intermediate, which can be further converted by specific synthases to downstream PGs, such as PGE 2 by PGE synthase and thromboxane A 2 by thromboxane synthase [3,5].
Macrophages are a group of cells with high phenotypic plasticity and functional diversity [6].The classical two subsets are grouped as M1-and M2-like macrophages.While M1-like macrophages, in vitro polarized by lipopolysaccharides (LPS) and IFN-γ, produce proinflammatory mediators, nitric oxide, and reactive oxygen intermediates, M2-like macrophages are generated in vitro upon exposure to IL-4 and are associated with the resolution phase of inflammation [7].Differentiation into M1 and M2 subtypes is associated with differential expression of AA cascade enzymes resulting in a phenotypically distinct lipid mediator (LM) profile upon pathogen stimulation, reflecting the central role of macrophage subpopulations in pro-and anti-inflammatory immune responses upon pathogen exposure [8,9].
Among microbial pathogens, the yeast Candida albicans is an increasing cause of infections tightly related to the innovation of immunosuppressive and invasive medical therapies that easily breach the physiological immune barrier and provide a bridge for fungal infections [10,11].The commensal C. albicans can cause disease, ranging from superficial mucosal infections to severe, systemic candidiasis.The shift from a harmless colonizer to a lifethreatening pathogen is a complex process that has been only partially understood [12,13].To date, C. albicans pathogenicity has been strongly associated with the morphological transition from yeast to hyphae [11,14].While both morphological forms are found during host infections, strains unable to form hyphae are less virulent [14,15].In addition to the morphological transition itself being considered an important virulence feature, this transition is accompanied by virulence-enhancing processes such as the expression of surface proteins, cell wall remodeling, the secretion of host epithelium-degrading enzymes and proteins, and the expression of the peptide toxin candidalysin [16][17][18][19].As part of the human inflammatory arsenal against invading C. albicans, the interplay between yeast or hyphae and human immune cells in terms of LM formation is only rudimentarily understood [20,21].
In the current report, we investigate the eicosanoid formation in both subpopulations of macrophages, neutrophils, and in 5-LOX/FLAP expressing HEK293 cells that are challenged by C. albicans.We further elucidate differences in yeast and hyphae stimulation and unravel the molecular mechanisms that provoke LM formation.

Blood cell isolation
Experiments with primary human blood cells were approved by the ethical review committee of the University Hospital Jena, Germany (license number: 4968-11/16).Leukocyte concentrates from human peripheral blood of healthy donors were purchased from the Transfusion Medicine, University Hospital Jena.Neutrophils and monocytes were isolated as described previously [22].Briefly, leukocytes were isolated using dextran sedimentation (2.5% dextran) and subsequently separated by centrifugation on density cell separation medium Histopaque 1077.Neutrophils were isolated from the granulocyte/erythrocyte fraction by hypotonic erythrocyte haemolysis and were washed twice with ice-cold PBS and resuspended in PBS (pH 7.4).In order to obtain monocytes, PBMC were washed twice with ice-cold PBS and resuspended in (PBS, pH 7.4; 1 mM Ca 2+ and 0.5 mM Mg 2+ ).Cell viability was determined by Trypan Blue on a Beckmann Vi-Cell cell counter.Cells were seeded into cell culture flasks for 1 h at 37°C for monocyte separation.The supernatant was removed and cells were seeded in 1640 (RPMI-1640) supplemented with 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin for subsequent differentiation and polarization.

Macrophage differentiation and polarization
Monocytes Briefly, neutrophils were isolated using dextran sedimentation (2.5% dextran) and subsequent density centrifugation on cell separation medium (Histopaque-1077), followed by hypotonic erythrocyte hemolysis.Neutrophils were washed twice with ice-cold PBS and resuspended in PBS (pH 7.4).

C. albicans culture
C. albicans (ATCC SC5314) [24] and all C. albicans mutant strains are listed in Table 1.C. albicans glycerol stocks were inoculated on YPD-agar (1% yeast-extract (m/v), 1% peptone (m/v), and 2% dextrose (m/m)).For experiments, single colonies were picked and incubated in a YPD medium at 30°C and 180 rpm overnight.Subsequently, overnight cultures were centrifuged and resuspended with PBS (pH 7.4) twice before adjustment to the required cell density.Yeast cells were transferred to RPMI (without phenol red) for hyphae induction (120 min at 37°C).Hyphae cultures were centrifuged, resuspended, and adjusted to the required cell density using Neubauer improved counting chamber.

Determination of LM formation in macrophages via UPLC-MS/MS
Monocyte-derived macrophages (MDMs) were seeded with RPMI-1640 into six-well plates prior to polarisation described above (2 × 10 6 cells/well).The medium was aspirated and MDMs were stimulated with C. albicans yeast or hyphae at indicated multiplicity of infections (MOI) in PBS+ (pH 7.4; 1 mM [Ca 2+ ]).To collect LM, the supernatant was removed and mixed with 2 mL of ice-cold methanol containing 10 μL of deuterium labeled internal standard (200 nM d8-5S-hydroxyeicotetraenic acid [HETE], d4-LTB 4 , d5-LXA 4 , d5-RvD 2 , d4-PGE 2 , and 10 μM d8-AA; Cayman Chemicals) for means of quantification and sample normalization.Sample purification was performed as previously described [25].In brief, samples were transferred to -20°C for at least 60 min to ensure protein precipitation and subsequently centrifuged at 1200 × rpm for 10 min, at 4°C.The supernatant was diluted with 8 mL acidified H 2 O (0.5 mM HCl), followed by solid phase extraction on Sep-Pak Vac 6cc 500 mg/6 mL C18-columns (Waters).After loading the sample, the column was washed with water and n-hexane, and LMs were eventually eluted with methylformate.Samples were evaporated under nitrogen (TurboVap LV; Biotage) and resuspended in 100 μL 50% methanol for ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as described [25].LMs were separated chromatographically applying an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm; Waters) at 50°C and at a 0.3 mL/min flow rate using MilliQ water (A) and methanol (B) both acidified with 0.01% acetic acid, starting with 42% of B, increasing up to 86% at 12.5 min that was followed by isocratic elution at 98% till 15.5 min.Eicosanoids were analyzed with an Acquity UPLC system (Waters) directly interfaced into the electrospray ionization Turbo V Source of a QTrap 5500 Mass Spectrometer (Sciex).Analysis was performed in the negative ion mode using multiple reaction monitoring with analytes being scheduled in specific retention time (RT) windows.Deuterated internal standards were detected using the following transitions and RTs: m/z 327.3→116.Quantification was performed by applying linear calibration curves with r 2 values of 0.998 or higher (for fatty acids: 0.95 or higher) for each LM as described [25].

Lactate dehydrogenase assay
To assess the cytotoxic effects of C. albicans (SC5314) yeast and hyphae on human cells, cell membrane integrity was quantified by determination of activity of the cytoplasmic enzyme lactate dehydrogenase (LDH) in the supernatants.Macrophages (2 × 10 6 cells/mL) and HEK_5-LOXFLAP cells (2 × 10 6 cells/mL) were incubated with C. albicans (SC5314) yeast or hyphae at MOI 10, vehicle control, and triton X-100 (0.2%) as full lysis control for 0.5, 1, 2, and 24 h.LDH release from disintegrated cells was measured by CytoxTox96 KIT (PROMEGA GmbH) according to the manufacturer's instructions.The values are presented as a percentage of the full lysis control Triton X-100 (0.2%).

Immunofluorescence microscopy
Immunofluorescence microscopy was applied to analyze the subcellular localization of 5-LOX and FLAP under stimulation with C. albicans (SC5314) yeast or hyphae.Macrophages (1 × 10 6 cells/well) and HEK_5-LOX/FLAP cells (1 × 10 6 cells/well) were seeded on coverslips in 12-well plates 2 days prior to the experiment.The supernatant was removed and cells were stimulated in PBS+ (PBS pH 7.4; 1 mM CaCl 2 ) with C. albicans at MOI 10 or vehicle control (PBS+) for 60 min (37°C; 5% CO 2 ).Cells were fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 min, followed by permeabilization in acetone on ice for 5 min and Triton X-100 (0.25% in PBS (m/v); pH 7.4) for 10 min.The slides were incubated with primary antibodies mouse monoclonal anti-5-LOX 1:100 and rabbit polyclonal anti-FLAP 1:150) in nonimmune goat serum overnight.Samples were incubated with secondary antibodies (Alexa Fluor 488 goat anti-rabbit (1:1000) and Alexa Fluor 555 goat anti-mouse (1:1000) fluorophore-labelled) in nonimmune goat serum for 30 min at 25°C in the dark and slides were mounted with DAPI-containing ProLong Diamond Antifade Mounting medium on glass slides.Samples were visualized using Zeiss Axiovert 200 M microscope and a Plan-APOCHROMAT 40×/1.3Oil DIC (UV)Vis-IR 0.17/∞ objective (Carl Zeiss) coupled with AxioCam MR camera (Carl Zeiss).Displayed Microscopic pictures show a 50 × 50 μm section of macrophages and a 20 × 20 μm section of HEK_5-LOXFLAP cells of the full image.

Statistics
Results are displayed as means + SEM of n observations, with n representing the number of experiments performed with different donors.Graphs were created and validated with GraphPad Prism 7.04 software (GraphPad Software).A value of p < 0.05 was considered statistically significant.

LM biosynthesis provoked by C. albicans yeast or hyphae in M1 and M2 macrophages
To further increase our knowledge of Candida-induced eicosanoid formation, we investigated the LM formation in monocyte-derived M1 and M2 macrophages and 5-LOX activation in immortalized stably transfected HEK_5-LOX/FLAP cells upon interaction with C. albicans yeast and hyphae.
Among the various branches of the AA cascade, AA can be converted by the 5-LOX/FLAP pathway to 5-H(p)ETE, which is then further metabolized to LTA 4 the unstable intermediate and substrate for LTA 4 hydrolase, to form the pro-inflammatory and chemoattractant LTB 4 .In cells, LTB 4 is degraded to 20-OH-LTB 4 (Fig. 1A).COX-1/2 metabolizes AA to PGH 2 , which is further converted to PGs and thromboxane depending on the tissue (Fig. 1A).M1 and M2 macrophages are obtained in vitro following differentiation of monocytes and subsequent polarization with LPS/IFNγ and IL-4, respectively, resulting in differential expression of AA cascade enzymes and consequently in different LM profiles.Western blot revealed protein expression of enzymes involved in AA metabolism in both M1 and M2 macrophages (Fig. S1).5-LOX and low levels of COX-1 are expressed in both subpopulations, whereas FLAP expression was strongly reduced in M2 macrophages, attenuating the 5-LOX pathway.Conversely, COX-2 was strongly expressed in LPS-polarized M1 macrophages.
M1 and M2 macrophages were stimulated with C. albicans yeast and hyphae for 60 min and phenotype-specific eicosanoid formation was analyzed via UPLC-MS/MS (Fig. 1B and F; Figs.S2  and S3).LTB 4 products (sum of LTB 4, t-LTB 4 , and 20-OH-LTB 4 ) and PGE 2 were chosen as representatives for the activation of the 5-LOX and COX biosynthetic pathways, respectively.C. albicans yeast failed to activate the 5-LOX pathway in M1 and M2 macrophages.However, a small but significant increase in PGE 2 formation was observed in yeast-stimulated macrophages.In contrast, total eicosanoid biosynthesis was significantly increased in M1 macrophages upon stimulation with C. albicans hyphae, as reflected by the formation of LTB 4 products and PGE 2 .Low levels of PGE 2 comparable to yeast-activated macrophages were detected in M2 macrophages upon stimulation with C. albicans hyphae, with total LM formation in M2 macrophages reaching less than a quarter of the LM levels detected in M1 macrophages.Note that IL-4 polarization of M2 macrophages strongly induces 15-LOX-1 expression [26], which shifts the LM biosynthesis towards 15-LOX-derived LM, a phenomenon seen in plenty of reports [8].Nevertheless, unexpectedly neither yeast nor hyphae of C. albicans stimulate 15-LOX product formation.
Cellular LT formation is dependent on the nuclear colocalization of 5-LOX and its helper protein FLAP [4].By monitoring the subcellular distribution of these proteins, we found that exclusively in hyphae-challenged M1 macrophages 5-LOX translocated and co-localized with FLAP at the nuclear membrane (Fig. 1C and G).Interestingly, in M2 macrophages and under stimulation with both morphologies 5-LOX remained disseminated within the nucleus (Fig. 1G), explaining the lack of LT formation.Both cPLA 2 and 5-LOX are stimulated by [Ca 2+ ] i mobilization [27,28].Therefore, changes of intracellular [Ca 2+ ] i levels were continuously monitored during the 60 min incubation period stimulated by C. albicans yeast or hyphae in both macrophage phenotypes (Fig. 1D and H).Although the increase in [Ca 2+ ] i -mobilization under fungal stimulation was not significant, C. albicans hyphae induced a slightly stronger increase in intracellular [Ca 2+ ] compared with the yeast cells.To exclude cytotoxic effects and macrophage membrane disintegration as the underlying cause of eicosanoid formation, potential cell damage of C. albicans yeast or hyphae-stimulated macrophages was monitored by LDH activity in supernatants.No significant LDH release was observed during the first 2 h of fungi-macrophage interaction but strongly increased after 24 h in both M1 and M2 macrophages (Figure 1E and I).In conclusion, LT formation was solely induced by C. albicans hyphae in M1 macrophages, and PGE 2 biosynthesis was stimulated in M1 and M2 macrophages by yeast and hyphae, but to a much higher extent in hyphae-stimulated M1 macrophages.

C. albicans hyphae provoke MAPKs activation in M1 and M2 macrophages
Stimulation of mitogen-activated protein kinases (MAPKs) is globally involved in the activation of cPLA 2 , 5-LOX, and ultimately in the expression of COX-2 [29,30].While 5-LOX can be activated through phosphorylation at Ser 271 and Ser 663 via p38-MAPKdependent MAKAPK-2 and ERK1/2, respectively, cPLA 2 activity is induced by phosphorylation at Ser505 via ERK1/2 [31].Western blot analysis confirmed p38-MAPK and ERK1/2 phosphorylation in response to C. albicans hyphae in both macrophage phenotypes (Fig. 2A and B).The progression of phosphorylation differed between the MAPKs but was phenotype-independent.While the level of p38-MAPK phosphorylation remained constant over 180 min, ERK1/2 phosphorylation peaked after 45 min of stimulation and decreased continuously over the next 135 min.

Hyphae-triggered eicosanoid formation is dependent on hyphal-associated protein expression
Based on the observation that C. albicans yeast cells failed to stimulate sufficient eicosanoid formation in M1 and M2 macrophages, we questioned whether hyphal-associated virulence factors activate the 5-LOX and COX pathways.Utilizing yeast-locked C. albicans mutant strains is an elegant approach to assess the importance of morphological and transcriptomic adaptations during the transition from yeast to hyphae.The yeast-locked mutant C. albicans efg1 / /cph1 / is unable to filament and express hyphal-associated genes [15].C. albicans hgc1 / mutant shows defective hyphal formation but still expresses hyphal-associated proteins, including hyphal wall protein 1 (Hwp1), Ece1, Hyr1, and Als3 [32].Consequently, these strains differ in their proteinexpression profile.All C. albicans strains were treated under hyphae-inducing conditions.In M1 macrophages 5-LOX and COX activity was significantly reduced upon stimulation with C. albi-cans efg1 / /cph1 / compared with C. albicans wild-type, as reflected by a significant decrease in LTB 4 and PGE 2 formation (Fig. 3, Fig. S4).Similarly, LTB 4 and PGE 2 formation in M2 macrophages was attenuated upon stimulation with C. albicans efg1 / /cph1 / compared with wild-type, but as mentioned above (Fig. 1B and F) starting from a less abundant level of LM formation than in M1 macrophages.However, co-incubation of macrophages with the hgc1 / C. albicans mutant still resulted in eicosanoid formation comparable to that of the wildtype strain (Fig. 3, Fig. S4).Since the hgc1 / mutant cannot form true hyphae, yet is still able to express hyphal-associated proteins, this suggests a strong involvement of the latter in hyphae-induced LM biosynthesis.

C. albicans hyphae stimulate 5-LOX activation in HEK_5-LOX/FLAP cells
During disseminated candidiasis, the fungus infects vital tissues during the late stages of infection.Drastic immune activation by the infected tissue cells not only drives protective immunity, but particularly, in the case of the kidney, leads to uncontrolled neutrophil influx [33].Here, we used HEK293 cells stably expressing 5-LOX and FLAP (HEK_5-LOX/FLAP) [23] to exclude innate immune-specific recognition pathways as underlying mechanisms for fungal-induced eicosanoid formation.These experiments allowed us to extend the validity of our results obtained in professional innate immune cells and prove them as universal pathogenicity mechanisms that may contribute to continuous inflammation.We monitored 5-LOX activation in HEK_5-LOX/FLAP cells by determining their cellular distribution using immunofluorescence microscopy.Similar to previous observations in macrophages, C. albicans hyphae strongly induced 5-LOX activation, visible in translocation, after 60 min of co-culture, whereas C. albicans yeast failed in this respect (Fig. 4A).Intracellular Ca 2+ mobilization as 5-LOX activator was monitored for 60 min in HEK_5-LOX/FLAP cells stimulated with both C. albicans morphologies.Surprisingly, in contrast to macrophages (Fig. 1D) and neutrophils [21], both yeast and hyphal cells of C. albicans failed to stimulate a detectable Ca 2+ -influx (Fig. 4B), suggesting the requirement of more precise activation factors for 5-LOX translocation.To exclude fungal cytotoxicity and to ensure membrane integrity during incubation, LDH activity in supernatants was examined.In contrast to macrophages and neutrophils [21], HEK_5-LOX/FLAP cells showed slightly but not significantly increased LDH levels during the first 2 h of incubation with C.  albicans yeast and hyphae, leading to a significant LDH release after 24 h of treatment (Fig. 4C).This observation suggests an expected higher susceptibility of HEK_5-LOX/FLAP cells compared with immunocompetent cells, but also rules out cytotoxic effects as trigger for 5-LOX activation within the first 2 h of interaction.

Deletion of HWP1 in C. albicans results in loss of 5-LOX activation in HEK cells and neutrophils, but not in macrophages
Based on our findings in macrophages (Fig. 3) and neutrophils [21], we investigated the influence of the hyphaelocked mutant strains efg1 / /cph1 / and hgc1 / on 5-LOX activation in HEK_5-LOX/FLAP cells.Both yeast-locked strains efg1 / /cph1 / and hgc1 / were unable to stimulate 5-LOX translocation and co-localization with FLAP (Fig. 5A) suggesting that hyphal formation is a prerequisite for 5-LOX translocation in the engineered HEK cell system.
One of the most relevant hyphal surface proteins is the Hwp1, which facilitates attachment to the target cell and is required for cell-to-cell adhesion [16,34].C. albicans hwp1 / and its revertant hwp1 / +HWP1 were used to evaluate the involvement of hyphal adhesins in 5-LOX activation in HEK_5-LOX/FLAP cells.The HWP1-deficient strain hwp1 / failed to induce 5-LOX translocation, whereas C. albicans hwp1 / +HWP1 stimulated 5-LOX translocation comparable to the wild-type (Fig. 5B).Thus, C. albicans appears to require the mature hyphal morphology with full Hwp1 expression at the fungal surface to trigger 5-LOX activation in stably transfected HEK_5-LOX/FLAP cells.Motivated by this finding, we stimulated macrophages and neutrophils with C. albicans hwp1 / and its revertant.Surprisingly, whereas in macrophages the absence of the Hwp1 did not disrupt a proper eicosanoid biosynthesis (Fig. 5C), in neutrophils LTB 4 formation was significantly reduced by hwp1 / and was restored by the revertant hwp1 / +HWP1 (Fig. 5D), which confirms Hwp1as essential surface protein to facilitate LT biosynthesis.Another virulent hyphae-associated protein is the cytolytic peptide candidalysin, which is expressed and released during filamentous growth.However, the deletion mutant extent of cell elongation 1 protein (ece1 / ), incapable of expressing candidalysin, showed comparable virulence regarding eicosanoid formation in M1 macrophages as the wild-type strain (Fig. 5E).Comparable findings were reported before in neutrophils [21].These obser-vations let us conclude that 5-LOX activation and accompanied LT formation are clearly associated with hyphae-specific protein expression but differ between different immunocompetent cells.Motivated by the finding that hyphae-specific protein expression plays a crucial role in hyphae-induced eicosanoid formation, we evaluated other surface proteins that are exclusively expressed in hyphae.Our focus turned toward the hyphae wall protein Hyr1 [35] and the surface protein Als3, belonging to the agglutinin-like sequence (Als) family [17,36].However, the deletion mutants hyr1 / and als3 / showed comparable potential to activate eicosanoid biosynthesis as the wild-type strain (Fig. 5E).

Discussion
In the current study, we investigated C. albicans-induced eicosanoid formation in macrophage phenotypes and HEK cells while unraveling the underlying inducing mechanisms.We demonstrated that hyphae in particular, but not yeast cells, induce LT and PG formation predominantly in M1 macrophages and that the expression of hyphal-associated proteins plays a crucial role in this process.In HEK cells overexpressing 5-LOX and FLAP and in neutrophils, 5-LOX activation and LT formation were also exclusively induced by hyphae and strongly associated with the expression of the surface protein Hwp1.
A successful human immune response to an invading pathogen requires unambiguous recognition of the micro-organism, followed by transmission and amplification of the proinflammatory signal to ensure the involvement of other mediators and cells of the immune system.Pathogens have developed sophisticated strategies to invade the host while evading the immune system.Among them, the fungus C. albicans undergoes a characteristic morphogenetic transition from yeast to hyphae during infection.This morphological change is not only associated with increased virulence [18,37] but is also an essential survival strategy of the fungus to adapt to the changing environment of the host and complicate immunological clearance [38,39].Hyphal formation per se is acknowledged as pathogenicity mechanism, but equally important for pathogenicity is the expression of hyphal-associated genes such as ECE1 and HWP1.While the extent of cell elongation protein 1 (Ece1) is the precursor of the amphiphilic peptide toxin candidalysin [18], Hwp1 is associated with adhesion and biofilm formation [16,40].Hwp1 is reported to serve as a substrate for mammalian transglutaminases, covalently linking C. albicans hyphae to host cells [16].Moreover, HWP1-deficient C. albicans strains showed attenuated virulence properties [16,41].We have shown that the formation of lipid mediators and the activation of 5-LOX in human macrophages and HEK_5-LOX/FLAP cells after stimulation by C. albicans is closely associated with the successful filamentation of the fungus.Using yeast-locked (efg1 / /cph1 / ) and hyphal-impaired (hgc / ) mutant strains of C. albicans, we confirmed this key finding and linked the activation to hyphal-associated proteins.This phenomenon has already been shown recently by us in neutrophils [21] and suggests that the host specifically mounts a distinct immune response to host-hyphal interactions in terms of the release of pro-inflammatory LM.In this report, we were able to extend our knowledge by identifying Hwp1 as the crucial surface protein that stimulates LT formation in neutrophils and HEK_5-LOX/FLAP cells.Most interestingly, while 5-LOX activation in neutrophils and HEK_5-LOX/FLAP cells is dependent on Hwp1, the expression of other hyphal-associated proteins appears to be involved in macrophages.Neither other adhesins, such as Hyr1 and Als3, nor the hyphal-specific toxin candidalysin seem to play a role in the stimulation of eicosanoid formation in macrophages.Such cell-type-specific stimulation of LM biosynthesis has been shown before for Staphylococcus aureus where LM biosynthesis in neutrophils is induced by phenol-soluble modulins, but in macrophages, it is stimulated by the pore-forming toxin alphahemolysin [9,42].Thus, it seems that different immune cells are equipped with distinct activation mechanisms stimulating LM biosynthesis.It should be noted that synergistic activation may also be required for eicosanoid formation in macrophages.A similar observation has been made during oropharyngeal infection by C. albicans, where both Hyr1 and Als3 are required for stimulation of c-Met and EGFR to develop full virulence and epithelial cell invasion [43].Future studies will have to show which hyphal-associated proteins or what kind of interplay between different actors stimulate LT or PG biosynthesis in macrophages.
During host invasion, C. albicans hyphae can breach mucosal and epithelial surfaces.Epithelial cells recognize the fungal pathogen and initiate damage response pathways [37].These signalling cascades lead to the release of chemokines, cytokines, and antimicrobial peptides that activate the immune system and promote the recruitment of neutrophils and macrophages [37,44].Hereby, macrophages are key players in fungal immune recognition.Tissue-resident macrophages (TRMs) were proven to function as sentinel cells, crucial during the early stages of C. albicans dissemination into organs [1,45,46].Previous studies have highlighted the relevance of TRM populations in various organs, such as the spleen and liver for C. albicans eradication as well as attenuation of fungal dissemination and filamentation [47,48].Shortly after C. albicans tissue invasion, the immune cell population substantially increases, mainly originating from neutrophils and MDM migrating toward the side of inflammation [33,49].Both TRMs and MDMs possess a distinct amatory of defense mechanisms against fungal pathogens, such as phagocytic clearance and subsequent secretion of cytokines or oxygenated lipids to amplify the proinflammatory signal.Eicosanoids, namely LTs and PGs tailor such an innate immune response.They are synthesized in myeloid cells in a cell-type distinct manner and mainly act locally through G-protein coupled receptors [50,51].Hereby, LTB 4 is a strong proinflammatory chemoattractant, induces ROS formation, and stimulates the release of chemokines from neutrophils and macrophages [8,52,53].PGE 2 , abundant in inflammatory environments, enhances inflammation by causing local vasodilation and attracting leukocytes.It also has immunosuppressive effects, such as stimulating the formation of IL-10.[54][55][56][57].Thus, we identified the cellular mechanism of how C. albicans hyphae accelerate inflammation by pushing biosynthesis of inflammatory lipid mediators.In addition, early reports indicated that PGE 2 enhances serum-induced germination and hyphae formation, whereas COX inhibition reduces biofilm formation and yeast-to-hyphae transition [58][59][60].It is therefore conceivable that small amounts of PGE 2 , already produced by yeast cells, then trigger the formation of hyphae and lead to dramatically increased inflammatory conditions through increased biosynthesis of LT and PG.However, the role of LM in C. albicans infection remains controversial in the scientific literature.While Peters et al. [61] could show that during co-infection with C. albicans and S. aureus treatment with non-selective COX-inhibitor indomethacin reduced mortality in mice, Jayaraja in vivo.[49] have demonstrated the importance of LTs and PGs for sufficient fungal clearance and survival in mice.Extended studies linking tissue and serum LM concentrations to pathophysiological circumstances are needed to evaluate the role of LM in Candida infections.Furthermore, our study showed that eicosanoid formation was significantly reduced in M2 macrophages compared with M1 macrophages, despite comparable activation of MAPK ERK1/2 and p38 MAPK.Both macrophage phenotypes differ in the expression profile of AA metabolizing enzymes resulting in a strongly reduced FLAP and COX-2 expression in M2 cells [25].It should be further considered that GM-CSF, used as a supplement for M1 macrophage differentiation, is known to upregulate dectin-1 expression [62], a receptor activated by the fungal cell wall component β-glucan [63] and known to be involved in hyphal-induced LT formation in neutrophils [21].Interestingly, polarization by IL-4 stimulates 15-LOX-1 expression in M2 macrophages [25], but 15-HETE and corresponding metabolites formed from other polyunsaturated fatty acids such as docosahexaenoic acid or eicosapentaenoic acid were not detected.This indicates that 15-LOX-1 in M2 MDM is not stimulated by either yeast or hyphae.
During disseminated candidiasis in mice, the murine kidney is among the primary target organs of C. albicans.This susceptibility can be linked to a delayed neutrophil and monocyte influx into kidney tissue compared to other organs [33,64].Presupposing myeloid cells and kidney tissue cells differ in their activation toward C. albicans yeast and hyphae, we questioned the response of stably transfected HEK_5-LOX/FLAP cells toward both morphologies [37,44].Interestingly, we observed a C. albicans hyphae-exclusive stimulating effect on 5-LOX.Additionally, the tested HEK cell line showed, comparable to neutrophils, an HWP1-related 5-LOX activation.As HEK-293 cells are no original carriers of 5-LOX and FLAP, it should be stressed, that HEK_5-LOX/FLAP cells represent an artificial approach, originally developed to study 5-LOX and FLAP interaction.However, our data indicate a susceptibility of HEK cells toward C. albicans hyphae via Hwp1 with corresponding activation of intracellular signaling cascades.
In conclusion, we report that C. albicans filamentation is essential for the activation of eicosanoid biosynthesis in M1 macrophages, neutrophils, and HEK_5-LOX/FLAP.For neutrophils and HEK cells, Hwp1 was identified as an essential hyphae surface protein required for 5-LOX activation and LT formation in the host cells.Whether the activation of the AA machinery contributes to detrimental immunopathology or enhances host-driven clearance of the pathogen is likely niche-dependent and remains to be elucidated.In the future, in vivo, infection models will be able to demonstrate the effects of activating eicosanoid biosynthesis on the C. albicans infection process in mammals, which could not be presented in this study.Nevertheless, the in vitro data highlights a strong specificity of the human immune system toward recognizing C. albicans filamentation.Thus, any intervention influencing hyphal growth will impact host-immune responses.

Table 1 .
C. albicans strains used in this study.