ABSTRACT
IL-17A has emerged as a key player in the pathologies of inflammation, autoimmune disease, and immunity to microbes since its discovery two decades ago. In this study, we aim to elucidate the activity of IL-17A in the protection against Cryptococcus neoformans, an opportunistic fungus that causes fatal meningoencephalitis among AIDS patients. For this purpose, we examined if C. neoformans infection triggers IL-17A secretion in the in vitro setting using RAW264.7 murine macrophage cells, and in vivo using wildtype C57BL/6 mice. In addition, an enhanced green fluorescence protein (eGFP) reporter and a knockout (KO) mouse models were used to track the source of IL-17A secretion and explore the protective function of IL-17A, respectively. Our findings showed that both in vivo and in vitro models of C. neoformans infection demonstrated induction of abundant IL-17A secretion. By examining the lung bronchoalveolar lavage fluid (BALF), mediastinal lymph node (mLN) and spleen of the IL-17A– EGFP reporter mice, we showed that intranasal inoculation with C. neoformans promoted leukocytes lung infiltration. A large proportion (~50%) of the infiltrated CD4+ helper T cell population secreted EGFP, indicating vigorous TH17 activity in the C. neoformans–infected lung. The infection study in IL-17A–KO mice, on the other hand, revealed that absence of IL-17A marginally boosted fungal burden in the lung and accelerated the mouse death. Therefore, our data suggest that IL-17A, released predominantly from TH17 cells in vivo, is essential in providing a protective immunity against C. neoformans infection.
INTRODUCTION
The opportunistic pathogenic basidiomycete Cryptococcus neoformans is an encapsulated yeast commonly found in bird excrement worldwide (1). The infection is often asymptomatic in healthy individuals but causes severe pulmonary cryptococcosis and life-threatening meningoencephalitis in immunocompromised patients. C. neoformans has gained attention in recent years as it is a major cause of death among patients who have advanced acquired immunodeficiency syndrome (AIDS) (2). Hence, it is important to study the host interaction with this pathogen as 30-60% of the patients who have cryptococcal meningitis succumb to cryptococcosis infection within one year despite antifungal therapy (3).
The early interaction between innate immune cells and cryptococci is critical in determining the outcome of cryptococcosis (4). The presence of C. neoformans cells or its major capsular polysaccharide glucuronoxylomannan promotes NF-κB nuclear translocation and activation in the macrophages (5), which subsequently induces secretion of cytokines including tumor necrosis factor alpha (TNF-α) and transforming growth factor β (TGF-β) (6). Activated macrophage or other phagocytes are able to eliminate most of the engulfed pathogens via formation of phagolysosome, nitric oxide and reactive oxygen activities. As with other successful pathogens such as Helicobacter pylori, C. neoformans has evolved a number of elaborate strategies to evade immune destruction by macrophages (7). The encapsulated C. neoformans can defend itself against the onslaught of macrophages through non-lytic expulsion (8, 9), a process that involves alteration of host cell Arp2/3 complex-mediated actin polymerization and phagosome pH (10, 11), thereby allowing the pathogen to survive and propagate within macrophages. C. neoformans-activated macrophage often displays an M1-like phenotype (12) whose polarization is highly plastic depending on external signals such as cytokines (13). C. neoformans intervenes in the polarization of macrophages by using Ssa1, a heat shock protein 70 homolog to drive macrophage development toward the alternative M2 phenotype that is defective in the fungal clearance (14). These processes impair the host innate immune system and promote the invasion and dissemination of C. neoformans in the host.
The essential role of T cells in the host immune response to C. neoformans has been well-studied using T cell depletion mouse model (15-17). In the era of two distinct helper T (TH) paradigm, TH1 or TH2, most findings are in agreement that protective immune response against the fungus is principally driven by TH1 cells, whereby IL-12 and IL-18 potentiate TH1 polarization and interferon-γ (IFN-γ) and TNF-α release which contribute to fungal clearance (18-20). Some studies suggest the involvement of TH2 as IL-4 is detected in high quantity in mice infected with highly virulent strain at 2 to 3 weeks post infection (21, 22). A dominant TH2 cytokine profile has been associated with increased cryptococcal proliferative potential (23). IL-4 and IL-13 released by TH2 or eosinophils in lung could also cause fatal allergic inflammation during bronchopulmonary mycosis (24, 25), a reaction which is dampened by IL-23 through an IL-17A–independent and –dependent mechanisms (26). Following the recent breakthrough in T cell subsets discovery, a relatively new T cell subset, TH17, has also been implicated in the immune response to fungus (27, 28). A study has also challenged the role played by IL-17A in anti-fungal response and claimed that IL-17A promotes the fungal infection (29), as such, the nature and role of these T cell subsets require further investigations. TH17 cell is characterized by its hallmark RORγt transcription factor and IL-17 secretion. Its differentiation from naïve CD4+ T cells is induced in the presence of IL-6 and TGF-β during inflammatory response. IL-23 is another important inducer for IL-17A as the IL-17A production was strongly impaired in the IL-23p19 deficient mice (30). C. gattii, a highly virulent cryptococcal species is able to attenuate both TH1 and TH17 by suppressing IL-12 and IL-23 genes transcription (31).
TH17 is not the sole source of IL-17A as it can also be released by other cells such as macrophages, NK cells, and neutrophils (32). IL-17A elicits inflammatory response by recruiting neutrophils, but does not contribute to classical macrophage activation as seen in pulmonary cryptococcosis induction in the mouse model (33). TH17 cells release a panel of other cytokines in addition to IL-17 such as IL-17F, IL-22, and IL-23. A full picture of regulatory mechanism as to how this subset of T cell interacts and eliminates the fungal infection requires further investigation. In this study, we examined the association of IL-17A with C. neoformans infection by using both in vitro and in vivo infection models. The main focus of our study lies on identifying the source of IL-17A secretion and determining its protective role in C. neoformans– infected mice. Using enhanced green fluorescence protein (eGFP) reporter mouse model, we showed that lung infiltrating TH17 cells are likely the predominant source of IL-17A. Data from a knockout (KO) mouse model supports a protective function of the IL-17A against C. neoformans infection.
RESULTS
C. neoformans infection induces IL-17A production in both in vitro and in vivo models
To examine if IL-17A gene transactivation is induced by C. neoformans infection, RAW264.7 mouse macrophage cells were first cultured with C. neoformans H99 strain at MOI 5:1 for 24 hours followed by quantitative real-time PCR. Intriguingly, the relative expression level of IL-17 to β-actin was drastically increased at 4.4–fold in the C. neoformans–infected cells compared to the non–infected control (Fig. 1). The relative expression levels for other cytokines, i.e. IL-6 and IFN-γ were also increased at 3.2– and 2.0–fold respectively, indicating initiation of pro-inflammatory response along with TH1– and TH17–inducing cytokines upon C. neoformans infection.
To investigate if C. neoformans infection–mediated IL-17A secretion occurs in vivo, C57BL/6 mice were intranasally inoculated with four different strains of C. neoformans (H99, S48B, S68B and H4) at 2 × 105 cells for 14 days before collecting serum for cytokine measurement (Fig. 2). Consistent with the data from in vitro infection, an elevated serum level of IL-17A was detected in the C. neoformans–infected mice (Fig. 2A). We noted that the serum IL-17A level was correlated with the degree of virulence of different C. neoformans strains (34), whereby the highest amount of serum IL-17A was observed in the group of mice infected with the most virulent C. neoformans H99 strain (115 ± 12 pg/ml). This was followed by moderate serum IL-17A level observed in the mice infected with less virulent environmental strains, S48S (89 ± 3 pg/ml) and S68B (75 ± 2 pg/ml), and lowest level of serum IL-17A was noted in the mice infected with non-virulent strain H4 (24 ± 1 pg/ml) strains, compared to control uninfected mice (<20 pg/ml). The level of serum IL-23 was also elevated in all C. neoformans–infected mice, i.e. H99 (67 ± 5 pg/ml), S48S (78 ± 3 pg/ml), S68B (36 ± 1 pg/ml) and H4 (47 ± 5 pg/ml) compared to <20 pg/ml in the mock control (Fig. 2B). This suggests IL-23–IL-17A axis pathway plays a major role in the host immunity against C. neoformans infection. On the other hand, the serum IL-17F levels were only scarcely increased in mice infected with C. neoformans H99 and S48B strains (Fig. 2C), while no noteworthy induction was observed for other cytokines (MIP-3α, IL-21, IL-31 and IL-33) examined (data not shown).
Intranasal C. neoformans inoculation causes leukocytes lung infiltration
To examine the importance of IL-17 in providing immunity to C. neoformans infection, we utilized a mouse model harboring IRES-EGFP-SV40-polyA signal sequence cassette after the stop codon of Il-17a gene in which the EGFP is co-expressed in the IL-17A–producing cells. Mice were intranasally administrated with 20 μl of control PBS or C. neoformans (2 × 105 cells) in suspension, and splenic, mediastinal lymph nodes (mLN), bronchoalveolar lavage fluid (BALF) cells were inspected after 4 weeks (Fig. 3). Total numbers of cells were significantly increased in the BALF (4.8 × 106 ± 1.0 × 106 versus 4.6 × 104 ± 1.0 × 104 cells, *P=0.0135) and mLN (3.0 ×105 ± 2.0 × 104 versus 1.5 × 105 ± 1.9 × 104 cells, **P=0.0017) of the C. neoformans H99– infected mice versus the control group (Fig. 3A and 3B). No significant increased numbers of cells were observed in the spleen after intranasal C. neoformans H99 infection (5.6 × 107 ± 7.1 × 106 versus 4.7 × 107 ± 6.1 × 106, P=0.46), suggesting localized infection (Fig. 3C).
In the BALF from the C. neoformans–infected mice, active immune response was noted as all types of leukocytes examined, except CD8+ T cells demonstrated an average of 2-fold increment (Fig. 3D). No significant differences of the cell constituents were observed in lymph node (data not shown). In spleen, the percentages of innate immune cells i.e. macrophages (CD11b+F4/80+), neutrophils (CD11b+Gr1hi) and inflammatory monocytes (CD11b+Gr1int) were increased at approximately 3– to 6–fold. On the contrary, percentages of T (both CD4+ and CD8+) cells were slightly reduced at 1.2– to 1.3–fold (data not shown).
Increased IL-17A–producing T cells in the lung of C. neoformans H99-infected mice
In the GFP in IL-17A–EGFP reporter mice, IL-17A–producing cells can easily be identified as they display EGFP fluorescence, hence this mouse model was utilized to determine the main source of IL-17 during C. neoformans infection. Our result showed that there was no profound increase of GFP+ cells amongst macrophage, neutrophil or inflammatory monocytes populations in the C. neoformans–infected mice (data not shown). On the contrary, significant increases of GFP+ cells were observed among the T cells (Fig. 4). Major source of IL-17A was derived from CD3+CD4+ but not CD3+CD8+ T cells. Almost half (54.2 ± 11.6 %, ***P=0.0008) of the total lung infiltrated CD3+CD4+ T cells recovered in BALF collected from C. neoformans H99-infected mice were GFP+, compared to only 4.8 ± 0.8% in the control (Fig. 4A). In the mLN, the percentages of GFP+ CD3+CD4+ cells were approximately 4–fold greater at 21.7 ± 1.4 % (P<0.0001) cells, compared to 5.1 ± 0.6 % in control mice (Fig. 4B). The percentages of GFP+ cells among CD3+CD4+ population were also marginally increased (17.53 ± 4.1 %, P=0.016) among the splenic CD4+ T cells compared to 6.1 ± 0.6 % in the control (Fig. 4C).
IL-17 provides protective immunity to pulmonary C. neoformans infection
A knock out mouse model was then applied to examine the protective role of IL-17 to host during C. neoformans infection. Intranasal inoculation of C. neformans in wildtype C57/BL6 mice and IL-17A–KO resulted in mice death starting from day 26 and 24, respectively. In IL-17A-KO mice, more than 80% (5 out of 6) mice died on day 34 whereas in wildtype control, this was observed at day 40 (Fig. 5A). The amounts of fungal cells in the local infection site (lung) as well as systemic infection (brain) were assessed. CFU counts in the lung derived from IL-17KO mice stayed at 657 ± 92, a higher level compared to 473 ± 119 in the wildtype control, whereas CFU count in the brain was 237 ± 39 compared to 133 ± 30 in the control mice (Fig. 5B). Hence, the absence of IL-17A accelerated mice death as a result of an increased CFU count, suggesting its protective role against C. neoformans invasion.
DISCUSSIONS
Increasing prevalence of mortality attributed to cryptococcal meningitis in the immunocompromised patients underscores the importance of elucidating the host defense– pathogen interaction. In this study, we focused on (i) determining the expression and source of elevated IL-17A from different types of immune cells using a IL-17A–GFP reporter animal model and, (ii) investigating the role of IL-17A in protective immunity using a IL-17A–KO mouse model. Our data demonstrated elevated serum levels of TH17 cytokines, i.e. IL-17A and IL-23 in the infected wild-type C57BL/6 mice; and proposed the lung–infiltrating TH17 subset as the major source for IL-17A secretion at the lung upon pulmonary C. neoformans infection. In addition, we also showed that the absence of IL-17A resulted in a greater fungal burden and accelerated death of the infected mice, which implies a protective role of the potent IL-17A response at an early stage of C. neoformans infection.
Although a high level of IL-17A was detected in the in vitro infection model using RAW264.7 macrophage cell line in this study, similar level of expression was not observed in the macrophages of in vivo animal infection model with the eGFP reporter system. Classical macrophage activation is not affected in the IL-17A–depleted mice upon pulmonary cryptococcal stimulation, hence IL-17A may not be derived from the activated macrophages (33). Instead, other cells like neutrophils and T cells may serve as the predominant leukocytic source of IL-17A (35). Human monocytes derived from healthy donors exhibited an extensive modification of transcriptome level upon incubation with C. neoformans, in particular the genes related to TNF-α, NF-κB, Jak-Stat and toll-like receptors pathways (36). Pro-inflammatory cytokine IL-1β is one of the top up-regulated cytokines in the C. neoformans–infected macrophages, and a deficiency in IL-1R1 results in defective TH17 cytokines secretion (37). This suggests that in the in vivo condition, macrophages may not directly release IL-17A but could instead be involved in indirect induction of IL-17A secretion from other immune cells through the release of pro-inflammatory cytokines such as IL-1β.
Most studies thus far pinpoint the association of TH1-type cytokine responses with protective immunity against pulmonary cryptococcosis (38-40). The cytokines in response were mainly those of TH1 subsets (IFN-γ, TNFα, IL-8), whereas moderate increases were also observed in TH2 (IL-4) and TH17 cytokines (IL-17A) (41). A predominant TH1 and/or TH17 cytokine profile limits the growth of C. neoformans and C. gattii, whereas a TH2 cytokine profile promotes intracellular fungus proliferation (23). In humans, it has also been reported that cryptococcal-specific CD4+ T-cell response is predominantly a TH1 type response with minimal involvement of TH2 and TH17 cells (41). However, patients with higher IFN-γ or TNF-α production showed greater level of IL-17A level in their cerebrospinal fluid (CSF). These patients demonstrated lower fungal burdens and faster clearance of C. neoformans infection, suggesting that both TH1 and TH17 responses worked cooperatively to provide optimal immunity against pulmonary cryptococcosis.
Our findings from IL-17A-KO mice experiment are in concordance with previous studies which showed that pulmonary fungal burden was resolved at a slower rate, and the overall survival was not deteriorated due to IL-17A deficiency (33, 35). Besides, higher fungal dissemination to the brain was also observed in the surviving IL-17A–depleted mice, consistent with a previous finding (35). These data support that IL-17A participates in providing protective anti-cryptococcal host defenses through the suppression of fungal growth and dispersal. This observation is in line with other studies on several other fungal species (42-44). It was shown that a deficiency in IL-17A response results in increased susceptibility to oropharyngeal and disseminated candidiasis (27, 45). Decreased neutrophil infiltration, increased fungal burden, and exacerbated pathology were reported upon IL-17A neutralization in C. albicans and Aspergillus fumigatus infections (42-44). Toll IL-1R8 (TIR8), another negative regulator of TH17 response, has also been shown to reduce the susceptibility and immunopathology to candidiasis (46). Some studies, on the contrary, provide evidence that outcome of aspergillosis in human is independent of TH17 responses (29), and the IL-23/IL-17A–driven inflammation could impede antifungal immune resistance and promote infection of A. fumigatus (47). Hence, further investigation is necessary to validate the precise function of TH17 immunity towards fungal infection in humans.
In summary, our data suggest that IL-17A derived from the lung infiltrating TH17 in BALF and mLN, plays a supportive role in rendering protection to pulmonary C. neoformans infection. Understanding the host immune response during cryptococcal infection is essential for the development of immunomodulatory therapies.
MATERIALS AND METHODS
Fungal and cell culture
C. neoformans var. grubii (serotype A) H99 and RAW264.7 murine monocytic macrophage cell-line were obtained from American Type Culture Collection (ATCC). Environmental strains S48B, S68B and H4 were isolated from pigeon droppings, as described (48, 49). To start the culture, a small drop of fungal cell stock was streaked on the Sabouraud’s dextrose agar (SDA) and incubated at 37°C for 48 hours. Then, 2 to 3 single colonies from freshly prepared agar plate were inoculated into Sabouraud’s dextrose broth (SDB) and incubated at 37°C for 48 hours. RAW264.7 cells were cultured in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10 % fetal bovine serum and incubated at 37°C, 5% CO2. For in vitro infection, RAW264.7 cells were seeded at 5.0 × 105 cells/ml and infected with C. neoformans at multiplicity of infection (MOI) of 5 for 24 hours.
Quantitative real-time PCR
RNA was isolated from cells as described previously (7). Briefly, 1 ml TRIzol Reagent (Invitrogen, Carlsbad, CA) and 200 μl chloroform were added to cells and vortexed vigorously for 15 sec. The upper phase was collected and precipitated with isopropanol, washed with 70% ethanol and dissolved in RNase-free water. Then, cDNA was generated using MMLV reverse transcriptase (Life Technologies). Samples were amplified with SsoAdvanced™ SYBR® Green Supermix (Biorad) using StepOnePlus™ Real-Time PCR Systems (Life Technologies) using the following PCR cycling parameters: 95°C for 10 min, 40 cycles at 95°C for 15 sec each, and 60°C for 1 min. Data were analyzed using StepOne software version 2.3. Primer sequences used were IL-17A (5’-TCTCCACCGCAATGAAGACC-3’ and 5’-CACACCCACCAGCATCTTCT-3’), IL-6 (5’-CCTCTGGTCTTCTGGAGTACC-3’ and 5’-ACTCCTTCTGTGACTCCAGC-3’ and IFN-γ (5’-TTCTTCAGCAACAGCAAGGC-3’ and 5’-TCAGCAGCGACTCCTTTTCC-3’). The fold change for each transcript relative to β-actin housekeeping gene was calculated using the 2-ΔΔCT method.
Animals
Wild type C57BL/6, IL-17A–eGFP (C57BL/6-Il-17atm1Bcgen/J) and IL-17A–KO (STOCK Il-17atm1.1(ire)Stock/J) mice were obtained from Jackson Laboratory (Bar Harbor, ME). IL17A–eGFP mice contain an IRES-eGFP-SV40-polyA signal sequence cassette inserted after stop codon of Il-17a gene and express eGFP as a marker of IL-17A activity. Whereas IL-17A– KO mice contained abolished IL-17A expression due to insertional mutation of a codon optimized Cre-recombinase and a polyA signal into exon 1 of Il-17a gene. Groups of 4 to 6 mice at age 8-12 weeks old were used throughout the study. All mice were maintained in individually ventilated cages under specific pathogen free condition. Mice were euthanized with CO2 inhalation when they exhibited overt signs including hunched posture, fur ruffling, weakness, increased respiratory rate and difficulty breathing. This study has been approved by the Faculty of Medicine Ethics Committee for Animal Experimentation at the University of Malaya.
In vivo infection
Fresh cultures of C. neoformans were washed and harvested by centrifugation at 1800 ×g for 10 min. Cells were adjusted to 107 cells/ml in phosphate buffer saline (PBS) using a hemocytometer. Mice were first anesthetized with intraperitoneal injection of a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) before inoculated with intranasal pipetting of 20 μl (2 × 105 cells) yeast suspension. For survival study, each infected mouse was examined daily from 2 to 6 weeks post infection. For other study, mice were euthanized with CO2 inhalation at 28 days post-infection and serum was collected from blood. Lung was lavaged with 1.0 ml PBS and BALF was collected. Lung, mediatinal lymph nodes (mLN), spleen and brain were excised for further analysis.
CFU count
Brain and lungs from the mice were excised, weighed and homogenized in 1 ml PBS using glass slides. A total volume of 20 μl of the serially diluted homogenates (at 10, 100 and 1000 folds) were plated on SDA plates in duplicates and cultured at 30°C for 48 hours. Fungal load was quantified using colony forming unit (CFU) per ml by calculating yeast colonies on each plate.
Bioplex assay
Sera from each mouse were collected for measurement of cytokines using Bio-plex Pro Mouse Th17 assay (Bio-rad, CA, USA) which included the following cytokines: IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-31, IL-33 and MIP-3α according to the manufacturer’s instructions. The Multiplex bead working solution was diluted from 25× stock solution beads and 50 μl of it was added into each well followed by 50 μl of sample. Each cytokine standards and samples were assayed in duplicate as provided by manufacturer. Samples with microbeads were incubated at room temperature on a magnetic microplate shaker for 30 minutes. After incubation, Bio-Plex detection antibody working solution was then added, washed 3× with Bio-Plex wash buffer and finally 1× streptavidin-PE was added before reading the plate on the Bio-Plex 200 system (Bio-Rad). Cytokine concentrations from each tissue homogenates were calculated based on each cytokine standard curve.
Statistics
All statistical analyses were performed using GraphPad Prism 6. Analyses between groups were performed using Student’s t-test, whereby a P value of <0.05 was considered statistically significant.
ACKNOWLEDGEMENTS
This work was supported by University of Malaya Research Grant (RG525-13HTM) and Fundamental Research Grant Scheme (FRGS, FP014-2017A). Movahed E. was supported by a postgraduate research fund (PPP) from University of Malaya.