Pro-inflammatory response ensured by LPS and Pam3CSK4 in RAW 264.7 cells did not improve a fungistatic effect on Cryptococcus gattii infection

RAW 264.7 cell line and Cryptococcus gattii

The peritoneal macrophage cell line, RAW 264.7, was routinely grown in RPMI 1640 (GE Healthcare; Chicago, Illinois, USA) supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, and antibiotics and these cells were cultured at 37 °C in a humidified atmosphere containing 5% CO.

C. gattii strain R265 (VGII molecular genotype) was recovered on Sabouraud dextrose agar and incubated at 30 °C for 24 h. One loopful from a single colony was inoculated in Sabouraud dextrose broth and grown for 24 h at 30 °C with constant shaking (150 rpm). Yeast was harvested by centrifugation at 2000 xg for 10 min at 25 °C, washed in sterile phosphate-buffered saline (PBS), and counted using China ink in a Neubauer chamber. The concentration of the yeast in each in vitro infection is described in the figure legend.

Macrophage polarization/repolarization in response to Pam3CSK4-P3C4, LPS, and ArtinM

Synthetic triacylated lipoprotein (Pam3CSK4-P3C4) was purchased from Invivogen (catalog code: tlrl-pms; San Diego, CA, USA), and LPS was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). ArtinM was purified as described previously (Da Silva et al., 2020) from the saline extract of Artocarpus heterophyllus (jackfruit) seeds through affinity chromatography with immobilized carbohydrate columns. The endotoxin removal from ArtinM solution was performed as described previously (Da Silva et al., 2020).

RAW 264.7 cells were distributed in a 12-well microplate at a concentration of 1 × 10⁵ cells/mL. RAW 264.7 cells were incubated with LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or medium alone (Medium). After 24 h of incubation, the cell culture supernatants were collected to quantify the levels of TNF-α using enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, San Diego, CA, USA), and the RNA from macrophages was isolated by TRIzol Reagent (Life Technologies, Carlsbad, CA, USA).

The quantification of transcripts of iNOS and arginase-1 by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in M2 macrophages incubated with TLR2 or TLR4 agonists was performed using RAW 264.7 cells (1 × 10⁵ cells/mL) distributed in a 12-well microplate. Initially, macrophages were incubated with IL-4 (40 ng/mL), which is an inductor of the M2 phenotype, or Medium, as the first stimulus. After 24 h, the cells were submitted to a second stimulus composed of LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), IL-4 (40 ng/mL), or Medium for an additional 24 h.

Determining the levels of TNF-α in M2 macrophages after stimulation with TLR2 or TLR4 agonists

RAW 264.7 cells (1 × 10⁴ cells/mL) distributed in a 96-well microplate. The first stimulus was composed of LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (10, 40, or 80 ng/mL), or Medium. After 24 h of incubation the levels of TNF-α were measured, and an additional 24 h of incubation containing a second stimulus was performed as follows: fresh medium; cells previously stimulated with LPS, P3C4, ArtinM, IL-4, or Medium were restimulated with the same stimulus; macrophages polarized to the M2 subset by stimulation with IL-4 at concentrations of 10 ng/mL, 40 ng/mL, and 80 ng/mL received a second stimulus composed of LPS, P3C4, ArtinM, IL-4, or Medium . The cell culture supernatants were obtained to quantify the levels of TNF-α by ELISA.

Quantification of the levels of TNF-α in response to the effect of IL-4 in RAW 264.7 cells previously incubated with P3C4, LPS, or ArtinM

RAW 264.7 cells (1 × 10⁴ cells/mL) were distributed in a 96-well microplate. The first stimulus was composed of LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or Medium. After 24 h of incubation the levels of TNF-α were measured. An additional 24 h of incubation containing a second stimulus was performed as follows: fresh medium; cells previously stimulated with LPS, P3C4, ArtinM, IL-4, or Medium were restimulated with the same stimulus; macrophages incubated with LPS, P3C4, ArtinM, IL-4, or Medium received a second stimulus composed of IL-4 at concentrations of 10 ng/mL, 40 ng/mL, and 80 ng/mL. The levels of TNF-α were measured in cell culture supernatants.

Co-culture of C. gattii and RAW 264.7 cells stimulated with P3C4, LPS, or ArtinM

The RAW 264.7 cell line (2 × 10⁴ cells/mL) was plated in a 48-well microplate and rested for 4 h. These cells were incubated with LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or Medium for 24 h. These cells were co-cultured with C. gattii at a concentration of 2 × 10⁴ yeast/mL, or Medium as a negative control, and these conditions were considered a second stimulus. After 24 h of incubation, the levels of TNF-α were measured in the cell culture supernatant by ELISA.

In another assay, RAW 264.7 cells (2 × 10⁴ cells/mL) were co-cultured with live or heat-killed yeast of C. gattii at a concentration of 2 × 10³ yeast/mL in a 48-well microplate at 37 °C in a humidified atmosphere containing 5% CO₂. Concomitantly, macrophages received LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or Medium. The cell culture supernatant was collected after 24 h of incubation and the levels of TNF-α were quantified by ELISA.

The measurement of relative expression of iNOS and arginase-1 was performed in RAW 264.7 cells (1 × 10⁵ cells/mL) stimulated with LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or Medium, and concomitantly co-cultured with live yeast of C. gattii (1 × 10⁵ yeast/mL). After 24 h of incubation at 37 °C in a humidified atmosphere containing 5% CO₂, the RNA from macrophages was isolated by TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). The quantification of transcripts of iNOS and arginase-1 by qRT-PCR was performed as described below in the quantitative reverse transcription topic.

Fungistatic effect of RAW 264.7 cells stimulated with P3C4, LPS, or ArtinM on C. gattii

The murine RAW 264.7 cell line at a concentration of 1 × 10⁵ cells/mL was distributed in a 24-well microplate and rested for 4 h at 37 °C in a humidified atmosphere containing 5% CO₂. Macrophages were stimulated with LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or Medium for 24 h. Then, 2 × 10⁴ yeast/mL of C. gattii was added to the culture of previously stimulated macrophages, and the co-culture was incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. C. gattii yeasts were plated in the absence of macrophages, which were considered the control of growth of C. gattii under the same conditions. The monolayer culture was detached and macrophages were lysed using sterile water and the suspension was mixed with supernatant to quantify the growth of C. gattii by the colony forming unit (CFU) assay that was expressed as CFU/mL

Phagocytosis and antifungal activity of RAW 264.7 cells stimulated with P3C4, LPS, or ArtinM and co-cultured with C. gattii

RAW 264.7 cells at a concentration of 1 × 10⁴ cells/mL was distributed in a 24-well microplate and rested for 4 h at 37 °C in a humidified atmosphere containing 5% CO₂. Macrophages were stimulated with LPS (0.1 µg/mL), P3C4 (0.1 µg/mL), ArtinM (2.5 µg/mL), IL-4 (40 ng/mL), or Medium. After 24 h, live yeast of C. gattii (1 × 10⁵ yeast/mL) was added to the culture and the co-culture was incubated for 5 h at 37 °C. Then, the samples were washed twice with PBS to remove nonadherent yeast and the cells were lysed with sterile water. This suspension was plated onto Sabouraud agar plate for CFU quantification. For antifungal activity assay, once the samples were washed twice with PBS to remove nonadherent yeast a fresh medium was added, and incubation was continued for an addition 5 h. After incubation, cells were lysed with sterile water and the fungal cells were plated onto Sabouraud agar plate for CFU quantification.

Quantitative reverse transcription PCR and cytokine measurement

RNA from macrophages stimulated for 24 h was isolated using the TRIzol Reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. The total RNA was reverse-transcribed into complementary DNA (cDNA) using an iScript™ cDNA Synthesis Kit (Bio-Rad). Real-time PCR was performed in 10 µL reactions using 2x qPCRBIO SyGreen Mix Separate-ROX (PCRBiosystems; Wayne, PA, USA), cDNA (10-25 ng), and 0.3 µM of the primer mix. All reactions were performed on a Bio-Rad CFX96 Real-Time Detection System (Bio-Rad) under the following conditions: 95 °C for 2 min, and 40 cycles of 95 °C for 5 s/60 °C for 30 s. Gene expression was quantified using the Δ ΔCt method and normalized to β-actin expression. The PCR primers used were: β-actin (F-CCTAAGGCCAACCGTGAAAA, R-GAGGCATACAGGGACAGCACA), Ym-1 (F-TCACAGGTCTGGCA ATTCTTCTG, R-ACTCCCTTCTATTGGCCTGTCC), Arginase-1 (F-GTTCCCAGATGTACCAGGATTC, R-CGATGTCTTTGGCAGATATGC), Fizz1 (F-CCTGAGATTCTGCCCCAGGAT, R-TTCACTGGGA CCATCAGCTGG), and iNOS2 (F-CCGAAGCAAACATCACATTCA, R-GGTCTAAAGGCTCCGGGCT).

The supernatants were used for the quantification of TNF-a levels by ELISA according to the manufacturer’s protocol using Ab pairs purchased from BD Biosciences (Pharmingen, San Diego, CA, USA). Concentrations were determined relative to standard curves prepared from recombinant murine cytokines. Absorbances were read at 450 nm in a PowerWave X microplate scanning spectrophotometer (BioTek Instruments, Inc.).

Statistical analysis

Results are presented as means ± SD. All data were analyzed using GraphPad Prism v.7.0 (GraphPad Software, San Diego, CA, USA). The normality test of all statistical determinations were analyzed by the Shapiro–Wilk normality test. For datasets with a non-normal distribution, the Kruskal-Wallis test was used for experiments with three or more groups. Differences between the means of groups were evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test or the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Differences at p < 0.05 were considered statistically significant.

Role of TLR2 and TLR4 agonists in the polarization and repolarization of RAW 264.7 cells

Macrophage polarization occurs in different sources of macrophages and the microenvironment and innate immune receptors are major inductors in differentiation to M1 or M2 subsets (Mantovani et al., 2004; Wang, Liang & Zen, 2014). The modulation of M1- and M2-type macrophage polarization can be orchestrated by C. gattii compounds through interactions with receptors on the cell surface (Ueno et al., 2019). In this context, carbohydrates located in the C. gattii capsule can inhibit TLR2 and TLR4 signaling, which is a mechanism to subvert the host immune response. Then, we evaluated the effect of TLR2 and TLR4 agonists on the maintenance of polarized RAW 264.7 cells into the M1 subset over time during C. gattii infection. Initially, we demonstrated that P3C4, LPS, and ArtinM induced a significant production of TNF- α by RAW 264.7 cells after 24 and 48 h of incubation (Fig. 1A). The impact of TLR2 and TLR4 agonists in M1/M2 polarization was investigated by measuring the mRNA levels of M1/M2 markers, and RAW 264.7 cells stimulated with TLR2 and TLR4 agonists had a significant increase in the levels of iNOS transcripts (Fig. 1B), whereas the relative expression of arginase-1, Ym-1, and Fizz-1 was significantly altered in the presence of IL-4 alone (Figs. 1C–1E).

To understand the potential of TLR2 and TLR4 agonists in the modulation of M2 macrophages, the RAW 264.7 cell line was incubated with different concentrations of IL-4 and, after 24 h of incubation, the cells were stimulated with P3C4, LPS, or ArtinM. This hypothesis was investigated via the measurement of M1/M2 markers in polarized RAW 264.7 cells to the M2 phenotype that received a second stimulus with TLR2 and TLR4 agonists for 24 h. The transcripts of iNOS and arginase-1 were quantified by qRT-PCR and only LPS induced a significant augmentation of levels of iNOS mRNA in macrophages (Fig. 2A), whereas the levels of arginase-1 mRNA in macrophages stimulated with P3C4 or LPS had been lower than those incubated with Medium (Fig. 2B). These findings demonstrate that TLR2 and TLR4 agonists can act in macrophage repolarization from the M2 to M1 phenotype.

TNF-α production induced by TLR2 and TLR4 agonists in RAW 264.7 cells is not affected by live yeast of C. gattii

Previous studies have reported that C. gattii modulates the host immune response using capsule compounds and intracellular proteins released (Fonseca et al., 2010), and innate immune receptors are potential targets in the suppression of the immune response induced by C. gattii. The impact of TLR2 and TLR4 agonists in the activation of the RAW 264.7 cells was assayed in the presence of an inflammatory regulator, such as IL-4, which was evaluated using two different approaches as follows: (i) cells were incubated with IL-4 at different concentrations (10-80 ng/mL) and were submitted to a second stimulus of P3C4, LPS, or ArtinM (Fig. 3); (ii) cells were stimulated with LPS, P3C4, or ArtinM for 24 h, and a second stimulus with IL-4 (10 to 80 ng/mL) was performed (Fig. S1). In both approaches the production of TNF-α was measured, and we observed that macrophages previously incubated with IL-4 at different concentrations (10–80 ng/mL) showed high levels of TNF-α after 24 h of incubation with P3C4, LPS, or ArtinM as second stimulus, compared to Medium (Fig. 3, blue, green, and yellow bars). In addition, there was no significant difference in the levels of TNF-α induced by TLR2 and TLR4 agonists between macrophages previously incubated with IL-4 at different concentrations (Fig. 3, blue, green, and yellow bars). In the other hand, the first stimulus with LPS, P3C4, or ArtinM induced high levels of TNF-α in the RAW 264.7 cell after 24 h of incubation (Fig. S1, black bar), and these levels were reduced after a second stimulus with IL-4 in a dose-dependent manner (Fig. S1, blue, green, and yellow bars). Taken together, these findings suggest that the TLR2 and TLR4 agonists-activated RAW 264.7 cell induce a proinflammatory response even in the presence of inflammatory regulator.

The immunomodulation induced by TLR agonists in RAW 264.7 cells was also evaluated after co-culture with C. gattii yeasts. The macrophages were stimulated with TLR2 and TLR4 agonists in the absence of C. gattii for 24 h and an additional 24 h of incubation in the presence of C. gattii. The levels of TNF-α were measured in the culture supernatant, and the cells incubated with TLR2 and TLR4 agonists for 24 h and co-cultured with C. gattii as a second stimulus induced high levels of TNF-α in response to stimulation with LPS and P3C4, compared to the Medium (Fig. 4A). In other assay, the effect of C. gattii on the levels of TNF-α produced was also tested in the RAW 264.7 cells concomitantly stimulated with TLR2 or TLR4 agonists and co-cultured with C. gattii (live or heat-killed yeast), and after 24 h the levels of TNF-α were measured. The LPS and P3C4-activated RAW 264.7 cells reached high levels of TNF- α even in the presence of C. gattii, and the live yeast of C. gattii did not decrease the production of TNF-α induced by RAW 264.7 cells stimulated with TLR2 and TLR4 agonists (Fig. 4B). However, the levels of TNF-α induced by LPS stimulus had a significant increase in the presence of heat-killed yeast of C. gattii, compared to RAW 264.7 cells co-cultured with or without live yeast of C. gattii (Fig. 4B). Therefore, the improvement in the production of TNF-α by RAW 264.7 cells in response to LPS stimulus occurred by heat-killed yeast of C. gattii.

RAW 264.7 cells stimulated with TLR2 and TLR4 agonists had not a fungistatic effect on C. gattii infection due the alteration in the balance of iNOS/Arginase-1 expression

Proinflammatory mediators are required in several intracellular pathogen infections and previous studies have reported the importance of proinflammatory immune responses to fight cryptococcosis (Kumaresan, daSilva & Kontoyiannis, 2017). As LPS and P3C4 modulate RAW 264.7 cells to the M1 phenotype, this study evaluated the co-culture of RAW 264.7 cells with C. gattii in the presence of TLR2 and TLR4 agonists. The macrophages were incubated with LPS, P3C4, ArtinM, or Medium for 24 h, and infection with C. gattii yeasts (1:100 C. gattii/macrophage ratio) was performed. After 24 h of co-culture, the growth of C. gattii was quantified by CFUs and the results provided by co-incubation of unstimulated RAW 264.7 cells (Medium) with C. gattii did not significantly differ from those obtained after stimulation with LPS, P3C4, or ArtinM. The C. gattii culture alone in the absence of RAW 264.7 cells (C. gattii alone) was performed as control, and the growth of C. gattii yeasts in unstimulated RAW 264.7 cells (Medium) did not differ from those obtained from C. gattii alone (Fig. 5A). The outcome of C. gattii infection was not strongly associated with M1 macrophages polarized by TLR2 and TLR4 agonists.

To understand the effect of C. gattii infection in the polarization of RAW 264.7 cells induced by TLR2 and TLR4 agonists, these cells were stimulated with IL-4, LPS, P3C4, and ArtinM concomitantly or not (uninfected) with live yeast of C. gattii. After 24 h, the mRNA levels of iNOS and arginase-1 were measured by RT-PCR. C. gattii-infected RAW 264.7 cells after stimulation with TLR2 and TLR4 agonists had a significant decrease in the levels of iNOS transcripts compared to uninfected macrophages (Fig. 5B), whereas the relative expression of arginase-1 was significantly augmented in RAW 264.7 cells infected with C. gattii and stimulated with LPS (Fig. 5C). These findings demonstrated that the C. gattii infection is able to regulate the iNOS expression induced by TLR2 and TLR4 agonists, which can influence the microbicidal activity of macrophage favoring the growth of yeast.

RAW 264.7 cells stimulated with TLR2 and TLR4 agonists inhibit the growth of C. gattii in the early period of infection

This work also investigated the impact of TLR2 and TLR4 agonists in the phagocytic activity of RAW 264.7 cells in the presence of C. gattii. The cells were incubated with IL-4, IFN- γ, LPS, P3C4, or ArtinM for 24 h previously the addition of C. gattii, and after 5 h of in vitro infection the non-adherent yeasts were removed. The level of phagocytosis was performed by CFU and the activation of RAW 264.7 cells induced by TLR2 and TLR4 agonists and IFN-γ improved the phagocytosis of C. gattii, compared to unstimulated macrophages (Medium) (Fig. 6). Then, the capacity of RAW 264.7 cells to control the growth of C. gattii in response to stimulation with TLR2 and TLR4 agonists was performed. To evaluated the antifungal activity of macrophages previously stimulated the cells were submitted to conditions described above for phagocytosis assay. Once the non-adherent yeasts were removed, fresh medium was added and incubation was continued for an additional 5 h and macrophage lysate was plated for CFU determination. The quantification of C. gattii in RAW 264.7 cells previously incubated with medium or IL-4 had a significant increase compared to CFU quantified after phagocytosis (Fig. 6). However, the stimulation of RAW 264.7 cells with IFN-γ, LPS, P3C4, or ArtinM did not result a significant difference compared to the adherent yeast measured in the phagocytosis assay (Fig. 6). Then, pro-inflammatory immune response induced in RAW 264.7 cells by TLR2 and TLR4 agonists favors the uptake of C. gattii promoting a control of growth of yeast in the early stage of infection.