Effect of Antifungal-Treated Host Macrophages on Candida glabrata

Objective Candida glabrata (C. glabrata) causes infections associated with severe sepsis and high mortality. This study describes the effects of micafungin (MCF), itraconazole (ICZ), and amphotericin B (AmB) on the function of macrophages during C. glabrata infection. Methods RAW264.1 macrophages were treated with MCF, ICZ, or AmB and then challenged with C. glabrata. Cytokines from infected macrophage supernatants and the levels of superoxide dismutase (SOD) in macrophages were measured at different time points after phagocytosis. Results The activity of SOD was significantly increased in RAW264.1 cells that phagocytized C. glabrata and reached a peak level at 6 hours (P < 0.05). ICZ and AmB did not affect the SOD activity in cells that phagocytized C. glabrata versus that in untreated macrophage. C. glabrata stimulated macrophages to secrete cytokines. Neither ICZ nor AmB affected the secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), or tumor necrosis factor-α (TNF-α) by C. glabrata-infected macrophages. However, MCF downregulated the secretion of TNF-α by infected macrophages and reduced the SOD activity of C. glabrata compared with those in untreated controls. Conclusion Echinocandins may increase their antifungal efficacy by altering the innate immune response of macrophages and attenuating antioxidants of this organism.


Introduction
Candida is generally regarded as a nonpathogenic symbiotic microbe of human mucosal tissue that occasionally causes opportunistic infections. Previous epidemiological studies from 2005 showed that the proportion of candidemia caused by Candida glabrata (C. glabrata) in public hospitals did not exceed 5-8% [1,2]. However, with the increasing use of various iatrogenic catheters, immunosuppressive agents during organ transplantation and anticancer treatment, broad-spectrum antibiotics, and the increase in HIV infection, infections caused by Candida spp. have tended to be more invasive in deep organs and manifest as sepsis, fungemia, and other life-threatening conditions. Candidemia caused by C. glabrata accounts for 10.6% of fungemia [3].
Infection caused by C. glabrata has a high mortality rate of 40-70% [4]. In recent years, the success of azole antifungal agents for the control of fungal infection has been consistent with the increase in non-Candida albicans (C. albicans) infection, among which C. glabrata infection has increased by three times. According to the ARTEMIS Global Antifungal Monitoring Project, C. glabrata accounted for 10.2%-11.7% of all Candida spp. strains isolated from June 1997 to December 2007 [5]. A multicenter prospective survey of severe invasive Candida infections in China also showed that the incidence rate of C. glabrata in China was 12.9% [6]. It has been shown that C. glabrata is resistant to most antifungal drugs [7]. However, the involved mechanism is not fully understood.
Macrophages, as the first line of defense against invading pathogens, produce an oxidative stress response against pathogens while upregulating the expression of various proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α), thus improving the ability to kill pathogens. C. albicans survives in macrophages through the formation of hyphae and mycelia that penetrate the cell membrane [8][9][10], which causes significant toxicity to macrophages. C. glabrata cannot form hyphae and has no obvious toxicity to macrophages. Studies have found that C. glabrata does not cause significant cytotoxicity in macrophages within 48 hours but can survive in macrophages for a long time [11], indicating that C. glabrata can adapt to the harsh intracellular environment and evolve strategies for intracellular survival. e antioxidant system of C. glabrata can resist damage and can be used to avoid being killed by phagocytes. e antioxidant system of C. glabrata includes catalase, glutathione peroxidase, superoxide dismutase (SOD), and glutathione, and all of which are effectors of oxidative stress. SOD is very important for many pathogenic bacteria and fungi, such as C. albicans, Histoplasma capsulatum (H. capsulatum), and Cryptococcus neoformans (C. neoformans) [12][13][14]. It also plays an important role in the metabolism of C. glabrata and is related to the integrity of DNA and the prevention of aging [15]. In this study, we aimed to examine the role of macrophages in C. glabrata infection and treatment.

Macrophages.
e macrophage cell line RAW264.7 was a gift from the Department of Immunology of Kunming Medical University. e cells were cultured in a DMEM medium ( ermo Fisher, USA) with 10% FBS (Sangon Biotech, Shanghai, China) in an incubator containing 5% CO 2 at 37°C. For each experiment, macrophages were pretreated with each antifungal drug at less than each MIC value and the plate was then incubated for 24 hours. After this pretreatment, C. glabrata (2 × 10 6 /mL) was added into each culture flask with 1 mL macrophages (2 × 10 6 cells/mL) at an MOI � 1 [17]. e end points for the evaluation of each phagocytosis experiment are indicated in the Result section.

Determination of Cytokines and SOD Activities.
To quantify cytokines, IL-6, IL-8, and TNF-α in supernatants from medium or yeast-stimulated RAW264.1 macrophages were harvested by centrifugation and stored at −80°C until analysis.
Infected macrophages were washed with PBS (phosphate-buffered saline, 1.06 mM KH 2 PO4, 155.17 mM NaCl, 2.97 mM Na 2 HPO 4 -7H 2 O) ( ermo Fisher, USA) to remove extracellular yeast; monolayer cells were examined under a microscope to ensure that they were intact and free of extracellular yeast. Cells were then treated with cold deionized water to breakdown and release C. glabrata cells that were phagocytosed. e amounts of IL-6, IL-8, and TNF-α were measured by specific commercial ELISA kits (Solarbio, Beijing, China) according to the manufacturer's instructions. e activity of SOD was determined by a SOD activity detection kit according to the manufacturer's instructions (Solarbio, Beijing, China).

Statistics.
e results were obtained from at least three independent experiments and subjected to statistical analysis. Data are presented as the mean ± SD and were analyzed by ANOVA or Student's t-test by using Prism 8 software (GraphPad, San Diego, CA). e level of statistical significance was set at P < 0.05.

General Information.
A total of 10 clinical strains tested in this study were from adult patients recently diagnosed at our hospital. Among 10 infected patients, 6 patients were female. Of these, two women suffered from uterine fibroid, one from renal calculus and one from cervical carcinoma.
ree C. glabrata strains in the male group were isolated from patients with jaundice and intestinal obstruction. e source of 10 clinical strains and detailed clinical information are shown in Table 1.

Drug Sensitivity of Clinical Strains.
To determine the optimal concentration of each antifungal agent to examine macrophage activity, the susceptibility of 11 experimental strains to MCF, ICZ, and AmB was measured according the M27-A2 protocol of NCCLS and is shown in Table 2. Compared with the MICs of ATCC2001 for AmB (0.5 mg/L) and ICZ (0.125 mg/L), the MICs of only one strain (16K1128) for AmB and two strains (16K1132 and 17K1173) for ICZ were 4 times higher. We found that the MIC values of the four strains were at least 4-fold higher: 17K1150 (4fold), 17K1152 (8-fold), 16K1128 (15-fold), and 17K1173 (31-fold).

Effects of Antifungal Agents on SOD Activity in C. glabrata
after Phagocytosis. C. glabrata tends to "hide" or "escape" from killing by phagocytes via the upregulation of the antioxidant system. SOD is at the front line of this antioxidant mechanism, and it has been adopted by many pathogenic bacteria and fungi, including C. albicans, Histoplasma capsulatum (H. capsulatum), and Cryptococcus neoformans (C. neoformans). In this study, we found that the SOD activity in C. glabrata phagocytosed by macrophages gradually increased after 1 hour and peaked at 6 hours after infection. As shown in Figure 1, compared with the SOD level of nonphagocytized C. glabrata (0 h), the SOD level significantly increased at 1 hour after infection (P < 0.05). SOD levels in C. glabrata cells in macrophages, with or without antifungal treatment, began to decrease at 12 hours after infection. e mean SOD level of fungus-infected macrophages treated with MCF was lower than that of untreated fungus-infected macrophages, especially at 6 and 12 hours after infection (P < 0.05). However, there was no significant difference in SOD levels between untreated and fungus-infected macrophages pretreated with ICZ or AmB (P < 0.05).

Cytokines Secreted by C. glabrata-Infected Macrophages.
e production of IL-6, IL-8, and TNF-α can be induced by C. glabrata infection [18,19]. However, the levels of cytokines secreted by macrophages after C. glabrata infection were significantly lower than those after C. albicans infection [20]. In this study, we found that IL-6 increased 5.5-8.4 times within 2-28 hours macrophage infection by C. glabrata, as shown in Figure 2 Similar to SOD activity, at any point in time, there was no significant difference in IL-6 and IL-8 secretion between each antifungal-treated macrophages and untreated macrophages or among the drug pretreatment groups after fungal infection (P > 0.05). However, MCF-treated macrophages showed a slight decrease in TNF-α levels within 4-28 hours after infection but were still higher than those in untreated noninfected macrophages (t � 20.611, P < 0.05).

Discussion
e innate immune system plays an important role in defending against pathogens, such as the opportunistic pathogen C. glabrata. Infections caused by C. glabrata have become common and cause high morbidity and high mortality. However, there have been fewer studies on its pathogenesis compared with that of C. albicans [21,22]. Resisting macrophage killing is the first step employed by C. albicans to evade the immune system and cause disseminated infection. Cytokines secreted by macrophages recruit other types of immune cells to the infection site [11]. For example, the mycelium of C. albicans binds to tool-like receptor 2 (TLR2) on monocytes, thereby increasing the level of the anti-inflammatory cytokine IL-10 during infection [23]. Unlike C. albicans, blood infections caused by C. glabrata can persist for a long time in mice without  causing high levels of inflammation, even in immunocompetent mice; C. glabrata cannot be eliminated after a few weeks [24]. Our data showed that the levels of IL-6, IL-8, and TNF-α secreted by macrophages significantly increased after infection with C. glabrata, although they were lower than those reported for C. albicans infection, which is in consistence to report in previous work [20]. e effects of antimicrobial agents on mammalian cells, especially those of the host innate immune response, have recently been noted, as patients with severe disease may depend on the complex interactions between antibiotics and their activity on pathogens and host cells [25,26]. In this study, we selected three antifungal drugs to examine their possible side effects on macrophage function. ese three antifungals target different components of fungi. ICZ and AmB target the fungal cell membrane, and MCF inhibits β-1,3-glucan synthesis for the cell wall [27]. We used a concentration of half the MIC value of each drug to examine its possible effects on cytokine secretion and fungal SOD activity; the results showed that there was no change in the levels of IL-6 and IL-8 in antifungal-treated macrophages within 28 hours after fungal infection. In contrast, TNF-α decreased slightly in macrophages treated with MCF. It was reported that cytokines such as IL-8 and TNF-α are released from human monocytes after exposure to β-1,3-glucan by C. albicans infection [28]. e reason for the decrease in TNF-α may be that β-1,3-glucan is insufficient due to the effect of the echinocandin on β-1,3-glucan synthesis or fungicidal effects.
is hypothesis seems to differ from previous observations, which indicated that C. glabrata cells in replicative senescence in macrophages thickened their cell walls when treated with antifungal drugs [29]. e other mechanism may be mediated by hypoxia-induced β-glucan masking which is dependent on mitochondrial signaling and the cAMP-protein kinase pathway [30]. Since the levels of IL-8 and IL-6 in macrophages treated with MCF did not change, the possibility of decreasing secondary levels of proinflammatory cytokines secreted by macrophages induced by overexposure to β-1,3-glucan [31][32][33] is not yet clear.
Contrary to reports of the antioxidative activity of other fungi induced by ICZ or AmB [34,35], we found that the low concentration of these two antifungal agents had no effect on the SOD activity of phagocytized C. glabrata. Similar to the TNF-α response to MCF-treated macrophages, the SOD activity of phagocytized C. glabrata was reduced when macrophages were pretreated with MCF. Here, the unidentified macrophage effector induced by the echinocandin or the killing effect of the echinocandin is responsible for the decrease in SOD. However, this observation highlights that echinocandins may have antifungal effects by simultaneously inhibiting cell wall formation and reducing antioxidants of C. glabrata.
ese results support our hypothesis that echinocandins may directly affect the activity of human cells related to innate immunity. In fact, the effect of echinocandins on cytokines secreted by macrophages is related to the function of the endoplasmic reticulum [36]. However, we still lack adequate evidence to conclude that β-1,3-glucan from C. glabrata is associated with TNF-α production by 1 2 2 4 * * * * * * * * * * * Figure 1: SOD activity of C. glabrata in macrophages pretreated with three antifungal drugs. We measured the SOD activity of macrophages treated with MCF, ICZ, and AmB. e control group was not pretreated with antifungal agents. e SOD level after 1 hour was significantly increased compared with the SOD level of nonphagocytized C. glabrata (0 hour) ( * , P < 0.05). e mean SOD level of fungus-infected macrophages treated with MCF was lower than that of untreated fungus-infected macrophages, especially 6-12 hours after infection ( * * , P < 0.05). ere was no significant difference in SOD levels between the untreated and ICZ-or AmB-treated fungus-infected macrophage groups ( * * * , P > 0.05). 4 Canadian Journal of Infectious Diseases and Medical Microbiology macrophages. In addition, this study had several limitations. First, this study used only one dose of antifungal therapy. We evaluated the cytokines secreted by C. glabrata-infected macrophages, and the goal was to compare the difference between treatment with different antifungal drugs and untreated macrophages after treatment with only one concentration (1/2 MIC) of each drug for 24 hours. Small changes in cytokines between cells administered antifungal treatment and no treatment may have resulted from insufficient doses of each drug treatment. Second, the number of C. glabrata cells was not evaluated during the study. We observed that although the total content of antioxidant SOD increased at 6 hours after antifungal treatment, MCF caused a slight decrease in the SOD activity in phagocytized C. glabrata cells. Compared with the other two antifungal drugs and no treatment, the fungicidal effect of MCF may lead to a decrease in C. glabrata cells in macrophages. ird, the amount of fungal sample used in our study was very small. Further studies are required to verify the current results with a larger sample size. e persistent presence of C. glabrata and the weak inflammatory response in tissues remain one of the biggest challenges in the treatment of systemic fungal infections. As we observed in this study, all clinical specimens were isolated from pus or peritoneal drainage, and none of them were drawn from the blood, again indicating the chronic progression of C. glabrata. In the course of fungal infection, small differences in cytokines from macrophages treated with different antifungal drugs and untreated macrophages confirmed the safety of the three antifungal drugs on the immune response against C. glabrata. Our results also indicate that echinocandins tend to kill fungi via cell wall exposure and interfering with the response of host macrophages.

Summary
e survival and proliferation of C. glabrata in macrophages is an important factor in systemic infection, which depends on the ability of these fungi to respond effectively to the stressful environment and avoid immune defenses. It has long been recognized that C. glabrata's intrinsic resistance [37,38] to oxidative stress occurs through the high expression of detoxifying enzymes (catalase and superoxide dismutase) during infection [39]. Additionally, resistance to antifungals, including high-value antifungal agents such as echinocandins, has emerged in C. glabrata [40,41]. Current research is still focused on the interactions between organisms and antibacterial agents, and little is known about the immune effects of antifungals during infection. To understand the effect of antifungal drugs on C. glabratainfected macrophages, we pretreated macrophage cell lines with an azole, echinocandin, and AmB for 24 hours before C. glabrata infection. We found out that macrophages pretreatment with ICZ and AmB had no effect on cytokine production by macrophages or antioxidant SOD activity in phagocytized C. glabrata. However, cytokines and the activity of SOD decreased slightly after macrophages were pretreated with MCF.
is study suggests that there is a complex response to antifungal drugs in fungal cells and host defense mechanisms.

Data Availability
e data used to support the findings of this study are included within the article.
Ethical Approval e authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. is study was approved by the review board of First Affiliated Hospital of Kunming Medical University. All experiments were performed following the approved guidelines and regulations and were in accordance with the principles of the Declaration of Helsinki. e experimental protocols were approved by the institutional review board of Kunming Medical University. Patient samples were collected according to protocols approved by the respective institutional review boards.

Consent
Study participants gave written informed consent in accordance with the Declaration of Helsinki.

Conflicts of Interest
e authors declare that they have no conflicts of interest.