The human immune system is inextricably linked to metabolic systems. These two systems share common regulatory mechanisms that allow for coordination and extensive bidirectional communication (Odegaard and Chawla, 2013). Increased evidence supports the notion that metabolic reprogramming in immune cells is required for the response to microbial infections (Arts et al., 2016; Lachmandas et al., 2016; O’neill and Pearce, 2016; Domínguez-Andrés and Arts, 2017). Candida albicans is an opportunistic pathogen, which represents a severe threat to human life in immunocompromised individuals. Especially when C. albicans causes invasive fungal diseases, morbidity and mortality may exceed 50% even with antifungal therapy (Schultz et al., 2020).

The innate immune system is typically the first-line defense against C. albicans infection, and monocytes play a pivotal role in the process of antifungal immunity. (Qin et al., 2016; Grondman et al., 2019). Monocyte deficiency is associated with higher susceptibility to fungal infections in both mice and humans (Ngo et al., 2014; Ceesay et al., 2015; Domínguez-Andrés and Arts, 2017). When monocytes are stimulated and activated by pathogen-associated molecular patterns (PAMPs), glucose metabolism associated with ATP production is altered from aerobic oxidation to glycolysis (Palmer et al., 2016). While ensuring the energy supply for basic cellular processes, aerobic glycolysis fuels the production of nucleic acids and lipids required for the proliferation and differentiation of monocytes during the immune response (Raulien et al., 2017). However, not all the metabolic changes promote adequate immune responses. Previous studies have indicated that metabolic reprogramming after immune stimulation may be a major mechanism driving immune cell tolerance (Cheng et al., 2016; Kan and Michalski, 2018; Zhu et al., 2019). Moreover, the loss of metabolic plasticity affects the antifungal immune functions of monocytes (Grondman et al., 2019). Therefore, monocyte metabolic remodeling acts as a “double-edged sword” in regulation of the innate immune function.

In order to understand the role of cellular metabolism in the innate immune function of monocytes, human primary monocytes were stimulated with heat-killed C. albicans and proteomic and metabolomic analyses were performed. We found that stimulation of monocytes by C. albicans was accompanied by upregulation of glucose transporter 3 (GLUT3) and activation of the glycerophospholipid metabolism pathway. Upregulated GLUT3 expression has been preliminarily confirmed in monocytes from patients with C. albicans bloodstream infection (BSI) and GLUT3 knockdown by siRNA significantly reduced production of IL-1β in THP-1 cells. In addition, the glycerophospholipid metabolites could promote the localization and glucose uptake function of GLUT3 (Hresko et al., 2016). Our findings support the importance of GLUT3 in the complex network of glycerophospholipid metabolism and the innate immune responses against C. albicans.

C. albicans is the most common opportunistic fungal pathogen that causes a variety of human diseases, ranging from potential oral infections to life-threatening candidemia (Hasim et al., 2018). The development of immunotherapeutic approaches to boost host defense is urgently needed to reduce the mortality of C. albicans BSI (Pfaller and Diekema, 2007). Although increased evidence supports the interconnection between immunometabolism and fungal infections, we are just beginning to understand the metabolic reprogramming associated with the monocyte immune response and its impact on host defense against fungal infections (Tucey et al., 2018; Weerasinghe and Traven, 2020). Here, we describe the changes in proteins and metabolites in human monocytes after stimulation with heat-inactivated C. albicans. Briefly, we observed that monocytes stimulated with C. albicans for 24 h exhibited more significant changes in protein expression and metabolic activity than those stimulated for 12 h. Among these differentially expressed proteins in monocytes, the upregulation of GLUT3 may promote glucose transport and provide the energy needed for the innate immune response. Meanwhile, the structural stability and biological function of GLUT3 requires the participation of various glycerophospholipid metabolites, and concomitant with the activation of glycerophospholipid metabolism. Therefore, we speculate that GLUT3 is a critical intersection that participates in the innate immune response and glycerophospholipid metabolism.

Cellular glucose uptake is mediated by a family of GLUTs, which participate in immune and inflammatory responses and are critical for host defense against pathogens (Lang et al., 2020). GLUT3 is expressed and translocated to the cell membrane in response to a substantial increase in the energy demand associated with cell activation (Fu et al., 2004; Humphrey et al., 2004; Jones et al., 2006; Simpson et al., 2008). During infection, increased GLUT3 expression may help to redistribute glucose as a potential source of energy away from peripheral tissues and toward cells that mediate the immune response, and it is also essential for cell survival (Maratou et al., 2007). In addition, the differentiation of THP-1 monocytes into macrophages was associated with a significant induction of GLUT3 (Fu et al., 2004). GLUT3 is essential for monocytes to absorb energy for immune response function. However, few studies have focused on the relationship between GLUT3 expression and metabolic transformation in monocytes, especially glycerophospholipid metabolism.

Monocytes change their metabolic phenotype from oxidative to glycolytic metabolism when activated in humans (Palmer et al., 2016). However, based on our findings, there was no obvious activation of the pathways associated with glucose metabolism. Instead, the glycerophospholipid metabolism pathway, particularly the GPC pathway, was activated. Here, we briefly describe the basic features of these metabolites in the GPC pathway ( Figure 6B ). PtdCho is the most abundant phospholipid in mammalian cell membranes (Mountford and Wright, 1988; Sonkar et al., 2019). The first step in the GPC catabolic pathway is the hydrolysis of PtdCho to remove one fatty acid and produce 1-acyl GPC, which is subsequently converted to GPC by removing the second fatty acid, which is then converted to free choline (Cho) (Morash et al., 1988). In the synthesis pathway, free Cho is phosphorylated to phosphocholine (PC) and then converted to cytidinediphosphate (CDP)-choline to produce PtdCho, thus completing the GPC cycle (Sonkar et al., 2019).

The GPC metabolic pathway interacts with multiple biochemical pathways (Sonkar et al., 2019). PGAP is not only involved in glycerophosphate metabolism, but also is an important metabolite in glycolysis. Decreased expression of PGAP indicates that glycerol-3-phosphate (GA-3-P) tends to produce glycerol-3-phosphate (Gro-3-P) via dihydroxyacetone phosphate (DHAP), which promotes the synthesis of PS, PE, and GPC. As a product of fructose-1,6-bisphosphate (Fru-1,6-BP) in glycolysis, Gro-3-P is converted to phosphatidate (PA), which reversibly gets converted to diacylglycerol (DAG) or triacylglyceride (TAG), and then promotes the formation of lipid droplets. The DAG formed in this pathway was subsequently incorporated into the de novo synthesis of PtdCho (Sonkar et al., 2019). In addition, PEA participates in the pyruvate metabolism pathway and in the synthesis of PE and GPC. However, PEA and PGAP are indirectly involved in the synthesis of PS and PE, respectively, resulting in a decrease in their abundance ( Figure 6B ).

Previous studies have demonstrated that PE promoted GLUT3 activation in monocytes, and PS was found to be essential for GLUT3 function by activating and stabilizing its structure (Hresko et al., 2016). In this study, we found that PE and PS increased in monocytes after stimulation by heat-inactivated C. albicans, and both PE and PS were involved in the synthesis of GPC, promoting GPC cycling. These results suggest that PE and PS participate in the activation of GLUT3 via the GPC pathway, which contributes to the expression of GLUT3, localized at the cell membrane, thus facilitating immune response function. This prompted us to investigate the role of GLUT3 in the antifungal effector functions of monocytes against C. albicians infection. We found that downregulated GLUT3 significantly reduced the secretion of IL-1β in THP-1 cells stimulated with C. albicans. Pro-inflammatory cytokines IL-1β have been identified as integral components of human monocytes anti-fungal immune defenses (Ganesan et al., 2014; Gaidt et al., 2016).

The metabolic changes that we observed may correlate with the innate immune response after C. albicans stimulation. The downregulation proteins, such as ITGAM/CD11B, ITGB2/CD18, and CD14, involved in the innate immune pathway of monocytes, participate in the Toll-like receptor 4 (TLR4) signaling pathway. TLR4 is one of the receptors involved in the recognition of C. albicans and promotes the innate immune response (Ferwerda et al., 2008). TLR4 has been shown to be involved in proinflammatory responses in monocytes, macrophages, and dendritic cells, specifically, these immune cells combat C. albicans and require the release of cytokines that tend to rely on TLR4 pathway (Blasi et al., 2005; Qin et al., 2016; Wang et al., 2019). CD11B/CD18 (Mac-1/Complement Receptor 3, CR3) is an integrin molecule that is expressed on the surface of monocytes and plays an important role in their trafficking ability (Panni and Herndon, 2019). CD11B can modulate a variety of biological processes in innate immune cells, including the generation of type I interferon and proinflammatory cytokines (Khan et al., 2018). Notably, CD11b has also been implicated in the recognition of fungal β-glucan, which is a key PAMP located at the surface of C. albicans (Ballou et al., 2016). However, the downregulation of the inflammatory proteases caspase-8 (CASP8) levels may contribute to a reduction in the receptor-mediated apoptosis process (Mandal et al., 2020). A strong requirement for CASP8 and CR3 was also found for NLRP3 dependent IL-1β production in response to heat killed C. albicans (Ganesan et al., 2014).

Among the upregulated proteins that function in the innate immune response ( Figure S1 in the Supplementary Materials ), ISG15 is known as an immunomodulator that controls fungal infection and triggers the expression of proinflammatory cytokines (Dong et al., 2017; Liu and Lee, 2021). RAP2C is a member of the Ras GTPase superfamily, which acts as a molecular switch to regulate cellular movement, proliferation, differentiation, and apoptosis (Wang et al., 2020; Zhu et al., 2020). KCNAB2 is a voltage-gated potassium channel subunit beta-2 that promotes expression of the pore-forming alpha subunits at the cell membrane and plays an important role in the regulation of membrane potential, therefore, it is important for the absorption of glucose, amino acids, and long-chain fatty acids (Mcdaniel et al., 2001; Wongdee et al., 2009). Both KCNAB2 and GLUT3 are membrane channel proteins that are important for glucose absorption. These results indicate that elevated levels of ISG15, RAP2C, and KCNAB2 are beneficial to the immune response to resist C. albicans infection, however, whether these proteins are related to the metabolites of glycerophospholipids requires further study.

Of note, the GPC pathway was activated gradually over 24 h. Previous research based on transcriptomic analysis has confirmed that the expression levels of several main enzymes involved in the glycolysis pathways were significantly upregulated, but this enhancement occurred after stimulation with heat-inactivated C. albicans for 24 h (Domínguez-Andrés and Arts, 2017). In our study, the protein abundance of GLUT3 also increased significantly after 24 h of stimulation. Monocyte activation and immune responses are processes that require high energy consumption and the metabolic transformation of monocytes needs to go through an interim stage (Palmer et al., 2016). To allow cells to adapt to these changes, an energy deficiency period is necessary to reduce energy consumption and ensure cell survival, resulting in dampening of the innate immune response. The expression and localization of GLUT3 could provide a channel for increased glucose uptake by monocytes and mobilize the metabolism of glycerophospholipids as a part of the signal transduction pathway.

There is a complex regulatory network between the innate immune system and cell metabolism. Loss of cellular energy homeostasis may occur during the process of monocyte metabolic remodeling, eventually accounting for reduced antifungal immune response. Our findings suggest that GLUT3, as an intersection of glycerophospholipid metabolism and innate immunity, may promote energy absorption and restore the functional immune response. Clarifying the immunometabolic mechanism of the monocyte response to C. albicans infections is of great significance for exploring novel potential therapeutic targets.

The data presented in the study is deposited in ProteomeXchange, accession number: PXD024515.

The studies involving human participants were reviewed and approved by Ethics Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (JS-2782). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

XW conceived and designed the experiment. GZ and W-HY contributed reagents/materials/analysis tools. XW and J-TC performed the experiments. XW analyzed the data and wrote the manuscript. Y-CX, MX, and LZ revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (Grant No. 81971979, 81802049) and Beijing Key Clinical Specialty for Laboratory Medicine-Excellent Project (No. ZK201000).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.