A new breakthrough study by researchers from the Zhongnan Hospital of Wuhan University-China and Southern University of Science and Technology, Shenzhen-China has found that glutamine and pyruvate supplementation, and also the drug diabetic drug Rosiglitazone can reduce and prevent cytokine upregulation hyperinflammation as result of inflammation in macrophages induced by the SARS-CoV-2 virus, hence preventing disease severity and risk of mortality.
The study findings were published in the peer reviewed journal: iScience.
In various infectious diseases, cellular metabolism is intimately connected to the mechanisms of disease pathogenesis and the resulting pathology, as well as the host defense response. Although several recent clinical studies have suggested an association between plasma metabolites alteration and COVID-19 severity(Lam et al., 2021; Shen et al., 2020b; Song et al., 2020a; Su et al., 2020; Xiao et al., 2021), however, the immunometabolic regulation of leukocytes in lungs and its correlation with immune function and disease severity are currently unclear. In the present study, we depicted the immunometabolic landscape in lungs of COVID-19 patients.
Our data suggested that improper metabolic reprograming of macrophages exacerbated the imbalanced immune response such as hyperinflammatory and reduced endocytosis function.
These findings shed light on treating COVID-19 by immunometabolic modulation.
Metabolism is a key regulator of immune cell phenotype and function, but it remains difficult to investigate the metabolic status of individual cells in humans. Gene expression has been used as one of the indirect means to evaluate metabolic activity(Lee et al., 2012).
Recent developed computational algorithms allow for detection of metabolic pathway activity and metabolic flux based on metabolic gene expression using single-cell transcriptomic approach(Damiani et al., 2019; Xiao et al., 2019).
Here we took advantage of the integrated single-cell metabolic profiling platforms and depicted a global picture of immunometabolic profile in lungs of COVID-19 patients with different disease severity. Our data suggested that SARS-CoV-2 infection increased glycolysis but decreased most other core metabolic processes in BALF macrophages from severe COVID-19, which correlates with elevated inflammation and reduced antigen presentation and endocytosis functions.
Our study thus established the foundation to study metabolic state and functional phenotype of individual immune cells in clinical samples from COVID-19 or other diseases.
Previous studies have reported that SARS-CoV-2 infection can induce glycolytic reprogramming of isolated monocytes in vitro(Bhatt et al., 2022; Codo et al., 2020; Cory et al., 2021). Our research uncovered dramatic metabolic shift in BALF macrophages from mild to severe COVID-19 patients.
However, analysis using paired PBMCs indicated minor changes of metabolic activities of peripheral blood monocytes between mild and severe COVID-19 patients. This may be caused by the lack of direct exposure to SARS-CoV-2 virus of the circulating monocytes in vivo. In lung tissue, where direct exposure of macrophages to SARS-CoV-2 occurs,
we indeed found the increased level of glycolysis pathways from both mild and severe COVID-19 patients. Interestingly, the citrate cycle, oxidative phosphorylation and fatty acid degradation were inhibited in macrophages from severe COVID-19 patients compared to patients with mild diseases.
The much higher levels of SARS-CoV-2 particles in lungs of severe patients may lead to the glycolytic reprogramming but inhibition of mitochondrial oxidative metabolism of macrophages.
This was consistent with reports showing that SARS-CoV-2 infection suppresses OXPHOS and alters mitochondrial function in monocytes and cell lines in vitro(Codo et al., 2020; Cortese et al., 2020; Wang et al., 2020).
These results highlight the importance to study the metabolic and immunological events in SARS-CoV-2 infected lungs to further understand the mechanisms of immune dysfunction and immunopathogenesis.
Based on our single-cell metabolism study, we validated in vitro that glutamine and pyruvate supplementation or glycolysis inhibition by 2-DG reduced the levels of proinflammatory cytokines and chemokines in human primary macrophages stimulated with SARS-CoV-2 or TLR7/8 agonist.
These results were consistent with the findings that pro-inflammatory macrophages undergo metabolic reprogramming toward aerobic glycolysis(Galvan-Pena and O’Neill, 2014).
We used 2- DG as a model to inhibit glycolysis. High dose of 2-DG (10mM) was reported to suppress both glycolysis and mitochondrial metabolism, while low dose of 2-DG (< 1.25mM) slightly increased oxidative phosphorylation in bone marrow derived macrophages(Wang et al., 2018), indicating the dose-dependent effect of 2-DG.
We found here that low dose of 2-DG (1mM) can significantly suppress proinflammatory cytokines production by macrophages stimulated with either SARS- CoV-2 or R848. Consistently, 5mM of 2-DG treatment reduced SARS-CoV-2-induced proinflammatory cytokines by monocytes in vitro(Codo et al., 2020).
Amino-acid starvation also resulted in defective anti-inflammatory polarization in macrophages(Kimura et al., 2016). Considering that hospitalized COVID-19 patients presented malnutrition(Clemente-Suarez et al., 2021) and patients with severe COVID-19 exhibited a sharp drop in blood nutrients such as amino acid(Su et al., 2020), we proposed that amino acid supplementation in patients with severe COVID-19 might alleviate inflammatory cytokines production by macrophages.
Lipid metabolism plays a critical role in the differentiation and function of macrophage(Chawla et al., 2001). It was reported that anti-inflammatory macrophages trigger a metabolic program of increased oxidative phosphorylation and fatty acid oxidation(Galvan-Pena and O’Neill, 2014). Our single-cell metabolism analysis showed that reduced lipid metabolism in macrophages from bronchoalveolar of severe COVID-19.
The nuclear receptor PPARγ is known to regulate lipid metabolism in many tissues and it’s the target of anti-diabetic thiazolidinedione drugs(Ahmadian et al., 2013). In macrophages, PPARγ has been shown to play important roles in inflammation and metabolism(Hevener et al., 2007; Odegaard et al., 2007). PPARγ signaling controls lipid uptake(Chawla et al., 2001; Odegaard et al., 2007) and intracellular metabolism(Odegaard et al., 2007).
Impairment in PPARγ expression and lipid metabolism have been shown to be associated with blocked anti-inflammatory macrophage polarization(Kang et al., 2018). We found that expression of PPARγ was inhibited in macrophages from patients with severe COVID-19 as reported(Desterke et al., 2020), or in human primary macrophages stimulated with SARS-CoV-2 in vitro.
Most importantly, we proved that activating of PPARγ signaling in vitro by rosiglitazone, an anti-diabetic drug, enhanced lipid metabolism and phagocytosis functions of macrophages, and reduced proinflammatory cytokines and chemokines production in human primary macrophages stimulated with SARS-CoV-2 or TLR7/8 agonist.
The results indicated that rosiglitazone may reduce hyperinflammation in macrophages by regulating lipid metabolism. PPARγ was important for alveolar macrophages development and funciton (Schneider et al., 2014). It was reported that PPARγ in alveolar macrophages limits inflammation and promotes tissue recovery after respiratory viral infection (Huang et al., 2019).
Depletion and altered antigen presentation of alveolar macrophages was also reported to correlate with the severe COVID-19 (Chen et al., 2022). It will be interesting to explore the role of PPARγ in alveolar macrophages function in the context of COVID-19 in the future.
Patients with insulin resistance usually have enhanced inflammation such as increased levels of IL-6 and TNF-α(Esser et al., 2014), and other symptoms that reduced their ability to respond properly to the infection with SARS-CoV-2. Most recently, a large COVID-19 related population study showed that patients with type 2 diabetes prescribed with glucose lowering drugs including metformin, sodium-dependent glucose transporters 2 inhibitors, and sulfonylureas had a lower risk of COVID-19-related mortality(Khunti et al., 2021).
Although the study indicated that pre- infection prescription for thiazolidinediones had minor effect on reducing the risk of COVID-19- related mortality in people with type 2 diabetes, it will still be interesting to explore the anti-inflammatory properties of rosiglitazone in severe COVID-19 patients with or without diabetes. 2- DG which targeting glycolysis has also been approved for phase II and phase III clinical trials in India(Huang et al., 2022).
Other report suggested that targeting metabolism by arginine, epacadostat (a potent and selective indoleamine 2,3-dioxygenase inhibitor), mycophenolic acid (an inosine monophosphate dehydrogenase inhibitor) reduced cytokine release in PBMCs of infected monkeys in vitro(Xiao et al., 2021). Taken together, our results and published data further support drug repurposing for targeting immunometabolism for COVID-19 treatment.
In summary, we characterized the immunometabolic landscape in lungs of COVID-19 patients and suggested that regulating metabolism by 2-DG and rosiglitazone might reduce cytokine release syndrome in patients with severe COVID-19. Our study has identified immunometabolism dysregulation as a mechanism for COVID-19 pathogenesis, and further facilitated the development of novel therapeutic for COVID-19.