key: cord-0773930-36oi0j3h
authors: Uehara, Ikuno; Kajita, Mitsuko; Tanimura, Atsuko; Hida, Shigeaki; Onda, Munehiko; Naito, Zenya; Taki, Shinsuke; Tanaka, Nobuyuki
title: 2‐Deoxy‐d‐glucose induces deglycosylation of proinflammatory cytokine receptors and strongly reduces immunological responses in mouse models of inflammation
date: 2022-02-24
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.940
sha: 159b5b15437f2183f769e5136dbe08060bb797d2
doc_id: 773930
cord_uid: 36oi0j3h

Anti‐proinflammatory cytokine therapies against interleukin (IL)‐6, tumor necrosis factor (TNF)‐α, and IL‐1 are major advancements in treating inflammatory diseases, especially rheumatoid arthritis. Such therapies are mainly performed by injection of antibodies against cytokines or cytokine receptors. We initially found that the glycolytic inhibitor 2‐deoxy‐d‐glucose (2‐DG), a simple monosaccharide, attenuated cellular responses to IL‐6 by inhibiting N‐linked glycosylation of the IL‐6 receptor gp130. Aglycoforms of gp130 did not bind to IL‐6 or activate downstream intracellular signals that included Janus kinases. 2‐DG completely inhibited dextran sodium sulfate‐induced colitis, a mouse model for inflammatory bowel disease, and alleviated laminarin‐induced arthritis in the SKG mouse, an experimental model for human rheumatoid arthritis. These diseases have been shown to be partially dependent on IL‐6. We also found that 2‐DG inhibited signals for other proinflammatory cytokines such as TNF‐α, IL‐1β, and interferon ‐γ, and accordingly, prevented death by another inflammatory disease, lipopolysaccharide (LPS) shock. Furthermore, 2‐DG prevented LPS shock, a model for a cytokine storm, and LPS‐induced pulmonary inflammation, a model for acute respiratory distress syndrome of coronavirus disease 2019 (COVID‐19). These results suggest that targeted therapies that inhibit cytokine receptor glycosylation are effective for treatment of various inflammatory diseases.

Inflammation is an adaptive response caused by harmful stimuli and conditions such as infection and tissue injury. Moreover, it has been suggested that chronic systemic inflammation, which causes various diseases, is caused by homeostatic imbalances in the physiological system. 1 The inflammatory response is orchestrated by proinflammatory cytokines such as interleukins (IL)-6 and IL-1β, and tumor necrosis factor (TNF)α. 2 Proinflammatory cytokines are multifunctional proteins that regulate cell death in inflammatory tissues, alter vascular endothelial permeability, recruit immune cells, and induce acute phase protein production. During the inflammatory process, tissue-resident and recruited macrophages are activated and secrete various types of chemokines and cytokines to trigger both innate and adaptive immune responses. 3 Cytokine signalling is controlled by multiple regulatory checkpoints that include feedback inhibition, which allows tissues to return to an immunological quiescent state.

However, dysregulated production of proinflammatory cytokines and their signalling molecules can be detrimental, which causes various human diseases. 4 The inflammatory response is coordinately regulated by proinflammatory cytokines such as IL-6, IL-1, and TNFα. 2 Upregulation of these cytokines has been observed in various chronic inflammatory and autoimmune disorders, and antibody-based therapy against these cytokines have been used to effectively treat various inflammatory diseases. 5 For example, targeting the IL-6 pathway by an anti-IL-6 antibody, anti-IL-6 receptor antibody, or the soluble form of IL-6 receptor gp130 has become an effective treatment for various rheumatic diseases, Castleman disease, and cytokine release syndrome, and it is partially effective for treatment of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis. 6 Blocking of TNFα or TNF receptor (TNFR) efficiently prevents the progression of rheumatoid arthritis, psoriasis, ankylosing spondylitis, and IBD. 7 In IBD, many studies have demonstrated that TNF-targeted therapies inhibit the activation and proliferation of pathological T cells, reduce inflammation, and support restoration of intestinal mucosa. 8 Blocking IL-1 is also effective to treat rheumatic diseases and highly coexisting inflammatory diseases such as cardiovascular disease and type 2 diabetes. 9 Despite such effectiveness, cytokine-targeted therapies can have detrimental effects. 6, 7 These effects suggest that the imbalance caused by the suppression of a single cytokine signal in the orchestrated control process by multiple cytokines may lead to adverse effects in the immune system. 19) . Some patients develop acute respiratory distress syndrome (ARDS) with progressive respiratory failure due to pulmonary edema caused by cytokine storms. 12 The terms cytokine storm and cytokine release syndrome were originally used to describe acute graft-versus-host disease following allogeneic hematopoietic cell transplantation. Recently, these terms have also been applied to a similar syndrome following chimeric antigen receptor T-cell therapy. 13 Cytokine storms are life-threatening systemic inflammatory syndromes characterized by elevated circulating cytokine levels and hyperactivation of immune cells. These storms can be triggered by pathogens, cancers, autoimmune responses, and various immunomodulatory therapies. Immune hyperactivation occurs after inappropriate triggering of immune responses, massive immune cell activation, increased pathogen burden such as sepsis, and prolonged immune activation. These immune responses induce elevated cytokine production (the cytokine storm), which result in hyperinflammation and multiorgan failure. 13 The levels of proinflammatory cytokines, which include interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)α, and interferon (IFN)γ, are elevated and are considered to play major immunopathological roles in patients with cytokine storms.

Because acute respiratory failure and sepsis induced by cytokine storms are the main causes of mortality among COVID-19 patients, 14 anti-cytokine therapeutic strategies, which include cytokine neutralization and cytokine receptor blockade, have been applied in patients with severe COVID-19. Treatment of COVID-19 patients with the IL-6 inhibitor tocilizumab decreases the risks of intubation and mortality. 15 The utility of antibody drugs that block inflammatory cytokines such as IL-6 is widely acknowledged, and these drugs are administered to patients with various inflammatory diseases such as rheumatoid arthritis. 16 However, in addition to targeting only one cytokine, there are several functional limitations of antibody drugs, which include inadequate pharmacokinetics and tissue accessibility as well as off-target effects on the immune system. 17 Moreover, high production costs limit the widespread use of antibody drugs.

In the present study, we found that the glycolytic inhibitor 2-deoxy-D-glucose (2-DG), a simple monosaccharide, attenuated cellular responses to IL-6 by inhibiting N-linked glycosylation of the IL-6 receptor gp130. 18 2-DG also blocked TNFα, IL-1β, and IFNγ signals, and efficiently alleviated a mouse model of inflammatory bowel disease and human rheumatoid arthritis and prevented death following lipopolysaccharide (LPS) shock, a mouse model of a cytokine storm, 19 and attenuated LPS-induced pulmonary inflammatory responses, a mouse model of ARDS. 20 

DSS with an average molecular weight of 36,000-50,000 Da (MP Biomedicals, Santa Ana, CA, USA) was administered to 8-week-old female C57BL/6 mice at a concentration of 2% (w/v) in drinking water as described previously. 22 2-DG (10 mg/mouse; Sigma-Aldrich) was intraperitoneally (i.p.) injected once a day. Body weight was measured every day. Control mice were treated similarly, but were provided with drinking water without DSS and received i.p. injections of PBS without 2-DG.

Laminarin (30 mg/mouse; Sigma-Aldrich) was injected i.p. once into 8-week-old female SKG mice. The mice were maintained in a specific pathogen-free environment with or without 2-DG in their drinking water. 23 Arthritis scores were determined by weekly inspection in a double-blinded manner and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of the wrist or ankle; and 1.0, severe swelling of the wrist or ankle.

Scores for all fingers and toes, wrists, and ankles were summed for each mouse.

Eight-week-old C57BL/6 mice were injected i.p. with 2-DG (20 mg/mouse) or PBS. Two hours later, LPS (0.8 mg/mouse; Sigma-Aldrich) was injected i.p. Survival rates were monitored for 4 days. Two days after LPS injection, several mice were sacrificed, and their upper lobes of left lungs were prepared for total RNA preparation.

Six-week-old male C57BL/6 mice were randomly divided into five groups (Mock, LPS, LPS+2DG, LPS+mPSL, and LPS+2DG+mPSL; n = 3 mice per group). Mice were injected i.p. with or without 20 mg 2-DG and 100 mg/kg mPSL. Subsequently, the mice were administered intratracheal injections of LPS (10 mg/kg) or physiological saline as described previously. 24 2-DG, mPSL, and PBS i.p. injections were administered every 24 h.

Cells were lysed in lysis buffer (1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 50 mM Tris-HCl, pH 7.5), with 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, and a protease inhibitor mixture (Nacalai Tesque, Kyoto, Japan).

Immunoblotting and immunoprecipitation were performed as described previously. 21

SDS-PAGE was performed as described previously. 21 Oligosaccharide and lectin staining was performed using a GP Sensor Kit (J-Oil Mills) with biotin-labeled ConA (J-Oil Mills) or wheat germ agglutinin (J-Oil Mills) following the manufacturer's standard protocols.

Cells were lysed in the lysis buffer described above except at pH 8.6.

Treatment with recombinant GP-F (TaKaRa Bio) was performed for 16 h following the manufacturer's protocol. 

In the DSS-induced colitis model, mice were sacrificed after drinking water that contained DSS for 7 days. Their colons were fixed in 

Total RNA was extracted from cultured cells using a Nucleospin 

The pStat3 Luc plasmid was obtained from SABiosciences (Frederick, MD, USA). Control plasmids phRL-TK (renilla luciferase reporter) and pNF-kB Luc have been described previously. 21

Peripheral blood samples from mice were collected in tubes and allowed to clot for 2 h at room temperature. Whole blood samples were centrifuged at 2000 × g at 4°C to obtain sera. A Quantikine mouse IL-6 kit and Quantikine mouse TNFα kit (R&D Systems) were used to quantitate serum cytokine levels in accordance with the manufacturer's protocols.

The lung wet/dry weight ratio was determined to assess pulmonary edema as described previously. 24 The posterior lobe of the right lung was collected, and its net weight was recorded. The lung was then heated for 72 h at 65°C and weighed to determine its dry weight.

Data were expressed as the mean ±standard deviation of at least three independent experiments. Statistical analyses of parametric data were performed using GraphPad Prism 7 software (GraphPad Software Inc.). Significant differences between two groups were determined using the unpaired two-tailed t-test. Multiple comparisons were performed using one-way or two-way analysis of variance were considered statistically significant unless otherwise indicated.

The expression profiling microarray data of mouse DSS colitis tissues has been deposited in the GEO database under the accession code GSE16 7598 available in the GEO repository (https://www.ncbi.

nlm.nih.gov/geo/query/ acc.cgi?acc=GSE16 7598). All other data that support the findings of this study are available from the corresponding author upon reasonable request.

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy. org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 25 Figure 1C ) and on western blots ( Figure 1D ) further supported its aglycosylated status. In addition to glycolysis-inhibiting activity, 2-DG reportedly inhibits mannose metabolism and mannose-mediated N-linked glycosylation. 36, 37 Metabolome analyses 38 showed that glucose-6-phosphate and guanosine diphosphate mannose, a substrate for N-linked glycosylation, had accumulated in 2-DG-treated cells ( Figure S2 ), which suggested that 2-DG inhibited glucose phosphoisomerase and mannosyltransferase. 37, 39 As shown in Figure 1E , global patterns of cellular protein glycosylation were not as dramatically affected by 2-DG treatment compared with gp130 glycosylation as shown by oligosaccharide and lectin staining of whole cell lysates. The gp130 protein has an unusually high turnover rate with a half-life of approximately 2 h, 40 and its expression had disappeared within a few hours following treatment with the protein synthesis inhibitor cycloheximide. The kinetics were comparable with those of F I G U R E 2 2-Deoxy-D-glucose (2-DG) alleviates dextran sodium sulfate (DSS)-induced inflammatory bowel disease. (A) Thioglycollate broth was injected intraperitoneally, and 4 days later, 20 mg 2-DG was injected. Following the injections, mouse peritoneal macrophages were prepared at the indicated times. Expression of gp130 was determined by immunoblotting. (B) Time course of body weight loss. C57BL/6 mice (n = 4) were treated with 2% DSS in drinking water with or without 10 mg 2-DG and 20 mg Mannose injections once a day for the indicated periods. Body weight was measured daily. Relative body weight (%) compared with day 0 is plotted with standard errors. Results were analysed using two-way ANOVA followed by Tukey's post hoc test. *p < .05 (DSS vs. DSS+2DG 4days), **p < .01 (DSS+2DG vs. DSS+2DG +Man 7days) and ****p < .0001(DSS vs. DSS+2DG 5,6,7day) were considered statistically significant. Experiments were repeated three times independently with similar results. (C) Histological sections of colon tissues (control, DSS treated for 7 days and DSS+2DG treated for 7 days) following haematoxylin and eosin staining (upper panel, ×180) and anti-F4/80 antibody staining (lower panel, ×180). (D) Quantitative real-time PCR analysis of interleukin (IL)-6, IL-1β, and haptoglobin (HP) mRNAs in colon tissues of control mice and mice treated with DSS, DSS+2-DG, or 2-DG for 7 days. Results were analyzed using one-way ANOVA followed by Tukey's post hoc test. ****p < .0001, **p < .01. Graphs are presented as the mean ±s.d. (n = 3) the molecular weight reduction of gp130 caused by 2-DG treatment ( Figure 1F ). We speculated that proteins with high turnover rates, such as gp130, were selectively affected by 2-DG. Aglycosylated gp130 appeared to be transported efficiently onto the cell surface, because anti-gp130 antibodies stained 2-DG-treated cells almost as strongly as untreated cells ( Figure S3 ). This observation was inconsistent with a previous report suggesting that N-linked glycosylation of gp130 is important for trafficking to the cell surface. 41 The basis for these discrepant observations remains unclear, but amino acid substitutions in the previous study 41 

Next, we examined the effects of 2DG on IL-6 signalling in mice. Figure 2B and Figure S9 ). This led us to speculate that inhibition of IL-6 responses alone could not account for the high therapeutic efficacy and anti-inflammatory activity of 2-DG.

C57BL6 mice were injected with thioglycollate broth, and 4 days later, 15 mg/mouse of 2-DG was administrated by oral gavage. (A) PECs from two mice at each time prepared at indicated periods after 2-DG administration were subjected to gp130 immunoblotting. (B) MCP-1 mRNA from peritoneal cells and livers of C57BL6 mice injected at 1 h before with 0.5 µg IL-6/mouse was subjected to quantitative RT-PCR. Results were analysed using one-way ANOVA followed by Tukey's post hoc test. ****p < .0001, ***p < .001, and **p < .01. (C) Arthritis scores of SKG mice (n=10) under specific pathogen-free conditions following a single intraperitoneal injection of 30 mg laminarin with or without 0.5% 2-DG in drinking water. Results were analysed using two-way ANOVA followed by Sidak's post hoc test. 

We further examined the effect of orally administered 2-DG on gp130 glycosylation and IL-6 responses in vivo. We observed a similar reduction in the molecular weight of gp130 in PECs isolated from mice administered oral 2-DG to that observed in vitro ( Figure 3A) . Moreover, MCP-1 expression at the mRNA level was upregulated to a lesser extent in PECs and in the liver of mice fed 2-DG that received i.p. injection of IL-6 than it was in those of Figure 3B ). The observation that 2-DG was effective when administered orally implies that 2-DG can be easily administered over long periods of time to treat chronic inflammation.

To assess this possibility, we used SKG mice that develop arthritis in a strictly IL-6-dependent manner. IL-1β and TNFα are considered as disease-accelerating factors. 46 ,47 SKG mice are regarded as an ideal model for human rheumatoid arthritis that is currently treated by injection of protein antagonists of TNFα, IL-1β, and IL-6. 28-30 Oral 2-DG was confirmed to be effective in reducing IL-6 responses in SKG mice in vivo ( Figure S10 ). SKG mice developed arthritis at approximately 5 weeks after injection of laminarin, a β-glucan known to trigger arthritogenic immune responses in these mice ( Figure 3C-E) . 46, 47 Arthritis severity, as represented by arthritis scores and swollen joints, was clearly reduced in animals administered oral 2-DG compared with that in untreated mice.

Accordingly, subsynovial infiltration of macrophages, plasma cells, and lymphocytes was far less prominent in 2-DG-treated mice than in control mice ( Figure 3F and Figure S11 ). The expression of laminarin-induced inflammatory markers haptoglobin (HP) 48 and serum amyloid A (SAA) 49 was suppressed in the liver following oral 2-DG administration ( Figure 3G ). The body weight increase was slower in control SKG mice, likely because of laminarin-induced inflammation. Notably, SKG mice treated with 1% (w/v) 2-DG in drinking water showed consistent increases in body weight over 10 weeks (Figure 3C , right) and no drastic changes in laboratory blood results ( Figure S12 ), which suggested that oral 2-DG was well-tolerated at this dose. The therapeutic efficacy of 2-DG was elevated as doses were increased up to 1%, but no increased efficacy was observed at a dose of 1.5% ( Figure S13 ). At concentrations higher than 2%, water consumption was decreased, making it difficult to achieve a higher dose treatment.

In inflammatory diseases, release of inflammatory cytokines leads to activation of immune cells and the production and release of additional cytokines. 50 Therefore, the in vivo therapeutic efficacy of 2-DG for inflammatory bowel disease and arthritis may also involve inhibition of other inflammatory cytokines. We therefore investigated the effects of 2-DG on activation of the transcription factor nuclear factor (NF)-κB in response to proinflammatory cytokines TNFα and IL-1β. 51 Similar to IL-6-induced STAT3 activity, NF-κB activity induced by TNFα and IL-1β was efficiently suppressed by 2-DG ( Figure 4A) . Notably, 2-DG treatment reduced the apparent molecular weight of the TNFR1 and suppressed ligand binding by the receptor (Figure 4B,C) . Activation of JAKs induced by IFNγ, an important cytokine for macrophage activation during inflammatory responses, 52 was also inhibited by 2-DG, which was accompanied by a reduction in the molecular weight of IFNγ receptor α-chain ( Figure 4D and E). Additionally, IFNα/β receptor was deglycosylated by 2-DG, but activation and phosphorylation of JAKs and their target STAT1 were not decreased significantly ( Figure S14 ).

Moreover, it has been shown that glycosylation of vascular endothelial growth factor receptor 2 is affected by kifunensine and castanospermine, inhibitors of glycoprotein processing enzyme that convert protein N-linked high mannose carbohydrates to complex oligosaccharides. 53 However, castanospermine and another mannosidase inhibitor, 1-deoxymannojirimycin, did not attenuate glycosylation of gp130 and IL-6 signal ( Figure S15 ). These results demonstrated the versatile and specific inhibitory activity of 2-DG against a wide range of proinflammatory cytokine signals, which likely underlies the high therapeutic efficacy of this compound in inflammatory disease models.

The above results indicated that 2-DG inhibits multiple cytokine signals, especially inflammatory cytokines involved in cytokine storms.

Therefore, we analysed the effects of 2-DG on LPS shock, a mouse model of the cytokine storm. 54 We found that pretreatment with 2-DG by injection was very effective to prevent death following LPS shock in mice ( Figure 5A ) by reducing LPS-induced serum IL-6

and TNFα production ( Figure 5B ) as well as mRNA expression of IL-6, IL-1β, and SAA in the liver ( Figure S16 ). Moreover, i.p. injection of LPS elevated expression of cytokines (TNFα) and chemokines [MCP-1 and 10 kDa interferon-gamma-induced protein (IP-10)] in the lungs as observed in COVID-19 patients. 55 Expression of these mRNAs was attenuated by coinjection of 2-DG ( Figure 5C ). These results suggest that 2-DG attenuates the clinical symptoms caused by cytokine storms. Furthermore, we analysed the effects of 2-DG in LPS-induced acute lung inflammation, a mouse model of ARDS. 56 As shown in Figure 5D , pulmonary infiltration of inflammatory cells, which included macrophages, was attenuated by coinjection On the basis of these results, we think that only a limited number of proteins, such as inflammatory cytokine receptors, may be affected by the once-daily administration of 2-DG, which has a rapid degradation rate. Therefore, although 2-DG has low pharmacological specificity, effects other than its anti-inflammatory properties may not be significant, considering that no serious adverse events were observed in a clinical study, 58 see the next paragraph) using a human dose equivalent to that used in mice in this study. In addition, we observed that long-term oral administration of 2-DG in mice was well-tolerated. Before 2-DG can be used for the treatment of inflammatory diseases, more detailed in vivo analyses of the dose, frequency of administration, and other adverse events of 2-DG are required.

Because nutrient and energy deprivation are considered an efficient method to suppress the growth of cancer cells, whose main energy source is aerobic glycolysis, 2-DG may be an effective anticancer agent. 59 We believe that 2-DG may be beneficial for short-term treatment of severe cytokine-related diseases that require hospitalization, such as severe cytokine storms and ARDS. We also showed that 2-DG was effective for the treatment of inflammatory bowel disease and rheumatoid arthritis when administered at a high dose. In addition, another glycoprotein inhibitor, tunicamycin, also effectively suppressed colonic tissue destruction and macrophage infiltration related to DSS-induced inflammation in mice ( Figure S8 ), but its high cytotoxicity makes it difficult to use for therapy. Therefore, it will be necessary to develop glycosylation inhibitors that are effective at lower doses after oral administration. In addition, marked changes in cellular metabolism also alter the phenotype of macrophages: M1 macrophages were shown to be dependent on glycolysis. 62 Therefore, the inhibition of glycolysis by 2-DG might partly affect the activity of these cells. Further analysis is needed to clarify this issue. Furthermore, in this study, we focused on proinflammatory cytokines, especially IL-6 and TNFα, but to clarify the overall anti-inflammatory effects of 2-DG, it will be important to examine other proinflammatory cytokines and anti-inflammatory cytokines such as IL-10, whose receptor has been reported to be highly glycosylated. 63 Glucocorticoids are currently considered the standard of care for patients with severe COVID-19. 12 However, glucocorticoid therapy in patients with severe COVID-19-related ARDS is associated with increased mortality and delayed viral clearance, which suggests that treatment timing, dosage, and COVID-19 severity determine the outcomes of immunomodulatory therapies. 64 In the present study, we found that glucocorticoids inhibited LPS-induced pulmonary infiltration, lung edema, and the expression of chemokines relevant to COVID-19. Similar effects were observed following 2-DG administration and combined therapy with glucocorticoids and 2-DG almost completely suppressed these effects. We believe that the small number of animals used in this study may have been a limitation.

However, considering the effects of 2-DG on the inflammatory disease models, a therapeutic effect on LPS pneumonia is conceivable.

Although detailed analysis of the immunomodulatory effect of 2-DG has not been performed, our results suggest that 2-DG alone or in combination with glucocorticoids may be effective for treatment of severe COVID-19. Glucocorticoid is also used for treatment of IBD and rheumatoid arthritis. 65, 66 In the present study, although we did not investigate the combined effect of 2-DG on glucocorticoid treatment in our model mice, the results of LPS pneumonia suggest that these models may provide synergistic therapeutic effects and reduced glucocorticoid use. We believe it is necessary to analyse this aspect in the future. Moreover, because the present study mainly analysed the effect of 2-DG on inflammatory disease models, we believe that a detailed analysis of the 2-DG dose response in these mice should be performed in the future for clinical application. Because the actual efficacy against COVID-19 is still speculative, future research will be required to elucidate the potential clinical application of glycosylation agents, including 2-DG.

While anti-proinflammatory cytokine therapies are clinically effective, such as engineered antibodies and soluble cytokine receptors, these protein therapies require multiple injections and are quite expensive, which hinder their use as the primary choice to treat various inflammatory diseases. 28, 67 In practical terms, orally bioavailable, easy-to-synthesize drugs directed against diverse proinflammatory cytokine systems would be preferable. In this regard, 2-DG is a promising anti-inflammatory compound, although a disadvantage is the high dose required for activity. Development of further derivatives may result in increased efficacies and fewer adverse effects. Related compounds with more specific inhibitory effects on receptor N-glycosylation and fewer effects on glycolysis would meet such a requirement.

The animal experiment protocol was approved by the Ethics 

We are grateful to Keiko Kawauchi, Yoshinori Abe, Wataru Nakajima, and Tomio Yabe for their helpful suggestions and Miho Kawagoe, Hiroko Hiroike, Toshimi Takatera, Yumi Asano, and Seiko Egawa for F I G U R E 5 2-Deoxy-D-glucose (2-DG) alleviates lipopolysaccharide (LPS)-induced shock and LPS-induced acute lung inflammation. (A) Eight-week-old B6 mice were intraperitoneally (i.p.) injected LPS (0.8 mg/mouse) with (n = 15) or without (n = 15) 20 mg 2-DG. Two hours after injection, these mice were intraperitoneally injected 0.8 mg LPS. Survival was monitored for 4 days and analyzed by the Kaplan-Meier method; data were compared between the two groups using the Log-rank (Mantel-Cox) test. p < .0001. (B) Two hours after LPS injection as described in (A), serum concentrations of interleukin (IL)-6 and tumor necrosis factor (TNF)α were measured by ELISAs. Results were analysed using one-way ANOVA followed by Tukey's post hoc test. ****p < .0001, ***p < .001, and *p < .05. Graphs are presented as the mean ±s.d. (n = 6). (C) Quantitative real-time PCR analysis of monocyte chemotactic protein-1 (MCP-1), 10 kDa interferon-gamma-induced protein (IP-10), and tumor necrosis factor (TNF)α mRNAs in left lung tissue 2 days after LPS injection as described in (A). Significant differences were determined using the unpaired two-tailed t-test. ****p < .0001 and ***p < .001. (D) Mice were injected i.p. (n = 3) with or without 20 mg 2-DG and 100 mg/kg methylprednisolone (mPSL), Subsequently, 10 mg/kg LPS was injected intratracheally. Two days after LPS injection, histological sections of the left lung (Mock, LPS intratracheally injected, LPS+2DG treated, LPS+mPSL treated, and LPS+2DG+mPSL treated) were stained with haematoxylin and eosin (left panel, ×2 and ×20) and with the anti-F4/80 antibody (right panel, ×2 and ×20). Experiments were repeated three times independently with similar results. (E) LPS-induced lung edema as described in (D).

Results were analysed using one-way ANOVA followed by Tukey's post hoc test. ***p < .001, **p < .01, and *p < .05. (F) Quantitative realtime PCR analysis of MCP-1 and IP-10 mRNAs in left lung tissue 2 days after LPS injection as described in (D). Results were analysed using one-way ANOVA followed by Tukey's post hoc test. ****p < .0001, ***p < .001, **p < .01, and *p < .05 technical support. This study was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Uehara Memorial Foundation. We thank Mitchell Arico and J. Ludovic Croxford, PhD, from Edanz (https://jp.edanz.

com/ac) for editing a draft of this manuscript.

The authors declare no conflict of interest. 

This declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigor of preclinical research as stated in the BJP guidelines for design and analysis, immunoblotting, and animal experimentation, and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

No reproduced material from other sources was present in this study.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.

Nobuyuki Tanaka https://orcid.org/0000-0002-6373-2220

Origin and physiological roles of inflammation

Pattern recognition receptors and inflammation

Bacteria fighting back: how pathogens target and subvert the host innate immune system

Cytokines and chemokines: at the crossroads of cell signalling and inflammatory disease

Therapeutic antibodies for autoimmunity and inflammation

Targeting interleukin-6 signaling in clinic

Regulation of tumour necrosis factor signalling: live or let die

Current and emerging therapeutic targets for IBD

Treating rheumatological diseases and comorbidities with interleukin-1 blocking therapies

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Severe Covid-19

Analysis of the clinical characteristics of 77 COVID-19 deaths

Role for anti-cytokine therapies in severe coronavirus disease 2019

Interleukin-6: designing specific therapeutics for a complex cytokine

Therapeutic antibodies: successes, limitations and hopes for the future

Interleukin-6: from basic science to medicine-40 years in immunology

Genetic analysis of innate immunity

Molecular dynamics of lipopolysaccharide-induced lung injury in rodents

p53 regulates glucose metabolism through an IKK-NF-kB pathway and inhibits cell transformation

Dextran sulfate sodium (DSS)-induced colitis in mice

Local transplantation of adipose-derived stem cells has a significant therapeutic effect in a mouse model of rheumatoid arthritis

Astaxanthin prevents against lipopolysaccharide-induced acute lung injury and sepsis via inhibiting activation of MAPK/NF-kappaB

The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Catalytic receptors

THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Enzymes

Anti-inflammatory agents: present and future

Role of proinflammatory cytokines in the pathophysiology of osteoarthritis

Interleukin 6: from bench to bedside

The JAK-STAT signaling pathway: input and output integration

Molecular cloning and expression of an IL-6 signal transducer, gp130

Molecular cloning of a murine IL-6 receptor-associated signal transducer, gp130, and its regulated expression in vivo

Detailed studies on substrate structure requirements of glycoamidases A and F

N-glycans modulate the activation of gp130 in mouse embryonic neural precursor cells

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo

Under normoxia, 2-deoxy-D-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation

Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption

Dolichol-phosphate mannose synthase: structure, function and regulation

Biosynthesis and half-life of the interleukin-6 receptor and its signal transducer gp130

N-linked glycosylation is essential for the stability but not the signaling function of the interleukin-6 signal transducer glycoprotein 130

Proinflammatory cytokines in the pathogenesis of inflammatory bowel diseases

Interleukin-6 induces monocyte chemotactic protein-1 in peripheral blood mononuclear cells and in the U937 cell line

Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis

Oligosaccharides isolated from goat milk reduce intestinal inflammation in a rat model of dextran sodium sulfate-induced colitis

Distinct contribution of IL-6, TNF-alpha, IL-1, and IL-10 to T cell-mediated spontaneous autoimmune arthritis in mice

A role for fungal bglucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice

The carboxyl-terminal region of STAT3 controls gene induction by the mouse haptoglobin promoter

Serum amyloid A: an acutephase protein involved in tumour pathogenesis

Cytokines in inflammatory disease

Regulation and function of NF-kappaB transcription factors in the immune system

Exploring the full spectrum of macrophage activation

Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways

Cytokine storms: understanding COVID-19

SARS-CoV-2 infection: the role of cytokines in COVID-19 disease

Endothelial damage in acute respiratory distress syndrome

Different protein turnover of interleukin-6-type cytokine signalling components

A phase I dose-escalation trial of 2-deoxy-D-glucose alone or combined with docetaxel in patients with advanced solid tumors

2-Deoxy-d-glucose and its analogs: from diagnostic to therapeutic agents

Role of p53 in the regulation of the inflammatory tumor microenvironment and tumor suppression

A simple practice guide for dose conversion between animals and human

The metabolic signature of macrophage responses

Characterization of recombinant extracellular domain of human interleukin-10 receptor

COVID-19 pneumonia, and acute respiratory distress syndrome

Glucocorticoid therapy in inflammatory bowel disease: mechanisms and clinical practice

Views on glucocorticoid therapy in rheumatology: the age of convergence

Biologic drugs for rheumatoid arthritis in the Medicare program: a cost-effectiveness analysis