Google Scholar. Ding S, Jiang H, Fang J. Regulation of immune function by polyphenols. J Immunol Res. Zhou Y, Zheng J, Li Y, Xu DP, Li S, Chen YM, et al. Natural polyphenols for prevention and treatment of cancer. Mileo AM, Di Venere D, Linsalata V, Fraioli R, Miccadei S. Artichoke polyphenols induce apoptosis and decrease the invasive potential of the human breast cancer cell line MDA-MB J Cell Physiol.

Visioli F, De La Lastra CA, Andres-Lacueva C, Aviram M, Calhau C, Cassano A, et al. Polyphenols and human health: a prospectus. Crit Rev Food Sci Nutr. Dragan S, Andrica F, Serban MC, Timar R. Polyphenols-rich natural products for treatment of diabetes.

Curr Med Chem. Wang S, Moustaid-Moussa N, Chen L, Mo H, Shastri A, Su R, et al. Novel insights of dietary polyphenols and obesity. J Nutr Biochem. Ali F, Ismail A, Kersten S.

Molecular mechanisms underlying the potential antiobesity-related diseases effect of cocoa polyphenols. Mol Nutr Food Res. Magrone T, Russo MA, Jirillo E. Cocoa and dark chocolate polyphenols: from biology to clinical applications.

Front Immunol. Hossen MS, Ali MY, Jahurul MHA, Abdel-Daim MM, Gan SH, Khalil MI. Beneficial roles of honey polyphenols against some human degenerative diseases: a review.

Pharmacol Rep. Bhullar KS, Rupasinghe HP. Polyphenols: multipotent therapeutic agents in neurodegenerative diseases. Oxid Med Cell Longev. Yamagata K, Tagami M, Yamori Y. Dietary polyphenols regulate endothelial function and prevent cardiovascular disease.

Cardona F, Andres-Lacueva C, Tulipani S, Tinahones FJ, Queipo-Ortuno MI. Benefits of polyphenols on gut microbiota and implications in human health.

Tap J, Furet JP, Bensaada M, Philippe C, Roth H, Rabot S, et al. Gut microbiota richness promotes its stability upon increased dietary fibre intake in healthy adults. Environ Microbiol. Macpherson AJ, Harris NL.

Interactions between commensal intestinal bacteria and the immune system. Nat Rev Immunol. Pickard JM, Chervonsky AV. Intestinal fucose as a mediator of host-microbe symbiosis. J Immunol. Yang Y, Xu C, Wu D, Wang Z, Wu P, Li L, et al.

gammadelta T cells: crosstalk between microbiota, chronic inflammation, and colorectal cancer. Rescigno M. Intestinal microbiota and its effects on the immune system.

Cell Microbiol. Biragyn A, Ferrucci L. Gut dysbiosis: a potential link between increased cancer risk in ageing and inflammaging. Lancet Oncol. Zitvogel L, Galluzzi L, Viaud S, Vetizou M, Daillere R, Merad M, et al. Cancer and the gut microbiota: an unexpected link. Sci Transl Med. Sheflin AM, Whitney AK, Weir TL.

Cancer-promoting effects of microbial dysbiosis. Curr Oncol Rep. Sun J, Kato I. Gut microbiota, inflammation and colorectal cancer. Genes Dis. Jahani-Sherafat S, Alebouyeh M, Moghim S, Ahmadi Amoli H, Ghasemian-Safaei H. Role of gut microbiota in the pathogenesis of colorectal cancer; a review article.

Gastroenterol Hepatol Bed Bench. PubMed Abstract Google Scholar. Kawabata K, Yoshioka Y, Terao J. Role of intestinal microbiota in the bioavailability and physiological functions of dietary polyphenols. Ricciardiello L, Bazzoli F, Fogliano V.

Phytochemicals and colorectal cancer prevention—myth or reality? Nat Rev Gastroenterol Hepatol. Chow HH, Hakim IA. Pharmacokinetic and chemoprevention studies on tea in humans.

Pharmacol Res. Ju S, Ge Y, Li P, Tian X, Wang H, Zheng X, et al. Dietary quercetin ameliorates experimental colitis in mouse by remodeling the function of colonic macrophages via a heme oxygenasedependent pathway. Cell Cycle. Farinetti A, Zurlo V, Manenti A, Coppi F, Mattioli AV.

Mediterranean diet and colorectal cancer: a systematic review. Soldati L, Di Renzo L, Jirillo E, Ascierto PA, Marincola FM, De Lorenzo A. The influence of diet on anti-cancer immune responsiveness. J Transl Med. Ostan R, Lanzarini C, Pini E, Scurti M, Vianello D, Bertarelli C, et al.

Inflammaging and cancer: a challenge for the Mediterranean diet. Hardy TM, Tollefsbol TO. Epigenetic diet: impact on the epigenome and cancer. Kroemer G, Zitvogel L. Cancer immunotherapy in the breakthrough of the microbiota. Nosrati N, Bakovic M, Paliyath G.

Molecular mechanisms and pathways as targets for cancer prevention and progression with dietary compounds. Int J Mol Sci. Bhattacharyya S, Md Sakib Hossain D, Mohanty S, Sankar Sen G, Chattopadhyay S, Banerjee S, et al. Curcumin reverses T cell-mediated adaptive immune dysfunctions in tumor-bearing hosts.

Cell Mol Immunol. Zou T, Yang Y, Xia F, Huang A, Gao X, Fang D, et al. PLoS ONE. Yao J, Wei C, Wang JY, Zhang R, Li YX, Wang LS. Ohno M, Nishida A, Sugitani Y, Nishino K, Inatomi O, Sugimoto M, et al.

Nanoparticle curcumin ameliorates experimental colitis via modulation of gut microbiota and induction of regulatory T cells. Hong EH, Heo EY, Song JH, Kwon BE, Lee JY, Park Y, et al. Trans-scirpusin A showed antitumor effects via autophagy activation and apoptosis induction of colorectal cancer cells.

Zhang Z, Wu X, Cao S, Cromie M, Shen Y, Feng Y, et al. Chlorogenic acid ameliorates experimental colitis by promoting growth of akkermansia in mice. Cui X, Jin Y, Hofseth AB, Pena E, Habiger J, Chumanevich A, et al. Resveratrol suppresses colitis and colon cancer associated with colitis.

Cancer Prev Res. Churchill M, Chadburn A, Bilinski RT, Bertagnolli MM. Inhibition of intestinal tumors by curcumin is associated with changes in the intestinal immune cell profile. J Surg Res. Skyberg JA, Robison A, Golden S, Rollins MF, Callis G, Huarte E, et al.

Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression. J Leukoc Biol. Wang B, Wu C. Dietary soy isoflavones alleviate dextran sulfate sodium-induced inflammation and oxidative stress in mice.

Exp Ther Med. Wagner AE, Will O, Sturm C, Lipinski S, Rosenstiel P, Rimbach G. Ramiro-Puig E, Perez-Cano FJ, Ramos-Romero S, Perez-Berezo T, Castellote C, Permanyer J, et al.

Intestinal immune system of young rats influenced by cocoa-enriched diet. Ma B, Khazali A, Wells A. CXCR3 in carcinoma progression. Histol Histopathol. Abron JD, Singh NP, Murphy AE, Mishra MK, Price RL, Nagarkatti M, et al. Differential role of CXCR3 in inflammation and colorectal cancer.

Legitimo A, Consolini R, Failli A, Orsini G, Spisni R. Dendritic cell defects in the colorectal cancer. Hum Vaccin Immunother. Malietzis G, Lee GH, Jenkins JT, Bernardo D, Moorghen M, Knight SC, et al. Prognostic value of the tumour-infiltrating dendritic cells in colorectal cancer: a systematic review.

Cell Commun Adhes. Kajihara M, Takakura K, Kanai T, Ito Z, Saito K, Takami S, et al. Dendritic cell-based cancer immunotherapy for colorectal cancer. del Corno M, Scazzocchio B, Masella R, Gessani S. Regulation of dendritic cell function by dietary polyphenols.

Deng Z, Rong Y, Teng Y, Mu J, Zhuang X, Tseng M, et al. Broccoli-derived nanoparticle inhibits mouse colitis by activating dendritic cell AMP-activated protein kinase. Mol Ther. Del Corno M, Varano B, Scazzocchio B, Filesi C, Masella R, Gessani S.

Protocatechuic acid inhibits human dendritic cell functional activation: role of PPARgamma up-modulation. Cavalcanti E, Vadrucci E, Delvecchio FR, Addabbo F, Bettini S, Liou R, et al.

Administration of reconstituted polyphenol oil bodies efficiently suppresses dendritic cell inflammatory pathways and acute intestinal inflammation. De Santis S, Kunde D, Serino G, Galleggiante V, Caruso ML, Mastronardi M, et al.

Secretory leukoprotease inhibitor is required for efficient quercetin-mediated suppression of TNFalpha secretion. Ghattamaneni NKR, Panchal SK, Brown L. Nutraceuticals in rodent models as potential treatments for human Inflammatory Bowel Disease. Kaulmann A, Bohn T.

Bioactivity of polyphenols: preventive and adjuvant strategies toward reducing inflammatory bowel diseases-promises, perspectives, and pitfalls. Guan G, Lan S. Implications of antioxidant systems in inflammatory bowel disease.

Lee CY, Nanah CN, Held RA, Clark AR, Huynh UG, Maraskine MC, et al. Effect of electron donating groups on polyphenol-based antioxidant dendrimers.

Kalaiselvan I, Samuthirapandi M, Govindaraju A, Sheeja Malar D, Kasi PD. Olive oil and its phenolic compounds hydroxytyrosol and tyrosol ameliorated TCDD-induced heptotoxicity in rats via inhibition of oxidative stress and apoptosis.

Pharm Biol. Gosslau A, En Jao DL, Huang MT, Ho CT, Evans D, Rawson NE, et al. Effects of the black tea polyphenol theaflavin-2 on apoptotic and inflammatory pathways in vitro and in vivo. Zhang H, Tsao R. Dietary polyphenols, oxidative stress and antioxidant and anti-inflammatory effects.

Curr Opin Food Sci. Rodriguez-Ramiro I, Ramos S, Lopez-Oliva E, Agis-Torres A, Bravo L, Goya L, et al. Cocoa polyphenols prevent inflammation in the colon of azoxymethane-treated rats and in TNF-alpha-stimulated Caco-2 cells.

Br J Nutr. Rodriguez-Ramiro I, Ramos S, Bravo L, Goya L, Martin MA. Procyanidin B2 and a cocoa polyphenolic extract inhibit acrylamide-induced apoptosis in human Caco-2 cells by preventing oxidative stress and activation of JNK pathway.

Sahu BD, Kumar JM, Sistla R. Fisetin, a dietary flavonoid, ameliorates experimental colitis in mice: relevance of NF-kappaB signaling. Serra G, Incani A, Serreli G, Porru L, Melis MP, Tuberoso CIG, et al.

Olive oil polyphenols reduce oxysterols -induced redox imbalance and pro-inflammatory response in intestinal cells. Redox Biol.

Oz HS, Chen T, de Villiers WJ. Green tea polyphenols and sulfasalazine have parallel anti-inflammatory properties in colitis models. Saadatdoust Z, Pandurangan AK, Ananda Sadagopan SK, Mohd Esa N, Ismail A, Mustafa MR.

Larrosa M, Gonzalez-Sarrias A, Yanez-Gascon MJ, Selma MV, Azorin-Ortuno M, Toti S, et al. Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic metabolism.

Owczarek K, Hrabec E, Fichna J, Sosnowska D, Koziolkiewicz M, Szymanski J, et al. Flavanols from Japanese quince Chaenomeles japonica fruit suppress expression of cyclooxygenase-2, metalloproteinase-9, and nuclear factor-kappaB in human colon cancer cells.

Acta Biochim Pol. Bucio-Noble D, Kautto L, Krisp C, Ball MS, Molloy MP. Polyphenol extracts from dried sugarcane inhibit inflammatory mediators in an in vitro colon cancer model.

J Proteomics. Hu Q, Yuan B, Xiao H, Zhao L, Wu X, Rakariyatham K, et al. Polyphenols-rich extract from Pleurotus eryngii with growth inhibitory of HCT colon cancer cells and anti-inflammatory function in RAW Food Funct. Aboura I, Nani A, Belarbi M, Murtaza B, Fluckiger A, Dumont A, et al.

Protective effects of polyphenol-rich infusions from carob Ceratonia siliqua leaves and cladodes of Opuntia ficus-indica against inflammation associated with diet-induced obesity and DSS-induced colitis in Swiss mice.

Biomed Pharmacother. Farombi EO, Adedara IA, Ajayi BO, Ayepola OR, Egbeme EE. Kolaviron, a natural antioxidant and anti-inflammatory phytochemical prevents dextran sulphate sodium-induced colitis in rats.

Basic Clin Pharmacol Toxicol. Alabi QK, Akomolafe RO, Omole JG, Adefisayo MA, Ogundipe OL, Aturamu A, et al. Polyphenol-rich extract of Ocimum gratissimum leaves ameliorates colitis via attenuating colonic mucosa injury and regulating pro-inflammatory cytokines production and oxidative stress. Asensi M, Ortega A, Mena S, Feddi F, Estrela JM.

Natural polyphenols in cancer therapy. Crit Rev Clin Lab Sci. Orlich MJ, Singh PN, Sabate J, Fan J, Sveen L, Bennett H, et al. Vegetarian dietary patterns and the risk of colorectal cancers. JAMA Intern Med. Halmos EP, Gibson PR. Dietary management of IBD—insights and advice. Fini L, Piazzi G, Daoud Y, Selgrad M, Maegawa S, Garcia M, et al.

Montrose DC, Horelik NA, Madigan JP, Stoner GD, Wang LS, Bruno RS, et al. Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis. Pan P, Skaer CW, Wang HT, Stirdivant SM, Young MR, Oshima K, et al.

Ko JH, Sethi G, Um JY, Shanmugam MK, Arfuso F, Kumar AP, et al. The Role of resveratrol in cancer therapy. McFadden RM, Larmonier CB, Shehab KW, Midura-Kiela M, Ramalingam R, Harrison CA, et al. The role of curcumin in modulating colonic microbiota during colitis and colon cancer prevention.

Elbling L, Weiss RM, Teufelhofer O, Uhl M, Knasmueller S, Schulte-Hermann R, et al. Green tea extract and - -epigallocatechingallate, the major tea catechin, exert oxidant but lack antioxidant activities.

FASEB J. Araujo JR, Goncalves P, Martel F. Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutr Res. Khan HY, Zubair H, Ullah MF, Ahmad A, Hadi SM.

A prooxidant mechanism for the anticancer and chemopreventive properties of plant polyphenols. Curr Drug Targets. Mileo AM, Di Venere D, Miccadei S. Antitumour effects of artichoke polyphenols: cell death and ROS-mediated epigenetic growth arrest. Stem Cell Epigenet.

Khan HY, Zubair H, Faisal M, Ullah MF, Farhan M, Sarkar FH, et al. Plant polyphenol induced cell death in human cancer cells involves mobilization of intracellular copper ions and reactive oxygen species generation: a mechanism for cancer chemopreventive action. Venancio VP, Cipriano PA, Kim H, Antunes LM, Talcott ST, Mertens-Talcott SU.

anthocyanins exert anti-inflammatory activity in human colon cancer and non-malignant colon cells. Zitvogel L, Kepp O, Senovilla L, Menger L, Chaput N, Kroemer G. Immunogenic tumor cell death for optimal anticancer therapy: the calreticulin exposure pathway. Lee CS, Ryan EJ, Doherty GA.

Gastro-intestinal toxicity of chemotherapeutics in colorectal cancer: the role of inflammation. Golden EB, Apetoh L. Radiotherapy and immunogenic cell death. Semin Radiat Oncol. Steinman RM, Banchereau J.

Taking dendritic cells into medicine. Marin JJ, Sanchez de Medina F, Castano B, Bujanda L, Romero MR, Martinez-Augustin O, et al.

Chemoprevention, chemotherapy, and chemoresistance in colorectal cancer. Drug Metab Rev. Redondo-Blanco S, Fernandez J, Gutierrez-Del-Rio I, Villar CJ, Lombo F.

New insights toward colorectal cancer chemotherapy using natural bioactive compounds. Front Pharmacol. Yu Y, Kanwar SS, Patel BB, Nautiyal J, Sarkar FH, Majumdar AP. Elimination of colon cancer stem-like cells by the combination of curcumin and FOLFOX.

Transl Oncol. Patel BB, Sengupta R, Qazi S, Vachhani H, Yu Y, Rishi AK, et al. Curcumin enhances the effects of 5-fluorouracil and oxaliplatin in mediating growth inhibition of colon cancer cells by modulating EGFR and IGF-1R.

Aires V, Limagne E, Cotte AK, Latruffe N, Ghiringhelli F, Delmas D. Resveratrol metabolites inhibit human metastatic colon cancer cells progression and synergize with chemotherapeutic drugs to induce cell death. Abouzeid AH, Patel NR, Rachman IM, Senn S, Torchilin VP.

Anti-cancer activity of anti-GLUT1 antibody-targeted polymeric micelles co-loaded with curcumin and doxorubicin. J Drug Target. Jeng LB, Kumar Velmurugan B, Chen MC, Hsu HH, Ho TJ, Day CH, et al. Shakibaei M, Mobasheri A, Lueders C, Busch F, Shayan P, Goel A.

Curcumin enhances the effect of chemotherapy against colorectal cancer cells by inhibition of NF-kappaB and Src protein kinase signaling pathways.

Hamaya Y, Guarinos C, Tseng-Rogenski SS, Iwaizumi M, Das R, Jover R, et al. Hakim L, Alias E, Makpol S, Ngah WZ, Morad NA, Yusof YA. Gelam honey and ginger potentiate the anti-cancer effect of 5-FU against HCT colorectal cancer cells. Snyder V, Reed-Newman TC, Arnold L, Thomas SM, Anant S. Cancer stem cell metabolism and potential therapeutic targets.

Full-length blots are presented in Supplementary Figures 1 , 2. One of the primary mechanisms by which AMPK regulates cellular metabolism is through inhibition of mTOR, the major promoter of anabolic metabolism which is highly activated in response to LPS stimulation 29 — Having confirmed that carnosol and curcumin can activate AMPK, it was next investigated whether they might inhibit mTOR activity in LPS-stimulated DC.

The ribosomal protein S6 is phosphorylated downstream of mTOR activation and serves as a readout of mTOR activity. DC were treated with compound C, a pharmacological inhibitor of AMPK, for 1 h prior to incubation with carnosol or curcumin for 1 h, followed by stimulation with LPS for 1 h.

The expression of phospho-S6 was detected by Western blot. As expected, stimulation of human DC with LPS resulted in a strong increase in phospho-S6 expression, which was attenuated in DC treated with either carnosol or curcumin. However, the reduction of phospho-S6 expression by carnosol and curcumin was reversed with the addition of compound C Figure 3C.

Although AMPK signaling has been reported to regulate HO-1 expression in other cell types, there have been no reports of AMPK-dependent upregulation of HO-1 in DC. Therefore, to determine whether AMPK activation can upregulate HO-1 expression in human DC, DC were treated with increasing concentrations of AICAR for 24 h, after which the expression of HO-1 was detected by Western blot.

A dose-dependent increase of HO-1 expression was observed in AICAR-treated DC, with the greatest upregulation observed at 0. Following this, the contribution of AMPK to the upregulation of HO-1 by carnosol and curcumin was investigated.

DC were treated with compound C for 1 h prior to treatment with carnosol or curcumin. After 24 h, the expression of HO-1 was detected by Western blot.

As previously observed 19 , carnosol and curcumin increased the expression of HO-1 by DC, however, this increase was diminished in the presence of compound C Figure 3E. HO-1 is a known promoter of tolerogenic DC, as it is highly expressed in immature DC and limits their maturation in response to pro-inflammatory stimuli 21 — Upregulation of HO-1 by carnosol and curcumin was previously observed to limit the maturation of human DC stimulated with LPS As inhibition of AMPK via compound C was found to attenuate the induction of HO-1 by both carnosol and curcumin, it was next investigated whether AMPK inhibition could also reverse the effects of these polyphenols on DC maturation.

Human DC were treated with compound C for 1 h before addition of either carnosol or curcumin for a further 6 h to allow for the upregulation of HO-1 gene transcription and protein translation prior to stimulation with LPS.

After 24 h, expression of the maturation markers CD40 and CD83, and co-stimulatory molecules CD80 and CD86 was measured by flow cytometry. Consistent with previous observations 19 , carnosol treatment significantly reduced expression of CD83 and CD86 by LPS-stimulated DC, with a trend toward reduced CD40 also observed.

However, this effect was attenuated in the presence of compound C Figures 4A,C. Similarly, curcumin treatment significantly reduced the expression of CD40 and CD86 in LPS stimulated DC, with a trend toward reduced CD83 also observed. Again, this inhibition of surface marker expression by curcumin was reversed with the addition of compound C Figures 4B,D.

Treatment of LPS-stimulated DC with compound C alone did not increase the expression of DC surface markers. Figure 4. Inhibition of AMPK attenuates reduction of DC maturation markers by carnosol and curcumin.

Histograms depict expression of maturation markers in DC treated with A carnosol or B curcumin, with or without compound C, compared to controls from one representative experiment. Results shown are mean ± SEM of the measured Mean Fluorescence Intensities MFI , expressed as percentages of the vehicle control.

We have previously reported that treatment of human DC with carnosol or curcumin can maintain the capacity of DC to take up and process antigens after stimulation with LPS Following the observation that inhibition of AMPK signaling via compound C reversed the effects of carnosol and curcumin on the phenotypic maturation of DC, it was next determined whether their effects on functional DC maturation would also be attenuated.

DC were treated with compound C, carnosol or curcumin, and stimulated with LPS as before. After 24 h, DC were incubated with the model antigen DQ-Ovalbumin DQ-Ova for 20 min, and analyzed for antigen uptake by flow cytometry. As expected, stimulation of DC with LPS dramatically reduced their capacity to uptake antigen compared to immature DC.

Furthermore, both carnosol and curcumin treatment maintained the phagocytic capacity of LPS-stimulated DC similar to that of immature DC Figure 5A , as was observed previously However, addition of compound C to carnosol- and curcumin-treated DC significantly abrogated this effect Figures 5B,C.

Treatment of DC with compound C alone did not significantly alter their antigen uptake capacity following LPS stimulation. Taken together, these results confirm that the immunomodulatory effects of carnosol and curcumin on both phenotypic and functional DC maturation are dependent on their activation of AMPK.

Figure 5. Inhibition of AMPK attenuates the increased phagocytic capacity of LPS-stimulated DC treated with carnosol or curcumin. A Representative dot plots depicting DQ-Ova uptake by DC treated with carnosol, curcumin, and compound C from one representative experiment. Results shown are mean ± SEM percentages of DQ-Ova uptake in control-, carnosol- and curcumin-treated DC, with or without compound C.

Supplementation with immunonutrients such as polyphenols represents a novel strategy to modulate the immune response through dietary intervention. Cellular metabolism has emerged as a major modulator of immune cell function, yet there has been limited study into the effects of polyphenols on immunometabolism.

Here, we have investigated the activity of two plant-derived polyphenols, carnosol and curcumin, on the metabolism and downstream immune function of primary human DC. We demonstrate that the metabolic reprogramming which occurs in human DC upon LPS stimulation can be modulated by both carnosol and curcumin.

We also demonstrate that these polyphenols regulate metabolic signaling through activation of AMPK and an associated inhibition of mTOR activity. Furthermore, we describe a novel relationship between AMPK signaling and induction of the immunomodulatory enzyme HO-1 by carnosol and curcumin.

Together, this data demonstrates that regulation of metabolic signaling and function by naturally-derived polyphenols mediates their ability to promote tolerogenic DC. While a number of studies have investigated metabolic reprogramming in activated murine DC, studies assessing human DC metabolism are comparatively scarce.

LPS-stimulated BMDC have previously been observed to strongly upregulate aerobic glycolysis, and simultaneously downregulate oxidative phosphorylation via the action of iNOS-derived NO 10 — 12 , The results presented here demonstrate that human DC stimulated with LPS upregulate both glycolysis and oxidative phosphorylation within hours of activation.

Furthermore, a transient increase in the glycolytic reserve and spare respiratory capacity SRC of human DC was observed within 6 h post-LPS stimulation, which was absent at 24 h post-LPS. Therefore, it can be ascertained that while human DC also display increased glycolytic metabolism after activation, unlike BMDC, they also upregulate oxidative phosphorylation.

This disparity between murine and human DC is likely a result of their differing expression of iNOS, as human monocyte-derived DC do not readily express iNOS; however, some evidence suggests that certain human DC subsets can express iNOS in vivo , therefore the metabolic profile of these DC may differ from what is observed in vitro Interestingly, a recent study by Basit et al.

Thus, it is important to consider that differences in the metabolism of DC may exist in vivo vs. in vitro , between DC subsets, or due to the type of stimulus employed.

Further study of human DC under different conditions is required to delineate the impact of these variables on DC immunometabolism. Consistent with the results presented here, Malinarich et al.

have reported that monocyte-derived human DC matured with LPS are more glycolytic than immature DC, and do not downregulate oxidative phosphorylation However, they also observed a reduced glycolytic reserve and SRC in mature compared to immature DC; a finding which, in fact, agrees with these results, as the metabolism of DC was assessed 24 h after maturation with LPS, by which time the increased glycolytic reserve and SRC observed in this study was absent.

Interestingly, Everts et al. also observed an increase in the SRC of BMDC stimulated with LPS for 1 h, which was mediated by enhanced glycolytic flux into the Kreb's cycle This increased flow of pyruvate into the Kreb's cycle was found to produce citrate necessary for de novo fatty acid synthesis in the maturing DC, providing lipids required to expand the endoplasmic reticulum and Golgi membranes in anticipation of increased protein production Therefore, the transient increase in the glycolytic reserve and SRC of LPS-stimulated DC observed in this study may represent an early adaption of maturing DC to their new immunogenic functions, which is downregulated once adequate cellular remodeling has taken place.

Meanwhile, the mature DC continues to display higher basal rates of glycolysis and oxidative phosphorylation to meet its increased energy demands. Thus, this study expands the current understanding of human DC metabolism, and also underscores the importance of accounting for temporal changes when analyzing the metabolism of immune cells.

The results of this study also further support our previous work which described the anti-inflammatory properties of the polyphenols, carnosol and curcumin, in human DC The upregulation of glycolysis by BMDC in response to LPS has been demonstrated to promote their maturation, cytokine production and activation of T cells 10 — Interestingly, DC treated with carnosol or curcumin displayed a reduced basal rate of glycolysis, and failed to upregulate their glycolytic reserve after 6 h of LPS stimulation.

This reduced glycolytic flux was also manifest in the mitochondrial activity of carnosol- and curcumin-treated DC, as both polyphenols inhibited the increased SRC seen in response to LPS. Tolerogenic human DC have been reported to possess a greater capacity for oxidative phosphorylation and fatty acid oxidation, and are less glycolytic than mature DC Therefore, it is possible that the anti-inflammatory effects of carnosol and curcumin in human DC are at least partly mediated by their inhibition of glycolysis, resulting in a diminished glycolytic reserve and SRC and failure to meet the bio-energetic requirements of maturation.

Both carnosol and curcumin have previously been reported to activate AMPK in skeletal muscle and cancer cell lines 34 — In this study, carnosol and curcumin were found to activate AMPK in human DC.

Furthermore, polyphenol-induced activation of AMPK resulted in the inhibition of mTOR activation in LPS-stimulated DC. We also demonstrate that AMPK activation by carnosol and curcumin is required to mediate their immunomodulatory effects in human DC given that pharmacological inhibition of AMPK can reverse the observed reduction of DC maturation by these polyphenols.

In line with our study, Krawczyk et al. previously reported that AMPK signaling antagonizes the maturation of BMDC and inhibits their upregulation of glycolysis in response to LPS 10 , while Carroll et al.

found that AMPK-deficient BMDC display enhanced maturation and pro-inflammatory functions Signaling via AMPK has previously been implicated in the upregulation of HO-1 by certain drugs 26 , 27 , 38 , but there have been no such reports in human immune cells.

Here, AMPK activation was found to upregulate expression of HO-1 in human DC, while inhibition of AMPK attenuated the induction of HO-1 by carnosol and curcumin. This study is therefore the first to report an association between AMPK signaling and HO-1 expression in human DC, and that the upregulation of HO-1 by carnosol and curcumin is at least partially dependent on their ability to activate AMPK.

Indeed, a number of studies have identified cross-talk between AMPK and Nrf2, the major transcription factor in control of HO-1 expression 26 , 38 — 40 , hence it will be of interest to further explore the AMPK-Nrf2-HO-1 axis in the context of polyphenol-mediated immune modulation.

Interestingly, a number of xenobiotics, including various polyphenols, have been reported to activate AMPK via an increase in the AMP:ATP ratio; this is achieved by inhibition of the mitochondrial electron transport chain complexes Curcumin, in particular, has been shown to inhibit ATP synthase in mitochondrial preparations, thereby limiting ATP production and increasing the ratio of AMP to ATP Given that a number of polyphenols also appear to inhibit ATP synthase or complex I 24 , 43 , it is likely that carnosol acts in a similar fashion.

Therefore, elevation of AMP levels represents a probable mechanism by which carnosol and curcumin activate AMPK in human DC, however, further research is required to confirm this. In conclusion, our data describes the metabolic changes arising from the activation of human DC, and characterizes a hitherto-unidentified role for the HO-1 system in immunometabolism.

The data presented here supports a model whereby activation of AMPK by carnosol and curcumin leads to the upregulation of HO-1, which mediates the downstream immunomodulatory activity of these polyphenols in human DC Figure 6.

These results are also suggestive that the anti-inflammatory phenotype characteristic of immune cells with higher catabolic metabolism and AMPK signaling may arise from increased expression of HO-1, however future studies in HO-1 deficient cells are required to fully validate this hypothesis.

Although our study supports the use of the polyphenols carnosol and curcumin as potential immunonutrient supplements, translation of these results to a clinical setting requires careful consideration regarding drug formulation and administration. One of the caveats associated with these polyphenols is their poor solubility in aqueous solutions, which may limit their bioavailability by certain routes of administration.

Additionally, polyphenols have been described to undergo metabolic alterations during digestion via the intestinal microbiota, which could alter their metabolic and immunological properties as described here 44 — Efforts made to improve the oral bioavailability of polyphenols such as curcumin, or to utilize alternative routes of administration, have been met with success in pre-clinical studies and clinical trials 47 — It is hoped that future research can determine whether these polyphenols display similar effects on DC immunometabolism and function in an in vivo setting.

Research into the use of polyphenols as clinically relevant immunonutrient supplements has expanded greatly over the last number of years and our data highlighting specific effects on key cells relevant to inflammatory and autoimmune disease provides further evidence attesting to their use as potential immune modulating compounds.

Figure 6. Proposed model of AMPK-dependent modulation of human DC metabolism and immune function by carnosol and curcumin. Carnosol and curcumin are polyphenols which have been shown to inhibit components of the ETC, resulting in reduced ATP production and elevated AMP levels.

AMP activates AMPK, which results in downstream activation of Nrf2. Nrf2 translocates to the nucleus and induces transcription of HO HO-1 and its products can then act as antioxidants to neutralize ROS produced by mitochondrial metabolism, and downregulate DC maturation and pro-inflammatory functions.

Additionally, AMPK or HO-1 may mediate the reduced rate of glycolysis and SRC observed in carnosol- and curcumin-treated DC red dashed arrows.

NC, JF, and AD conceptualized and designed experiments. NC and HF performed experiments. NC, HF, and AD wrote the manuscript. 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.

This work was funded by the Health Research Board, Ireland Grant No: HRA-POR O'Neill LAJ, Kishton RJ, Rathmell J. A guide to immunometabolism for immunologists.

Nat Rev Immunol. doi: PubMed Abstract CrossRef Full Text Google Scholar. Magrone T, Jirillo E. Influence of polyphenols on allergic immune reactions: mechanisms of action. Proc Nutr Soc. Magrone T, Perez de Heredia F, Jirillo E, Morabito G, Marcos A, Serafini M.

Functional foods and nutraceuticals as therapeutic tools for the treatment of diet-related diseases. Can J Physiol Pharmacol. Magrone T, Antonio Russo M, Jirillo E. Role of immune cells in the course of central nervous system injury: modulation with natural products.

Curr Pharm Des. Lang A, Salomon N, Wu JCY, Kopylov U, Lahat A, Har-Noy O, et al. Curcumin in combination with mesalamine induces remission in patients with mild-to-moderate ulcerative colitis in a randomized controlled trial. Clin Gastroenterol Hepatol. Brück J, Holstein J, Glocova I, Seidel U, Geisel J, Kanno T, et al.

Sci Rep. Zhao HM, Xu R, Huang XY, Cheng SM, Huang MF, Yue HY, et al. Front Pharmacol. Liu L, Liu YL, Liu GX, Chen X, Yang K, Yang YX, et al.

Curcumin ameliorates dextran sulfate sodium-induced experimental colitis by blocking STAT3 signaling pathway. Int Immunopharmacol. Kelly B, O'Neill LA. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res. Krawczyk CM, Holowka T, Sun J, Blagih J, Amiel E, DeBerardinis RJ, et al.

Toll-like receptor-induced changes in glycolytic metabolism regulate dendritic cell activation. Jantsch J, Chakravortty D, Turza N, Prechtel AT, Buchholz B, Gerlach RG, et al. Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function.

J Immunol. Everts B, Amiel E, Huang SC-C, Smith AM, Chang C-H, Lam WY, et al. TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKε supports the anabolic demands of dendritic cell activation. Nat Immunol. CrossRef Full Text Google Scholar. Carroll KC, Viollet B, Suttles J. AMPKα1 deficiency amplifies proinflammatory myeloid APC activity and CD40 signaling.

J Leukoc Biol. Everts B, Amiel E, Van Der Windt GJW, Freitas TC, Chott R, Yarasheski KE, et al. Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells. Weinberg JB, Misukonis MA, Shami PJ, Mason SN, Sauls DL, Dittman WA, et al.

Human mononuclear phagocyte inducible nitric oxide synthase iNOS : analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages. PubMed Abstract Google Scholar.

Thomas AC, Mattila JT. Front Immunol. Thwe PM, Amiel E. The role of nitric oxide in metabolic regulation of dendritic cell immune function. Cancer Lett. Malinarich F, Duan K, Hamid RA, Bijin A, Lin WX, Poidinger M, et al.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells. Campbell NK, Fitzgerald HK, Malara A, Hambly R, Sweeney CM, Kirby B, et al.

Naturally derived heme-oxygenase 1 inducers attenuate inflammatory responses in human dendritic cells and T cells: relevance for psoriasis treatment. Wegiel B, Nemeth Z, Correa-Costa M, Bulmer AC, Otterbein LE.

Heme oxygenase a metabolic nike. Antioxid Redox Signal. Chauveau C, Rémy S, Royer PJ, Hill M, Tanguy-Royer S, Hubert FX, et al. Heme oxygenase-1 expression inhibits dendritic cell maturation and proinflammatory function but conserves IL expression.

Moreau A, Hill M, Thébault P, Deschamps JY, Chiffoleau E, Chauveau C, et al. Tolerogenic dendritic cells actively inhibit T cells through heme oxygenase-1 in rodents and in nonhuman primates. FASEB J. Al-Huseini LMA, Aw Yeang HX, Hamdam JM, Sethu S, Alhumeed N, Wong W, et al.

J Biol Chem. Kim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: mechanisms of action and physiological activities. Exp Mol Med. Cluxton D, Moran B, Fletcher JM. Differential regulation of human Treg and Th17 cells by fatty acid synthesis and glycolysis. Liu X, Peyton KJ, Shebib AR, Wang H, Korthuis RJ, Durante W.

Activation of AMPK stimulates heme oxygenase-1 gene expression and human endothelial cell survival. Am J Physiol Circ Physiol. Cho R-L, Lin W-N, Wang C-Y, Yang C-C, Hsiao L-D, Lin C-C, et al.

Biochem Pharmacol. Lin H, Yu CH, Jen CY, Cheng CF, Chou Y, Chang CC, et al. Adiponectin-mediated heme oxygenase-1 induction protects against iron-induced liver injury via a PPARα-dependent mechanism.

Am J Pathol. Tannahill GM, Curtis AM, Adamik J, Palsson-McDermott EM, McGettrick AF, Goel G, et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Cramer T, Yamanishi Y, Clausen BE, Förster I, Pawlinski R, Mackman N, et al. HIF-1α is essential for myeloid cell-mediated inflammation.

Byles V, Covarrubias AJ, Ben-Sahra I, Lamming DW, Sabatini DM, Manning BD, et al. The TSC-mTOR pathway regulates macrophage polarization. Nat Commun.

Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol. Basit F, Mathan T, Sancho D, de Vries IJM.

Upon completion of the assay the XF assay medium was removed and RIPA buffer was added to each well. Protein concentration was determined by the Pierce BCA assay ThermoFisher to ensure protein content was similar between all treatment wells.

Analysis of results was performed using Wave software Agilent Technologies. The rates of basal glycolysis, max glycolysis, glycolytic reserve, basal respiration, max respiration and respiratory reserve were calculated as detailed in Table 1.

Statistical analysis was performed using Prism 6 software GraphPad Software Inc. The current understanding of DC metabolism is largely based on murine studies, which have demonstrated that BMDC strongly upregulate aerobic glycolysis and downregulate oxidative phosphorylation upon TLR stimulation 10 — However, this engagement of Warburg metabolism has been reported to be dependent on NO produced by iNOS in BMDC Human monocyte-derived DC do not typically express iNOS, and therefore it is likely that their metabolic function may differ from that of BMDC A recent study investigating the metabolism of tolerogenic vs.

immunogenic human DC confirmed that LPS-matured DC do not undergo a switch to Warburg metabolism However, the metabolism of human DC was only assessed 24 h after stimulation. As the metabolic changes of BMDC have been observed to occur rapidly after TLR stimulation 12 , it was of interest in the present study to first characterize the metabolic changes of LPS-stimulated human DC over time.

Human DC were seeded into a Seahorse microplate and stimulated with LPS for 0, 1, 3, 6, or 24 h prior to placement into a Seahorse XF24 analyser.

The ECAR of LPS-stimulated DC was highest at 3 and 6 h post-LPS treatment, while the ECAR of DC 24 h post-LPS treatment was observed to be similar to that of unstimulated DC Figure 1A. The glycolytic profile of unstimulated vs. LPS-stimulated DC was assessed, and it was observed that the basal rate of glycolysis was increased in LPS-treated DC at all timepoints Figure 1B.

However, the maximum rate of glycolysis increased in LPS-stimulated DC after 1 h, and peaked at 3—6 h before returning to the unstimulated-DC baseline by 24 h post-LPS Figure 1C. This was reflected in the calculated glycolytic reserve of LPS-stimulated DC, which was greatest in DC 3—6 h post-LPS, whereas at 24 h post-LPS stimulation, DC displayed a glycolytic reserve similar to unstimulated DC Figure 1D.

Therefore, while stimulation of human DC with LPS results in a small but mostly sustained increase in the basal glycolytic rate, the increased glycolytic potential of LPS-stimulated DC appears to be transient, peaking at 3—6 h post-activation. Furthermore, the respiratory profiles of DC appeared to mirror their observed glycolytic activity; DC stimulated with LPS for 6 h displayed the highest OCR, while smaller increases in the OCR of DC 1, 3, and 24 h post-LPS were seen compared to unstimulated DC Figure 1E.

The basal respiratory rate of LPS-stimulated DC was higher than that of unstimulated DC at all timepoints, and was significantly increased in DC 6 h post-LPS treatment Figure 1F. Interestingly, the maximal respiratory rate Figure 1G and respiratory reserve Figure 1H were significantly increased in DC stimulated with LPS for 6 h compared to both unstimulated DC and DC treated with LPS for 1 or 24 h.

Taken together, these data indicate that, unlike murine DC, human DC upregulate both glycolytic metabolism and oxidative phosphorylation upon LPS-stimulation. However, this observed increase in DC metabolism peaks approximately 6 h post-activation.

Figure 1. Determination of the changes in glycolytic metabolism and oxidative phosphorylation over time in LPS-stimulated human DC.

A ECAR measurements over time for each LPS stimulation time-point. Data depicts one representative experiment. E OCR measurements over time for each LPS stimulation time-point.

Human DC were observed to undergo significant metabolic reprogramming during LPS stimulation, characterized by an increased basal rate of glycolysis and oxidative phosphorylation, and a temporary increase in glycolytic and respiratory capacity.

We have previously reported that the plant-derived polyphenols, carnosol and curcumin, inhibit the maturation and immune function of human DC Given that upregulation of cellular metabolism has been reported to be essential for BMDC maturation 10 — 12 , it was of interest to investigate whether treatment with carnosol and curcumin might alter the metabolic reprogramming observed in human DC upon stimulation with LPS.

As the greatest upregulation of glycolysis and oxidative phosphorylation was seen at 6 h post LPS stimulation, this timepoint was chosen to assess the action of carnosol and curcumin on DC metabolism.

Human DC were seeded into a Seahorse microplate and treated with carnosol or curcumin for 6 h prior to stimulation with LPS for a further 6 h.

As previously observed, the ECAR of LPS-stimulated DC was higher than that of unstimulated DC, whereas LPS-stimulated DC previously treated with either carnosol or curcumin displayed an ECAR similar to unstimulated DC Figure 2A. This was reflected in the basal rate of glycolysis, which was significantly reduced in curcumin-treated DC compared to control DC, and a trend toward reduced basal glycolysis was also seen in carnosol-treated DC Figure 2B.

The observed inhibition of glycolysis in carnosol- and curcumin-treated DC was more pronounced in the maximal rate of glycolysis Figure 2C and glycolytic reserve Figure 2D , which were significantly reduced with both polyphenols compared to control DC. The OCR of LPS-stimulated DC was also observed to be greater than that of unstimulated DC, and of carnosol- and curcumin-treated DC Figure 2E.

A slight reduction in the basal respiratory rate was observed in carnosol and curcumin treated DC compared to control DC, but this was not significant Figure 2F.

Conversely, a trend toward an increased maximal respiratory rate Figure 2G and respiratory reserve Figure 2H was observed in LPS-stimulated DC compared to unstimulated DC, which was reduced in DC previously treated with carnosol or curcumin.

Figure 2. Carnosol and curcumin reduce the upregulation of glycolysis and spare respiratory capacity of LPS-stimulated DC. A ECAR measurements over time for each treatment group.

E OCR measurements over time for each treatment group. Previous work from our laboratory has demonstrated that carnosol and curcumin exert extensive immunomodulatory and anti-inflammatory effects in human DC as a result of their upregulation of HO-1 expression The cellular energy sensor and master regulator of catabolic metabolism, AMPK, has been described to suppress glycolytic metabolism and pro-inflammatory responses in BMDC 10 , Furthermore, AMPK has been implicated in the induction of HO-1 expression in other cell types 26 — Thus, it was hypothesized that signaling via AMPK may regulate the inhibition of DC metabolism and induction of HO-1 by the polyphenols carnosol and curcumin.

To determine whether carnosol or curcumin treatment results in activation of AMPK in human DC, DC were treated with carnosol, curcumin, or AICAR, an AMPK agonist, for 1 h.

Phosphorylation, and therefore activation, of AMPK was detected by Western blot. Treatment with AICAR, carnosol and curcumin were all found to increase the activation of AMPK compared to control DC Figures 3A,B.

Figure 3. Carnosol and curcumin inhibit mTOR activity and upregulate HO-1 expression in human DC via activation of AMPK. A Primary human DC were incubated with AICAR 1 mM , carnosol 10 μM , curcumin 10 μM , or a vehicle control for 1 h.

Activation of AMPK was measured by Western blot. Expression of phospho-S6 was determined by Western blot. D Primary human DC were incubated with AICAR —1, μM for 24 h. Expression of HO-1 was detected by Western blot. E Primary human DC were incubated with compound C 5 μM for 1 h prior to treatment with carnosol 10 μM or curcumin 10 μM , or a vehicle control for 24 h.

All blots depict an individual donor and are representative of 3—7 independent experiments. Blots shown are derived from the same gel s ; membranes were first probed for the protein of interest and then re-probed for β-actin as a loading control. Full-length blots are presented in Supplementary Figures 1 , 2.

One of the primary mechanisms by which AMPK regulates cellular metabolism is through inhibition of mTOR, the major promoter of anabolic metabolism which is highly activated in response to LPS stimulation 29 — Having confirmed that carnosol and curcumin can activate AMPK, it was next investigated whether they might inhibit mTOR activity in LPS-stimulated DC.

The ribosomal protein S6 is phosphorylated downstream of mTOR activation and serves as a readout of mTOR activity. DC were treated with compound C, a pharmacological inhibitor of AMPK, for 1 h prior to incubation with carnosol or curcumin for 1 h, followed by stimulation with LPS for 1 h.

The expression of phospho-S6 was detected by Western blot. As expected, stimulation of human DC with LPS resulted in a strong increase in phospho-S6 expression, which was attenuated in DC treated with either carnosol or curcumin. However, the reduction of phospho-S6 expression by carnosol and curcumin was reversed with the addition of compound C Figure 3C.

Although AMPK signaling has been reported to regulate HO-1 expression in other cell types, there have been no reports of AMPK-dependent upregulation of HO-1 in DC. Therefore, to determine whether AMPK activation can upregulate HO-1 expression in human DC, DC were treated with increasing concentrations of AICAR for 24 h, after which the expression of HO-1 was detected by Western blot.

A dose-dependent increase of HO-1 expression was observed in AICAR-treated DC, with the greatest upregulation observed at 0. Following this, the contribution of AMPK to the upregulation of HO-1 by carnosol and curcumin was investigated.

DC were treated with compound C for 1 h prior to treatment with carnosol or curcumin. After 24 h, the expression of HO-1 was detected by Western blot. As previously observed 19 , carnosol and curcumin increased the expression of HO-1 by DC, however, this increase was diminished in the presence of compound C Figure 3E.

HO-1 is a known promoter of tolerogenic DC, as it is highly expressed in immature DC and limits their maturation in response to pro-inflammatory stimuli 21 — Upregulation of HO-1 by carnosol and curcumin was previously observed to limit the maturation of human DC stimulated with LPS As inhibition of AMPK via compound C was found to attenuate the induction of HO-1 by both carnosol and curcumin, it was next investigated whether AMPK inhibition could also reverse the effects of these polyphenols on DC maturation.

Human DC were treated with compound C for 1 h before addition of either carnosol or curcumin for a further 6 h to allow for the upregulation of HO-1 gene transcription and protein translation prior to stimulation with LPS. After 24 h, expression of the maturation markers CD40 and CD83, and co-stimulatory molecules CD80 and CD86 was measured by flow cytometry.

Consistent with previous observations 19 , carnosol treatment significantly reduced expression of CD83 and CD86 by LPS-stimulated DC, with a trend toward reduced CD40 also observed.

However, this effect was attenuated in the presence of compound C Figures 4A,C. Similarly, curcumin treatment significantly reduced the expression of CD40 and CD86 in LPS stimulated DC, with a trend toward reduced CD83 also observed.

Again, this inhibition of surface marker expression by curcumin was reversed with the addition of compound C Figures 4B,D. Treatment of LPS-stimulated DC with compound C alone did not increase the expression of DC surface markers.

Figure 4. Inhibition of AMPK attenuates reduction of DC maturation markers by carnosol and curcumin. Histograms depict expression of maturation markers in DC treated with A carnosol or B curcumin, with or without compound C, compared to controls from one representative experiment. Results shown are mean ± SEM of the measured Mean Fluorescence Intensities MFI , expressed as percentages of the vehicle control.

We have previously reported that treatment of human DC with carnosol or curcumin can maintain the capacity of DC to take up and process antigens after stimulation with LPS Following the observation that inhibition of AMPK signaling via compound C reversed the effects of carnosol and curcumin on the phenotypic maturation of DC, it was next determined whether their effects on functional DC maturation would also be attenuated.

DC were treated with compound C, carnosol or curcumin, and stimulated with LPS as before. After 24 h, DC were incubated with the model antigen DQ-Ovalbumin DQ-Ova for 20 min, and analyzed for antigen uptake by flow cytometry. As expected, stimulation of DC with LPS dramatically reduced their capacity to uptake antigen compared to immature DC.

Furthermore, both carnosol and curcumin treatment maintained the phagocytic capacity of LPS-stimulated DC similar to that of immature DC Figure 5A , as was observed previously However, addition of compound C to carnosol- and curcumin-treated DC significantly abrogated this effect Figures 5B,C.

Treatment of DC with compound C alone did not significantly alter their antigen uptake capacity following LPS stimulation. Taken together, these results confirm that the immunomodulatory effects of carnosol and curcumin on both phenotypic and functional DC maturation are dependent on their activation of AMPK.

Figure 5. Inhibition of AMPK attenuates the increased phagocytic capacity of LPS-stimulated DC treated with carnosol or curcumin.

A Representative dot plots depicting DQ-Ova uptake by DC treated with carnosol, curcumin, and compound C from one representative experiment. Results shown are mean ± SEM percentages of DQ-Ova uptake in control-, carnosol- and curcumin-treated DC, with or without compound C.

Supplementation with immunonutrients such as polyphenols represents a novel strategy to modulate the immune response through dietary intervention. Cellular metabolism has emerged as a major modulator of immune cell function, yet there has been limited study into the effects of polyphenols on immunometabolism.

Here, we have investigated the activity of two plant-derived polyphenols, carnosol and curcumin, on the metabolism and downstream immune function of primary human DC.

We demonstrate that the metabolic reprogramming which occurs in human DC upon LPS stimulation can be modulated by both carnosol and curcumin. We also demonstrate that these polyphenols regulate metabolic signaling through activation of AMPK and an associated inhibition of mTOR activity.

Furthermore, we describe a novel relationship between AMPK signaling and induction of the immunomodulatory enzyme HO-1 by carnosol and curcumin. Together, this data demonstrates that regulation of metabolic signaling and function by naturally-derived polyphenols mediates their ability to promote tolerogenic DC.

While a number of studies have investigated metabolic reprogramming in activated murine DC, studies assessing human DC metabolism are comparatively scarce. LPS-stimulated BMDC have previously been observed to strongly upregulate aerobic glycolysis, and simultaneously downregulate oxidative phosphorylation via the action of iNOS-derived NO 10 — 12 , The results presented here demonstrate that human DC stimulated with LPS upregulate both glycolysis and oxidative phosphorylation within hours of activation.

Furthermore, a transient increase in the glycolytic reserve and spare respiratory capacity SRC of human DC was observed within 6 h post-LPS stimulation, which was absent at 24 h post-LPS. Therefore, it can be ascertained that while human DC also display increased glycolytic metabolism after activation, unlike BMDC, they also upregulate oxidative phosphorylation.

This disparity between murine and human DC is likely a result of their differing expression of iNOS, as human monocyte-derived DC do not readily express iNOS; however, some evidence suggests that certain human DC subsets can express iNOS in vivo , therefore the metabolic profile of these DC may differ from what is observed in vitro Interestingly, a recent study by Basit et al.

Thus, it is important to consider that differences in the metabolism of DC may exist in vivo vs. in vitro , between DC subsets, or due to the type of stimulus employed. Further study of human DC under different conditions is required to delineate the impact of these variables on DC immunometabolism.

Consistent with the results presented here, Malinarich et al. have reported that monocyte-derived human DC matured with LPS are more glycolytic than immature DC, and do not downregulate oxidative phosphorylation However, they also observed a reduced glycolytic reserve and SRC in mature compared to immature DC; a finding which, in fact, agrees with these results, as the metabolism of DC was assessed 24 h after maturation with LPS, by which time the increased glycolytic reserve and SRC observed in this study was absent.

Interestingly, Everts et al. also observed an increase in the SRC of BMDC stimulated with LPS for 1 h, which was mediated by enhanced glycolytic flux into the Kreb's cycle This increased flow of pyruvate into the Kreb's cycle was found to produce citrate necessary for de novo fatty acid synthesis in the maturing DC, providing lipids required to expand the endoplasmic reticulum and Golgi membranes in anticipation of increased protein production Therefore, the transient increase in the glycolytic reserve and SRC of LPS-stimulated DC observed in this study may represent an early adaption of maturing DC to their new immunogenic functions, which is downregulated once adequate cellular remodeling has taken place.

Meanwhile, the mature DC continues to display higher basal rates of glycolysis and oxidative phosphorylation to meet its increased energy demands. Thus, this study expands the current understanding of human DC metabolism, and also underscores the importance of accounting for temporal changes when analyzing the metabolism of immune cells.

The results of this study also further support our previous work which described the anti-inflammatory properties of the polyphenols, carnosol and curcumin, in human DC The upregulation of glycolysis by BMDC in response to LPS has been demonstrated to promote their maturation, cytokine production and activation of T cells 10 — Wang, Kai.

Kai Wang. Journal of Immunology Research, , Vol. ISSN: EISSN: DOI: PMID: Allergic diseases. animal diseases. basic medicine. biochemical phenomena, metabolism, and nutrition. Catechin - immunology.

Cell biology. chemical and pharmacologic phenomena. Curcumin - metabolism. developmental biology. Environmental changes.

Environmental regulations. Environmental stress. Epigallocatechin gallate. Epigenesis, Genetic. food and beverages. Gene expression. Hypersensitivity - immunology. Immune response.

Immune system. Immunologic diseases. Inflammation - genetics. Inflammation - immunology. Immunomodulatory Effects of Dietary Polyphenols. Published Feb Biron, Christine A. Katze, Marcus J. Korth, G. Lynn Law, and Neal Nathanson, 41— Boston: Academic Press, Moticka, Edward J. Moticka, 9— Amsterdam: Elsevier, Hachimura S, Totsuka M, Hosono A.

Immunomodulation by food: impact on gut immunity and immune cell function. Biosci Biotechnol Biochem. Shimizu M. Multifunctions of dietary polyphenols in the regulation of intestinal inflammation. J Food Drug Anal.

Corrêa TAF, Rogero MM, Hassimotto NMA, Lajolo FM. Front Nutr. Published Dec Wu HJ, Wu E. The role of gut microbiota in immune homeostasis and autoimmunity.

Gut Microbes. Burkard M, Leischner C, Lauer UM, Busch C, Venturelli S, Frank J. J Nutr Biochem. Maeda-Yamamoto M. Curr Pharm Des.

Ding S, Jiang H, Fang J. Regulation of Immune Function by Polyphenols. J Immunol Res. Published Apr Neyestani, Tirang R. Rimbach G, Melchin M, Moehring J, Wagner AE. Polyphenols from cocoa and vascular health-a critical review. Int J Mol Sci. Published Nov Pérez-Cano FJ, Massot-Cladera M, Franch A, Castellote C, Castell M.

The effects of cocoa on the immune system. Front Pharmacol. Published Jun 4. Magrone T, Russo MA, Jirillo E. Cocoa and Dark Chocolate Polyphenols: From Biology to Clinical Applications. Front Immunol. Published Jun 9. Malaguarnera L. Influence of Resveratrol on the Immune Response.

Catanzaro M, Corsini E, Rosini M, Racchi M, Lanni C. Immunomodulators Inspired by Nature: A Review on Curcumin and Echinacea. Published Oct Jagetia GC, Aggarwal BB.