The Influence of Ketogenic Diets on Psoriasiform-Like Skin Inflammation

Nutrition is considered an essential factor in the management of inflammatory skin diseases such as psoriasis (Barrea et al., 2016Barrea L. Nappi F. Di Somma C. Savanelli M.C. Falco A. Balato A. et al.Environmental risk factors in psoriasis: the point of view of the nutritionistInt.J Environ Res Public Health. 2016; 13: 743Crossref Scopus (62) Google Scholar, Ford et al., 2018Ford A.R. Siegel M. Bagel J. Cordoro K.M. Garg A. Gottlieb A. et al.Dietary recommendations for adults with psoriasis or psoriatic arthritis from the Medical Board of the National Psoriasis Foundation.JAMA Dermatol. 2018; 154: 934-950Crossref PubMed Scopus (56) Google Scholar). A recent study identified saturated fatty acid (FA) intake as a significant risk factor for exacerbation of psoriasis, independent of adipocytokine levels, fat mass, and glucose homeostasis (Herbert et al., 2018Herbert D. Franz S. Popkova Y. Anderegg U. Schiller J. Schwede K. et al.High-fat diet exacerbates early psoriatic skin inflammation independent of obesity: saturated fatty acids as key players.J Invest Dermatol. 2018; 138: 1999-2009Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In line with this observation, another study showed that in mice fed a high-fat diet (60% of kcal from fat) in which there was still a substantial amount of carbohydrates (20% of kcal), T-cells accumulated in the skin and promoted psoriasis via IL-17A production (Nakamizo et al., 2017Nakamizo S. Honda T. Adachi A. Nagatake T. Kunisawa J. Kitoh A. et al.High fat diet exacerbates murine psoriatic dermatitis by increasing the number of IL-17-producing γδ T cells.Sci Rep. 2017; 7: 14076Crossref PubMed Scopus (36) Google Scholar). Ketogenic diet (KD; 75–80% kcal from fat, 5–10% kcal from carbohydrate, and 15–25% kcal from protein) increases ketone bodies such as beta-hydroxybutyrate, reduces blood glucose levels (Vidali et al., 2015Vidali S. Aminzadeh S. Lambert B. Rutherford T. Sperl W. Kofler B. et al.Mitochondria: the ketogenic diet-A metabolism-based therapy.Int J Biochem Cell Biol. 2015; 63: 55-59Crossref PubMed Scopus (86) Google Scholar) and can act anti-inflammatorily (Dupuis et al., 2015Dupuis N. Curatolo N. Benoist J.F. Auvin S. Ketogenic diet exhibits anti-inflammatory properties.Epilepsia. 2015; 56: e95-e98Crossref PubMed Scopus (96) Google Scholar, Nandivada et al., 2016Nandivada P. Fell G.L. Pan A.H. Nose V. Ling P.R. Bistrian B.R. et al.Eucaloric ketogenic diet reduces hypoglycemia and inflammation in mice with endotoxemia.Lipids. 2016; 51: 703-714Crossref PubMed Scopus (24) Google Scholar, Youm et al., 2015Youm Y.H. Nguyen K.Y. Grant R.W. Goldberg E.L. Bodogai M. Kim D. et al.The ketone metabolite beta-hydroxybutyrate blocks NLRP3 inflammasome-mediated inflammatory disease.Nat Med. 2015; 21: 263-269Crossref PubMed Scopus (804) Google Scholar). In humans, medium-chain triglyceride (MCT) rich KD induces higher beta-hydroxybutyrate levels compared to long-chain triglycerides (LCT), and MCTs also exert a more significant anti-inflammatory effect (Geng et al., 2016Geng S. Zhu W. Xie C. Li X. Wu J. Liang Z. et al.Medium-chain triglyceride ameliorates insulin resistance and inflammation in high fat diet-induced obese mice.Eur J Nutr. 2016; 55: 931-940Crossref PubMed Scopus (45) Google Scholar, Kono et al., 2004Kono H. Fujii H. Asakawa M. Maki A. Amemiya H. Hirai Y. et al.Medium-chain triglycerides enhance secretory IgA expression in rat intestine after administration of endotoxin.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G1081-G1089Crossref PubMed Scopus (38) Google Scholar, Papada et al., 2014Papada E. Kaliora A.C. Gioxari A. Papalois A. Forbes A. Anti-inflammatory effect of elemental diets with different fat composition in experimental colitis.Br J Nutr. 2014; 111: 1213-1220Crossref PubMed Scopus (21) Google Scholar). Because of its anti-inflammatory and anti-angiogenic effects, omega-3 (ω-3) rich nutrition is also of high interest for dietary interventions in inflammatory skin diseases (Calder, 2013Calder P.C. Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology?.Br J Clin Pharmacol. 2013; 75: 645-662Crossref PubMed Scopus (705) Google Scholar). Because KDs differ in composition from classic high-fat diets, and no studies have been performed to evaluate the effect of KDs on psoriasis, we aimed to elucidate the impact of different KDs, alone or supplemented with ω-3 FA on imiquimod (IMQ)-induced psoriasiform-inflammation in mice. After an adaption phase, all KDs (Supplementary Figure S1 and Table S1) caused significant elevation of blood beta-hydroxybutyrate levels compared to a standard diet (SD; Supplementary Figure S2a). Interestingly, blood glucose levels were increased in the LCT/MCT ± ω-3 cohorts on day 1 of IMQ treatment and dropped significantly on day 4, compared to the SD ± ω-3 control group (Supplementary Figure S2b). KD feeding (± ω-3) resulted in significant weight gains compared to the SD group (Supplementary Figure S2c). All animal experiments were approved by the local animal ethics committee according to Austrian legislation on animal experiments (20901-TVG/118/6-2017). Induction of psoriasis via IMQ (Locker et al., 2018Locker F. Vidali S. Holub B.S. Stockinger J. Brunner S.M. Ebner S. et al.Lack of galanin receptor 3 alleviates psoriasis by altering vascularization, immune cell infiltration, and cytokine expression.J Invest Dermatol. 2018; 138: 199-207Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar) led to increased erythema in mice fed with an LCT/MCT diet compared to SD-fed mice on day 4 (Figure 1a). The LCT/MCT group showed a trend toward increased cumulative disease severity compared to the SD group (8.2 ± 1.1 vs. 6.2 ± 0.7; P = 0.07), which was significant in comparison with the LCT group (8.2 ± 1.1 vs. 5.9 ± 1.7; P < 0.05) after 4 days of IMQ treatment (Figure 1d). Inflammatory patterns were not affected by ω-3 supplementation, as evidenced by the high cumulative disease severity in the LCT/MCT + ω-3 group on day 4 compared to the SD + ω-3 group (Figure 1d, e, and l). Interestingly the LCT/MCT KD ± ω-3 FA supplementation induced skin inflammation, even in Vaseline-treated mice, as illustrated by the significant increase in erythema, scaling, and thickening (Figure 1g–i) as well as the cumulative disease severity score compared to Vaseline-treated SD-fed mice on day 4 (Figure 1j and k). Because the SD ± ω-3 FA-fed mice showed no signs of skin inflammation we cannot exclude that the LCT/MCT ± ω-3 FA diets can also affect normal skin. LCT/MCT and LCT groups showed significantly higher skin levels of IL-17A mRNA than the SD group, after IMQ treatment (Figure 2a). Supplementation with ω-3 FA changed this pattern only slightly because the LCT + ω-3 group expressed higher levels of IL-17A mRNA compared to the SD + ω-3 control after IMQ treatment (Figure 2a). However, addition of ω-3 FAs to the LCT and LCT/MCT diets resulted in a significant increase of IL-1β mRNA compared with the SD + ω-3 group (Figure 2b), after IMQ treatment. The level of IL-12b mRNA was significantly lower only in the skin of the LCT/MCT + ω-3 group compared with the SD + ω-3 control (Supplementary Figure S4). Interestingly, all skin biopsies of Vaseline-treated mice fed KDs with or without ω-3 FAs showed increased levels of IL-17A mRNA (Figure 2a). In contrast, IL-1β mRNA was upregulated only when ω-3 FAs were added to the LCT/MCT-KD compared with SD + ω-3 (Figure 2b). LCT/MCT + ω-3 skin showed significantly higher neutrophil-derived myeloperoxidase levels in comparison with SD + ω-3 skin upon psoriasis induction and Vaseline treatment (Figure 2c). Accordingly, we detected a significantly higher number of NIMP-R14+ neutrophils in LCT/MCT + ω-3 skin compared to SD + ω-3 skin (P < 0.05) (Figure 2d and Supplementary Figure S3). CD31-positive vessels in skin sections did not differ between LCT KD, LCT/MCT KD, and SD-fed mice (± ω-3) 4 days after IMQ treatment. However, there was a significant difference between the SD and SD + ω-3 groups (83.2 ± 12.3 vs. 71.9 ± 6.0, P = 0.012) (Figure 2e). Vegf mRNA levels were not altered after 4 days of IMQ treatment (Figure 2f). We detected higher levels of IFN-γ (Figure 2g) in the blood of the LCT/MCT group than the SD group on day 4. Also, IL-6 levels tended to be higher in plasma of the LCT/MCT group compared to the SD group (Figure 2h) (P = 0.087). However, plasma IL-1β levels, among others, remained unaffected (Figure 2i and Supplementary Figure S5). Our data indicate that the low carbohydrate content in the KD might protect from the previously reported pro-inflammatory effects of saturated FA intake (Herbert et al., 2018Herbert D. Franz S. Popkova Y. Anderegg U. Schiller J. Schwede K. et al.High-fat diet exacerbates early psoriatic skin inflammation independent of obesity: saturated fatty acids as key players.J Invest Dermatol. 2018; 138: 1999-2009Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). However, the FA composition of KD needs to be taken in consideration, as supplementation with MCTs and ω-3 FAs exacerbate IMQ-induced psoriasis, as well as elicit basal skin alterations. As the IMQ model is transient future research should focus on the long-term effects of KDs in a chronic psoriasiform-like skin inflammation model such as the K14-IL-17A(ind/+) model, described by Croxford et al., 2014Croxford A.L. Karbach S. Kurschus F.C. Wörtge S. Nikolaev A. Yogev N. et al.IL-6 regulates neutrophil microabscess formation in IL-17A-driven psoriasiform lesions.J Invest Dermatol. 2014; 134: 728-735Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar. Datasets related to this article can be found at https://doi.org/10.17632/mnb67rtr2v.2, an open-source online data repository hosted at Mendeley Data. Felix Locker: https://orcid.org/0000-0003-3487-362X Julia Leitner: https://orcid.org/0000-0001-7287-2316 Sepideh Aminzadeh-Gohari: https://orcid.org/0000-0003-3976-4297 Daniela D. Weber: https://orcid.org/0000-0002-9825-6674 Philippe Sanio: https://orcid.org/0000-0002-2884-7268 Andreas Koller: https://orcid.org/0000-0001-6108-8411 René Günther Feichtinger: https://orcid.org/0000-0002-4215-8258 Richard Weiss: https://orcid.org/0000-0003-3185-7253 Barbara Kofler: https://orcid.org/0000-0002-1198-4776 Roland Lang: https://orcid.org/0000-0002-9750-0188 The authors declare no conflict of interest. The study was supported by the Paracelsus Medical University Research Fund ( PMU-FFF E-16/24/125-LAL ), TRANSMIT project ( EU H2020-MSCA-ITN-2016-722605 ) and the Austrian Research Promotion Agency ( 822782 /THERAPEP). Conceptualization, FL, RL, BK; Methodology, FL, BK; Investigation, FL, JL, SA, PS, AK, DDW, RGF, RW; Formal Analysis, FL; Visualization, FL; Writing – Original Draft, FL, RL, BK; Writing – Review & Editing, FL, RL, BK; Funding. Acquisition, FL, RL, BK; Supervision, RL, BK Six-week-old male C57BL/6N mice were kept under specific pathogen-free conditions and provided with food and water ad libitum. Mice receiving ketogenic diets (KDs) were gradually adapted to the diets over 10 days. All diets were supplemented with the same amounts of antioxidants, vitamins, and trace elements. The compositions of the experimental diets are provided in Supplementary Table S1. For the LCT/MCT KD, a 1.5:1 mixture of caprylic acid and capric acid was used as the MCT component. The diets were custom made by an animal food manufacturer (Sniff, Soest, Germany). The standard diet and KDs were also supplemented with 6% marine fish oil (18% eicosapentaenoic acid and 12% docoahexaenoic acid), resulting in 1.1% total eicosapentaenoic acid and 0.7% total docoahexaenoic acid. The amount of ω-3 was chosen according to a previous study (Yanai et al., 2014), and these diets are indicated as + ω-3 throughout the manuscript. KD feeding was initiated 7 days before IMQ treatment following adaption phase to ensure appropriate ketosis at the time of induction of psoriasiform-like inflammation (Supplementary Figure S1). One day before IMQ treatment, mice were shaved with an electric razor and depilated with depilation creme (Veet Sensitive, Reckitt Benckiser, Austria), and given a daily topical dose of 62.5 mg of commercially available 5% IMQ cream (Aldara; 3M Pharmaceuticals, Neuss, Germany) for 3 or 6 consecutive days (equivalent to a 3.125 mg/d of the active compound; Locker et al., 2018Locker F. Vidali S. Holub B.S. Stockinger J. Brunner S.M. Ebner S. et al.Lack of galanin receptor 3 alleviates psoriasis by altering vascularization, immune cell infiltration, and cytokine expression.J Invest Dermatol. 2018; 138: 199-207Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Control mice were similarly treated and given Vaseline cream (Fagron, Barsbüttel, Germany). For evaluation of skin inflammation, macroscopic images were recorded daily. On day 4 or 7 following the start of IMQ treatment, the mice were killed, and the dorsal skin was removed. To score the severity of inflammation in the dorsal skin, we used the scoring system based on the clinical Psoriasis Area and Severity Index (Locker et al., 2018Locker F. Vidali S. Holub B.S. Stockinger J. Brunner S.M. Ebner S. et al.Lack of galanin receptor 3 alleviates psoriasis by altering vascularization, immune cell infiltration, and cytokine expression.J Invest Dermatol. 2018; 138: 199-207Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar, van der Fits et al., 2009van der Fits L. Mourits S. Voerman J.S. Kant M. Boon L. Laman J.D. et al.Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.J Immunol. 2009; 182: 5836-5845Crossref PubMed Scopus (1166) Google Scholar). Erythema, scaling, and thickening (skin wave formation because of rete ridges) were scored independently on a scale from 0 to 4 on macroscopic images of the skin in a blinded fashion. The cumulative score (erythema + scaling + thickening) served as an indicator of the severity of inflammation (scale 0–12). The order in which individual mice were picked for IMQ treatment was random, and all diet specifications were encrypted to ensure blinding of the assessors. All experiments were approved by the local animal ethics committee according to Austrian legislation on animal experiments (20901-TVG/118/6-2017). Bodyweight was recorded, and blood glucose and beta-hydroxybutyrate levels were monitored using enzyme-based methods (Precision Xceed, Abbott Laboratories, North Chicago, IL) at the indicated time points (Supplementary Figure S1, green stars). Before killing, mice received an injection of 10 μl/g of anesthetic mix (Ketamine 20.5 mg/ml, Xylazine 5.4 mg/ml, Acepromazine 270 μg/ml) in saline solution. After checking for the absence of reflexes from the paw, heart puncture was performed, and blood was collected in specific tubes (Microtainer PST with lithium heparin; Becton Dickinson, Franklin Lakes, NJ). Plasma was collected and snap-frozen in liquid nitrogen. All frozen samples were stored at –80 °C until utilization. Total RNA from mouse skin biopsies was isolated using TRI-Reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer's instructions. A total of 2 μg of RNA was digested with DNase (Ambion, Frankfurt, Germany) and subsequently reverse-transcribed to cDNA using Maxima Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) and random hexamer primers according to the manufacturer's protocol. For quantitative real-time reverse transcriptase–PCR, iQ SYBR green supermix (Bio-Rad, Munich, Germany) was used according to the manufacturer's protocol. The relative expression of each gene (ΔCt) was calculated as differences between the threshold cycle (Ct) of the gene of interest and Ct of the murine housekeeping gene ribosomal protein L4. Primers and PCR conditions are provided in Supplementary Tables S2 and S3 (Locker et al., 2018Locker F. Vidali S. Holub B.S. Stockinger J. Brunner S.M. Ebner S. et al.Lack of galanin receptor 3 alleviates psoriasis by altering vascularization, immune cell infiltration, and cytokine expression.J Invest Dermatol. 2018; 138: 199-207Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). For immunohistochemical studies, formalin-fixed, paraffin-embedded skin sections were stained with the following antibodies: mCD31 (rabbit anti-mouse monoclonal, 1:75; Abcam, Cambridge, United Kingdom) and mNIMP-R14 (rat anti-mouse monoclonal, 1:100; Abcam). Antibodies were diluted in Dako antibody diluent with background-reducing components (Dako, Hamburg, Germany). For antigen retrieval, the sections were immersed overnight in 1 mM EDTA, 10 mM TRIS-HCl, pH 9.0, at 60 °C. The following secondary antibodies were used: labeled anti-rabbit Polymer EnVision immunoglobulins/horseradish peroxidase (ready to use; Dako, Glostrup, Denmark), rabbit anti-rat immunoglobulins/horseradish peroxidase (1:50 in antibody diluent; Dako). Quantification of immunohistological staining was performed by two independent researchers. Two sections per mouse were evaluated. Numbers of cells positive for CD31 (vessels) were counted per high-power field (400 × magnification), and four high-power fields were counted per section. The numbers of cells positive for NIMP-R14 (neutrophils) were counted in a total area of 9.4 mm2 (all cells in two high-power fields with 100 × magnification). Myeloperoxidase activity was assessed as an index of neutrophil infiltration according to Schierwagen (Schierwagen et al., 1990Schierwagen C. Bylund-Fellenius A.C. Lundberg C. Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity.J Pharmacol Methods. 1990; 23: 179-186Crossref PubMed Scopus (350) Google Scholar). Skin biopsies (8 mm in diameter) were punched out, weighed, snap-frozen in liquid nitrogen and processed as previously described (Schierwagen et al., 1990Schierwagen C. Bylund-Fellenius A.C. Lundberg C. Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity.J Pharmacol Methods. 1990; 23: 179-186Crossref PubMed Scopus (350) Google Scholar). Myeloperoxidase activity was determined by comparing the absorbance of the samples with a standard curve. The results were expressed as units myeloperoxidase per ml per biopsy. Mouse plasma levels of CCL2, GM-CSF, IFN-β, IFN-γ, IL-1α, IL-1β, IL-6, IL-10, IL-12 (p70), IL-17A, IL-23, IL-27, and TNF-α, were measured with the LEGENDplex mouse inflammation panel (BioLegend, San Diego, CA) according to the manufacturer's instructions. Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Prism, La Jolla, CA). Three independent experiments were performed. One-way analysis of variance and Tukey's multiple comparisons were applied for all data sets except for mouse scoring and quantitative real-time reverse transcriptase–PCR data, for which the non-parametric Kruskal-Wallis test was used, and all groups were compared to each other. In Figure 1a–d the statistical analysis between the groups was performed for the respective days. For the analysis of metabolic parameters Two-way ANOVA and Tukey's multiple comparisons were used, P < 0.05 was considered as statistically significant, and P values between 0.05–0.1 were considered to represent a trend.Supplementary Figure S2Metabolic parameters. (a, d) Blood beta-hydroxybutyrate, (b, e) blood glucose, and (c, f) body weight of (a, b; n = 18) imiquimod and (d–f; n = 7–9 [except day 7, n = 6]) vehicle-treated mouse groups over time. Values are depicted as mean ± standard error of mean. Statistical signficance was assessed using two-way analysis of variance and Dunnett's multiple comparison: *P < 0.05, **P < 0.01, ***P < 0.001. d, day; LCT, long-chain triglycerides; MCT, medium-chain triglycerides; SD, standard diet; ω-3, omega-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Neutrophil abundances in the skin. Representative images of skin sections stained with anti-NIMP-R14 antibody (brown) of Imiquimod-treated mice from day 4, Bar = 40 μm. LCT, long-chain triglycerides; MCT, medium-chain triglycerides; SD, standard diet; ω-3, omega-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S4mRNA expression analysis of skin sections of Imiquimod-treated mice on day 4. Values are depicted as mean ± standard error of the mean (n = 10–12). Statistical significance was assessed using non-parametric Kruskal-Wallis test. *P < 0.05. LCT, long-chain triglycerides; MCT, medium-chain triglycerides; SD, standard diet; ω-3, omega-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S5Plasma cytokine levels. Plasma cytokine levels in vehicle (n = 8–9) and IMQ-treated (n = 10–12) mice on day 4. Values represent mean ± standard error of the mean. Statistical significance was assessed using one-way analysis of variance and Dunnett's multiple comparisons were used. IMQ, imiquimod; LCT, long-chain triglycerides; MCT, medium-chain triglycerides; SD, standard diet; ω-3, omega-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Main components of the dietsIngredientsSDLCTLCT/MCTSD + ω 3 FALCT + ω-3 FALCT/MCT + ω-3 FACrude protein1Presented as % weight per kilogram of diet.16.113.913.916.113.913.9LCT1Presented as % weight per kilogram of diet.7.066.841.91.060.835.9MCT21Presented as % weight per kilogram of diet.00150015MCT101Presented as % weight per kilogram of diet.00100010Sugar1Presented as % weight per kilogram of diet.61.01.06.01.01.0Starch1Presented as % weight per kilogram of diet.51.21.01.051.21.01.0Crude fiber1Presented as % weight per kilogram of diet.10.09.99.910.09.99.9Crude ash1Presented as % weight per kilogram of diet.4.54.44.44.54.44.4Fish oil1,Presented as % weight per kilogram of diet.2Fish oil contained ∼18% C20:5 (EPA) and ∼12% C22:6 (DHA).———6.06.06.0BHT1Presented as % weight per kilogram of diet.0.0150.0150.0150.0150.0150.015Vitamin A3Presented as international units per kilogram of diet.150001500015000150001500015000Vitamin D3Presented as international units per kilogram of diet.150015001500150015001500Vitamin E4Presented as mg per kilogram of diet.150150150150150150Vitamin K4Presented as mg per kilogram of diet.202020202020Vitamin C4Presented as mg per kilogram of diet.303030303030Copper4Presented as mg per kilogram of diet.111111111111Abbreviations: BHT, butylated hydroxytoluene; LCT, long-chain triglycerides; MCT, medium-chain triglycerides; SD, standard diet; ω-3 FA, omega-3 fatty acids.1 Presented as % weight per kilogram of diet.2 Fish oil contained ∼18% C20:5 (EPA) and ∼12% C22:6 (DHA).3 Presented as international units per kilogram of diet.4 Presented as mg per kilogram of diet. Open table in a new tab Supplementary Table S2Primers used for expression analysis of the indicated genesGeneForward primer sequenceReverse primer sequencemRPL45′-GTATGGCACTTGGCGGAAGG-3′5′-TGCTCGGAGGGCTCTTTGG-3′mTNF-α5′-GGTCCCCAAAGGGATGAGAA-3′5′-CTCAGCCACTCCAGCTGCTC-3′mIL-12b5′-TCCTGAAGTGTGAAGCACCA-3′5′-GACAGAGACGCCATTCCACA-3′mIL-17A5′-AACCGTTCCACGTCACCCTG-3′5′-GTCCAGCTTTCCCTCCGCAT-3′mIL-225′-AGGTGGTGCCTTTCCTGACCA-3′5′-GCAGGTCCAGTTCCCCAATCG-3′mIL-235′-CCAGCGGGACATATGAATCTACTAA-3′5′-TGCAAGCAGAACTGGCTGTTG-3′mS100A75′-TCTCCCGAGACATGGCCTGAT-3′5′-CTGCCCAAAACGATGTTCCTGA-3′mVEGF5′-ACTGGACCCTGGCTTTACTGCT-3′5′-ATCGGACGGCAGTAGCTTCG-3′mIL-225′-AGGTGGTGCCTTTCCTGACCA-3′5′-GCAGGTCCAGTTCCCCAATCG-3′CCL55′-CAGCAGCAAGTGCTCCAATCTT-3′5′-TTCTTGAACCCACTTCTTCTCTG-3′mCD1635′-GACGACAGATTCAGCGACTT-3′5′-CCGAGGATTTCAGCAAGTCCA-3′mIL-105′-ACAGCCGGGAAGACAATAACT-3′5′-TTCCGATAAGGCTTGGCAAC-3′mFPR25′-GAGACCTCAGCTGGTTGTGCAG-3′5′-GCCCAGCACACCAAGGAAGAA-3′mKeratin 165′-AGCAGGAGATCGCCACCTA-3′5′-AGTGCTGTGAGGAGGAGTGG-3′mIL1-β5′-AAAGACGGCACACCCACCCT-3′5′-CCA GTT GGG GAA CTC TGC AGA C-3′mPPARG25′-GAGACCAACAGCCTGACGGG-3′5′-CACCGCTTCTTTCAAATCTTG-3′ Open table in a new tab Supplementary Table S3PCR cycling conditionsPCR typeCycling conditionsGeneral qPCR95 °C for 5 min, 40 cycles (97 °C for 15 sec, 62 °C for 30 sec, 72 °C for 10 sec), 72 °C for 5 minIL-23p19 qPCR95 °C for 5 min, 40 cycles (95 °C for 20 sec, 65 °C for 30 sec, 72 °C for 10 sec), 72 °C for 5 minAbbreviation: qPCR, quantitative reverse transcriptase in real time. Open table in a new tab Abbreviations: BHT, butylated hydroxytoluene; LCT, long-chain triglycerides; MCT, medium-chain triglycerides; SD, standard diet; ω-3 FA, omega-3 fatty acids. Abbreviation: qPCR, quantitative reverse transcriptase in real time.