Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8−/−) or heterozygous KO (Fut8+/−) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/− mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/− mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/− mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/− mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation. α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8−/−) or heterozygous KO (Fut8+/−) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/− mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/− mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/− mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/− mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation. Core fucosylation is catalyzed explicitly by α1,6-fucosyltransferase (Fut8) that transfers a fucose residue from GDP-fucose onto the innermost asparagine-linked GlcNAc through an α1,6-linkage in mammals (1García-García A. Ceballos-Laita L. Serna S. Artschwager R. Reichardt N.C. Corzana F. et al.Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase.Nat. Commun. 2020; 11: 973Crossref PubMed Scopus (39) Google Scholar). The biosynthesis of core fucosylation demands donor substrate GDP-fucose, which can be synthesized by two distinct pathways: the de novo pathway and the salvage pathway (Fig. 1A) (2Tu Z. Lin Y.N. Lin C.H. Development of fucosyltransferase and fucosidase inhibitors.Chem. Soc. Rev. 2013; 42: 4459-4475Crossref PubMed Scopus (18) Google Scholar). Under normal conditions, the de novo pathway produces up to 90% of GDP-fucose. When this pathway is disrupted, the salvage pathway compensates for the loss of GDP-fucose (3Adhikari E. Liu Q. Burton C. Mockabee-Macias A. Lester D.K. Lau E. L-fucose, a sugary regulator of antitumor immunity and immunotherapies.Mol. Carcinog. 2022; 61: 439-453Crossref PubMed Scopus (14) Google Scholar, 4Feichtinger R.G. Hüllen A. Koller A. Kotzot D. Grote V. Rapp E. et al.A spoonful of L-fucose-an efficient therapy for GFUS-CDG, a new glycosylation disorder.EMBO Mol. Med. 2021; 13e14332Crossref Scopus (8) Google Scholar). l-Fucose is a six-deoxy hexose monosaccharide that is abundantly present in plants and seaweed (5Staudacher E. Altmann F. Wilson I.B. März L. Fucose in N-glycans: from plant to man.Biochim. Biophys. Acta. 1999; 1473: 216-236Crossref PubMed Scopus (206) Google Scholar, 6Citkowska A. Szekalska M. Winnicka K. Possibilities of fucoidan utilization in the development of pharmaceutical dosage forms.Mar. Drugs. 2019; 17: 458Crossref PubMed Scopus (0) Google Scholar). In mammals, free l-fucose originating from dietary sources or the lysosomal catabolism of glycoproteins can directly serve as a substrate for GDP-fucose synthesis via the salvage pathway (4Feichtinger R.G. Hüllen A. Koller A. Kotzot D. Grote V. Rapp E. et al.A spoonful of L-fucose-an efficient therapy for GFUS-CDG, a new glycosylation disorder.EMBO Mol. Med. 2021; 13e14332Crossref Scopus (8) Google Scholar, 7Wang Y. Huang D. Chen K.Y. Cui M. Wang W. Huang X. et al.Fucosylation deficiency in mice leads to colitis and adenocarcinoma.Gastroenterology. 2017; 152: 193-205.e10Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The produced GDP-fucose is subsequently transported into the lumen of the Golgi apparatus through the GDP-fucose transporter and subjected as the donor substrate for fucosyltransferases, such as Fut8, that catalyzes core fucosylation (Fig. 1A) (8Moriwaki K. Noda K. Nakagawa T. Asahi M. Yoshihara H. Taniguchi N. et al.A high expression of GDP-fucose transporter in hepatocellular carcinoma is a key factor for increases in fucosylation.Glycobiology. 2007; 17: 1311-1320Crossref PubMed Scopus (71) Google Scholar). Therefore, exogenous l-fucose potentially enhances fucosylation levels. The variety of core fucosylation is intimately involved in the pathophysiological processes of numerous diseases, including pulmonary emphysema (9Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. et al.Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15791-15796Crossref PubMed Scopus (365) Google Scholar), schizophrenia (10Fukuda T. Hashimoto H. Okayasu N. Kameyama A. Onogi H. Nakagawasai O. et al.Alpha1,6-fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.J. Biol. Chem. 2011; 286: 18434-18443Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), cancers such as hepatocellular carcinoma (11Wang Y. Fukuda T. Isaji T. Lu J. Im S. Hang Q. et al.Loss of α1,6-fucosyltransferase inhibits chemical-induced hepatocellular carcinoma and tumorigenesis by down-regulating several cell signaling pathways.FASEB J. 2015; 29: 3217-3227Crossref PubMed Scopus (74) Google Scholar), non–small cell lung cancer (12Chen C.Y. Jan Y.H. Juan Y.H. Yang C.J. Huang M.S. Yu C.J. et al.Fucosyltransferase 8 as a functional regulator of nonsmall cell lung cancer.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 630-635Crossref PubMed Scopus (203) Google Scholar), pancreatic carcinoma (13Liang C. Fukuda T. Isaji T. Duan C. Song W. Wang Y. et al.α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as to cancer stemness features in pancreatic carcinoma.Biochim. Biophys. Acta Gen. Subj. 2021; 1865129870Crossref PubMed Scopus (10) Google Scholar), and antibody-dependent cellular cytotoxicity (14Larsen M.D. de Graaf E.L. Sonneveld M.E. Plomp H.R. Nouta J. Hoepel W. et al.Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.Science. 2021; 371eabc8378Crossref PubMed Scopus (207) Google Scholar, 15Sun Y. Li X. Wang T. Li W. Core fucosylation regulates the function of pre-BCR, BCR and IgG in humoral immunity.Front. Immunol. 2022; 13844427Google Scholar). It has been well established that neuroinflammation is a normal immune response resulting from numerous pathological injuries, including ischemia, toxins, infection, and trauma in the central nervous system (CNS) (16Leng F. Edison P. Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here?.Nat. Rev. Neurol. 2021; 17: 157-172Crossref PubMed Scopus (1003) Google Scholar). Neuroinflammation cascades primarily depend on the activation of microglial cells, which are the CNS-resident innate immune cells stemming from the yolk sac macrophages (17Rebelo A.L. Gubinelli F. Roost P. Jan C. Brouillet E. Van Camp N. et al.Complete spatial characterisation of N-glycosylation upon striatal neuroinflammation in the rodent brain.J. Neuroinflammation. 2021; 18: 116Crossref PubMed Scopus (20) Google Scholar, 18Gomez Perdiguero E. Klapproth K. Schulz C. Busch K. Azzoni E. Crozet L. et al.Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1514) Google Scholar, 19Prinz M. Jung S. Priller J. Microglia biology: one century of evolving concepts.Cell. 2019; 179: 292-311Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). In addition, upon pathological stimuli or neuronal insults, microglia secrete proper concentrations of immune mediators, that is, interleukin-6 (IL-6), IL-1β, nitric oxide, and tumor necrosis factor-alpha to coordinate neuroinflammation (19Prinz M. Jung S. Priller J. Microglia biology: one century of evolving concepts.Cell. 2019; 179: 292-311Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar, 20Xu X. Hu P. Ma Y. Tong L. Wang D. Wu Y. et al.Identification of a pro-elongation effect of diallyl disulfide, a major organosulfur compound in garlic oil, on microglial process.J. Nutr. Biochem. 2020; 78108323Crossref PubMed Scopus (11) Google Scholar). Among numerous proinflammatory cytokines, IL-6 is the most pivotal cytokine for acute-phase responses (14Larsen M.D. de Graaf E.L. Sonneveld M.E. Plomp H.R. Nouta J. Hoepel W. et al.Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.Science. 2021; 371eabc8378Crossref PubMed Scopus (207) Google Scholar). It can be produced by various types of cells, including microglia, astrocytes, neurons, macrophages, and B cells (21Hayashi M. Takai J. Yu L. Motohashi H. Moriguchi T. Yamamoto M. Whole-body in vivo monitoring of inflammatory diseases exploiting human interleukin 6-luciferase transgenic mice.Mol. Cell. Biol. 2015; 35: 3590-3601Crossref PubMed Scopus (29) Google Scholar, 22Sanchis P. Fernández-Gayol O. Vizueta J. Comes G. Canal C. Escrig A. et al.Microglial cell-derived interleukin-6 influences behavior and inflammatory response in the brain following traumatic brain injury.Glia. 2020; 68: 999-1016Crossref PubMed Scopus (20) Google Scholar). IL-6 is a multifunctional and pleiotropic cytokine and is associated with multiple sclerosis (23Maimone D. Guazzi G.C. Annunziata P. IL-6 detection in multiple sclerosis brain.J. Neurol. Sci. 1997; 146: 59-65Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), experimental autoimmune encephalomyelitis (24Sanchis P. Fernández-Gayol O. Comes G. Escrig A. Giralt M. Palmiter R.D. et al.Interleukin-6 derived from the central nervous system may influence the pathogenesis of experimental autoimmune encephalomyelitis in a cell-dependent manner.Cells. 2020; 9: 330Crossref PubMed Google Scholar), perioperative neurocognitive disorders (25Hu J. Feng X. Valdearcos M. Lutrin D. Uchida Y. Koliwad S.K. et al.Interleukin-6 is both necessary and sufficient to produce perioperative neurocognitive disorder in mice.Br. J. Anaesth. 2018; 120: 537-545Abstract Full Text Full Text PDF PubMed Google Scholar), and depression (26Kelly K.M. Smith J.A. Mezuk B. Depression and interleukin-6 signaling: a Mendelian Randomization study.Brain Behav. Immun. 2021; 95: 106-114Crossref PubMed Scopus (12) Google Scholar). In the CNS, proinflammatory IL-6 signaling is mainly mediated via trans-signaling with soluble IL-6 receptor (sIL-6R) that subsequently constitutes a complex with glycoprotein 130 kDa (gp130) and activates downstream Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway (27Escrig A. Canal C. Sanchis P. Fernández-Gayol O. Montilla A. Comes G. et al.IL-6 trans-signaling in the brain influences the behavioral and physio-pathological phenotype of the Tg2576 and 3xTgAD mouse models of Alzheimer's disease.Brain Behav. Immun. 2019; 82: 145-159Crossref PubMed Scopus (21) Google Scholar, 28Campbell I.L. Erta M. Lim S.L. Frausto R. May U. Rose-John S. et al.Trans-signaling is a dominant mechanism for the pathogenic actions of interleukin-6 in the brain.J. Neurosci. 2014; 34: 2503-2513Crossref PubMed Scopus (175) Google Scholar). Previous studies have confirmed that many types of cytokine and immune receptors, such as transforming growth factor beta 1 receptor (9Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. et al.Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15791-15796Crossref PubMed Scopus (365) Google Scholar), B-cell receptor (15Sun Y. Li X. Wang T. Li W. Core fucosylation regulates the function of pre-BCR, BCR and IgG in humoral immunity.Front. Immunol. 2022; 13844427Google Scholar), T-cell receptor (15Sun Y. Li X. Wang T. Li W. Core fucosylation regulates the function of pre-BCR, BCR and IgG in humoral immunity.Front. Immunol. 2022; 13844427Google Scholar, 29Fujii H. Shinzaki S. Iijima H. Wakamatsu K. Iwamoto C. Sobajima T. et al.Core fucosylation on T cells, required for activation of T-cell receptor signaling and induction of colitis in mice, is increased in patients with inflammatory bowel disease.Gastroenterology. 2016; 150: 1620-1632Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), integrin α3β1 (30Zhao Y. Itoh S. Wang X. Isaji T. Miyoshi E. Kariya Y. et al.Deletion of core fucosylation on alpha3beta1 integrin down-regulates its functions.J. Biol. Chem. 2006; 281: 38343-38350Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and epidermal growth factor receptor (31Wang X. Gu J. Ihara H. Miyoshi E. Honke K. Taniguchi N. Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling.J. Biol. Chem. 2006; 281: 2572-2577Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), contain core fucosylation that differently regulates their biological functions. Fut8 homozygous KO (Fut8−/−) mice exhibit a schizophrenia-like phenotype with a decrease in working memory (10Fukuda T. Hashimoto H. Okayasu N. Kameyama A. Onogi H. Nakagawasai O. et al.Alpha1,6-fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.J. Biol. Chem. 2011; 286: 18434-18443Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and long-term potentiation (32Gu W. Fukuda T. Isaji T. Hang Q. Lee H.H. Sakai S. et al.Loss of α1,6-fucosyltransferase decreases hippocampal long term potentiation: IMPLICATIONS FOR CORE FUCOSYLATION IN THE REGULATION OF AMPA RECEPTOR HETEROMERIZATION AND CELLULAR SIGNALING.J. Biol. Chem. 2015; 290: 17566-17575Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Curiously, patients suffered from a complete loss of core fucosylation because of biallelic Fut8 mutations (33Ng B.G. Xu G. Chandy N. Steyermark J. Shinde D.N. Radtke K. et al.Biallelic mutations in FUT8 cause a congenital disorder of glycosylation with defective fucosylation.Am. J. Hum. Genet. 2018; 102: 188-195Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) that showed growth retardation, and severe developmental and neurological impairment, which were quite similar to the abnormal phenotypes observed in the Fut8−/− mice (9Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. et al.Dysregulation of TGF-beta1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15791-15796Crossref PubMed Scopus (365) Google Scholar, 10Fukuda T. Hashimoto H. Okayasu N. Kameyama A. Onogi H. Nakagawasai O. et al.Alpha1,6-fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.J. Biol. Chem. 2011; 286: 18434-18443Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). While the pathophysiology of schizophrenia has not yet been fully elucidated, several studies suggest that neuroinflammation leading to glial dysfunction could contribute to the pathogenesis of schizophrenia (34Howes O.D. McCutcheon R. Inflammation and the neural diathesis-stress hypothesis of schizophrenia: a reconceptualization.Transl. Psychiatry. 2017; 7e1024Crossref Scopus (172) Google Scholar, 35Pasternak O. Kubicki M. Shenton M.E. In vivo imaging of neuroinflammation in schizophrenia.Schizophr. Res. 2016; 173: 200-212Crossref PubMed Scopus (99) Google Scholar). Our previous study revealed that core fucosylation negatively regulated the sensitivity of glia to inflammatory stimuli (36Lu X. Zhang D. Shoji H. Duan C. Zhang G. Isaji T. et al.Deficiency of α1,6-fucosyltransferase promotes neuroinflammation by increasing the sensitivity of glial cells to inflammatory mediators.Biochim. Biophys. Acta Gen. Subj. 2019; 1863: 598-608Crossref PubMed Scopus (0) Google Scholar). The initial activation status of glial cells in the neuroinflammation induced by lipopolysaccharide (LPS) was significantly enhanced in the Fut8−/− mice, compared with WT (Fut8+/+) mice. Notably, the degree of neuroinflammation in the Fut8 heterozygous KO (Fut8+/−) mice ranged between that of Fut8+/+ and Fut8−/− mice, presumably because of haploinsufficiency of Fut8. Consistently, the Fut8+/− mice exhibited greater sensitivity to cigarette smoke–induced emphysema than the Fut8+/+ mice (37Gao C. Maeno T. Ota F. Ueno M. Korekane H. Takamatsu S. et al.Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: implication of aberrant transforming growth factor-β signaling and matrix metalloproteinase gene expression.J. Biol. Chem. 2012; 287: 16699-16708Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In the present study, we explore the importance of core fucosylation and its participation in neuroinflammation, utilizing the bacterial artificial chromosome (BAC)–based human IL-6 gene (hIL6)–driven firefly luciferase reporter transgenic mice that we previously generated (21Hayashi M. Takai J. Yu L. Motohashi H. Moriguchi T. Yamamoto M. Whole-body in vivo monitoring of inflammatory diseases exploiting human interleukin 6-luciferase transgenic mice.Mol. Cell. Biol. 2015; 35: 3590-3601Crossref PubMed Scopus (29) Google Scholar). After crossbreeding the hIL6-BAC-Luc mice with the Fut8+/− mice, we obtained the Fut8::hIL6-Luc compound transgenic mice. Using this mouse strain, we quantitatively monitored the neuroinflammation induced by LPS. We found that the Fut8+/−::hIL6-Luc mice showed a higher neuroinflammatory response than Fut8+/+::hIL6-Luc transgenic mice. Exogenous l-fucose ameliorated the LPS-induced neuroinflammatory responses, including glial cell activation and several cytokine expressions in the Fut8+/− mice. Considering that l-fucose is a natural and nontoxic food ingredient, such as seaweed (38Choi S.S. Lynch B.S. Baldwin N. Dakoulas E.W. Roy S. Moore C. et al.Safety evaluation of the human-identical milk monosaccharide, l-fucose.Regul. Toxicol. Pharmacol. 2015; 72: 39-48Crossref PubMed Scopus (26) Google Scholar), the present study proposes its potential utility for the treatment or prevention of neuroinflammation. It has been known that the Fut8+/− mice exhibit a reduced amount of Fut8 and consequently diminish the core fucosylation level compared with the Fut8+/+ control mice. To investigate whether exogenous l-fucose improves the core fucosylation level in Fut8+/− mice, we fed the mice by oral gavage with different doses of l-fucose twice a day for 2 weeks, as shown in Figure 1B. Expectedly, the expression levels of core fucosylation in brain tissues detected by lectin blotting with Lens culinaris agglutinin (LCA), which preferentially recognizes core fucose (39Matsumura K. Higashida K. Ishida H. Hata Y. Yamamoto K. Shigeta M. et al.Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose.J. Biol. Chem. 2007; 282: 15700-15708Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), were lower in Fut8+/− mice than in Fut8+/+ mice (Fig. 1C). Similarly, the expression levels of Fut8 protein were also lower in Fut8+/− mice (Fig. 1C). The decreased levels of core fucosylation in the Fut8+/− mice were significantly rescued by l-fucose administration at 12 or 36 mg/day (Fig. 1C). Consistently, the results obtained from mass spectrometry (MS) analysis (Fig. S1) also showed that the levels of major N-glycans containing core fucose in the hippocampus of Fut8+/− mice were lower than that in Fut8+/+ mice (Fig. 1D). As anticipated, the decreased levels of core fucosylation in the hippocampus were partially rescued by exogenous l-fucose (Fig. 1D). Furthermore, HPLC separation of nucleotide sugars demonstrated that the levels of GDP-fucose increased after l-fucose administration at 12 or 36 mg/day (Fig. S2). Surprisingly, the level of GDP-fucose was decreased in the Fut8+/− mice compared with in Fut8+/+ mice. These results demonstrate that exogenous l-fucose can increase the modification of core fucosylation in the brain tissues in vivo. Next, we evaluated the therapeutic efficacy of l-fucose against neuroinflammation via the inflammation-monitoring system, namely whole-body in vivo monitoring employing the hIL6-BAC-Luc transgenic system (WIM-6 system), which can evaluate different levels of inflammatory responses by examining the intensities of luciferase luminescence (21Hayashi M. Takai J. Yu L. Motohashi H. Moriguchi T. Yamamoto M. Whole-body in vivo monitoring of inflammatory diseases exploiting human interleukin 6-luciferase transgenic mice.Mol. Cell. Biol. 2015; 35: 3590-3601Crossref PubMed Scopus (29) Google Scholar). The luciferase luminescence was analyzed using in vivo imaging system (IVIS) 4 h after the LPS treatment. The IVIS results showed that the luciferase luminescence in brain tissues was markedly increased in Fut8+/−::hIL6-Luc mice than that of Fut8+/+::hIL6-Luc mice (Fig. 2, A and B). Very interestingly, the 14-day constant l-fucose pretreatment (Fig. 1B) dramatically reduced the bioluminescence (Fig. 2, A and B). Subsequently, we also performed an ex vivo imaging to ask whether l-fucose diminishes the LPS-induced neuroinflammation. Consistent with the IVIS results, the ex vivo results showed a partial reduction of the hIL6-Luc luminescence upon l-fucose administration (Fig. 2, C and D). These results (Fig. 2, C and D) suggest that the effects of l-fucose were nondose responsive. The exact reason for this phenomenon needs further clarification. We hypothesize that Fut8 may preferentially utilize the GDP-fucose originating from the exogenous l-fucose (40Sosicka P. Ng B.G. Pepi L.E. Shajahan A. Wong M. Scott D.A. et al.Origin of cytoplasmic GDP-fucose determines its contribution to glycosylation reactions.J. Cell Biol. 2022; 221e202205038Crossref PubMed Scopus (5) Google Scholar), the 12 mg/day l-fucose dosage might reach a saturation effect. In contrast, the 36 mg/day l-fucose dosage produced more GDP-fucose (Fig. S2), potentially exceeding the capacity for GDP-fucose utilization by Fut8 enzyme in Fut8+/− mice. This surplus could be utilized by other fucosyltransferases to modify N-glycan antennae. In addition, a higher dose of l-fucose might alter the sugar metabolism. These together may lead to some unexpected outcomes. The underlying mechanisms require further elucidation. These results indicate that l-fucose can ameliorate the neuroinflammation induced by LPS. To further explore the efficacy of l-fucose administration on neuroinflammation, we examined expression levels of several cytokines and mediators related to inflammation. We administered the different doses (0.5, 1, and 2 mg/kg) of LPS as an inflammatory stimulus to the Fut8::hIL6-Luc mice. RT–PCR and real-time PCR assays revealed that the LPS administration induced the IL-6 and luciferase mRNA expression dose-dependently in the Fut8+/− and Fut8+/+ mice (Fig. 3, A–C). The induced IL-6 and luciferase mRNA expression were significantly higher in the Fut8+/− mice than in the Fut8+/+ mice (Fig. 3, A–C). Of note, the basal mRNA expression of IL-6 and luciferase in the absence of the LPS stimulus was higher in the Fut8+/− mice than in the Fut8+/+ mice (Fig. 3, A–C). This result is consistent with the previous observation that Fut8−/− mice showed a spontaneous increase in microglial activation in vivo (36Lu X. Zhang D. Shoji H. Duan C. Zhang G. Isaji T. et al.Deficiency of α1,6-fucosyltransferase promotes neuroinflammation by increasing the sensitivity of glial cells to inflammatory mediators.Biochim. Biophys. Acta Gen. Subj. 2019; 1863: 598-608Crossref PubMed Scopus (0) Google Scholar). In the subsequent experiments, we selected the dose at 1 mg/kg of LPS as an inducer of the neuroinflammatory model since both 1 and 2 mg/kg of LPS induced significantly different levels of IL-6 and luciferase mRNA between Fut8+/− and Fut8+/+ mice. Since exogenous l-fucose rescued the core fucosylation level and neuroinflammation in the cerebral tissues of Fut8+/− mice, as described previously, we examined whether l-fucose administration could alleviate the inflammatory responses induced by LPS. Consistent with the IVIS results, the RT–PCR and real-time PCR results showed that the induction of IL-6 and luciferase expression by LPS were much higher in the Fut8+/− mice than in the Fut8+/+ mice. The induction was significantly suppressed by l-fucose administration (Fig. 3, D–F). The results in Figure 3F did not align with the luciferin signals in Figure 2. This discrepancy could be attributed to two factors (1) the nonlinear correlation between the expression levels of luciferase and luciferin signals because luciferase is an enzyme and (2) a compromised blood–brain barrier in Fut8+/− mice, potentially leading to improved penetration of LPS and/or luciferin into brain parenchyma, since the deficiency of Fut8 may suppress the expression of vascular endothelial growth factor receptor-2 and subsequently affect angiogenesis (41Wang X. Fukuda T. Li W. Gao C.X. Kondo A. Matsumoto A. et al.Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice.J. Biochem. 2009; 145: 643-651Crossref PubMed Scopus (38) Google Scholar). Further investigation is needed to elucidate the detailed mechanisms. Given that LPS injection may also increase proinflammatory markers, we further assessed mRNA expression levels of tumor necrosis factor-alpha, IL-1β, and inducible nitric oxide synthase in the brain tissues. Again, the results showed that the expression levels of these proinflammatory cytokines were higher in the Fut8+/− mice than in the Fut8+/+ mice. The induction by LPS was significantly suppressed in an l-fucose dose-dependent manner (Fig. 3, G and H). These results demonstrate that a lower core fucosylation leads to enhanced neuroinflammatory status and higher sensitivity to inflammatory stimulators, which can be corrected by exogenous l-fucose administration. Microglia, which account for approximately 10% of brain cells, play a pivotal role in active immune defense (42Glass C.K. Saijo K. Winner B. Marchetto M.C. Gage F.H. Mechanisms underlying inflammation in neurodegeneration.Cell. 2010; 140: 918-934Abstract Full Text Full Text PDF PubMed Scopus (2688) Google Scholar, 43Kreisel T. Frank M.G. Licht T. Reshef R. Ben-Menachem-Zidon O. Baratta M.V. et al.Dynamic microglial alterations underlie stress-induced depressive-like behavior and suppressed neurogenesis.Mol. Psychiatry. 2014; 19: 699-709Crossref PubMed Scopus (486) Google Scholar). Upon pathogen invasion or inflammatory stimuli, microglia transit to an activated state and generate inflammatory mediators to participate in the immune res