A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway.
The milk pentasaccharide LNFPIII has therapeutic action for metabolic and autoimmune diseases and prolongs transplant survival in mice when presented as a neoglycoconjugate. Within LNFPIII is the Lewisx trisaccharide, expressed by many helminth parasites. In humans, LNFPIII is found in human milk and also known as stage-specific embryonic antigen-1. LNFPIII-NGC drives alternative activation of macrophages and dendritic cells via NFκB activation in a TLR4 dependent mechanism. However, the connection between LNFPIII-NGC activation of APCs, TLR4 signaling and subsequent MAP kinase signaling leading to anti-inflammatory activation of APCs remains unknown. In this study we determined that the innate receptor CD14 was essential for LNFPIII-NGC induction of both ERK and NFkB activation in APCs. Induction of ERK activation by LNFPIII-NGC was completely dependent on CD14/TLR4-Ras-Raf1/TPL2-MEK axis in bone marrow derived dendritic cells (BMDCs). In addition, LNFPIII-NGC preferentially induced the production of Th2 "favoring" chemokines CCL22 and matrix metalloprotease protein-9 in a CD14 dependent manner in BMDCs. In contrast, LNFPIII-NGC induces significantly lower levels of Th1 "favoring" chemokines, MIP1α, MIP1β and MIP-2 compared to levels in LPS stimulated cells. Interestingly, NGC of the identical human milk sugar LNnT, minus the alpha 1-3 linked fucose, failed to activate APCs via TLR4/MD2/CD14 receptor complex, suggesting that the alpha 1-3 linked fucose in LNFPIII and not on LNnT, is required for this process. Using specific chemical inhibitors of the MAPK pathway, we found that LNFPIII-NGC induction of CCL22, MMP9 and IL-10 production was dependent on ERK activation. Over all, this study suggests that LNFPIII-NGC utilizes CD14/TLR4-MAPK (ERK) axis in modulating APC activation to produce anti-inflammatory chemokines and cytokines in a manner distinct from that seen for the pro-inflammatory PAMP LPS. These pathways may explain the in vivo therapeutic effect of LNFPIII-NGC treatment for inflammation based diseases.
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, 30602, United States of America; Department of Microbiology, University of Chicago, Chicago, Illinois, 60637, United States of America.
Smanla
S
Tundup
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, 30602, United States of America.
Leena
L
Srivastava
Department of Biochemistry and Organic Chemistry, Uppsala University, Uppsala, Sweden.
Thomas
T
Norberg
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, 30602, United States of America.
Wendy
W
Watford
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, 30602, United States of America; Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, 30602, United States of America.
Donald
D
Harn
D000606:Amino Sugars / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000494:pharmacology*; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D002460:Cell Line; D054422:Chemokine CCL22 / Q000235:genetics / Q000276:immunology; D054405:Chemokine CCL3 / Q000235:genetics / Q000276:immunology; D054407:Chemokine CCL4 / Q000235:genetics / Q000276:immunology; D054426:Chemokine CXCL2 / Q000235:genetics / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation; D006001:Glycoconjugates / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000276:immunology; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology; D011134:Polysaccharides / Q000737:chemistry / Q000494:pharmacology*; D061251:Primary Cell Culture; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000276:immunology; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000276:immunology; D015398:Signal Transduction; D058408:Th1-Th2 Balance / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology
D000606:Amino Sugars; D000893:Anti-Inflammatory Agents; C515483:Ccl22 protein, mouse; C496534:Ccl3 protein, mouse; C496535:Ccl4 protein, mouse; D054422:Chemokine CCL22; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D054426:Chemokine CXCL2; C404915:Cxcl2 protein, mouse; D006001:Glycoconjugates; D018950:Lipopolysaccharide Receptors; D016328:NF-kappa B; D011134:Polysaccharides; D011518:Proto-Oncogene Proteins; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C434666:lacto-N-fucopentaose III; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; C489843:Map3k8 protein, mouse; D020780:Matrix Metalloproteinase 9