Host defense against many invading Gram-negative bacteria (GNB) depends upon innate immune recognition of endotoxin (E), unique, abundant and generally conserved glycolipids of GNB. Optimal TLR4- dependent responses are exquisitely sensitive and robust, but also self-limited. How this is normally achieved and why regulation of TLR4 activation may go awry under various patho-physiological conditions is incompletely known. The most potent inflammatory responses to E require certain canonical structural features of E and ordered protein-E and protein-protein interactions involving the LPS-binding protein (LBP), CD14, MD-2, and Toll-like receptor (TLR) 4. MD-2 plays an essential role in regulation of TLR4 activation by E, by bridging recognition of E, normally initiated by LBP and CD14, to receptor activation or antagonism. Our identification and characterization of monomeric E.MD-2 complexes as potent TLR4 agonists or antagonists, depending on E and/or MD-2 structure, have clearly demonstrated that it is the properties of the E.MD-2 complex, not E alone, that specifies TLR4 activation or receptor occupancy without activation. The unique structural and functional properties of these complexes suggest novel approaches to investigate how endotoxin induces TLR4 activation, how differences in E structure can dictate differences in TLR4 responsiveness, and how these new mechanistic insights may be translated to the development of novel TLR4-directed immunomodulators. Growing evidence of a broader array of molecules that can act as TLR4 agonists or antagonists, including host and/or diet-derived lipids, suggest many other circumstances in which the regulation or dysregulation of MD-2/TLR4 function could be pivotal in host physiology and patho-physiology. We will make use of novel synthetic, biochemical, structural biological, and functional approaches (e.g., metabolically labeled native and synthetic E and lipid A variants, recombinant wild-type and mutant human E-binding proteins and TLR4 ectodomain, monoclonal antibodies, models of airway infection and inflammation) to: 1) Further define the structural properties of ligands that promote binding to MD-2 and the action of ligand.MD-2 complexes as TLR4 agonists or antagonists;2) Define more specifically the structural properties of E.MD-2 complexes that activate TLR4;and 3) Better define in vivo actions in the murine airway of wt and variant E(lipdA).Md-2.
TLR4-dependent cell activation by endotoxin plays a critical role in host response and defense against many Gram-negative bacteria. The ubiquity of endotoxin in the surrounding environment and the apparent existence of other microbe- and host-derived agonists/antagonists of TLR4 suggest an even broader role of TLR4 in health and disease. Better understanding of how endotoxin induces TLR4 activation and how differences in endotoxin structure dictate differences in TLR4 responsiveness is likely to lead to a better understanding of how mammalian hosts, including humans, normally respond effectively against invading Gram-negative bacteria and how certain Gram-negative bacterial pathogens can evade/resist these normally efficient host responses. The new mechanistic insights may also be translated to the development of novel TLR4-directed immuno-modulators whose application would likely include, but not be limited, to Gram-negative bacterial infections.
|Hoppe Parr, Kimberly A; Ha?ina, Suzana; Kilburg-Basnyat, Brita et al. (2017) Modification of sample processing for the Limulus amebocyte lysate assay enhances detection of inflammogenic endotoxin in intact bacteria and organic dust. Innate Immun 23:307-318|
|Vašl, Jožica; Oblak, Alja; Peternelj, Tina T et al. (2016) Molecular Basis of the Functional Differences between Soluble Human Versus Murine MD-2: Role of Val135 in Transfer of Lipopolysaccharide from CD14 to MD-2. J Immunol 196:2309-18|
|Calabrese, Valentina; Cighetti, Roberto; Peri, Francesco (2015) Molecular simplification of lipid A structure: TLR4-modulating cationic and anionic amphiphiles. Mol Immunol 63:153-61|
|Krasity, Benjamin C; Troll, Joshua V; Lehnert, Erik M et al. (2015) Structural and functional features of a developmentally regulated lipopolysaccharide-binding protein. MBio 6:e01193-15|
|Peri, Francesco; Calabrese, Valentina (2014) Toll-like receptor 4 (TLR4) modulation by synthetic and natural compounds: an update. J Med Chem 57:3612-22|
|Gioannini, Theresa L; Teghanemt, Athmane; Zhang, DeSheng et al. (2014) Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins. Immunol Res 59:3-11|
|Cighetti, Roberto; Ciaramelli, Carlotta; Sestito, Stefania Enza et al. (2014) Modulation of CD14 and TLR4·MD-2 activities by a synthetic lipid A mimetic. Chembiochem 15:250-8|
|Guinan, Eva C; Palmer, Christine D; Mancuso, Christy J et al. (2014) Identification of single nucleotide polymorphisms in hematopoietic cell transplant patients affecting early recognition of, and response to, endotoxin. Innate Immun 20:697-711|
|Barker, Jason H; Kaufman, Justin W; Zhang, De-Sheng et al. (2014) Metabolic labeling to characterize the overall composition of Francisella lipid A and LPS grown in broth and in human phagocytes. Innate Immun 20:88-103|
|Peri, Francesco (2013) Clustered carbohydrates in synthetic vaccines. Chem Soc Rev 42:4543-56|
Showing the most recent 10 out of 40 publications