title: Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF, unlike IL-12p40 secretion, is Toll-like receptor 2 dependent.
abstract: Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.
authors.affiliation: Laboratory of Plasma Derivatives, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
authors.forename: Li-Yun
authors.initials: LY
authors.lastname: Huang
authors.forename: Julio
authors.initials: J
authors.lastname: Aliberti
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Leifer
authors.forename: David M
authors.initials: DM
authors.lastname: Segal
authors.forename: Alan
authors.initials: A
authors.lastname: Sher
authors.forename: Douglas T
authors.initials: DT
authors.lastname: Golenbock
authors.forename: Basil
authors.initials: B
authors.lastname: Golding
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000502:physiology*; D002004:Brucella Vaccine / Q000008:administration & dosage / Q000276:immunology*; D002003:Brucella abortus / Q000276:immunology*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D006358:Hot Temperature; D006801:Humans; D007274:Injections, Intraperitoneal; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000378:metabolism; D053762:Interleukin-12 Subunit p40; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B / Q000378:metabolism; D021122:Protein Subunits / Q000096:biosynthesis* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D011971:Receptors, Immunologic / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000943:Antigens, Differentiation; D002004:Brucella Vaccine; D053762:Interleukin-12 Subunit p40; C507779:MYD88 protein, human; D008562:Membrane Glycoproteins; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D021122:Protein Subunits; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C495371:TLR9 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12