Brucellosis 2003 proceedings - PHIDIAS

Short Oral Communications
Immunology, pathogenesis and host-pathogen interaction
cells transfected with cDNAs encoding TLR-4, TLR-2, CD14 and MD-2, together with
a reporter gene 5 x NF-kB coupled to the firefly luciferase gene. The assay of the kBtranscriptional
activity elicited by LPS from E. coli, Brucella melitensis, Brucella
abortus, Yersinia enterocolítica, and Ochrobactrum anthropi was addressed in this
sytem. LPS from enterobacteriae such as E. coli and Yersinia enterocolítica
produced a significant increase of kB-transcriptional activity on cells expressing TLR-
4, CD14 and MD-2, whereas the remaining LPS failed to do so, even though the cells
coexpressed TLR-2 as well. Attempts to address by ribonuclease protection assay
some of the sets of genes, the expression of which could be influenced by LPS,
showed a strong induction of chemokines (MIP-1a, MIP-1b, IL-8, and RANTES) by
enterobacteria LPS, whereas a weaker expression was observed in response to
Brucella spp and Ochrobactrum anthropi LPS. These data indicate that the different
chemical structures of LPS might explain the induction of distinct proinflammatory
effects through TLR or through other signalling pathway(s).
PO5- Brucella abortus ACTIVATES DENDRITIC CELLS AND SPLENOCYTES TO
SECRETE IL-12P40 AND TNFa VIA DIFFERENT TOLL-LIKE RECEPTORS.
Li-Yun Huang, Julio Aliberti, Cynthia Leifer, and Basil Golding. CBER, FDA, and LPD NIAID,
Bethesda, MD, 20892, USA.
Cattle and humans are susceptible to infection with the Gram-negative
intracellular bacterium Brucella abortus (BA). Heat-killed BA (HKBA) is a strong Th-1
adjuvant and carrier. Previously, we have demonstrated that dendritic cells (DC)
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, MyD88, in the activation of DCs following stimulation by HKBA. Our results
show that HKBA-mediated induction of IL-12p40 and TNFa 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. In serum, TNFa
responses to HKBA were TLR4-independent; however, the response in TLR2
deficient mice was significantly delayed and reduced, although not completely
abolished. Interestingly, the IL-12p40 response to HKBA, as well as the upregulation
of CD40, CD80, and CD86 in DC, is both TLR2- and TLR4-independent, suggesting
that other TLR may mediate HKBA recognition in these APC. Studies using reporter
cell lines confirmed the results seen with knockout mice, namely TLR2, but not TLR4,
can mediate cellular activation by HKBA. In addition, HEK293 cells, which do not
respond to HKBA, were made responsive by transfecting TLR2, but not TLR1, TLR4,
TLR6 or TLR9. Taken together, our data demonstrate that MyD88-dependent
pathways are crucial for DC activation by HKBA and that TLR2 plays a role in TNFa,
but not IL-12p40 pathways activated by this microbial product. Ongoing studies are
aimed at identifying the possible molecular partners for DC recognition of HKBA.
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Brucellosis 2003 International Research Conference