Results II Mice in vivo

1. Fig. 1. TLR2 and TLR4 are strongly up-regulated on both monocytes and neutrophils of patients (n=36) with severe melioidosis compared to healthy controls (n=32). Fig. 3. Reduced lung inflammation in TLR2 KO mice 72 hour after infection. Representative lung histology of wildtype, TLR2 KO and TLR4 KO mice at 72 hours after inoculation with B. pseudomallei, showing significantly less inflammation, pleuritis, peribronchial inflammation, oedema, endothelialitis and necrosis in the TLR2 KO mice compared to WT and TLR4 KO mice. The insets are representative pictures of immunostaining for granulocytes, showing dense granulocytic infiltrations and confirming reduced inflammation and granulocyte influx in theTLR2 KO mice. Magnification, x20. Wildtype TLR2 KO TLR4 KO Detrimental role of Toll-like receptor (TLR)-2 in Gram-negative sepsis caused by Burkholderia pseudomallei  Wiersinga WJ*, Wieland CW*, Dessing MC*, Leendertse M*, Cheng AC‡, Limmathurotsaku D§, Chieraku W§, de Vos AF*, Florquin S†, Woods DE¶, Dondorp AM§, White N§, Day NP§, Peacock SJ§, van der Poll T* *Center for Infection and Immunity Amsterdam and †Department of Pathology, AMC, Amsterdam, the Netherlands, ‡Menzies School of Health Research, Darwin, Australia, §Wellcome Trust, Bangkok, Thailand, ¶Department of Microbiology, University of Calgary, Canada Results I Patients • Patients with culture proven melioidosis demonstrated increased expression of TLR1, TLR2 and TLR4 on the cell surface of circulating monocytes and granulocytes (FACS analysis, p < 0.05-0.001) and increased TLR1, TLR2, TLR4, TLR5 and TLR10 mRNA levels in peripheral blood cells (p < 0.001) when compared with health controls. Fig. 5. Decreased B. pseudomallei outgrowth in the organs of TLR2 KO (A,B), but not TLR4 KO mice (C,D). The amount of B. pseudomallei CFU’s in the lungs and blood of WT (open bars), TLR2 KO (dashed bars) and TLR4 KO (closed bars) mice are depicted at 24, 48 and 72h after inoculation with B. pseudomallei. Data are mean ± SEM. N = 8 per group. * P < 0.05; ***P<0.0001. Results II Mice in vivo • Relative to wild type mice, TLR2 gene deficient mice displayed a markedly improved host defense during experimental melioidosis induced by intranasal inoculation of B. pseudomallei, as reflected by decreased bacterial loads in their lungs (p < 0.0005) and blood (p < 0.05), lower TNFα and IL-10 concentrations (p < 0.05), reduced lung inflammation (PA score; p < 0.001) and a strong survival advantage (p <0 .001). Background Toll-like receptors • Toll-like receptors (TLRs) play an essential role in host defense against microorganisms by virtue of their capacity to detect pathogens and initiate the immune response. • TLR2 is seen as the most important receptor for Gram-positive bacteria, while TLR4 is regarded as the Gram-negative TLR. Melioidosis • Melioidosis is a severe infection caused by the Gram-negative bacterium Burkholderia pseudomallei that is endemic in South-East Asia and is characterized by pneumonia, multiple abscesses and high mortality. Aim • To characterize the expression and function of TLRs in septic melioidosis. • Surprisingly, TLR4 gene deficient mice were indistinguishable from wild type mice in this model with respect to bacterial outgrowth and survival. Methods Patient study • 36 patients with sepsis caused by B. pseudomallei and 32 healthy controls were enrolled at the Sapprasittiprasong Hospital, Ubon Radchathani, Thailand. Mice in vitro studies • Whole blood and alveolar macrophages of WT and TLR2 and TLR4 KO mice were stimulated ex vivo with HK B. pseudomallei and LPS. Mice in vivo studies • Wildtype and TLR2 and TLR4 KO C57BL/6 mice were inoculated with 500 CFU B. pseudomallei intranasally and sacrificed at 24, 48 and 72 hrs. Mice in vitro • Blood and alveolar macrophages obtained from TLR2 or TLR4 deficient mice released less TNFα than blood and alveolar macrophages from wild type mice upon stimulation with B. pseudomallei in vitro (p < 0.05). Conclusions • The expression of a whole repertoire of TLRs is upregulated in leukocytes of patients with septic melioidosis. • Although both TLR2 and TLR4 contribute to cellular responsiveness to B.pseudomallei in vitro, only TLR2 impacts on the immune response of the intact host in vivo. • Inhibition of TLR2 may be a novel treatment strategy in melioidosis. Fig. 2. Membrane TLR2 and TLR4 are required for responsiveness of alveolar macrophages and whole blood to B. pseudomallei. Isolated alveolar macrophages and whole blood of WT, TLR2 and TLR4 KO mice (n=8 per group) were incubated with medium (control), LPS (250 ng/ml) or heat-killed B. pseudomallei (target:effector ratio 1:10) for 16 hours before TNFα was measured. * p < 0.05; **p < 0.001; ***p < 0.0001. Fig. 4. Enhanced survival in TLR2 KO mice. Survival after intranasal inoculation with B. pseudomallei in WT (closed squares), TLR2 KO mice (A, open circles) and TLR4 KO mice (B, open circles). * P < 0.0001 vs WT mice. N = 12 per group. For additional information please contact: Joost Wiersinga, MD Center for Infection and Immunity Amsterdam Academic Medical Center, University of Amsterdam Meibergdreef 9, room G2-132 1105 AZ Amsterdam, the Netherlands Email: w.j.wiersinga@amc.uva.nl