TD-BF01: Innate immunity to microorganisms I. Toll receptors (adapted from Takeuchi, O. et al. (1999) Immunity 11:443; Kawai, T. et al. (1999) Immunity 11:115; Hemmi, H. et al. (2000) Nature 408:740; Muzio, M. et al. (2000) J.Immunol. 164:5998) To fight against microorganisms, vertebrates use an innate immune defense system that normally exists before infection in all individuals and can be activated only minutes after infection. Recognition of microorganisms is due to «Toll» receptors present on cells of the immune system that specifically recognize microorganism components on bacteria, viruses, fungi (lipopolysaccharide LPS, lipoarabinomanan LAM, lipopeptides, peptidoglycan or ADN). Here, we study the mechanisms of action of such Toll receptors, and their role in the innate immune response. Part 1 Human leukocyte sub-population T and B lymphocytes, Th1 and Th2 lymphocytes, monocytes, polynuclears (PMN), dendritic cells (DC), and natural killer cells (NK) were isolated and cultured in vitro in the absence or presence of the stimuli as indicated during 3 hours. Total cell RNA was extracted and «Northern Blot» were performed in order to detect transcripts for Toll receptors 1 to 5 (TLR 1-5) (Figure 1). Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 February 5, 2005 1
Figure 1 : TLR1 to 5 expression in different cell types of the immune system. Question 1. What can be concluded regarding the expression of Toll receptors by these different cell types? Why were some cell types treated with PHA or LPS? What is the action of PHA or LPS treatment on Toll receptor expression? Part 2 In order to study in vivo the role of Toll 2 and 4 (TLR2 and TLR4), wild-type (wild-type), TLR2 deficient (TLR2-/-) or TLR4 deficient (TLR4-/-) mouse strains received high doses of LPS (such doses normally induce a toxic shock in the wild-type mouse strain leading to death), and survival was followed (Figure 2). Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 February 5, 2005 2
Figure 2 : Mice received a 1 mg LPS injection intraperitonally. Mortality was followed for 6 days. Question 2. What can you conclude from this experiment? Macrophages from wild-type, TLR2-/- or TLR4-/- mice were cultured in the absence or presence of IFNγ, and treated with 1 ng/ml LPS derived from Salmonella minnesota or lipid A derived from E. coli, during 24 hours. IL-6, nitric oxid (NO 2 - ) TNF-α production was measured in the culture supernatants (Figure 3). Figure 3 : (ND : not detected) Question 3. Why were IL-6, TNF-α and nitric oxide concentrations measured? What is the role of these molecules during the innate immune response? Question 4. What is the role of IFNγ? Question 5. What can be concluded on the respective role of TLR2 and TLR4 receptors in the response to LPS and lipid A? The response to wall components of gram positive bacteria (Staphylococcus aureus, Corynebacterium diphteriae and Nocardia coeliaca) was next studied. Macrophages from wild-type, TLR2-/- or TLR4-/- mice were cultures for 24 hours in the absence or in the Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 February 5, 2005 3
Figure 4 : presence of increasing concentrations of gram + bacterial wall components. TNF-α production is measured in culture supernatants (Figure 4). Question 6. Comment the results of this experiment. What conclusions can you draw regarding Toll receptor specificity? TLR9, another Toll receptor was later identified. The authors studied the in vivo role of this receptor, by using TLR9 deficient mice (TLR9-/-). Macrophages of wild-type (wild type) or TLR9-/- mice were cultured in the absence or in the presence of IFNγ, and treated with bacterial DNA (CpG ODN), LPS or peptidoglycan (PGN). TNF-α, IL-6 and IL-12 production was measured in culture supernatants (Figure 5). Survival of TLR9-/- mice in response to injection of a high dose of bacterial DNA was also evaluated (Figure 6). Figure 5 : Figure 6 : Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 February 5, 2005 4
Question 7. Describe the results obtained in these experiments. What new finding can be drawn by these two experiments? Part 3 Then, the authors identified the intracellular molecular mechanisms involved during TLR2 and TLR4 activation. Macrophages from wild-type, TLR2-/- or TLR4-/- mice were treated during 20 minutes with 1 ng/ml of LPS from S. minnesota (LPS) or 10 µg/ml of PGN from S. aureus. Cells were lyzed. Lysates were immunoprecipitated with an anti-irak1 antibody and kinase activity of IRAK protein was measured in vitro (Auto). In parallel, an anti-irak Western blot was performed (WB) (Figure 7A et B). Activation of NF-κB in nuclear extracts was then determined at different times after LPS or PGN treatment, by performing a gel-shift assay (Figure 7C et D). Question 8. Describe the gel shift assay technique. Why was it necessary to perform the anti-irak Western blot? What conclusion can be drawn from results shown in Figure 7? Authors described that MyD88 deficient mice do not respond to LPS, nor to any bacterial compound, such as PGN, lipoproteins or hypomethylated bacterial DNA. Thus, macrophages from a MyD88 deficient mouse, whether in the presence of LPS or lipid A, do not produce IL-6, TNF-α, or nitric oxide. Macrophages from wild-type or MyD88-/- mice were treated for 10, 20 and 60 minutes with 2 µg/ml of lipid A. Cells were then lyzed and lysates were immunoprecipitated with an anti- IRAK antibody and IRAK kinase activity was measured in vitro (Auto). In parallel, an anti- IRAK Western blot was performed (WB) (Figure 8). Figure 7 : (C et D): nuclear extracts were incubated with a probe containing a binding-site for NF-κB. Inducible complexes containing NF-κB are indicated by arrows. Figure 8 : Question 9. Based on your knowledge and on the results of the experiments above, recapitulate the transduction pathway of Toll receptors (see Figure 9 and Figure 10). Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 February 5, 2005 5
Figure 9 : Aderem, A., and Ulevitch, R. J. (2000) Nature 406:782. Figure 10 : Imler, J.-L., and Hoffmann, J. A. (2003) Nature Immunol. 4:105. Institut Pasteur in Ho Chi Minh City, Vietnam, January 24 February 5, 2005 6