The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors

Abstract

Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cell-wall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-alpha production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-alpha production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.

Figure 1

TLR2 and TLR6 are required for the macrophage TNF-α response to yeast and Gram-positive bacteria. Macrophages transfected with DN-TLR6 or DN-TLR2 were stimulated with the indicated microbes, and the production of TNF-α was measured by flow cytometry. The density plots (Top) correlate the level of DN-TLR expression (GFP, x axis) with the production of TNF-α (y axis). By using the gates indicated in the zymosan-stimulated density plot (dotted rectangles), histograms were generated for TNF-α production by DN-TLR- expressing and nonexpressing cells. Thin lines indicate TNF-α production by macrophages that did not express DN-TLRs. Thick lines indicate TNF-α production by cells expressing DN-TLR6 (Middle) or DN-TLR2 (Bottom). Dotted lines indicate the background level of TNF-α production in unstimulated cells. The y axes indicate relative cell number.

Figure 2

Macrophages use TLR2, TLR4, and TLR6 to induce the production of TNF-α in response to different microbial components. (A) Mock-transfected RAW-TT10 macrophages were stimulated with the indicated doses of peptidoglycan, Pam₃CSK₄ lipopeptide, or LPS, and the production of TNF-α was measured by flow cytometry. The y axis indicates the percent maximal TNF-α production for each stimulus. Maximal TNF-α production was induced by 10 μg/ml peptidoglycan, 100 ng/ml Pam₃CSK₄ lipopeptide, or 10 ng/ml LPS, and these doses were used in B. (B) Macrophages transfected with dominant negative forms of TLR2, TLR4, or TLR6 were stimulated with the indicated microbial components, and the production of TNF-α was measured by flow cytometry. Thin lines indicate TNF-α production by macrophages that did not express DN-TLRs. Thick lines indicate TNF-α production by cells expressing DN-TLR6 (Top), DN-TLR2 (Middle), or DN-TLR4 (Bottom). Dotted lines indicate the background level of TNF-α production in unstimulated cells. The y axes indicate relative cell number.

Figure 3

TLR6 is enriched on macrophage phagosomes and physically associates with TLR2. (A) V5-epitope-tagged TLR6, HA-epitope-tagged TLR2, and HA-epitope-tagged TLR1 were localized by immunofluorescence microscopy in transiently transfected RAW macrophages after the phagocytosis of zymosan or IgG-coated sheep erythrocytes [EIgG (arrows)] for 5 min. (B) CHO cells were transfected with HA-epitope-tagged TLR2 with or without V5-epitope-tagged TLR6 and were stimulated, where indicated, with peptidoglycan (10 μg/ml for 10 min). TLR2 was detected by Western blotting in the cell lysates, and by co-immunoprecipitation (IP) with TLR6. A truncated form of TLR2 (lacking a cytoplasmic tail, HA-TLR2Δ) also was coimmunoprecipitated with TLR6. (C) CHO cells were transfected with V5-epitope-tagged TLR6 and chimeric receptors composed of the extracellular domain of CD4 fused to the transmembrane domain and the cytoplasmic tail of TLR1 (CD4-TLR1), TLR2 (CD4-TLR2), TLR4 (CD4-TLR4), or TLR6 (CD4-TLR6). The CD4-TLR chimeras were detected by Western blotting in the cell lysates, but did not coimmunoprecipitate with TLR6.

Figure 4

TLR6 cooperates with TLR2 in mediating proinflammatory signals. (A) The relative activity of an NF-κB luciferase reporter was assessed in CHO cells transiently expressing the indicated TLR constructs without further stimulation, or after stimulation by the indicated bacterial products. (B) RAW-TT10 macrophages were transfected with the indicated CD4-TLR chimeric constructs, and the production of TNF-α was measured by flow cytometry. The representative density plot shows that expression of CD4-TLR4 results in the production of TNF-α. Histograms were generated by using the gates shown. Thin lines indicate TNF-α production by macrophages that did not express the indicated CD4-TLRs (gate 1). Thick lines indicate TNF-α production by cells expressing CD4-TLRs (gate 2). The y axes of the histograms indicate relative cell number, and the x axes indicate TNF-α production on a log scale identical to that shown (on the y axis) in the density plot.