Therefore, SIGNR1 is widely involved in immune responses to pathogens in cooperation with other PRRs. In this study, we investigated RO4929097 the roles of SIGNR1

in recognizing and inducing cellular responses to zymosan, HK- and live C. albicans. We found that SIGNR1 enhanced Syk-dependent oxidative burst response possibly in cooperation with Dectin-1. We first examined the binding to microbe particles using soluble forms of SIGNR1 and Dectin-1 tagged with an N-terminal Strep-tag II sequence. When tetramers were formed by preincubating with PE-Strep-Tactin at 37°C, soluble SIGNR1 (sSIGNR1) tetramer bound more to the microbes than that at 4°C, although soluble Dectin-1 (sDectin-1) bound equally to HK-C. albicans (Fig. 1A). Based on these observations, tetramers formed at 37°C were used in the subsequent experiments. Although both SIGNR1 and Dectin-1 recognized zymosan, as reported 23, 27, the amount of sSIGNR1 binding was much higher than that of sDectin-1 (Fig. 1B, left panels). Moreover, sDectin-1 bound comparably to zymosan and HK-microbes, but much less to live C. albicans, as reported 27. In contrast, sSIGNR1 equally bound not only to zymosan and HK-C. albicans but also live microbes (Fig. 1B, left panels). Furthermore, the binding of sSIGNR1, but not sDectin-1, was EDTA- and mannan-sensitive (Fig. 1B, right panels and data not shown). Less binding of sDectin-1 to live microbes PF-562271 price was also confirmed by immunofluorescence

microscopy, in which sDectin-1 bound to the surface of killed microbes, but stained mainly budding scars and occasionally showed a spotty staining pattern on live microbes (Fig. 1C). Since oxidative burst is crucial for Mϕ functions in response to microbes, we measured the oxidative burst response using RAW264.7 cells transfected with SIGNR1 cDNA Atorvastatin (RAW-SIGNR1) or control plasmid (RAW-control). Parental RAW264.7 cells lack SIGNR1 expression. First, RAW-SIGNR1 and RAW-control cells were confirmed to express comparable levels of Dectin-1 (Fig. 2A). RAW-SIGNR1 cells showed a markedly higher response than the RAW-control cells (Fig. 2B). Although

this elevated response in the RAW-SIGNR1 cells was partially reduced by depletion of zymosan, and TLR2 ligand, PAM3CSK4 was ineffective in either inducing the response by itself (Fig. 2B) or elevating the response by depleted zymosan (Fig. 2C). Antagonistic anti-TLR2 mAb (T2.5) showed no effect on the oxidative burst of RAW-SIGNR1 to zymosan or depleted zymosan (Fig. 2D). These results implied that SIGNR1 plays a role in the induction of the oxidative burst independently of TLR2, this being consistent with previous reports 13, 14. Considering the role of Dectin-1 in oxidative burst 13, 14, it is possible that SIGNR1 utilizes the Dectin-1-dependent pathway, although both of these lectins can independently recognize zymosan/HK-C. albicans. To confirm this possibility, the effects of various inhibitors were examined in response to HK-C. albicans, since HK-C.