Dectin-1-Mediated DC-SIGN Recruitment to Contact Sites.

At host-pathogen contact sites with , Dectin-1 activates pro-inflammatory signaling, while DC-SIGN promotes adhesion to the fungal surface. We observed that Dectin-1 and DC-SIGN collaborate to enhance capture/retention of under fluid shear culture conditions. Therefore, we devised a cellular model system wherein we could investigate the interaction between these two receptors during the earliest stages of host-pathogen interaction. In cells expressing both receptors, DC-SIGN was quickly recruited to contact sites (103.15% increase) but Dectin-1 did not similarly accumulate. Once inside the contact site, FRAP studies revealed a strong reduction in lateral mobility of DC-SIGN (but not Dectin-1), consistent with DC-SIGN engaging in multivalent adhesive binding interactions with cell wall mannoprotein ligands. Interestingly, in the absence of Dectin-1 co-expression, DC-SIGN recruitment to the contact was much poorer-only 35.04%. These data suggested that Dectin-1 promotes the active recruitment of DC-SIGN to the contact site. We proposed that Dectin-1 signaling activates the RHOA pathway, leading to actomyosin contractility that promotes DC-SIGN recruitment, perhaps via the formation of a centripetal actomyosin flow (AMF) directed into the contact site. Indeed, RHOA pathway inhibitors significantly reduced Dectin-1-associated DC-SIGN recruitment to the contact site. We used agent-based modeling to predict DC-SIGN transport kinetics with ("Directed + Brownian") and without ("Brownian") the hypothesized actomyosin flow-mediated transport. The Directed + Brownian transport model predicted a DC-SIGN contact site recruitment (106.64%), similar to that we observed experimentally under receptor co-expression. Brownian diffusive transport alone predicted contact site DC-SIGN recruitment of only 55.60%. However, this value was similar to experimentally observed DC-SIGN recruitment in cells without Dectin-1 or expressing Dectin-1 but treated with RHOA inhibitor, suggesting that it accurately predicted DC-SIGN recruitment when a contact site AMF would not be generated. TIRF microscopy of nascent cell contacts on glucan-coated glass revealed Dectin-1-dependent DC-SIGN and F-actin (LifeAct) recruitment kinetics to early stage contact site membranes. DC-SIGN entry followed F-actin with a temporal lag of 8.35 ± 4.57 s, but this correlation was disrupted by treatment with RHOA inhibitor. Thus, computational and experimental evidence provides support for the existence of a Dectin-1/RHOA-dependent AMF that produces a force to drive DC-SIGN recruitment to pathogen contact sites, resulting in improved pathogen capture and retention by immunocytes. These data suggest that the rapid collaborative response of Dectin-1 and DC-SIGN in early contact sties might be important for the efficient acquisition of yeast under flow conditions, such as those that prevail in circulation or mucocutaneous sites of infection.