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Complement receptor (CR) 3 is a bona fide phagocytic receptor for Borrelia burgdorferi, the causative agent of Lyme borreliosis 1, 2. CR3 is also the receptor for the opsonin iC3b, a final degradation product of the complement component C3 3, 4. The transit of B. burgdorferi through the blood during the dissemination phase exposes the spirochete to serum components, including complement 5. B. burgdorferi contains proteins with anti-complement activities that trigger the inactivation of C3b through the binding of factor H (FH) on the surface of the bacterium 6. This could lead to the accumulation of the opsonin, iC3b, and the enhanced phagocytosis of the spirochete. To test whether the presence of serum would increase the phagocytosis of B. burgdorferi, we incubated bone marrow-derived macrophages (BMMs), generated as described 1, with heat-inactivated (HI; 56 oC, 30 min) or active normal mouse sera (NMS). Phagocytosis protocols were followed according to published reports 1, 2. The presence of 10% HI serum resulted in phagocytosis levels comparable to those in the absence of serum (Fig. ​(Fig.1A,B).1A,B). However, the presence of 10 % NMS enhanced the capacity of macrophages to internalize B. burgdorferi (Fig. ​(Fig.1A).1A). The same effect was observed on RAW264.7 (RAW) cells (Fig. ​(Fig.1C).1C). Thus, the contact of B. burgdorferi with serum components results in their enhanced phagocytic uptake. Next, we assessed whether the increased phagocytic activity associated with the presence of serum was due to C3-derived opsonin generation. We used sera from C3-deficient mice (C3 KO), in which B. burgdorferi dissemination is enhanced compared to WT mice 5. The presence of C3 KO serum abrogated the increased phagocytosis observed in the presence of NMS, both in BMMs (Fig. ​(Fig.1A,B)1A,B) and RAW cells (Fig. ​(Fig.11C).

Phagocytosis of Borrelia burgdorferi, the causative agent of Lyme disease, is mediated partly by the interaction of the spirochete with Complement Receptor (CR) 3. CR3 requires the GPI-anchored protein, CD14, in order to efficiently internalize CR3-B. burgdorferi complexes. GPI-anchored proteins reside in cholesterol-rich membrane microdomains, and through its interaction with partner proteins, help initiate signaling cascades. Here, we investigated the role of CD14 on the internalization of B. burgdorferi mediated by CR3. We show that CR3 partly colocalizes with CD14 in lipid rafts. The use of the cholesterol-sequestering compound methyl-β-cyclodextran completely prevents the internalization of the spirochete in CHO cells that co-express CD14 and CR3, while no effect was observed in CD11b-deficient macrophages. These results show that lipid rafts are required for CR3-dependent, but not independent, phagocytosis of B. burgdorferi. Our results also suggest that CD14 interacts with the C-lectin domain of CR3, favoring the formation of multi-complexes that allow their internalization, and the use of β-glucan, a known ligand for the C-lectin domain of CR3, can compensate for the lack of CD14 in CHO cells that express CR3. These results provide evidence to understand the mechanisms that govern the interaction between CR3 and CD14 during the phagocytosis of B. burgdorferi.