Search

Macrophages are tissue-resident phagocytes that play critical roles in immune response and tissue homeostasis. They have a tremendous capacity to internalize objects of various sizes ranging from nanoscale viral particles to micron sized bacteria and tumor cells. This phenomenon, termed phagocytosis for the uptake of large particles, or endocytosis for the uptake of small particles, is integral to the immune response in multicellular organisms. Macrophages express FcγRs, which are tyrosine kinase receptors that bind IgG on an opsonized target. Binding of IgG Fc domain to the extracellular domain of an FcγR triggers signaling cascades that coordinate internalization of the opsonized target, generation of reactive oxygen species and release of cytokines. FcγR mediated phagocytosis of large particles has been characterized in macrophages and dendritic cells, but an understanding of FcγR endocytosis of small particles is limited. Insight into FcγR endocytosis in macrophages will be useful in the design and targeting of therapeutic antibodies for treatment of cancers and viral infections. Here, ~100nm fluorescent PEGylated liposomes displaying surface biotin antigen and antibiotin IgG2a were used to investigate FcγR trafficking. Internalization of liposomes occurred by FcγR dependent endocytosis with no contributions from macropinocytosis. Rather, liposomes associated with clathrin on the plasma membrane and within the cytoplasm at early time points, demonstrating that the endocytic process involved the participation of clathrin. Internalized IgG2a-liposome complexes were trafficked to the limiting membrane of macropinosomes where IgG was segregated from cargo liposomes in a pH dependent manner. Contrary to trafficking of CSF-1 in macrophages, segregated IgG and liposomes were excluded from the lumen of macropinosomes and did not undergo immediate intraluminal budding, demonstrating that lysosomes did not immediately destroy the immune complex. Segregated IgG was recycled back to the cell surface where it was capable of phagocytosing new biotinylated SRBCs. Although macropinosomes and liposomes formed a multivesicular object, three-color live cell microscopy showed that fluid phase marker movement did not predict movement of liposomes to the lysosome. Lysosomes rapidly internalized luminal content of macropinosomes via piranhalysis (or squidlysis), but interacted with IgG-liposome complexes by kiss-and-run events resulting in early transition of fluid phase marker and delayed transition of liposome cargo into lysosomal compartments. Thus, we show that the macropinosome has a novel role in organizing antigen-antibody segregation in macrophages. The contributions of murine FcγRs (I, IIb, and III) and FcRγ to endocytosis of IgG2aliposomes were delineated using single and multiple receptor knockouts in macrophages. Knockouts were created by means of synthetic gRNAs targeting FcγRs and FcRγ in FLM cells harvested from CRISPR/Cas9 transgenic mice. Analysis of particle uptake showed that FcγRs drive differential internalization of IgG2a-liposomes with binding and uptake of particles heavily dependent on high affinity FcγR I and FcRγ. Knockout of three receptors namely FcγR I, IIb, and III abolished binding and uptake of particles. Analysis of fluid phase uptake in knockout lines showed that FcγR I, III, IIb&III, FcRγ, and TKO were defective for macropinosome formation, whilst FcγR IIb knockouts produced more than twice the number of macropinosomes compared to parental cells. Together, these demonstrate that activating FcγRs may have a novel role in driving constitutive macropinosome formation attenuated by inhibitory FcγR IIb.

Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects granulocytes and causes human granulocytic anaplasmosis (HGA). This bacterium requires cholesterol for host cell infection in vitro and incorporates exogenous cholesterol into its membrane. However, the role of blood cholesterol in HGA in vivo and how A. phagocytophilum acquires cholesterol inside the host cells are still unknown. To understand the role of host cholesterol in A. phagocytophilum infection in vivo, we analyzed the effects of a high-cholesterol diet and reduced apolipoprotein E (apo E) activity on A. phagocytophilum infection in mice. A high-cholesterol diet significantly increased A. phagocytophilum bacteria burden in the spleen, liver, and blood of apoE-deficient (apoE -/-) mice and also induced a significant elevation in the mRNA expression of MIP-2 and a MIP-2 receptor, CXCR2, in the spleen in apoE-/- mice fed a high-cholesterol diet. These results suggest that high blood cholesterol levels facilitate A. phagocytophilum infection and up-regulate a proinflammatory chemokine and its receptor, which may contribute to HGA pathogenesis. In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. It is unknown how A. phagocytophilum, a cholesterol-dependent bacterium, interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in HL-60 cells. Cellular free cholesterol was enriched in A. phagocytophilum inclusions. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL) uptake pathway, rather than de novo cholesterol biosynthesis. The uptake of fluorescence-labeled LDL was enhanced in infected cells. A. phagocytophilum infection up-regulated LDLR expression at both the mRNA and protein levels by stabilizing LDLR mRNA via the 3’ UTR region, but not through activation of the sterol regulatory element binding proteins. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication. Extracellular signal–regulated kinase (ERK) was persistently activated by A. phagocytophilum infection in HL-60 cells, especially at middle-late infection stage, and the ERK activation is required for A. phagocytophilum infection. We demonstrate that Raf-1 is actively phosphorylated by A. phagocytophilum infection and this phsphorylation may activate the ERK. Up-regulation of LDLR mRNA by A. phagocytophilum was inhibited by a MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. Ehrlichia ewingii is the most recently recognized human ehrlichiosis agent and is the only Ehrlichia sp. known to infect neutrophils. In the blood or in ex vivo culture, neutrophils generally have a short life span. In the present study, we investigated the effect of E. ewingii infection on spontaneous apoptosis of neutrophils. E. ewingii infection significantly delayed dog neutrophil apoptosis during ex vivo culture. By using the fluorescent mitochondrial dyes Mitotracker Red 580 and JC-1, we found that E. ewingii infection stabilized mitochondrial integrity by maintaining mitochondrial membrane potential in neutrophils. These results suggest that E. ewingii delays spontaneous apoptosis of neutrophils via stabilization of host cell mitochondria.