Macrophages play a critical role in innate immunity by killing invading pathogens and eliminating apoptotic cells. Macrophage ABCA1 expression dampens MyD88-dependent TLR signaling by facilitating FC efflux and reducing plasma membrane lipid rafts12–14
. However, whether ABCA1 expression also impacts macrophage function, such as bacterial killing or chemotaxis, is less clear. In this study, we demonstrate that myeloid cell-specific ABCA1 deficiency renders mice more resistant to Lm infection and macrophage Lm infection down-regulates cholesterol export proteins. We also show that macrophage ABCA1 deletion accelerates macrophage migration toward chemoattractants, which may partially explain the enhanced bacterial killing in MSKO mice. Collectively, our data suggest that deletion of macrophage ABCA1 enhances macrophage function.
Gram-positive, facultative intracellular bacterium Lm is a widely used model of intracellular bacterial infection15
. Bacteria are first internalized into a vacuole, also known as a phagosome. In the vacuoles, Lm secretes the pore-forming toxin Listeriolysin O and phospholipase C, which lyse phagosomal membranes and allow Lm to escape into cytosol. In the cytosol, the bacteria rapidly replicate, and recruit and polymerize host cell actin. The polymerization of actin at one pole of the cell produces energy to propel bacterial entry into neighboring cells. Lm infection stimulates macrophage MyD88-dependent secretion of IL-12 and TNF-α, which stimulates natural killer (NK) cells to generate IFN-γ, enhancing macrophage bactericidal action26, 27
. Mice lacking MyD88, IL-12, or IFN-γ are more susceptible to Lm infection27, 28
, indicating the important role of MyD88-dependent cytokine production in Lm clearance. Since our previous studies established that ABCA1-deficient macrophages are hyper-sensitive to MyD88-dependent TLR stimulation14
, we hypothesized that MSKO mice may have enhanced Lm clearance with increased pro-inflammatory cytokine production due to exaggerated MyD88-dependent TLR response to Lm infection. In support of this hypothesis, we observed significantly better clearance of Lm in MSKO mice. However, the cytokine profile in mice at 36 h or 3 days post infection showed no differences in MSKO mice, suggesting that MyD88-dependent cytokine production induced by Lm infection does not play a significant role in the increased Lm clearance in MSKO mice. Alternatively, increased efficiency of Lm clearance in MSKO mice may have allowed a more rapid return of plasma cytokines to basal levels.
Compared to MSKO mice, WT mice infected with Lm showed marked hepatic triglyceride and cholesterol accumulation. In rodents, infection and inflammation stimulate adipose tissue lipolysis, and increase de novo
hepatic fatty acid and cholesterol synthesis, coupled with suppression of fatty acid oxidation, decreased LDL clearance and conversion of cholesterol to bile acids29
. In our study, the more severe Lm infection in WT vs. MSKO mice may have led to greater adipose tissue lipolysis, resulting in increased hepatic lipogenesis (fatty acid, triglyceride, and cholesterol) and lipid accretion. However, the marked hepatic lipid accumulation in Lm-infected mice resulted in down-regulation of de novo
lipogenic genes (e.g. HMGCoA synthase, SREBP-1c, SCD1 and ACC1), and increased ACAT2 to reduce FC accumulation in liver by conversion of FC to CE (Online Figure I
). The more efficient clearance of Lm in MSKO vs. WT mice reduced the inflammatory state of the liver and completely abrogated the increase in hepatic neutral lipid content ().
Macrophages and neutrophils are two major innate immune cells responsible for Lm killing during the early phase of infection. Our results demonstrate that 3 days post infection, MSKO mice had significantly more monocytes/macrophages and neutrophils in the liver than WT mice, indicating an improved local microenvironment for bacterial clearance. We hypothesized that increased monocytes/macrophages in Lm-infected MSKO liver might result from: 1) decreased hepatic immune cell apoptosis, 2) increased migration of Ly6Chigh
monocytes from bone marrow22
, 3) increased hepatic chemokine production, and/or 4) increased chemotaxis of MSKO macrophages to the liver. Our data indicate that the increased macrophage and neutrophil accumulation in livers of MSKO mice is not due to the decreased apoptosis of hepatic leukocytes or enhanced recruitment of Ly6Chigh
inflammatory monocytes from bone marrow to the bloodstream. We observed a significant elevation in hepatic MCP-1 and MIP-2 production and a marked increase in positive staining for MCP-1 in perivascular leukocyte infiltrates, in MSKO vs. WT mice at 36 h post Lm infection. MIP-2 is a potent neutrophil chemoattractant. Signaling via its receptor CCR2, MCP-1 is a potent monocyte/macrophage chemoattractant and is readily detected in liver and spleen of Lm-infected mice30
. Mice lacking the CCR2 receptor are highly susceptible to Lm infection22, 31
. Thus, increased hepatic MCP-1 and MIP-2 expression and increased MCP-1-producing leukocyte infiltration in MSKO mice may partially explain the more rapidly recruitment of monocytes/macrophages and neutrophils to the foci of infection, leading to a more efficient pathogen clearance. Macrophage MCP-1 production can be induced by WT Lm, but not by heat-killed or Listeriolysin O deficient Lm, even though macrophage TNF-α and IL-12p40 production is similar30, 32
. These results suggest that Lm-induced MCP-1 production requires cytosolic invasion by bacteria, is independent of classic TLR-MyD88 signaling, and likely is mediated via unknown cytosolic receptors. If so, enhanced hepatic production of MCP-1, and perhaps MIP-2, in Lm-infected MSKO mice may result both from up-regulated MyD88-dependent TLR signaling and MyD88-independent cytosolic receptor signaling.
ABCA1 has been implicated in macrophage chemotaxis, although results have been conflicting16, 24
. Using resident peritoneal macrophages, Francone et al16
observed a significant increase in chemotactic migration of Abca1−/−
macrophages, suggesting that ABCA1 expression limits macrophage migration toward chemokines. In contrast, using thioglycollate-elicited peritoneal macrophages, Pagler et al24
found no difference in macrophage migration toward chemokines between ABCA1-deficient and WT macrophages. Instead, they found that Abca1−/−
vs. WT macrophages had increased plasma membrane FC and defective redistribution of sterol to the outer leaflet, which resulted in increased Rac1 signaling and impaired chemotaxis. Using both elicited PMs or BMDMs, we consistently observed a significant increase in chemotaxis of MSKO macrophages in response to MCP-1 and MIP-1α, consistent with Francone's finding. Meanwhile, our in vivo
migration assay further supported enhanced chemotaxis of MSKO vs. WT macrophages. The enhanced chemotaxis in ABCA1-deficient macrophages provides another potential explanation for increased monocyte/macrophage infiltration in Lm-infected MSKO mice, although the underlying mechanism is unknown and still under investigation.
Expression of ABCA1, especially in macrophages, is highly regulated by LXR19
, a nuclear receptor with an established role in lipid metabolism. Interestingly, one function of LXR is to regulate apoptosis inhibitor of macrophages (AIM, also known as SPα) to reduce macrophage apoptosis in response to Lm infection20, 21
. Gram-negative bacteria, or bacterial products such as LPS, down-regulate LXR targeted gene expression related to lipid metabolism25
, although the physiological role of this down-regulation remains unknown. In our current study, we observed that Lm infection, like Gram-negative bacterial infection, markedly reduced expression of ABCA1, ABCG1, apoE, and other LXR-responsive genes, mainly at the post-transcriptional level. Notably, macrophages lacking ABCA1 and/or ABCG1 are pro-inflammatory12–14
. Thus, acute bacterial infection not only activates LXR to up-regulate SPα expression to prevent macrophages from apoptosis, but also lowers cholesterol transporter gene expression to mount a more robust inflammatory response that aids host defense against infections. However, infection may also worsen chronic inflammation-related diseases, such as atherosclerosis, by exaggerating foam cell formation due to down-regulation of cellular lipid export proteins.
In summary, we have shown that ABCA1 deficiency in myeloid cells protects the host against bacterial infection by accelerating macrophage chemotaxis and increasing chemokine expression in local infected tissue. Bacterial infection also down-regulates macrophage LXR-induced cholesterol export proteins. Thus, ABCA1 not only plays a key role in lipid metabolism, but also in innate immunity, suggesting a more diverse functional role for this transporter than originally envisioned. Our study also suggests that diminished myeloid cholesterol efflux enhances macrophage function and facilitates innate immunity against acute pathogen infection.