Environ Res. 2016 Apr;146:173-84. doi: 10.1016/j.envres.2015.12.027.
Validation of research trajectory 1 of an Exposome framework: Exposure to benzo(a)pyrene confers enhanced susceptibility to bacterial infection.
Ryan S. Clarka, Samuel T. Pellomb,c, Burthia Bookerb, Aramandla Rameshc, Tongwen Zhangd, Anil Shankerc, Mark Maguirea , Paul D. Juareze, Patricia Matthews-Juareze, Michael A. Langstonf, Maureen Y. Lichtveldg and Darryl B. Hoodd*
aDepartment of Neuroscience and Pharmacology, Meharry Medical College, Nashville, TN 37208
bDepartment of Microbiology, Meharry Medical College, Nashville, TN 37208
cDepartment of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208
dDivision of Environmental Health Sciences, College of Public Health, The Ohio State University, Columbus, OH 43210
eDepartment of Family and Preventive Medicine, Meharry Medical College, Nashville, TN 37208
fDepartment of Electrical Engineering and Computer Science, University of Tennessee, Knoxville, TN 37996
gDepartment of Global Environmental Health Sciences, School of Public Health & Tropical Medicine, Tulane University, 1440 Canal Street, New Orleans, LA 70112
The exposome provides a framework for understanding elucidation of an uncharacterized molecular mechanism conferring enhanced susceptibility of macrophage membranes to bacterial infection after exposure to the environmental contaminant benzo(a)pyrene, [B(a)P]. The fundamental requirement in activation of macrophage effector functions is the binding of immunoglobulins to Fc receptors. FcγRIIa (CD32a), a member of the Fc family of immunoreceptors with low affinity for immunoglobulin G, has been reported to bind preferentially to IgG within lipid rafts. Previous research suggested that exposure to B(a)P suppressed macrophage effector functions but the molecular mechanisms remained elusive. The goal of this study was to elucidate the mechanism(s) of B(a)P-exposure induced suppression of macrophage function by examining the resultant effects of exposure-induced insult on CD32-lipid raft interactions in the regulation of IgG binding to CD32. The results demonstrate that exposure of macrophages to B(a)P alters their lipid raft integrity by decreasing membrane cholesterol 25% while increasing CD32 into non-lipid raft fractions. This robust diminution in membrane cholesterol and 30% exclusion of CD32 from lipid rafts causes a significant reduction in CD32-mediated IgG binding to suppress essential macrophage effector functions. Such exposures across the lifespan would have the potential to induce an immunosuppressive endophenotypes in vulnerable populations.
KEYWORDS: Benzo(a)pyrene; FcγRII (CD32) antibody; Immune suppression; Lipid rafts; Membrane integrity
Polycyclic aromatic hydrocarbons (PAHs) are a group of potent environmental toxicants formed by incomplete combustion of organic materials. It is well known that the PAH family of global environmental contaminants targets and suppresses virtually every component of cell-mediated and humoral immune response systems.[1-5] The mechanism(s) by which PAH’s modulate this apparent immunosuppression is poorly understood, and previous studies used animal models to evaluate possible mechanistic links. Data from animal studies suggest that AhR ligands such as B(a)P and 2, 3, 7, 8, tetrachloro, dibenzo-p-dioxin (TCDD) suppress immunity by their ability to compromise virtually every stage of lymphocyte development, activation, and effector function [6, 7].
B(a)P is the archetypical member in the family PAHs. We hypothesized that the mechanism for B(a)P exposure-induced suppression of macrophage effector functions occur via disruption of lipid raft architecture thereby causing a decrease in CD32a within lipid rafts. We determined the cellular location of CD32a in the presence and absence of IgG, and examined the effect of B(a)P, MβCD, and nystatin on CD32a-IgG binding. Fig. 1B depicts such an interrogation of the effects of exposure to B(a)P to deplete lipid raft cholesterol concentration and the resulting accumulation of B(a)P metabolites. The results demonstrate that in the absence of IgG, CD32a is mostly found outside lipid rafts and subsequently translocates to lipid rafts in the presence of IgG (Fig. 1A). As indicated in Fig. 1B, our results suggest that exposure to B(a)P significantly suppresses CD32a association with lipid rafts as compared to controls, leading to reduced IgG-CD32a binding. Collectively, these findings suggest that intact lipid rafts are paramount for IgG-CD32a binding and effector function signaling. These findings also support a role for B(a)P exposure in developmental immunotoxicity in that these exposures have the capacity to result in suppression of macrophage effector function. Subsequently, the disruption of lipid raft integrity results in reduced IgG-CD32a binding and thus, an exposure-induced immunosuppressive endophenotype that is observed in vulnerable populations living in high risk communities.
Figure 1. Proposed mechanism for B(a)P exposure-induced suppression of macrophage effector function.
Macrophages are cells of the innate immune system that constitute the first line of defense against many infectious diseases. To determine the effects of exposure to B(a)P on peripheral blood monocyte differentiation, monocytes were cultured with M-CSF in the presence or absence of B(a)P. When cultured in the presence of B(a)P, M-CSF-treated blood monocytes tended to appear undifferentiated morphology and expressed low levels of macrophage markers. This results indicated B(a)P exposure inhibits the differentiation of M-CSF treated blood monocytes into macrophages.
Mature macrophages function in promoting the clearance of invading microorganisms by respiratory burst activity, phagocytosis, then excretion. An assessment of whether the effects of B(a)P exposure during M-CSF stimulation were functionally significant was undertaken. As seen in Fig. 2A, M-CSF stimulated monocytic cell cultures treated with B(a)P appeared a dose-dependent, statistically significant reduction in the expression of CD68, a macrophage surface marker, to quantify the number of differentiated macrophages in the cell culture performing phagocytosis. The effects of B(a)P exposure on the respiratory burst activity of M-CSF stimulated cells and untreated cells were analyzed by exposing the cells to PMA to trigger reactive oxygen species production. High dose B(a)P exposure was found to reduce significantly respiratory burst activity following PMA treatment when compared to the unexposed, non PMA-stimulated cells (Fig. 2B). In monocytic cells cultured in the presence of M-CSF and/or B(a)P, there was no significant difference in reactive oxygen species levels of PMA-stimulated non PMA-stimulated cells (Fig. 2B). It is important to note that in the untreated cell cultures, there was a basal level of reactive oxygen species production.
Figure 2. Exposure to B(a)P decreases macrophage phagocytic and respiratory burst activity. (A) Quantification of phagocytic activity at 4°C and 37°C. Results presented are values ± SE from at least five independent experiments. (*, p < 0.05) compared to DMSO-treated cells. B) The quantification of reactive oxygen species intermediate production and cells expressing CD68 when cultured in the absence or presence of M-CSF, 1, 5 or 10μM B(a)P and stimulated with or without PMA. Results presented are ± SE from at least five independent experiments. (*, p < 0.05) compared to DMSO-treated cells.
To discern the ability of B(a)P exposure to impact lipid raft homeostasis and CD32-IgG complex binding, we determined the levels of IgG co-localization with lipid rafts and IgG complex binding to CD32. Monocytic cells were cultured with or without M-CSF or co-treated with M-CSF and B(a)P for 6 days. Cells were incubated with 10μg/mL hHAIgG and CD32 expression and IgG binding were assessed by flow cytometry. As can be seen in Fig. 3A, the CD32 expression and IgG binding in monocytic cell cultures stimulated with M-CSF are quantified. To verify IgG binds to CD32 within lipid rafts, monocytic cells were plated in MatTek dishes and cultured. Cells were stained with cholera toxin subunit B-FITC labeled (CTαB) and IgG. The association of IgG with lipid rafts was determined by confocal immunofluorescence microscopy (Fig. 3B). As evident in Fig. 4B, when compared to the untreated monocytic cell cultures, 10μM B(a)P exposure reduces colocalization of IgG with the ganglioside GM1, a well-established lipid raft marker. To interrogate further IgG-CD32 binding, cells were grown as previously described and quantified by flow cytometry analysis. Again, when compared to the untreated cells, the co-treatment of M-CSF and 10μM B(a)P was found to be effective in suppressing IgG-CD32 binding in monocytic cells (Fig. 3C). IgG-CD32 binding also was assessed after monocytic cells were cultured, harvested and treated with either 10mM MβCD (depletes membrane cholesterol) or 30μg/mL nystatin (increases membrane cholesterol). A statistically significant decrease in the IgG binding of cells treated with B(a)P, MβCD, or nystatin was observed as compared to the control (Fig.3D). It should be noted that although disruption of lipid rafts with B(a)P or MBCD had no effect on CD32 expression, we did observe an effect on CD32 distribution.
Figure 3. Exposure to B(a)P disrupts lipid raft homeostasis. (A) Quantification of IgG complex binding to CD32 in untreated CD14+ monocytic cells. (B) Representative confocal immunofluorescence micrographs of monocytic cells treated with IgG (hHAIgG-10μg/mL) for 30 min, stained for ganglioside 1 with CTαB-AF555 (Red) and for CD32-PE (Green) mean fluorescent intensity was determined by nikon imaging software. (C) Representative flow cytometry histograms of IgG and CD32 expression in monocytic cells exposed to MCSF or MCSF+B(a)P. (D) Measurement of IgG complex binding to CD32 in monocytic cells exposed to either B(a)P (10μM), MβCD, or nystatin. Results presented the mean (±SE) from at least three independent experiments (*, p <0.05) compared with untreated control cells.
We investigated the effects of B(a)P exposure on CD32 translocation event. Toward this end, monocytic cells were co-treated with 10μM and M-CSF, the then cells were lysed and placed in a discontinuous sucrose gradient and centrifuged for 18 h to fractionate the lipid rafts. Ten 1ml fractions were collected, the DRM fractions were determined by CD59 staining, a lipid raft marker, and analyzed for CD32 expression in the absence or presence of IgG. The fractions were also analyzed by liquid-liquid extraction and HPLC to quantify B(a)P and metabolites. The dot blot results of indicated that in the presence of IgG, a higher percentage of CD32 is found within the lipid raft fractions as compared to the percentage of CD32 in the non-raft fractions (Fig. 4A). In the absence of IgG, there is a reduced quantity of CD32 found in raft fractions; in the presence of IgG, CD32 also is distributed at higher amounts in the non-raft fractions when compared to CD32 distribution in non-raft fraction in the presence of IgG (Fig. 4B). A cholesterol oxidase assay measured the amount of cholesterol in each fraction and determined that fractions 5-8 contained the highest amount of cholesterol (Fig. 4C). As expected, the highest quantities of B(a)P and metabolites were found within the lipid raft fractions. It was interesting to note that the lipid raft fractions of cells co-treated with M-CSF and B(a)P contained the highest amount of cholesterol and B(a)P metabolites, indicating a possible relationship between B(a)P and cholesterol(Fig. 4D).
Figure 4. Exposure to B(a)P results in decreased IgG-CD32 binding within lipid raft regions. (A) DRM fractionation and analysis of CD32 distribution in monocytic cells treated with M-CSF and 10 μM B(a)P for 6 days. CD59 was used as a marker for DRM fractions. (B) Quantification of CD32 band intensities present in each fraction (C) A cholesterol oxidase assay measured the amount of cholesterol in each fraction. (D) In parallel, the 1 mL fractions were analyzed by liquid-liquid extraction and HPLC analysis to measure B(a)P metabolite concentration.
These findings validate research trajectory 1 of the Public Health Exposome in the area of physiologic disruption (e.g. immune response) representing translational knowledge gained (on a continuum) from basic science research. Specifically, these findings demonstrate the critical role of exposure to traffic related air pollution (including PAHs) across gestation and describe an immuno-toxicological molecular level mechanism that is particularly relevant during critical windows of development. Combined with our findings over the last decade, the collective data suggest that exposure to benzo(a)pyrene containing environmental contaminants during gestation has the capacity to result in suppression of macrophage effector function by disrupting lipid raft integrity to result in reduced IgG-CD32a binding, inducing an immunosuppressive endophenotype that can lead to disparate health outcomes in vulnerable populations across the lifespan.
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