Benzo(a)pyrene attenuates the pattern-recognition-receptor induced proinflammatory phenotype of murine macrophages by inducing IL-10 expression in an aryl hydrocarbon receptor-dependent manner

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene (BaP) are environmental contaminants known to be immunosuppressive. Most effects of BaP towards immune cells are thought to be mediated through activation of the aryl hydrocarbon receptor (AhR). The AhR is a ligand-activated transcription factor, which plays a critical modulatory role in various cells during immune response. Macrophages are key players in innate immunity against intracellular bacteria and are discussed to be a target of AhR-mediated immune regulation. However, so far there is only incomplete knowledge about the effects of BaP on activated macrophages and whether these effects are AhR-dependent in each case. Using murine bone marrow-derived macrophages (BMMs) stimulated with heat-killed salmonellae as a source of different pathogen-associated molecular patterns (PAMPs) for stimulation of different pattern recognition receptors (PRRs) as an in-vitro model, we studied the immunomodulatory effects of low-dose BaP exposure. PRR-activated BMMs produced nitric oxide (NO) and a spectrum of proinflammatory cytokines, i.e. tumor necrosis factor (TNF)-α interleukin (IL)-1β IL-6, and IL-12 but also the anti-inflammatory cytokine IL-10. While BaP exposure suppressed the production of proinflammatory cytokines, the secretion of IL-10 was augmented. Moreover, BaP exposure increased the expression of major histocompatibility complex class II (MHC-II), CD14, Fcγ receptor I (FcγRI/CD64), or CD86, enhanced NO production and phagocytosis what may be beneficial for phagocytosis and killing of microbial pathogens. Of note, without PRR activation low-dose BaP exposure has little influence on the macrophage phenotype. BMMs from AhR-deficient (Ahr −/−) mice were widely refractory to BaP-induced modulation of cytokine production, surface marker expression, and functional properties in response to PAMPs stimulation, indicating that these effects are dependent on AhR. In summary, these data suggest that induction of AhR-mediated signalling pathways by BaP may attenuate the proinflammatory phenotype of PRR-activated BMMs, while activating IL-10-mediated anti-inflammatory properties but also enhancing uptake and killing of pathogens as well as antigen presentation. Together these features imply a favourable role of BaP exposure for macrophage functions in an ongoing immune response. However, the strong induction of IL-10 may lead to defective pathogen clearance and subsequently to chronic persistent infection. This concept suggests an inhibitory rather than a supporting influence of environmental BaP on immunity to infection or cancer and also emphasises the important regulatory role of AhR in immunity and inflammation

Characterization of the murine myeloid precursor cell line MuMac-E8

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies

Immunophenotyping of MuMac-E8 cells.

<p>MuMac-E8 cells were stained with fluorochrome-labeled monoclonal antibodies recognizing the surface marker proteins CD11b, F4/80 (A), CD14, and CD64 (B) in order to identify the functional phenotype of this cell line. It could be shown that MuMac-E8 cells in general express CD11b and that the majority of cultured MuMac-E8 cells express also F4/80 (91%). The number of CD11b⁺/F4/80⁺ cells increased following co-incubation with heat-killed <i>S</i>.E. (96%; A, right-hand panel). CD11b⁺/F4/80⁺ MuMac-E8 cells revealed high or moderate surface expression of CD14 or CD64, respectively. Moreover, the expression of both CD14 and CD64 on CD11b⁺/F4/80⁺ MuMac-E8 cells was additionally inducible by stimulation with <i>Salmonella</i> antigen (B).</p

Optimum cell culture conditions and morphology of cultured MuMac-E8 cells.

<p>By means of real-time cell analysis using the xCELLigence system the optimum cell density (n = 12 per value of cell density, mean ± SD; A) and the optimum serum supplementation for cell synchronization through serum deprivation (B) were determined. Typical morphology of MuMac-E8 cells in culture was shown by inverse phase contrast microscopy (Axiovert, Zeiss). The majority of the cells were found to grow adherent until confluence (C). Visualisation of cell spreading after fluorescence staining of F-actin with Phalloidin-Alexa was performed by confocal laser-scanning microscopy (LSM 510 Meta, Zeiss) (D). Figure was reprint from Biochemica, 4, 14–16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113743#pone.0113743-Pfefferkorn1" target="_blank">[12]</a> with permission from the journal.</p

Experimental strategy for gene expression analyses.

<p>MuMac-E8 cells harvested from bulk culture were synchronized by serum deprivation for 48 h. The cell cycle was re-entered by supplementation of the culture medium with 10% FCS. At indicated time points total RNA was isolated for subsequent gene expression analysis by real-time RT-PCR.</p

Overview of the expression of investigated marker genes.

<p>Overview of the expression of investigated marker genes.</p

Sequences of primers and corresponding UPL probes for selected marker genes.

<p>Sequences of primers and corresponding UPL probes for selected marker genes.</p

Phagocytic activity of MuMac-E8 cells.

<p>For measurement of phagocytic potential, MuMac-E8 cells were harvested and 1×10⁶ cells were incubated for 2 h with 2×10⁷ FITC-labeled heat-killed salmonellae. Afterwards cells were washed 4 times with HBSS and the uptake of bacteria was assessed by imaging flow cytometry (Amnis FlowSight system, Merck Millipore). In this representative experiment 44% of the cells revealed a positive signal (A). By parallel imaging it could be confirmed that the positive fluorescence signal coincided with phagocytosis of salmonellae. White arrows show cells with internalized FITC-labeled bacteria (B).</p

Kaplan-Meier survival curve and number of leukocytes in peripheral blood after transplantation of MuMac-E8 cells into mice.

<p>(A) Survival of lethally irradiated mice after administration of different cell numbers of MuMac-E8. Animals that have received a transplantation dose of 2×10⁶ cells survived up to 17 days. Following the administration of 1×10⁶ the survival decreased down to 14 days. The control animals died within 12 days. (n = 4 per group) (B) Level of leukocytes in peripheral blood determined by analysis of blood, n = 4 per group, mean ± SD. Leukocytes decreased within 13 days post transplantation in all groups (no engraftment).</p