The Response of Human Macrophages to β-Glucans Depends on the Inflammatory Milieu

<div><p>Background</p><p>β-glucans are fungal cell wall components that bind to the C-type lectin-like receptor dectin-1. Polymorphisms of dectin-1 gene are associated with susceptibility to invasive fungal infection and medically refractory ulcerative colitis. The purpose of this study has been addressing the response of human macrophages to β-glucans under different conditions mimicking the composition of the inflammatory milieu in view of the wide plasticity and large range of phenotypical changes showed by these cells, and the relevant role of dectin-1 in several pathophysiological conditions.</p><p>Principal Findings</p><p>Serum-differentiated macrophages stimulated with β-glucans showed a low production of TNFα and IL-1β, a high production of IL-6 and IL-23, and a delayed induction of cyclooxygenase-2 and PGE₂ biosynthesis that resembled the responses elicited by crystals and those produced when phagosomal degradation of the phagocytic cargo increases ligand access to intracellular pattern recognition receptors. Priming with a low concentration of LPS produced a rapid induction of cyclooxygenase-2 and a synergistic release of PGE₂. When the differentiation of the macrophages was carried out in the presence of M-CSF, an increased expression of dectin-1 B isoform was observed. In addition, this treatment made the cells capable to release arachidonic acid in response to β-glucan.</p><p>Conclusions</p><p>These results indicate that the macrophage response to fungal β-glucans is strongly influenced by cytokines and microbial-derived factors that are usual components of the inflammatory milieu. These responses can be sorted into three main patterns i) an elementary response dependent on phagosomal processing of pathogen-associated molecular patterns and/or receptor-independent, direct membrane binding linked to the immunoreceptor tyrosine-based activation motif-bearing transmembrane adaptor DNAX-activating protein 12, ii) a response primed by TLR4-dependent signals, and iii) a response dependent on M-CSF and dectin-1 B isoform expression that mainly signals through the dectin-1 B/spleen tyrosine kinase/cytosolic phospholipase A₂ route.</p></div

Production of PGE₂.

<p>(A, C, E and F) Macrophages were primed with 10 ng/ml LPS or (B) 100 U/ml IFNα and IFNγ for 3 hours or left untreated, and then stimulated with different additions at a concentration of 1 mg/ml. 10 µM CA-074, 2 µM cytochalasin D, and 20 µg/ml PVNO were added before the stimuli. The supernatants were collected for the assay of PGE₂. Results represent mean ± S.D. of 6 to 7 independent experiments. Veh, indicates vehicle, *indicates <i>p</i><0.05.</p

Oligonucleotide primers used for real time RT-PCR and ChIP.

<p><i>mrc1</i> stands for the mannose receptor gene.</p

Proposed mechanisms involved in zymosan uptake and signaling in human macrophages.

<p>Zymosan particles can activate Syk via dectin-1 engagement and through the adaptor protein DAP12. After internalization into phagosomes, TLR2 recognition and cathepsin B leakage may occur. In the presence of M-CSF, the expression of dectin-1 B isoform is increased and allows for an enhanced receptor-dependent binding. If the LPS/TLR4 cascade is activated, an additional, concomitant mechanism of NF-κB activation will be triggered. cPLA₂ is activated by dectin-1/Syk-dependent mechanisms involving MAPK-dependent Ser-505 phosphorylation and Ca²⁺-dependent membrane translocation, whereas the induction of COX-2 depends mainly on κB-dependent transcriptional regulation. IFNγ signaling leads to the activation of the promoter of the inducible microsomal isoform of prostaglandin E synthase (<i>pges</i>) by an IFN-stimulated response element (ISRE)-dependent or GAS (interferon-γ activated sequence)-dependent mechanism. The dotted lines indicate the steps associated with phagocytic cargo processing. Ac-H3 indicates acetylated histone H3.</p

Expression of receptors.

<p>(A) The expression of different receptors was assayed by flow cytometry. The panel represents a typical experiment of two. (B) Expression of dectin-1 mRNA. The image shows a 40 cycles PCR carried out to show the minor band corresponding to dectin-1 A isoform that was not observed with a lower number of cycles. The high number of PCR cycles explains the difficulty to assess in the image the increased expression of dectin-1 B isoform in M-CSF differentiated macrophages over serum-differentiated macrophages that could be detected in real-time RT-PCR. The identification of the different isoforms was carried out by DNA sequencing on both strands of the RT-PCR product. This is a representative experiment of two. (C) Real-time RT-PCR was carried out with reverse primers in exon 5 and exon 6 to assay dectin-1 A and B isoforms, as well as with primers to assay DC-SIGN, the mannose receptor, and TLR2 in 7 day differentiated macrophages and DC. <i>gadph</i> was assayed as a load control and as a reference in real-time RT-PCR. Results represent mean ± S.D. of 5 experiments. *Indicates <i>p</i><0.05.</p

Zymosan uptake by macrophages and DC.

<p>(A, B and C) Macrophages and DC were incubated with Alexa Fluor® 488-labeled zymosan at the concentration of 5 particles per cell at 37°C for the times indicated and the uptake of particles assayed by flow cytometry. Results in (A) show representative experiments in macrophages differentiated in the presence of human serum or M-CSF for seven days and then treated for 3 hours with 10 ng/ml LPS or left untreated prior to the addition of zymosan particles. (B) Zymosan uptake by DC. Experiments in (C) were conducted to compare percentage of phagocytosing cells and MFI in serum and M-CSF differentiated macrophages. Ops-Zym indicates serum opsonised zymosan. Results show a representative experiment of three independent ones with a similar trend.</p

Release of [³H]AA.

<p>Cells were differentiated and stimulated as indicated. Anti-CD32A antibody was used at the concentration of 10 µg/ml 30 min prior to the addition of the stimuli. Zymosan (Zym), Ops-Zym, depleted zymosan (Dep-Zym), and β-glucan (β-glu) particles were used at the concentration of 1 mg/ml. Immune complexes (IC) were used at the concentration of 100 µg/ml. Results represent mean ± S.D. of 5 to 6 independent experiments. *Indicates <i>p</i><0.05.</p

Effect of <i>clec7a</i> deletion on the response to β-glucans.

<p>(A) BMDM from WT and <i>dectin-1^−/−</i> mice were used for the release of [³H]AA after incubation for 1 hour with the indicated additions at the concentration of 1 mg/ml. (B) The production of PGE₂ was assayed after 24 hour in BMDM preincubated for 3 hours with 10 ng/ml LPS or left untreated. (C) Production of IL-6 assayed 24 hours after the addition of the stimuli. Results indicate mean ± S.D. of 4 to 6 independent experiments. *Indicates <i>p</i><0.05 as compared to WT mice.</p

Activation of NF-κB proteins by LPS and zymosan.

<p>(A) Macrophages were treated for up to 5 h with 10 ng/ml LPS and RelA/p65 was assayed in the cytoplasm and nucleus. (B) Macrophages were primed for 3 h with LPS and then stimulated for different times. (B–C) The nuclear fractions were used for the assay of RelA/p65, c-Rel, and p50. (D) Binding of RelA/p65 and c-Rel, and histone H3 acetylation of the <i>ptgs2</i> promoter were assayed in the presence and absence of LPS priming 2 h after stimulation. These are representative experiments of at least two showing identical results.</p

Eicosanoid release by macrophages.

<p>Human macrophages were primed for 3 hours with LPS and stimulated for 24 hours with zymosan. Results represent mean values of one experiment with duplicate samples.</p