The effect of reactive oxygen species on monocytes and macrophages

The myeloid immune system is the first line of defence against infections. The acute inflammatory response encompasses the recruitment of immune cells to the site of inflammation, the production of cytokines, phagocytosis of foreign material and the release of reactive oxygen species (ROS) to kill pathogens. The latter is accomplished by the NADPH oxidase which generates superoxide anions from molecular oxygen and releases them against pathogens. Previously, it was shown that monocytes, but not macrophages and dendritic cells (DCs), lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 which are required for the efficient repair of damaged DNA bases and single-strand breaks (SSBs) during base excision repair (BER). In addition, the double-strand break repair protein DNA-PKcs is also not expressed (Bauer et al. 2011; Briegert and Kaina 2007). The attenuated BER and non-homologous end-joining pathways sensitise monocytes to DNA oxidising and alkylating agents while DNA repair competent macrophages and DCs are resistant. As ROS are potent genotoxins, it was addressed whether the oxidative burst of myeloid cells led to DNA damage in the ROS-producing cells (auto-intoxication). It was shown that phorbol 12-myristate 13-acetate (PMA) can stimulate the NADPH oxidase via upstream activation of the protein kinase C. PMA-mediated stimulation of myeloid immune cells, i.e. granulocytes, macrophages and monocytes, led to an increase in intracellular ROS levels. The extracellular ROS burst was detected using a chemiluminescence-based assay. In contrast to T lymphocytes, myeloid immune cells produced massive ROS in a time-dependent manner. It was shown that monocytes, but not macrophages are killed by their own ROS; following PMA treatment and triggering the oxidative burst, cells exhibited oxidative DNA damage and SSB formation followed by cell death. Surviving monocytes were unable to differentiate into macrophages. Furthermore, it was shown that ROS from adjacent cells were also strong enough to damage monocytes (so called ‘killing in trans’). Co-culture experiments with stimulated macrophages or granulocytes and unstimulated monocytes led to DNA damage in monocytes, subsequent activation of the DNA damage response and ultimately monocytic cell death. DNA repair competent macrophages were resistant and did not die. These findings strengthen the hypothesis that the BER-defect of monocytes functions as a regulatory feedback loop in the acute immune response by removing monocytes from the site of inflammation and thus dampening the immune reaction. The analysis of the regulation of the BER protein XRCC1 in monocytes and macrophages was another aspect of this work. It was shown that the demethylation inhibitor 2-hydroxy-glutarate led to an attenuated XRCC1 signal during cytokine-induced differentiation into macrophages. However, the XRCC1 promoter analysis showed no differential methylation pattern between the two cell types. Using a reverse chromatin immunoprecipitation assay coupled with mass spectrometry, potential transcription factors responsible for XRCC1 expression were screened for. Promising candidates were CTCF and AP-1 factors, which were also found by in silico analysis of the promoters of PARP-1, DNA-PK and ligase IIIα. The findings of DNA repair defects in monocytes were extended to neutrophilic granulocytes. It was shown that neutrophils also display severe defects in DNA repair affecting BER and double-strand break repair. Similar to monocytes they do not express XRCC1, PARP-1 and ligase IIIα. Furthermore, DNA damage signalling factors like ATM, p53 and γH2AX were also not detectable. The data indicate a lineage-specific phenomenon.Das myeloische Immunsystem ist die erste Abwehr bei Infektionen. Die akute Entzündungs-reaktion umfasst die Rekrutierung von Immunzellen zum Entzündungsherd, die Zytokin-Produktion, die Phagozytose vom Fremdmaterial und die Freisetzung von reaktiven Sauerstoff-spezies (ROS), um Pathogene abzutöten. Letzteres wird durch die NADPH-Oxidase vermittelt, die Superoxid-Anionen aus molekularem Sauerstoff generiert und gegen Pathogene freisetzt. Frühere Studien haben gezeigt, dass den Monozyten – nicht aber den Makrophagen oder dendritische Zellen (DC) – die DNA-Reparaturproteine XRCC1, Ligase IIIα und PARP-1 fehlen, welche für die effiziente Basen-Exzisionsreparatur (BER) bei geschädigten DNA-Basen und Einzelstrangbrüchen (ESB) notwendig sind. Des Weiteren wird das Doppelstrangbruch-Reparaturprotein DNA-PKcs nicht exprimiert. (Bauer et al. 2011; Briegert and Kaina 2007). Die beeinträchtigte BER und nicht-homologe Endverknüpfung sensitivieren Monozyten gegenüber oxidierenden und alkylierenden Substanzen, während DNA-reparaturkompetente Makrophagen und DC resistent sind. Da ROS stark genotoxisch sind, wurde untersucht, ob der ‚oxidative burst‘ myeloischer Zellen in den Produzenten zu DNA-Schäden führt (Autointoxikation). Es wurde gezeigt, das Phorbol-12-Myristat 13-Acetat (PMA) die NADPH-Oxidase mittels Aktivierung der Proteinkinase C stimuliert. PMA-vermittelte Stimulation der myeloischen Immunzellen, i.e. Granulozyten, Monozyten und Makrophagen, führte zu einem Anstieg an intrazellulären ROS. Der extra-zelluläre ‚burst‘ wurde über einen Chemilumineszenz-Ansatz gemessen. Im Gegensatz zu T Lymphozyten, produzieren myeloische Immunzellen viel ROS in einer Zeit-Wirkungsbeziehung. Es wurde gezeigt, dass Monozyten, nicht aber Makrophagen, durch ihre eigenen ROS getötet werden; der durch PMA-Behandlung ausgelöste ‚oxidative burst‘ führte in Monozyten zu oxidativen DNA-Schäden und ESB, gefolgt von Zelltod. Die überlebenden Monozyten konnten nicht mehr zu Makrophagen ausdifferenzieren. Zusätzlich konnte gezeigt werden, dass ROS von benachbarten Zellen stark genug waren, um Monozyten zu schädigen (sog. killing in trans). Co-Kulturexperimente mit aktivierten Makrophagen oder Granulozyten mit nicht-aktivierten Monozyten führten zu DNA-Schäden in Monozyten, der Aktivierung ihrer DNA-Schadensantwort und letztlich zum monozytären Zelltod. DNA-reparaturkompetente Makrophagen waren resistent und starben nicht. Die Ergebnisse untermauern die Hypothese, dass der monozytäre BER-Defekt eine negative Rückkopplungsschleife in der akuten Immunantwort ist, indem er die Monozyten vom Entzündungsherd entfernt und damit die Immunreaktion abschwächt. Ein weiterer Teil dieser Arbeit betraf die Analyse der Regulation des BER-Proteins XRCC1 in Monozyten und Makrophagen. Es wurde gezeigt, dass der Demethylierungsinhibitor 2-Hydroxyglutarat zu einem abgeschwächten XRCC1-Signal während der Zytokin-vermittelten Ausdifferenzierung zu Makrophagen führte. Die XRCC1-Promoteranalyse zeigte jedoch keine Unterschiede im Methylierungsgrad der beiden Zelltypen. Mittels reverser Chromatin-Immun-präzipitation und anschließender Massenspektrometrie wurden potentielle Transkriptions-faktoren verantwortlich für die XRCC1-Expression gesucht. Vielversprechende Kandidaten waren CTCF und AP-1-Faktoren, die auch in der in silico-Analyse der Promotoren von PARP-1, DNA-PK und Ligase IIIα gefunden wurden. Die Erkenntnisse über DNA-Reparaturdefekte in Monozyten wurden auf neutrophile Granulozyten ausgeweitet. Neutrophile wiesen massive Defekte in ihrer DNA-Reparatur, besonders BER und Doppelstrangbruchreparatur, auf. Ähnlich den Monozyten wurden XRCC1, PARP-1 und Ligase IIIα nicht exprimiert. Des Weiteren konnten DNA-Schadensantwortproteine wie ATM, p53 oder γH2AX nicht detektiert werden. Die Daten lassen ein Abstammungslinie-bedingtes Phänomen vermuten

Death of Monocytes through Oxidative Burst of Macrophages and Neutrophils: Killing in Trans

<div><p>Monocytes and their descendants, macrophages, play a key role in the defence against pathogens. They also contribute to the pathogenesis of inflammatory diseases. Therefore, a mechanism maintaining a balance in the monocyte/macrophage population must be postulated. Our previous studies have shown that monocytes are impaired in DNA repair, rendering them vulnerable to genotoxic stress while monocyte-derived macrophages are DNA repair competent and genotoxic stress-resistant. Based on these findings, we hypothesized that monocytes can be selectively killed by reactive oxygen species (ROS) produced by activated macrophages. We also wished to know whether monocytes and macrophages are protected against their own ROS produced following activation. To this end, we studied the effect of the ROS burst on DNA integrity, cell death and differentiation potential of monocytes. We show that monocytes, but not macrophages, stimulated for ROS production by phorbol-12-myristate-13-acetate (PMA) undergo apoptosis, despite similar levels of initial DNA damage. Following co-cultivation with ROS producing macrophages, monocytes displayed oxidative DNA damage, accumulating DNA single-strand breaks and a high incidence of apoptosis, reducing their ability to give rise to new macrophages. Killing of monocytes by activated macrophages, termed <i>killing in trans</i>, was abolished by ROS scavenging and was also observed in monocytes co-cultivated with ROS producing activated granulocytes. The data revealed that monocytes, which are impaired in the repair of oxidised DNA lesions, are vulnerable to their own ROS and ROS produced by macrophages and granulocytes and support the hypothesis that this is a mechanism regulating the amount of monocytes and macrophages in a ROS-enriched inflammatory environment.</p></div

PMA-induced ROS formation.

<p>(A) Cells were stained with 10 μM CM-H2DCFDA immediately before treatment with PMA or t-BOOH for 30 min. PMA triggered intracellular ROS production similar to the positive control t-BOOH. (B) Extracellular ROS produced by monocytes and macrophages following treatment with PMA was measured via chemiluminescence with luminol plus HRP and then quantified. (C) The extracellular ROS production of monocytes and macrophages measured over time showed different kinetics. PMA was added to the cells at zero time and chemiluminescence was measured thereafter as described. Data are the mean of at least three independent experiments ± SEM, 1-way ANOVA, Dunnett's Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001.</p

Model of regulation of the immune response by killing of monocytes <i>in trans</i>.

<p>Since monocytes are not only precursors of macrophages but also dendritic cells (DC), their selective killing may also have an impact on the amount of DCs and DC-mediated responses during infection and inflammation.</p

XRCC1 expression during macrophage maturation and poly(ADP-ribose) formation after hydrogen peroxide treatment in monocytes and monocyte-derived macrophages.

<p>(A) Representative images of immunofluorescence staining of XRCC1 in monocytes differentiating into macrophages through GM-CSF treatment over a period of 6 days. (B) Quantification of the mean fluorescence signal of XRCC1. Each dot represents the fluorescence intensity of a single cell. (C) Representative images of the immunofluorescence staining of PAR in monocytes and monocytes that were differentiated into macrophages (at day 3). The control displayed no PAR signal whereas cells treated with 1 mM H₂O₂ for 5 min showed increased levels of PAR in macrophages on day 3 of differentiation. (D) Quantification of the mean fluorescence signal of PAR. Each dot represents the fluorescence intensity of a single cell. Data are from two independent experiments with at least 50 cells counted for each sample ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, ****p < 0.0001.</p

Apoptosis of monocytes and macrophages co-cultured with non-activated and activated macrophages.

<p>(A) Monocytes were co-cultured with un-treated (Control), solvent-treated (Mph-Solvent) or PMA-activated (Mph-PMA) macrophages and apoptosis was measured 48 h later. Monocytes and macrophages were also concomitantly exposed to PMA (PMA) and apoptosis was measured after 48 h. Exposure of both cell types to PMA exacerbated the killing effect in monocytes. (B) Macrophages themselves showed in the same experimental setting no toxicity after short-term treatment with PMA (Mph-PMA) and only a moderate increase in cell death after direct exposure to PMA (PMA). (C) Monocytes were co-cultured with solvent-treated or PMA-activated macrophages in the absence or presence of the ROS scavenger DMTU (10 mM). In the presence of DMTU, they showed significantly less cell death than monocytes co-cultured with macrophages in the absence of ROS scavenger. Data are the mean of at least four independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001.</p

8-oxo-guanine (8OHdG) formation, DNA break induction and apoptosis in monocytes and macrophages following ROS burst resulting from PMA treatment.

<p>(A) The initial oxidative DNA damage was measured in monocytes and macrophages using an FPG-modified alkaline Comet assay. Cells were treated with PMA for 15 min and then incubated for additional 45 min in PMA free medium. Monocytes and macrophages showed similar levels of initial DNA damage. (B) In parallel, 8OHdG was detected via immunostaining. Both monocytes and macrophages displayed clear 8OHdG staining after PMA treatment compared to the solvent control. (C) DNA strand breaks were measured at different times after the ROS burst was induced by 15 min PMA treatment. Monocytes displayed increasing levels of strand breaks over time compared to macrophages. (D) Monocytes and macrophages were treated with PMA for 15 min and cell death was measured 48 h later. Monocytes displayed increased apoptosis compared to macrophages, which were resistant. Control, untreated cells; solvent, DMSO control (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170347#sec002" target="_blank">material and methods</a>). Data are the mean of at least three independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, **p < 0.01, ***p < 0.001.</p

The DNA damage response was activated in monocytes co-cultured with activated macrophages for 24 h.

<p>(A) Monocytes displayed pATM immunostaining when they were co-cultured with activated macrophages (Mph-PMA), but not with non-activated macrophages (Mph-Solvent). (B) Monocytes showed increased 53BP1 foci formation after co-culture with activated macrophages. (C) There was stabilisation of p53 protein as well as phosphorylation of p53 at position Ser46 and phosphorylation of CHK2 in monocytes exposed to activated macrophages (Mph-PMA).</p

Monocytes in a co-culture setting with activated macrophages display oxidative DNA damage.

<p>(A) Monocytes were co-cultured with solvent-treated (Mph-Solvent) or PMA-activated (Mph-PMA) macrophages for 1 h before 8OHdG was detected. Monocytes exposed to activated macrophages displayed increased 8OHdG signals. (B) 1 h after co-culture with activated macrophages monocytes were analysed for oxidative DNA damage using the FPG-modified alkaline Comet assay. Monocytes exposed to PMA-activated macrophages (Mph-PMA) displayed increased DNA damage. Data are the mean of four independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, **p < 0.01.</p

LPS/bzATP-activated macrophages and PMA-activated granulocytes produce ROS that kills monocytes in co-culture.

<p>(A) ROS production in LPS/bzATP-activated macrophages. LPS/bzATP was added to the cells at zero time. (B) Apoptosis of monocytes co-cultured for 24 h with LPS/bzATP-activated macrophages. (C) Extracellular ROS produced by granulocytes as a function of time following addition of PMA to the medium. The kinetics were similar to ROS produced by activated monocytes. T cells are shown for comparison; they do not produce ROS. (D) Monocytes were co-cultured with solvent-treated (Granulo-Solvent) or PMA-activated granulocytes (Granulo-PMA) and apoptosis was measured in monocytes after 48 h. Data are the mean of four independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, ***p < 0.001.</p