LPS protects macrophages from AIF-independent parthanatos by downregulation of PARP1 expression, induction of SOD2 expression, and a metabolic shift to aerobic glycolysis

Highlights

• High concentrations of hydrogen peroxide causes PARP-1 mediated necrotic cell death (parthanatos) in macrophages.

• Apoptosis inducing factor (AIF) is not required for hydrogen peroxide-induced parthanatos in macrophages.

• LPS-stimulated macrophages are protected from hydrogen peroxide-induced cell death.

• LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD⁺ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.

Abstract

In inflamed tissues or during ischemia-reperfusion episodes, activated macrophages produce large amounts of reactive species and are, thus, exposed to the damaging effects of reactive species. Here, our goal was to investigate the mechanism whereby activated macrophages protect themselves from oxidant stress-induced cell death. Hydrogen peroxide-treated mouse bone marrow-derived macrophages (BMDM) and THP-1 human monocyte-derived cells were chosen as models. We found a gradual development of resistance: first in monocyte-to-macrophage differentiation, and subsequently after lipopolysaccharide (LPS) exposure. Investigating the mechanism of the latter, we found that exposure to intense hydrogen peroxide stress causes poly(ADP-ribose) polymerase-1 (PARP-1) dependent programmed necrotic cell death, also known as parthanatos, as indicated by the protected status of PARP-1 knockout BMDMs and the protective effect of the PARP inhibitor PJ-34. In hydrogen peroxide-treated macrophages, however, apoptosis inducing factor (AIF) proved dispensable for parthanatos; nuclear translocation of AIF was not observed. A key event in LPS-mediated protection against the hydrogen peroxide-induced AIF independent parthanatos was downregulation of PARP1 mRNA and protein. The importance of this event was confirmed by overexpression of PARP1 in THP1 cells using a viral promoter, which lead to stable PARP1 levels even after LPS treatment and unresponsiveness to LPS-induced cytoprotection. In BMDMs, LPS-induced PARP1 suppression lead to prevention of NAD⁺ depletion. Moreover, LPS also induced expression of antioxidant proteins (superoxide dismutase-2, thioredoxin reductase 1 and peroxiredoxin) and triggered a metabolic shift to aerobic glycolysis, also known as the Warburg effect. In summary, we provide evidence that in macrophages intense hydrogen peroxide stress causes AIF-independent parthanatos from which LPS provides protection. The mechanism of LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD⁺ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.

Introduction

The mononuclear phagocyte system is comprised of macrophages, monocytes, and dendritic cells. While resident macrophages populate most tissues prenatally, monocytes are also recruited to the gut mucosa and skin under disease-free conditions, and can differentiate to macrophages in these tissues [1], [2]. Inflammatory signals can recruit monocytes to virtually all tissue niches where exposure to colony stimulating factor 1 (CSF1) and granulocyte-macrophage colony stimulating factor (GM-CSF) stimulate the monocytes to differentiate into macrophages [3]. Depending on the composition of the tissue environment, macrophages may develop various different phenotypes. Two extreme states of a continuous macrophage polarization spectrum are usually referred to as M1 and M2 macrophages [4].

Inflammatory stimuli, such as lipopolysaccharide (LPS), IFNγ, and TNFα, trigger M1 differentiation (classically activated macrophage), while interleukin-4 (IL-4) and/or interleukin-13 (IL-13) induce alternative (M2) differentiation. LPS, the most studied M1 signal, acts mostly via TLR4 receptors, and utilizes MyD88 and Toll-interleukin 1 receptor domain containing adaptor protein (Tirap)-mediated signaling pathways. LPS stimulates the production of a plethora of inflammatory cytokines and mediators under the control of NFκB, AP1, STAT1, IRFs, and members of the early growth response (EGR) family [4].

The M1 macrophage is a central cell type in the generation and propagation of inflammation, while M2 cells play a role in the resolution of inflammation. In addition to cytokines and chemokines, macrophages produce reactive oxygen and nitrogen species (ROS and RNS, respectively), which are important in the antimicrobial weaponry of macrophages [5]. The primary source of ROS in monocytes/macrophages is the multisubunit protein, NADPH oxidase-2 (NOX2) [6]. Monocytes and macrophages produce superoxide radicals and hydrogen peroxide. Macrophages also produce a key oxidant product, peroxynitrite, via a reaction between nitric oxide and superoxide [5]. (M1 macrophages overexpress inducible nitric oxide synthase (iNOS) and produce large amounts of nitric oxide.)

Operating in the very center of inflammation, macrophages are not only the main source, but also targets of ROS/RNS. Thus, macrophages must resist cell death-inducing stimuli from ROS/RNS in order to maintain the inflammatory environment (e.g. in the fight against microbes). On the other hand, understanding the mechanisms of macrophage cell death and resistance to cell death may be important when therapeutic suppression of inflammation is needed (in chronic inflammation, ALI, ARDS, atherosclerosis or sepsis). Macrophage death, e.g. in response to oxidative stress or microbial infections, may include apoptosis, necroptosis, autophagic cell death, or pyroptosis [7], [8]. Although several oxidative species, including hydroxyl radical and peroxynitrite, can cause DNA strand breakage [9], [10], [11], the role of parthanatos, a DNA damage-induced cell death, in macrophage cell fate regulation is not well understood.

Parthanatos is mediated by the nuclear DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) [12]. PARP-1 is rapidly activated by DNA single strand breaks and cleaves NAD⁺ to nicotinamide and ADP-ribose. PARP1 then attaches ADP-ribose to suitable protein acceptors near DNA nicks and builds a branched poly-ADP-ribose (PAR) polymer to initiate the repair process [13]. PAR polymers are then cleaved off from proteins by poly(ADP-ribose) glycohydrolase (PARG) and shuttled to the mitochondria where they trigger nuclear translocation of the mitochondrial protein apoptosis-inducing factor (AIF), the final executioner of parthanatos [14], [15]. PARP-1-mediated cell dysfunction and cell death has been demonstrated in various diseases, such as diabetic endothelial dysfunction, stroke, myocardial ischemia reperfusion injury, and many forms of inflammation [16]. The role of PARP-1 in macrophages, with special regard to the different functional states of these cells, however, has not yet been investigated.

The goal of the present study was to examine the oxidant stress sensitivity of macrophage cells and how this sensitivity is affected by the polarization state of the cells. Our results show that monocyte to macrophage differentiation is accompanied by increased oxidative stress resistance, which increases further upon LPS stimulation, while M2 differentiation had no effect. Furthermore, we show that a key event in the mechanism of oxidative stress resistance is the downregulation of PARP1 expression, upregulation of antioxidant enzymes (SOD2) and proteins, and metabolic reprogramming towards aerobic glycolysis.