Activation of an appropriate innate immune response to bacterial infection is critical to limit microbial spread and generate cytokines and chemokines to instruct appropriate adaptive immune responses. Recognition of bacteria or bacterial products by pattern recognition molecules is crucial to initiate this response. However, it is increasingly clear that the context in which this recognition occurs can dictate the quality of the response and determine the outcome of an infection. The cross talk established between host and pathogen results in profound alterations on cellular homeostasis triggering specific cellular stress responses. In particular, the highly conserved integrated stress response (ISR) has been shown to shape the host response to bacterial pathogens by sensing cellular insults resulting from infection and modulating transcription of key genes, translation of new proteins and cell autonomous antimicrobial mechanisms such as autophagy. Here, we review the growing body of evidence demonstrating a role for the ISR as an integral part of the innate immune response to bacterial pathogens.

Prior studies demonstrate the activation of poly-(ADP-ribose) polymerase 1 (PARP1) in various pathophysiological conditions, including sepsis. We have assessed the effect of olaparib, a clinically used PARP1 inhibitor, on the responses of human peripheral blood leukocytes (PBMCs) obtained from healthy volunteers in response to challenging with live bacteria, bacterial lipopolysaccharide (LPS), or oxidative stress (hydrogen peroxide, H2O2). The viability of PBMCs exposed to olaparib or to the earlier generation PARP inhibitor PJ-34 (0.1–1000 µM) was monitored using Annexin V and 7-aminoactinomycin D. To evaluate the effects of olaparib on the expression of PARP1 and its effects on protein PARylation, PBMCs were stimulated with Staphylococcus aureus with or without olaparib (1–10 μM). Changes in cellular levels of nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP), as well as changes in mitochondrial membrane potential (MMP), were measured in PBMCs exposed to H2O2. Bacterial killing was evaluated in PBMCs and polymorphonuclear leukocytes (PMNs) incubated with S. aureus. Cytokine production was measured in supernatants using a cytometric bead array. Reactive oxygen species (ROS), nitric oxide (NO) production, and phagocytic activity of monocytes and neutrophils were measured in whole blood. For ROS and NO production, samples were incubated with heat-killed S. aureus; phagocytic activity was assessed using killed Escherichia coli conjugated to FITC. Olaparib (0.1–100 µM) did not adversely affect lymphocyte viability. Olaparib also did not interfere with PARP1 expression but inhibits S. aureus-induced protein PARylation. In cells challenged with H2O2, olaparib prevented NAD+ and ATP depletion and attenuated mitochondrial membrane depolarization. LPS-induced production of TNF-α, MIP-1α, and IL-10 by PBMCs was also reduced by olaparib. Monocytes and neutrophils displayed significant increases in the production of ROS and NO after stimulation with S. aureus and phagocytic (E. coli) and microbicidal activity, and these responses were not suppressed by olaparib. We conclude that, at clinically relevant concentrations, olaparib exerts cytoprotective effects and modulates inflammatory cytokine production without exerting adverse effects on the cells’ ability to phagocytose or eradicate pathogens. The current data support the concept of repurposing olaparib as a potential experimental therapy for septic shock.

Cystathionine β-synthase (CBS), CSE (cystathionine γ-lyase) and 3-mercaptopyruvate sulfurtransferase (3-MST) have emerged as three significant sources of hydrogen sulfide (H2S) in various forms of mammalian cancer. Here, we investigated the functional role of CBS’ and 3-MST’s catalytic activity in the murine breast cancer cell line EO771. The CBS/CSE inhibitor aminooxyacetic acid (AOAA) and the 3-MST inhibitor 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) were used to assess the role of endogenous H2S in the modulation of breast cancer cell proliferation, migration, bioenergetics and viability in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). CBS and 3-MST, as well as expression were detected by Western blotting; H2S production was measured by the fluorescent dye AzMC. The results show that EO771 cells express CBS, CSE and 3-MST protein, as well as several enzymes involved in H2S degradation (SQR, TST, and ETHE1). Pharmacological inhibition of CBS or 3-MST inhibited H2S production, suppressed cellular bioenergetics and attenuated cell proliferation. Cell migration was only inhibited by the 3-MST inhibitor, but not the CBS/CSE inhibitor. Inhibition of CBS/CSE of 3-MST did not significantly affect basal cell viability; inhibition of 3-MST (but not of CBS/CSE) slightly enhanced the cytotoxic effects of oxidative stress (hydrogen peroxide challenge). From these findings, we conclude that endogenous H2S, generated by 3-MST and to a lower degree by CBS/CSE, significantly contributes to the maintenance of bioenergetics, proliferation and migration in murine breast cancer cells and may also exert a minor role as a cytoprotectant.

Poly(ADP-ribose) polymerase 1 (PARP1), as a potential target for the experimental therapy of acute lung injury (ALI), was identified over 20 years ago. However, clinical translation of this concept was not possible due to the lack of clinically useful PARP inhibitors. With the clinical introduction of several novel, ultrapotent PARP inhibitors, the concept of PARP inhibitor repurposing has re-emerged. Here, we evaluated the effect of 5 clinical-stage PARP inhibitors in oxidatively stressed cultured human epithelial cells and monocytes in vitro and demonstrated that all inhibitors (1–30 µM) provide a comparable degree of cytoprotection. Subsequent in vivo studies using a murine model of ALI compared the efficacy of olaparib and rucaparib. Both inhibitors (1–10 mg/kg) provided beneficial effects against lung extravasation and pro-inflammatory mediator production—both in pre- and post-treatment paradigms. The underlying mechanisms include protection against cell dysfunction/necrosis, inhibition of NF-kB and caspase 3 activation, suppression of the NLRP3 inflammasome, and the modulation of pro-inflammatory mediators. Importantly, the efficacy of PARP inhibitors was demonstrated without any potentiation of DNA damage, at least as assessed by the TUNEL method. These results support the concept that clinically approved PARP inhibitors may be repurposable for the experimental therapy of ALI.