Mycobacterium abscessus (M.abs) is a rapidly growing mycobacterial species that infects macrophages, and is an important pathogen in patients with cystic fibrosis. We studied the early stages of M.abs infection of macrophages, with emphasis on the role of heme-oxygenase-1 (HO-1) in this infection. THP-1 cells were activated using TPA into macrophage-like cells and infected with M.abs for different time points. M.abs infection robustly induced HO-1 expression in the THP-1 cells. Production of HO-1 was p38 MAPK-dependent, as p38 inhibitors suppressed HO-1 induction. Pretreatment with HO-1 inhibitors tin-protoporphyrin (SnPP) significantly inhibited M.abs growth inside macrophages. Furthermore, inhibiting HO-1 using HO-1 siRNA or the HO-1 upstream signaling molecule; Nrf2 using Nrf2 siRNA resulted in similar inhibition of M.abs. In contrast, inducing HO-1 did not increase M.abs intracellular growth above control. Products of HO-1 metabolism of heme are bilirubin, biliverdin, carbon monoxide (CO) and iron. The addition of either bilirubin or biliverdin, but not CO, completely restored the SnPP inhibitory effect and partially that with HO-1 siRNA. To understand the mechanisms, we used Syto-62 labeled M.abs to infect macrophages. Interestingly, HO-1 inhibition promoted M.abs-containing phagosome fusion with lysosomes, which should enhance M.abs killing. M.abs infection enhanced THP-1 ROS production as demonstrated by increased DHE, DCF fluorescence, and EPR signal. HO-1 inhibition further increased ROS production in infected macrophages. Our results indicate that HO-1 induction is important for M.abs growth during the early stages of infection, and that the HO-1 products bilirubin and biliverdin, perhaps through modulation of intracellular ROS levels, may be involved.

Studies have shown an increased risk for a variety of cancers, specifically brain cancer, in healthcare workers occupationally exposed to ionizing radiation. Although the mechanisms mediating these phenomena are not fully understood, ionizing radiation-mediated elevated levels of reactive oxygen species (ROS), oxidative DNA damage, and immune modulation are likely involved. A group of 20 radiation exposed workers and 40 sex- and age-matched non-exposed control subjects were recruited for the study. We measured superoxide (O₂•⁻) levels in whole blood of healthcare workers and all other measurements of cytokines, oxidative DNA damage, extracellular superoxide dismutase (EcSOD) activity and reduced/oxidized glutathione ratio (GSH/GSSG) in plasma. Levels of O₂•⁻ were significantly higher in radiation exposed workers compared to control. Similarly, a significant increase in the levels of interleukin (IL)-6, IL-1α and macrophage inflammatory protein (MIP)-1α in radiation exposed workers compared to control was observed, while there was no significance difference in the other 27 screened cytokines. A significant positive correlation was found between MIP-1α and O₂•⁻ levels with no correlation in either IL-6 or IL-1α. Further, a dose-dependent relationship with significant O₂•⁻ production and immune alterations in radiation exposed workers was demonstrated. There was no statistical difference between the groups in terms of oxidative DNA damage, GSH/GSSG levels, or EcSOD activity. Although the biologic significance of cytokines alterations in radiation exposed workers is unclear, further studies are needed for determining the underlying mechanism of their elevation.

Pseudomonas aeruginosacauses lung infections in patients with cystic fibrosis (CF). ThePseudomonasquinolone signal (PQS) compound is a secretedP. aeruginosavirulence factor that contributes to the pathogenicity ofP. aeruginosa. We were able to detect PQS in sputum samples from CF patients infected withP. aeruginosabut not in samples from uninfected patients. We then tested the hypothesis that PQS induces oxidative stress in host cells by determining the ability of PQS to induce the production of reactive oxygen species (ROS) in lung epithelial cells (A549 and primary normal human bronchial epithelial [NHBE]) cells and macrophages (J774A.1 and THP-1). ROS production induced by PQS was detected with fluorescent probes (dichlorodihydrofluorescein diacetate, dihydroethidium, and MitoSOX Red) in conjunction with confocal microscopy and flow cytometry. PQS induced ROS production in lung epithelial (A549 and NHBE) cells and macrophages (J774A.1 and THP-1 cells). NHBE cells were sensitive to PQS concentrations as low as 500 ng/ml. PQS significantly induced early apoptosis (P< 0.05,n= 6) in lung epithelial cells, as measured by annexin/propidium iodide detection by flow cytometry. However, no change in apoptosis upon PQS treatment was seen in J774A.1 cells. Heme oxygenase-1 (HO-1) protein is an antioxidant enzyme usually induced by oxidative stress. Interestingly, incubation with PQS significantly reduced HO-1 and NrF2 expression in A549 and NHBE cells but increased HO-1 expression in J774A.1 cells (P< 0.05,n= 3), as determined by immunoblotting and densitometry. These PQS effects on host cells could play an important role in the pathogenicity ofP. aeruginosainfections.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis. Tumor hypoxia plays an active role in promoting tumor progression, malignancy, and resistance to therapy in PDAC. We present evidence that nab-paclitaxel–gemcitabine (NPG) and/or a hypoxic tumor microenvironment (TME) up-regulate heme oxygenase-1 (HO-1), providing a survival advantage for tumors. Using PDAC cells in vitro and a PDAC mouse model, we found that NPG chemotherapy up-regulated expression of HO-1 in PDAC cells and increased its nuclear translocation. Inhibition of HO-1 with ZnPP and SnPP sensitized PDAC cells to NPG-induced cytotoxicity (p < 0.05) and increased apoptosis (p < 0.05). Additionally, HO-1 expression was increased in gemcitabine-resistant PDAC cells (p < 0.05), and HO-1 inhibition increased GEM-resistant PDAC sensitivity to NPG (p < 0.05). NPG combined with HO-1 inhibitor inhibited tumor size in an orthotopic model. In parallel, HO-1 inhibition abrogated the influx of macrophages and FoxP3+ cells, while increasing the proportion of CD8+ infiltration in the pancreatic tumors. These effects were mediated primarily by reducing expression of the immunosuppressive cytokine IL-10.

Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form, accounting for more than 90% of all pancreatic malignancies. In a previous study, we found that hypoxia and chemotherapy induced expression of Heme Oxygenase-1 (HO-1) in PDAC cells and tissues. Arsenic trioxide (ATO) is the first-line chemotherapeutic drug for acute promyelocytic leukemia (APL). ATO increases the generation of reactive oxidative species (ROS) and induces apoptosis in treated cells. The clinical use of ATO for solid tumors is limited due to severe systemic toxicity. In order to reduce cytotoxic side effects and resistance and improve efficacy, it has become increasingly common to use combination therapies to treat cancers. In this study, we used ATO-sensitive and less sensitive PDAC cell lines to test the effect of combining HO-1 inhibitors (SnPP and ZnPP) with ATO on HO-1 expression, cell survival, and other parameters. Our results show that ATO significantly induced the expression of HO-1 in different PDAC cells through the p38 MAPK signaling pathway. ROS production was confirmed using the oxygen-sensitive probes DCFH and DHE, N-acetyl cysteine (NAC), an ROS scavenger, and oxidized glutathione levels (GSSG). Both ATO and HO-1 inhibitors reduced PDAC cell survival. In combined treatment, inhibiting HO-1 significantly increased ATO cytotoxicity, disrupted the GSH cycle, and induced apoptosis as measured using flow cytometry. ATO and HO-1 inhibition modulated autophagy as shown by increased expression of autophagy markers ATG5, p62, and LC3B in PDAC cells. This increase was attenuated by NAC treatment, indicating that autophagy modulation was through an ROS-dependent mechanism. In conclusion, our work explored new strategies that could lead to the development of less toxic and more effective therapies against PDAC by combining increased cellular stress and targeting autophagy.