Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption

Abstract

Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses.

Keywords: Innate immune function; Inorganic arsenic; Macrophage; PBA; ROS; UPR.

Fig. 1

Arsenic trioxide activates UPR signaling in murine macrophage Raw 264.7 cells. Reverse transcriptase PCR analysis of UPR responsive genes in ATO-treated Raw 264.7 cells was performed. For this, cells were treated with ATO at two different concentrations (0.2 and 2 μM) for 60 and 180 min. Band intensity showing transcriptional expression levels of GRP78 and GRP94 (A-I), CHOP and ATF4 (A-II) and spliced XBP-1 and HRD1 (A-III) is depicted. Graphs indicate the densitometry analysis of band intensity in terms of fold change. 18sRNA was used as endogenous control. (B-I) Western blots analysis of UPR signaling proteins (p-PERK, p-eIF2α, ATF4 and CHOP) in ATO (0.2 and 2 μM)-treated Raw 264.7 cells for 3 h. (B-II) Relative densitometry analysis of band intensity expressed in terms of fold change. β-Actin was used as an endogenous control. (C) Immunofluorescence staining showing nuclear localization of ATF4 in ATO (0.2 and 2 μM for 3 h)-treated Raw 264.7 cells. Nuclei were stained with Hoechst 33342 dye. Arrows indicate the migration and localization of ATF4 from cytosol to nucleus in ATO-treated cells as compared to saline-treated control cells. (D) Western blot analysis of ATO-induced translocation and activation of ATF4 and its downstream molecule CHOP. Fraction purity was confirmed by α/β-tubulin and TATA binding protein for cytoplasmic and nuclear fractions respectively. Data are expressed as Mean ± SE of at least three independent samples. Statistical significance was determined using Student’s t test. *P > 0.05, **P > 0.01 and ***P > 0.001 show significance levels.

Fig. 2

Arsenic trioxide-mediated ROS generation and UPR signaling activation are inter-dependent. ROS generation was assessed using fluorescent DCFH-DA dye. (A-I) Raw 264.7 cells were exposed to ATO (0.2 and 2 μM) treatment for 1.5 and 3 h alone as well as in the presence of NAC (10 mM for 24 h) and PBA (1 mM for 24 h). Fluorescence intensity was measured at excitation wavelength at 485 nm and emission wavelength at 528 nm by micro-plate reader and changes were calculated as percent of control. (A-II) Representative microphotographs showing ATO-induced ROS generation in Raw 264.7 cells. Images were snapped using upright fluorescence microscope. The positive control (Post. control) group consisted of Raw 264.7 cells pretreated (1 h) with 500 μM of H₂O₂. (B) Cells were pre-incubated with NAC (10 mM for 2 h and 10 mM for 24 h) followed by ATO (2 μM for 3 h) treatment. UPR regulating proteins p-PERK, GRP78, GRP94, p-eIF2α and CHOP were assessed by western blot analysis. (C-I) Raw 264.7 cells were pretreated with PBA and co-treated with thapsigargin at concentrations 1 mM and 2.5 μM for 24 h and 3 h respectively. Following various pretreatments/co-treatments, cells were incubated with ATO (2 μM for 3 h) and levels of proteins including p-PERK, GRP78, p-eIF2α and CHOP were examined by western blot analysis. (C-II) Relative densitometry analysis of band intensity expressed in fold change. β-Actin was used as an endogenous control. Saline-treated cells served as control in all experiments. Data are expressed as Mean ± SE of at least three independent samples. Statistical significance was determined using Student’s t test. #,*P > 0.05, ##,**P > 0.01 and ###,***P > 0.001 show significance levels. * indicates significant level when compared to saline-treated control cells. # indicates significant level when compared to ATO-treated cells.

Fig. 3

UPR signaling regulates arsenic trioxide-inhibited bacterial engulfment and clearance of E. coli (K-12 strain) BioParticles® fluorescents conjugated bacteria by murine macrophage. (A) Raw 264.7 cells were treated with ATO (2 μM for 16 h) alone as well as in combination of PBA (1 mM for 24 h) followed by incubation with latex beads coated with fluorescently labeled rabbit-IgG for 45 min at 37 °C. Engulfed fluorescent-beads were observed using a fluorescent microscope. Intensity of green fluorescence indicates the engulfed beads. (B) Mean fluorescence intensity (MFI) of engulfed fluorescence beads were recorded in 96 wells black bottom plate at excitation 485 nm and emission at 535 nm by microplate reader. Saline-treated cells receiving an identical treatment with similar procedure as described above except for the incubation temperature which was 4 °C instead of 37 °C served as negative control. (C) Infection load of opsonized E. coli (K-12 strain) BioParticles® fluorescents conjugated bacteria in Raw 264.7 cells-treated with ATO (2 μM for 24 h) in the presence and absence of PBA and NAC were observed under fluorescent microscopy. At the end of treatment, cells were incubated with fluorescent tagged E. coli bacteria (Bacteria:cells 50:1 ratio) at 37 °C for 2 h and baseline was imaged under the fluorescent microscope. Cells were further incubated at 37 °C to complete the process of bacterial clearance. After 24 h, cells were recaptured for fluorescent imaging. (C-I) Saline-treated control cells effectively cleared the fluorescence tagged E. coli bacteria within 24 h. (C-II) ATO-treated macrophage could not clear this bacterial load and continued to show the presence of fluorescent tagged opsonized bacteria. (C-III & IV) PBA and NAC pretreatment of Raw 264.7 cells restored the ATO-impaired clearance/phagocytosis of engulfed bacteria.

Fig. 4

Diminished cytokine production in ATO-treated Raw 264.7 cells was dependent on UPR signaling: Cytokine production was quantified by real time quantitative PCR (qRT-PCR) using SYBR Green methodology. Transcription levels of IL-1β, TNF-α, IL-6, TGF-β, and IL-10 were determined and presented in terms of fold change. (A) Cells received five different treatments. (1) Control-treated with normal saline, (2) LPS-treated with 250 ng/ml for 3 h, (3) ATO-treated with 2 μM for 3 h, (4) pre-treated with ATO followed by LPS, and (5) pretreated with PBA (1 mM for 24 h) followed by ATO and LPS. ATO-treatment to murine macrophage reduced the cytokine production alone as well as in combination of LPS stimulation. PBA pretreatment to the macrophage restored the cytokine production of ATO and LPS-induced cytokine production. Data are expressed as Mean ± SE of at least three independent samples. Statistical significance was determined using Student’s t test. #,*P > 0.05, ##,**P > 0.01 and ###,***P > 0.001 show significance levels. * indicates significant level when compared to saline-treated control cells. # indicates significant level when compared to LPS-treated cells. @ indicates significant level when compared to ATO + LPS-treated cells.

Fig. 5

Arsenic treatment impaired macrophage functions are UPR regulated: ATO which causes endoplasmic reticulum stress, induces UPR signaling and ROS concomitantly in macrophage. In addition, ATO-treated macrophage show impairment in innate immune functions including diminished phagocytosis of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. However, treatment of macrophage with a chemical chaperon, 4-phenylbutyric acid attenuated both UPR signaling and ROS generation in these cells. Furthermore, this treatment afforded significant protection against ATO-mediated impairment of macrophage functions. Similar protective effects against ATO-mediated impairment in innate immune functions were noted following treatment of these cells with antioxidant NAC. Interestingly the mechanism by which NAC affords protection against immunotoxicity of ATO also involves diminution in UPR signaling pathway.