HMGB1-RAGE Signaling Plays a Role in Organic Dust-Induced Microglial Activation and Neuroinflammation

Abstract

Occupational exposure to contaminants in agriculture and other industries is known to cause significant respiratory ailments. The effect of organic dust on lung inflammation and tissue remodeling has been actively investigated over many years but the adverse effect of organic dust-exposure on the central vital organ brain is beginning to emerge. Brain microglial cells are a major driver of neuroinflammation upon exposure to danger signals. Therefore, we tested a hypothesis that organic dust-exposure of microglial cells induces microglial cell activation and inflammation through HMGB1-RAGE signaling. Mouse microglial cells were exposed to organic dust extract showed a time-dependent increase in cytoplasmic translocation of high-mobility group box 1 (HMGB1) from the nucleus, increased expression of receptor for advanced glycation end products (RAGE) and activation of Iba1 as compared to control cells. Organic dust also induced reactive oxygen species generation, NF-κB activation, and proinflammatory cytokine release. To establish a functional relevance of HMGB1-RAGE activation in microglia-mediated neuroinflammation, we used both pharmacological and genetic approaches involving HMGB1 translocation inhibitor ethyl pyruvate (EP), anti-HMGB1 siRNA, and NOX-inhibitor mitoapocynin. Interestingly, EP effectively reduced HMGB1 nucleocytoplasmic translocation and RAGE expression along with reactive oxygen species (ROS) generation and TNF-α and IL-6 production but not NF-κB activation. HMGB1 knockdown by siRNA also reduced both ROS and reactive nitrogen species (RNS) and IL-6 levels but not TNF-α. NOX2 inhibitor mitoapocynin significantly reduced RNS levels. Collectively, our results demonstrate that organic dust activates HMGB1-RAGE signaling axis to induce a neuroinflammatory response in microglia and that attenuation of HMGB1-RAGE activation by EP and mitoapocynin treatments or genetic knockdown can dampen the neuroinflammation.

Keywords: HMGB1; RAGE; microglia; neuroinflammation; organic dust.

Figure 1.

Ethyl pyruvate (EP) and mitoapocynin (MA) reduce organic dust extract (ODE)-exposure induced Iba1 expression. Cells exposed to media (control) or lipopolysaccharide (LPS) or peptidoglycan (PGN) or ODE followed by either vehicle or EP or MA were stained for Iba1 (microglial activation marker). A, Compared to controls, ODE-exposed cells showed higher amounts of Iba1 staining at 6, 24, and 48 h (upper panel, A). Compared to vehicle, both EP- and MA-treatment reduced the ODE-induced increase in Iba1 staining at 6, 24, and 48 h (B, middle and C, lower panel respectively). Immunocytochemistry data for LPS and PGN are not shown and micrometer bar = 20 µm. B and C, Mean intensity of Iba1 staining was measured using HCImage software. B, Compared to control, LPS or PGN or ODE-exposure resulted in increased staining intensity for Iba1 at 6 (LPS alone), 24, and 48 h (LPS, PGN, and ODE). Compared to controls, EP treatment significantly reduced the staining intensity for Iba1 at 6 (LPS), 24, and 48 h for LPS, PGN and ODE. C, Compared to control, LPS or PGN or ODE-exposure resulted in increased staining intensity for Iba1 at 6, 24, and 48 h. Compared to controls, MA treatment significantly reduced the staining intensity for Iba1 at 6 and 24 (LPS, PGN and ODE) and 48 h (PGN and ODE respectively).

Figure 2.

Mitoapocynin (MA) treatment reduces organic dust extract (ODE)-exposure induced reactive nitrogen specie (RNS) production. Cells exposed to media (control) or ODE were stained for 3-NT (RNS). A, Compared to controls, ODE-exposed cells showed higher amounts of 3-NT staining (RNS) at 6, 24, and 48 h (upper panel). Compared to vehicle, MA treatment reduced ODE-induced increase in 3-NT (RNS) staining at 6, 24, and 48 h (lower panel). Immunocytochemistry data for LPS and PGN are not shown and micrometer bar = 20 µm. B, Mean intensity of 3-NT staining was measured and compared to control, LPS (6, 24, and 48 h) or PGN (6, 24, and 48 h) or ODE (6 and 24 h) exposure increased the staining intensity for 3-NT. Compared to controls, MA treatment significantly reduced the staining intensity for 3-NT at 6, 24, and 48 h for LPS and PGN but not ODE at 48 h. C, Griess assay was performed to measure nitrite levels as a readout of secreted RNS levels at 12 h. Compared to controls, LPS and ODE-exposure increased the secretion of RNS. Compared to vehicle, MA treatment significantly decreased nitrite levels at 12 h.

Figure 3.

Ethyl pyruvate (EP) reduces organic dust extract (ODE)-exposure induced reactive oxygen species (ROS) production. Cells exposed to media (control) or ODE were stained for gp91 phox (source of ROS). A, Compared to controls, ODE-exposed cells appear to contain higher amounts of gp91 phox (source of ROS) at 6 and 24 h (upper panel, A). Compared to vehicle, EP treatment reduced ODE-induced increase in gp91 phox (ROS) staining at 6 and 24 h (lower panel, B). Immunocytochemistry data for LPS and PGN are not shown and micrometer bar = 20 µm. B, Mean intensity of gp91 phox staining was measured using HCImage software. Compared to control, LPS (24 and 48 h) or PGN (24 and 48 h) or ODE (6 and 24 h) exposure resulted in increased staining intensity for gp91 phox. Compared to controls, EP treatment significantly reduced the staining intensity for gp91 phox at 6 h (#, p < .05, ODE), 24 h (LPS, PGN, and ODE), and 48 h (LPS). C, Dichlorodihydrofluorescein diacetate (DCFDA) assay was performed to measure DCF fluorescence as a readout of secreted ROS levels at 6, 24, and 48 h. Compared to controls, LPS, PGN, and ODE-exposure increased the secretion of ROS. Compared to vehicle, EP treatment significantly decreased ROS levels at 24 and 48 h for LPS, PGN, and ODE.

Figure 4.

Ethyl pyruvate (EP) reduces organic dust extract (ODE)-induced high-mobility group box 1 (HMGB1) nucleocytoplasmic translocation. Cells treated medium (control) or lipopolysaccharide (LPS), peptidoglycan (PGN), and ODE followed by either vehicle (Ringer’s solution) or EP were stained with anti-HMGB1 (Cy3, red) and anti-RAGE (receptor for advanced glycation end products; FITC, green) antibodies and nuclei were identified with DAPI (blue). Data for LPS and PGN treated cells are not shown here. A, Compared to media treated control cells (0 h), ODE (1%) treated cells at early time point showed HMGB1 expression (cy3, red) restricted to the nucleus (6 h, arrow, upper panel). Compared to control and ODE (6 h) treated cells, ODE-treated cells at later time points (24 and 48 h) show nucleocytoplasmic translocation (double arrows) and secretion into extra-cellular space (48 h, arrow head). EP treatment (lower panel, A) reduces ODE-induced nucleocytoplasmic translocation and secretion of HMGB1 into extra-cellular space. With EP treatment HMGB1 expression mostly remains in the nucleus (arrows, lower panel, A). Micrometer bar = 20 µm. B, Compared to controls, LPS, PGN, and ODE-treated cells showed significantly higher HMGB1 translocation (24 and 48 h). Compared to medium, EP treatment significantly reduced HMGB1 translocation (24 and 48 h). C–H, Cell culture supernatants, cytoplasmic, and nuclear fractions from control and ODE-treated cells were processed for western blotting analysis to quantify HMGB1 and loading control proteins. β-Actin served as a loading control for cell culture supernatant and cytoplasmic fractions whereas Lamin-B1 served as a control for nuclear fraction. Densitometry and statistical data analysis were performed on normalized bands (n = 4). C–D, Compared to controls, nuclear fractions from ODE treated cells contained higher levels of HMGB1 protein. Following EP treatments, the amount of HMGB1 protein in the nuclear fractions increased at 24 and 48 h. E–F, Compared to controls, ODE treated cells showed higher levels of HMGB1 in the cytoplasmic fractions. Compared to Ringer’s solution (vehicle), EP treatment significantly reduced the HMGB1 levels at 48 h. G–H, Compared to controls, ODE-exposure significantly increased the secretion of HMGB1 into cell culture supernatant at 24 and 48 h. Compared to Ringer’s solution, EP treatment significantly reduced the HMGB1 levels (below detection limit) at 6, 24, and 48 h.

Figure 5.

Ethyl pyruvate (EP) treatment reduces organic dust extract (ODE)-exposure induced increase in receptor for advanced glycation end products (RAGE) expression. Cells treated with medium (control) or LPS, PGN, and ODE followed by either vehicle (medium) or EP were stained with anti-RAGE (FITC, green) antibody. A, Compared to medium, ODE-exposed cells showed an increase in RAGE expression at 6, 24, and 48 h (A, upper panel). Compared vehicle (medium), treatment with EP reduced the expression of RAGE at 6, 24, and 48 h (A, lower panel). Immunocytochemistry data for LPS and PGN are not shown and micrometer bar = 20 µm. B, The mean intensity of staining for RAGE was measured using (HCImage software). Compared to controls, LPS or PGN or ODE-exposed cells showed an increase in RAGE staining intensity at 6, 24, and 48 h. Compared to vehicle, EP treatment reduced the RAGE staining intensity. C–D, Western blotting was performed on cell lysates using anti-RAGE and anti-β-actin (loading control) antibodies. Densitometry and statistical analysis of normalized bands was conducted. Compared to controls ODE-exposed cells showed an increase in the expression of RAGE at 24 and 48 h. Compared to the vehicle (medium), EP treatment significantly reduced the expression of RAGE at 24 and 48 h (n = 4, A–D).

Figure 6.

Organic dust extract (ODE)-exposure results in nuclear translocation of NF-κB p65. Cells exposed to media (control) or lipopolysaccharide (LPS) or peptidoglycan (PGN) or ODE were stained with anti-NF-κB p65 antibody (cy3) and DAPI staining to identify nuclei. A, Compared to controls (cytoplasmic NF-κB, white arrows), ODE-exposed cells showed nuclear translocation of NF-κB p65 at 6, 24, and 48 h (white arrowheads, lower panel in A (i)). Neither vehicle or nor EP had any significant effect on nuclear translocation of NF-κB p65 (lower panel of A (ii)). Immunocytochemistry data for LPS and PGN are not shown and micrometer bar = 20 µm. C and D, Western blot analysis on whole cell lysate using anti-κB p65 and anti-β-actin (loading control) was performed. Densitometry and statistical analysis of normalized band intensities indicated that, compared to controls, ODE-exposed cells showed an increase in NF-κB levels at 48 h. Neither vehicle nor EP treatment had any significant effect on NF-κB levels.

Figure 7.

Ethyl pyruvate (EP) reduces organic dust extract (ODE)-exposure induced proinflammatory cytokines. Culture supernatants from cells treated media (control) or ODE were processed for various cytokine ELISA. A, Compared to medium, ODE-exposed cell culture supernatants secreted increased amounts of TNF-α. Compared to vehicle, EP treatment decreased the levels of TNF-α at 6 and 24 h. B, Compared to medium, ODE-exposed cell culture supernatants secreted increased amounts of IL-6 at 6 and 24 h. Compared to vehicle, EP treatment decreased the levels of IL-6 at 6 and 24 h. C, IL-10 levels did not change between control and ODE-treated groups at any time points and EP treatment did change IL-10 levels. D, Compared to medium, ODE-exposed cells secreted increased amounts of TGF-β1 at 48 h and EP treatment did not change ODE-induced TGF-β1 production.

Figure 8.

Transfection of microglia with anti-HMGB1 siRNAs reduces high-mobility group box 1 (HMGB1) mRNA and protein levels. Cells treated with DsiRNAs (R1, R2, R3, NC) or TYE 563 (transfection control) were processed for whole cell lysate preparation or fixed with paraformaldehyde respectively. A, Following qRT-PCR R2 (10 nm) or R3 (10 nm) and R1 (10 nm) or R2 (10 nm) significantly reduced mRNA expression of HMGB1 at 24 and 48 h respectively. B–C, Following Western blot analysis R2 (10 nm) or R3 (10 nm) and R1 (10 nm), R2 (10 nm) or R3 (10 nm) reduced HMGB1 expression at 24 and 48 h respectively. D, After 48 h, immunofluoroscence shows successful transfection with TYE563 DsiRNA (white arrows) in the cytoplasm of microglia and the nucleus is stained in blue with DAPI (n = 3, A–D).

Figure 9.

Transfection of microglia with anti-HMGB1 siRNAs reduces RAGE protein levels. Following the siRNA-mediated reduction in high-mobility group box 1 (HMGB1) mRNA, receptor for advanced glycation end products (RAGE) (42 kD) and housekeeping protein α-tubulin (50 Kd) bands (n = 3) are shown (A). Normalized densitometry values show that, compared to negative control (NC) siRNA, anti-HMGB1 siRNA treatment (R1, R2, and R3) reduced the expression of RAGE protein levels at 24 and 48 h (#, anti HMGB1 siRNA effect, n = 3, B).

Figure 10.

Transfection of microglia with anti-HMGB1 (high-mobility group box 1) siRNAs reduces organic dust extract (ODE)-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) production. Compared to medium treated control cells, cells treated with ODE (with media or negative control [NC] siRNA) produced significantly increased amounts of ROS (A) and RNS (B). Following anti-HMGB1 siRNA treatment, ODE-induced ROS and secreted nitrite (representing RNS) production significantly decreased (n = 4, A and B).

Figure 11.

Transfection of microglia with anti-HMGB1 (high-mobility group box 1) siRNAs and effect on organic dust extract (ODE)-induced cytokine production. Compared to medium treated control cells, cells treated with ODE (with saline or negative control [NC] siRNA) produced significantly increased amounts of TNF-α (B) and IL-6 (A). Following anti-HMGB1 siRNA treatment, ODE-induced TNF-α (B) did not change whereas IL-6 production significantly decreased (A, n = 4, both A and B).

Figure 12.

Organic dust extract (ODE)-exposure of microglia leads to sustained inflammation via nucleocytoplasmic translocation of high-mobility group box 1 (HMGB1) and HMGB1-RAGE signaling whereas ethyl pyruvate (EP) or anti-HMGB1 siRNA treatment abrogates ODE-induced inflammation. ODE-exposure of microglial cells (1) leads to the production of reactive species (reactive oxygen species (ROS) and reactive nitrogen species (RNS) 2), nucleocytoplasmic translocation of HMGB1 (3) as well as HMGB1-RAGE (4) signaling leading to sustained levels of nuclear NF-κB p65 (5) to feed the inflammation (left side of the figure). EP or anti-HMGB1 siRNA or mitoapocynin (MA) treatment leads to a reduction in reactive species and inflammatory mediators promoting resolution of inflammation (right side of the figure).