Journal: Brain, behavior, and immunity

Article Title: HMGB1/IL-1β Complexes Regulate Neuroimmune Responses in Alcoholism

doi: 10.1016/j.bbi.2017.10.027

Figure Lengend Snippet: Recombinant HMGB1 (rHMGB1) and recombinant IL-1β (rIL-1β) were at 4C for 24 hours to form HMGB1/IL-1β complexes. Hippocampal entorhinal brain slice culture (HEC) were incubated with HMGB1 (17nM) alone, IL-1β alone (600pm or 6nM), HMGB1/IL-1β (17nM/600pM or 17nM/6nM) for 24 hours. Innate immune gene induction was assessed by RT-PCR. (A) Co-immunoprecipitation was performed of synthetic HMGB1/IL-1β complexes to assess for stable complex formation. The entire western blot of eluate confirmed complex formation is shown. Eluate was probed for either anti-IL-1β (800 channel, red) or anti-HMGB1 (700 channel, green) antibodies. Bands consistent with the eluted HMGB1 and IL-1β were seen consistent with successful synthetic complex formation. (B) iNOS gene induction by HMGB1/IL-1β complexes. HMGB1 alone did not induce iNOS. IL-1β alone (600pM and 6nM) increased iNOS by 712% and 2981% respectively (Figure 5B). HMGB1/IL-1β complexes increased iNOS expression by 1.7- 2-fold above IL-1β alone (1452% and 4941% at 600pM and 6nM). (C) IL-1β induced its own gene induction to 2367% and 11,621% of control (600pM and 6nM respectively). HMGB1/IL-1β complexes increased IL-1β gene induction by 1.7 to 3.5 fold above IL-1β alone (8379% and 19,916% above control, 600pM and 6nM IL-1β respectively). (D) IL-1 receptor antagonist (IL-1 RA) reduced the HMGB1/ILβ induction of iNOS by 64% while returning TNFα and IL-1β to control levels. **p<0.01, ***p<0.001, N=3 per group.

Article Snippet: For FLAG-HMGB1 transfection, the FLAG-HMGB1 (N-terminal tagged) regular plasmid construct was obtained from VectorBuilder™.

Techniques: Recombinant, Slice Preparation, Incubation, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Expressing

Journal: Brain, behavior, and immunity

Article Title: HMGB1/IL-1β Complexes Regulate Neuroimmune Responses in Alcoholism

doi: 10.1016/j.bbi.2017.10.027

Figure Lengend Snippet: HMGB1 and IL-1β immunofluorescence were visualized in mouse cortex by confocal microscopy (90×) in control and ethanol (6g/kg, i.g.) treated mice (A) HMGB1 and IL-1β co-localized to a degree in the cytoplasm of controls and ethanol treated mice. DAPI colabeling of nuclei shows that colocalization occurred in the cell cytoplasm. Arrows denote sites of visible colocalization (B) Analysis of sites of visible colocalization was assessed for accuracy using Pearson’s method. Pearson’s correlation values for representative sites of visible colocalization were determine and were mostly between 0.7–0.8 indicating colocalization. (C) Representative images of individual subjects. White arrows denote sites of colocalization. Ethanol treatment caused a robust increase in cytoplasmic HMGB1 and IL-1β co-localization 12 hours after treatment. At 24 hours post-ethanol treatment, cytoplasmic HMGB1 and IL-1β co-localization was observed to a lesser degree (D) High magnification from a 12 hour ethanol treated subject (number 2, boxed region) showing punctate sites of colocalization. HMGB1 (green) is primarily nuclear. Yellow puntate cytoplasmic colocalization sites are highlighted by arrowheads. (E) Quantification of HMGB1+/IL-1+ colocalized staining showed a near 2-fold increase in HMGB1/IL-1β co-localization in mouse cortex at 12 hours; 1-way ANOVA F(2,6)=13.2, p<0.0064; Sidak’s post-test 57 vs 33.3, Ethanol 12h vs. control, *p<0.05, N=3 subjects per group. Inserts show high magnification images of selected regions. Arrowheads denote HMGB1/IL-1+ colocalization.

Article Snippet: For FLAG-HMGB1 transfection, the FLAG-HMGB1 (N-terminal tagged) regular plasmid construct was obtained from VectorBuilder™.

Techniques: Immunofluorescence, Confocal Microscopy, Staining

Journal: Brain, behavior, and immunity

Article Title: HMGB1/IL-1β Complexes Regulate Neuroimmune Responses in Alcoholism

doi: 10.1016/j.bbi.2017.10.027

Figure Lengend Snippet: Mice were treated with ethanol (6g/kg, i.g). HMGB1 protein levels were measured at different time points by ELISA and Immunohistochemistry (IHC). (A) Whole brain HMGB1 increased within 1 hour (173% increase, p<0.05) and stabilized to control levels by 48h (F(6,31)=1.290, p<0.03) (B) Quantification of IHC of HMGB1 showed increased cortical HMGB1 after binge ethanol. HMGB1+immunoreactive (IR) cells were increased by 1.7-fold up to 24 hours after ethanol treatment. (C) Representative images of HMGB1 IHC in mouse cortex after ethanol showing increased numbers of HMGB1+IR cells. (D) Binge-ethanol (6g/kg) caused a transient increase in plasma HMGB1 (F(6,29)=0.7107, p<0.0003). HMGB1 levels peaked at 12 hours after ethanol (226% increase, p<0.001). N=4–11 mice per time point, 1-way ANOVA with Sidak’s multiple comparisons *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: For FLAG-HMGB1 transfection, the FLAG-HMGB1 (N-terminal tagged) regular plasmid construct was obtained from VectorBuilder™.

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Brain, behavior, and immunity

Article Title: HMGB1/IL-1β Complexes Regulate Neuroimmune Responses in Alcoholism

doi: 10.1016/j.bbi.2017.10.027

Figure Lengend Snippet: Mice were treated with either water or ethanol (6g/kg, i.g). Brain protein was isolated and western blot performed. (A) Multiple bands stained positive for HMGB1. On a separate blot of the same samples, HMGB1 antigenic peptide (10µg/mL) was pre-incubated with HMGB1 antibody prior to overnight incubation with the blot to identify specific HMGB1 bands. Arrows show bands with specific HMGB1 staining that disappear after pre-incubation of primary antibody with HMGB1 immunogenic peptide. Western blots shown in A are not for quantitation, but to show that preincubation with exogenous HMGB1 reduces heterocomplex detection (B) HMGB1 antibody validation by western blot of mouse-derived BV2 microglia cell lysates with siRNA for HMGB1. Mouse BV2 microglia were incubated with siRNA (100–500nM) as described in Methods. Similar to mouse brain, multiple HMGB1+ bands were observed both at 29kD and at higher molecular weights (arrowheads). With increasing concentrations of siRNA to HMGB1, each of these bands disappeared though GAPDH remained similar, indicating specific antibody staining at both 29kD and higher molecular weights. (C) Fluorescent Western Blot of brain protein stained for both HMGB1 and IL-1β. Composite overlay shows a ~65kD band was positive for both HMGB1 and IL-1β (C) Western blot for IL-1β showing an IL-1β+ band at ~65kD. Pre-incubation of the IL-1β peptide with the immunogenic peptide caused disappearance of the ~65kD band indicating antigen specificity (D) Schematic of co-immunoprecipitation (Co-IP) assessment for HMGB1/IL-1β complex formation. Agar beads coupled with anti-HMGB1 antibodies to precipitate for HMGB1. Elution of HMGB1 from anti-HMGB1 complexes allowed for detection of HMGB1/IL-1β complexes by western blot. (E) Immunoprecipitation for HMGB1 was performed. Western blots on the eluate for IL-1β and HMGB1 found both IL-1β and HMGB1 in the eluate consistent with the presence of HMGB1-IL-1β heterocomplexes in mouse brain.

Article Snippet: For FLAG-HMGB1 transfection, the FLAG-HMGB1 (N-terminal tagged) regular plasmid construct was obtained from VectorBuilder™.

Techniques: Isolation, Western Blot, Staining, Incubation, Quantitation Assay, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Brain, behavior, and immunity

Article Title: HMGB1/IL-1β Complexes Regulate Neuroimmune Responses in Alcoholism

doi: 10.1016/j.bbi.2017.10.027

Figure Lengend Snippet: Rat hippocampal-entorhinal slice culture (HEC) sections were transfected with either control or a FLAG-HMGB1 plasmid as described in the Methods. Sections were then treated with either saline, LPS (100ng/mL) or Ethanol (100mM) for 24 hours. Slice culture media was collected and co-immunoprecipitation was performed for FLAG-HMGB1 and IL-1β to assess the release of FLAG-HMGB1/IL-1β heterocomplexes. (A) Western blot was performed on the eluate and probed with either anti-FLAG or anti-IL-1β antibodies. FLAG-HMGB1 was detected in the eluate at the molecular weight expected, ~25–29kD. Cleaved, active IL-1β was also detected in eluate consistent with HMGB1/IL-1β complex detection. (B) Quantitation of OD of IL-1β and FLAG-HMGB1 found that LPS and ethanol increased the secretion of IL-1β with FLAG-HMGB1 146% and 143% respectively relative to control, *p<0.05, t-test, N=2 culture wells per group. (C) Schematic illustrating ethanol-induced secretion of HMGB1/IL-1β complexes and their resulting enhance immune activation.

Article Snippet: For FLAG-HMGB1 transfection, the FLAG-HMGB1 (N-terminal tagged) regular plasmid construct was obtained from VectorBuilder™.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Molecular Weight, Quantitation Assay, Activation Assay

Journal: Brain, behavior, and immunity

Article Title: HMGB1/IL-1β Complexes Regulate Neuroimmune Responses in Alcoholism

doi: 10.1016/j.bbi.2017.10.027

Figure Lengend Snippet: Postmortem human hippocampal brain tissue was obtained from the New South Wales Tissue Bank. Protein was isolated from frozen tissue and assessed for IL-1β and HMGB1 protein level by ELISA. (A) Human alcoholics showed a 42% increase in IL-1β above healthy controls: 404.5 ± 51.5 vs 573.4 ± 99.2pg/mg, Control vs Ethanol, *p<0.05, paired two-tailed t-test, N=10 per group. (B) Human alcoholics showed a 24% increase in HMGB1 (157.8 ±24.7 vs 195.5± 60.1 ng/mg, Control vs Ethanol, *p<0.05, paired two-tailed t-test, N=10 per group. Note: Data published previously from this same cohort of patients in a different form in Coleman et al J Neuroinflammation 2017. (C) Co-Immunoprecipitation for HMGB1 and IL-1β was performed on postmortem human hippocampus from healthy controls and alcoholics. Eluate was probed for either HMGB1 or IL-1β. HMGB1+ bands were observed at the expected 25–29kD MW. An IL-1β+ band was also observed at ~17kD consistent with cleaved IL-1β, indicating the presence of HMGB1-IL-1β complexes. Quantification of optical densitometry of each band found an increased ratio of IL-1β to HMGB1 in eluate consistent with increased HMGB1-IL-1β complex formation in the hippocampus of human alcoholics. Alcoholics showed a 275% increase in IL-1β association with HMGB1 relative to control. *p<0.05, paired t-test, N=4 per group (D) Theoretical model of enhanced HMGB1/IL-1β signaling

Article Snippet: For FLAG-HMGB1 transfection, the FLAG-HMGB1 (N-terminal tagged) regular plasmid construct was obtained from VectorBuilder™.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Immunoprecipitation