Bik promotes proteasomal degradation to control low-grade inflammation

Although chronic low-grade inflammation does not cause immediate clinical symptoms, over the longer term, it can enhance other insults or age-dependent damage to organ systems and thereby contribute to age-related disorders, such as respiratory disorders, heart disease, metabolic disorders, autoimmunity, and cancer. However, the molecular mechanisms governing low-level inflammation are largely unknown. We discovered that Bcl-2–interacting killer (Bik) deficiency causes low-level inflammation even at baseline and the development of spontaneous emphysema in female but not male mice. Similarly, a single nucleotide polymorphism that reduced Bik levels was associated with increased inflammation and enhanced decline in lung function in humans. Transgenic expression of Bik in the airways of Bik-deficient mice inhibited allergen- or LPS-induced lung inflammation and reversed emphysema in female mice. Bik deficiency increased nuclear but not cytosolic p65 levels because Bik, by modifying the BH4 domain of Bcl-2, interacted with regulatory particle non-ATPase 1 (RPN1) and RPN2 and enhanced proteasomal degradation of nuclear proteins. Bik deficiency increased inflammation primarily in females because Bcl-2 and Bik levels were reduced in lung tissues and airway cells of female compared with male mice. Therefore, controlling low-grade inflammation by modifying the unappreciated role of Bik and Bcl-2 in facilitating proteasomal degradation of nuclear proteins may be crucial in treating chronic age-related diseases.

Genetically Engineered Mouse Facility following standard methods and Institutional Animal Care and Use Committee-approved protocols and bred with CCSP-rtTA mice at LRRI.To conditionally express Bik in the lung, transgenic mice were fed with doxycycline-containing diet.
Exposures of Mice to Cigarette Smoke or Lipopolysaccharide (LPS): Bik +/+ and bik -/- littermates were bred from the respective heterozygote mice at the Lovelace Respiratory Research Institute under specific pathogen-free conditions and genotyped as described previously (20).Male and female bik +/+ and bik -/-mice at 6 -8 wk of age were exposed to CS for 6 h/d, 5 d/wk, for 3 weeks, in H1000 chambers as described (64).For all experiments, type 2R4F research cigarettes (Kentucky Tobacco Research and Development Center) were used.
Exposure concentrations were kept at 250 mg TPM/m 3 except for the first wk of exposure, during which the mice were exposed to 100 mg TPM /m 3 .Control animals were housed in similar exposure chambers and exposed to filtered air (FA).
For LPS (LPS, Sigma) exposures, mice were lightly anesthetized with isoflurane and intranasally instilled with 5 or 50µg LPS.Mice were euthanized four hours following LPS instillation.For the house dust mite (HDM, Greer) exposures, TetoBik-and tetoBik+ mice were instilled intranasally with 50 µg Dermatophagoides Pteronyssinus and BAL fluid was analyzed for neutrophil numbers 5 days later.bik -/-mice were instilled with 50 ug HDM intranasally daily for 5 consecutive days.
On days 6 and 7, mice were intranasally treated with 10 µM of control TAT peptide, BH3 WT Bik peptide, or BH3 mutant Bik peptide.BAL fluids were analyzed for inflammatory cell numbers.In a separate experiment, mice were sensitized with HDM via intranasal instillation on days 1 and 8 and subsequently challenged with HDM 5 d/wk for 4 wks and kept on doxycycline diet during this time (32).
Six weeks old (TetO)7-Bik mice and their littermates were kept with 400 mg/l doxycycline water until they reach the age of 25 weeks.Lung tissues were harvested, inflated, and fixed with zinc formalin.H&E-stained lung tissues were analyzed for changes in alveolar space using ImageJ software.
Tissue Processing, Lavage and Lung Morphology: Mice were euthanized, tracheas cannulated, left main stem bronchi temporarily clamped, and right lungs lavaged three times with 0.5 ml phosphate-buffered saline (PBS).The bronchoalveolar lavage fluid (BALF) was assessed for the number of inflammatory cells and the non-cellular supernatant cytokines for levels of chemokines and cytokines.The left lung was inflated with 4% buffered formalin at a constant hydrostatic pressure of 25 cm for 6 h.Lungs were fixed further by immersion in fixative for 48-96 h.The fixed left lung lobes were trimmed, processed for histology, sectioned, and stained with hematoxylin and eosin (H&E) as described.(81) Briefly, depending on the size of the lung, four or five slices were prepared and numbered from the proximal (slice 1) to the distal (slice 4 or 5) end.The slices were then embedded in paraffin and tissues sections (5 μm thick) prepared and stained with Alcian Blue and Hematoxylin-Eosin (Sigma, St. Louis, MO, USA) as per manufacturer's instructions.Emphysema was described as irregular, multifocal expansion of alveolar airspaces and alveolar ducts due to destruction of parenchymal tissue.Morphometry was used to quantify emphysema as described previously.(82)Digital images of the H&E stained slides were captured using an Olympus NanoZoomer (Hamamatsu Photonics) slide scanner with a 20x objective lens to scan the slides.Volume-weighted mean volumes of alveoli were estimated by the method of point sampled intercepts (83).This method combines measurements of both mean volume and variability of size of the specified parenchymal spaces.The volume weighted mean volumes of alveoli can be estimated on a single section and the estimation is unbiased without shape assumptions (84,85).The VisioMorph module of VisioPharm analysis software (VisioPharm, Denmark) was used to determine alveolar volume or mean cord length.

BAL Cell Counts:
BAL was centrifuged at 1000g at 4°C for 5 minutes and supernatant was collected and stored in aliquots at -80°C for cytokine analyses.The cell pellet was diluted in 5 ml of media and BAL cell number was enumerated using a hematocytometer, cytospin slides were prepared with 50,000 cells, stained with hematoxylin/Giemsa, and cell differential quantified as described previously (86).
RNA Isolation and qRT-PCR: RNA was isolated from HAECs using TRI reagent (Sigma-Aldrich, St. Louis, MO) and mRNA was isolated following manufacturer's instructions.cDNA was synthesized using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA).TaqMan real time PCR was conducted to measure the expression of candidate genes using the ΔCT method with CDKN1β, β-Actin, or 18s as the endogenous control.The expression data are presented as the average from 3 cDNAs independently made from one mRNA sample.
Immunoblotting and Immunoprecipitation: Cytosolic and nuclear extracts were isolated as previously described (20).Briefly, cells were lysed in the presence of a buffer containing 10mM Tris (pH 8), 60mM KCL, 1mM EDTA, 0.5%NP-40, 1mM DTT, and a protease inhibitor cocktail (Sigma).Following centrifugation at 2000g, supernatants were collected (cytosolic) and pellets were subjected to high salt buffer extraction (20mM Tris pH 8, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl 2, 25% glycerol, 1mM DTT, and a protease inhibitor cocktail).After centrifugation at 10,000g, supernatants were collected as the nuclear fraction.To isolate cytosolic, mitochondrial, ER, nuclear, NP-40, and nuclear pellet, a fractionation protocol was utilized.Briefly, cells were trypsinized and collected by centrifugation.They were then resuspended in a hypotonic buffer (10 mM HEPES, 1mM EGTA, and 25mM KCL) for 10 minutes and centrifuged (supernatant was removed and discarded).Cells were then resuspended in an isotonic buffer (10mM HEPES, 250 mM Sucrose, 1mM EGTA, and 25 mM KCL) and Dounce homogenized.After centrifugation, the pellet (nuclear) was incubated in a hypertonic buffer (50 mM HEPES, 50mM KCL, and 300 mM NaCl) an NP-40-based buffer (20 mM Tris pH 8, 100 mM NaCl, 1% NP-40, 3 mM EDTA, 2 mM DTT, and a protease inhibitor cocktail), and the final pellet was dissolved in a buffer containing SDS (2%) and DTT (50 mM).Meanwhile, the supernatant after the Dounce homogenizer step was centrifuged at 28,000 rpm for 10 minutes and the pellet saved (mitochondrial pellet).The supernatant was then centrifuged at 48,000 rpm for 1 hour.The supernatant was designated as cytosolic fraction and final pellet was designated as ER fraction.
To isolate soluble nuclear fractions, we sequentially lysed cell components by extracting the cell fractions with buffers involving various detergents and ionic strength.Briefly, cell pellets were first incubated on end-over-end rotator for 10 min at 4 °C with ice-cold lysis buffer A (1 mM MgCl2, 1mM Na3VO2, 10 mM KCl, 50 mM HEPES (pH 7.4), 1 M Hexylene glycol, 1% protease inhibitor cocktail).Following centrifugation at 2000g for 10 min, supernatants were collected (cytosolic).
The nuclear pellet #1 was incubated on ice with ice-cold lysis buffer (10 mM NaCl, 1 mM MgCl2, 10 mM KCl, 50 mM HEPES (pH 7.4), 1% Igepal, 1 M Hexylene glycol, and protease inhibitor cocktail) for 30 min and centrifuge at 7000 g for 10 min at 4 °C to collect the nuclei.The supernatant is collected as perinuclear fraction #1.This fraction contains the proteins that are loosely associated with the nuclei.The nuclear pellet #2 was resuspended in lysis buffer containing 150 mM NaCl, 50 mM HEPES, 1% Igepal, 1 M Hexylene glycol, and protease inhibitor cocktail, incubated on ice for 30 min, and centrifuge at 7000 × g for 10 min at 4 °C to collect the nuclei.The supernatant was collected (perinuclear fraction# 2).This fraction contains the proteins that are tightly associated with the nuclei.Finally, the nuclear pellet #3 was incubated on ice for 30 min with buffer containing 400 mM NaCl, 50 mM HEPES, 0.5% sodium deoxycholate, 0.15 sodium dodecyl sulfate, 7 μL of benzonase and protease inhibitor cocktail and resuspend.
The sample was centrifuged at 7800xg for 10 min at 4 °C to remove the nuclear proteins from the non-soluble pellet.The supernatant was collected, and this fraction contains the nuclear proteins and was used to analyze proteins by Western blot.
Protein samples were immunoprecipated using protein A agarose beads (Catalogue no.9863P, Cell Signaling Technology) conjugated to p65 or Bcl-2 antibody according to manufacturer's instructions (Thermo Scientific).All samples were separated by SDS-PAGE and subsequently analyzed by the following antibodies: Bik (Catalogue no.ab52182, Abcam, Rabbit polyclonal; are contained in Supplementary Table 1. The dual luciferase reporter (DLR) assay (Promega, Madison, Wisc.) was used to test the impact of rs738276 alleles on promoter activity.Plasmids were co-transfected with the Renilla luciferase plasmid for normalization purposes.Cells were grown in 24-well culture plates until 70-80% confluent.The plasmids were transfected with TransIT-2020 (Mirus Bio, Madison, Wis.) using manufacturer's protocol, and harvested and assayed for luciferase activity twenty-four hours after transfection.
The BIK gene comprises four introns and five exons spanning a region of about 20 kb, with a total unprocessed transcript length of 18.97kb (including UTRs).The noncoding first exon precedes a 13.2kb first intron that comprises the majority of the unprocessed transcript.A promoter region for BIK was defined based on ChIP-seq data from the ENCODE project that indicates a 1947 bp region roughly symmetrical about the transcription start site with a H3K4 tri-methylation, DNase hypersensitivity, and binding of multiple transcription factors in various cell types (Supplementary Figure 3A), consistent with the presence of a promoter.Similar to the promoter region, the 750bp intronic region where rs738276 is localized, demonstrates a conservation across all placental mammals (Supplementary Figure 3A) that is comparable to the exonic regions (not shown).
Because rs738276 was not in LD with other SNPs and was located within a potential promoter region, we cloned the region upstream of a luciferase reporter gene to investigate its function.
Three luicferase reporter constructs were tested: (1) The ~2kb upstream promoter region and terminating at 980 from the transcriptional start site (pPRO).( 2) The 750bp conserved sequence immediately downstream of the pPRO promoter with either rs738276 A or G allele (p750A/G).( 3) The endogenous BIK splice acceptor at the end of the 750bp sequence of p750A/G resulting in splicing of intronic sequence (p750A/G-SA) (Supplementary Figure 3B).The point mutation A/G was constructed by site-directed mutagenesis and verified by sequencing.The pPRO construct demonstrated strong promoter activity relative to the empty vector in a variety of cell types of lung epithelial origin; ranging from 28-fold increase in N1 cells to 493-fold in H1299 (Supplementary Figure 3C).Compared with previously identified BIK promoters that comprised the 2 kb upstream region and ending at basepair +203 or -78 relative to the transcription start site (91) the pPRO construct showed a drastically increased promoter activity (Supplementary

Figure 3C
).These findings point to the importance for transcriptional activity of the BIK intronic region that is located between +203 to +980 from the transcriptional start site.However, luciferase activity was drastically reduced when the p750A/G constructs were inserted downstream of the pPRO sequence, likely due to the introduction of an inappropriate translation start site (data not shown).However, inserting the endogenous intron 1 BIK splice acceptor downstream of the 750bp conserved region (p750A/G-SA) restored the luciferase activity to that observed in pPRO construct (data not shown).
Proteomics Analysis: Mouse airway epithelial cells from bik +/+ and bik -/-mice were grown to 100% confluency.Nuclear fractions were isolated after removing the cytosolic and perinuclear fractions and immunoprecipitated using anti-Bcl-2 antibody.Immunoprecipitates were run on SDS-PAGE gels and protein bands were cut and submitted for proteomic analysis.Identified Chromosome Immunoprecipitation Assay: Primary HAECs from individuals homozygous for either rs738276 A or G allele were grown on 10 cm culture plate.At 75% confluency, cells were fixed with 1% formaldehyde, and the reaction was quenched using 1.25M glycine and scraped into cold PBS containing protease inhibitors.ChIP was performed using the Magna ChIP G kit (MAGNA0002; EMD Millipore) as described by the manufacturer.Briefly, immunoprecipitation was performed by incubating sonicated nuclear fractions overnight at 4°C with 10 µg of an anti-IRF-1 (catalogue no.sc-497) and normal mouse immunoglobulin G (IgG; sc-2025) (both from Santa Cruz Biotechnology).The genomic DNA fragments in the immunoprecipitated samples were analyzed by PCR by using a primer sets for the Bik intronic region (Forward primer 5′-TACAAAACACCACTGGGCCT -3′ and reverse primer 5′-GAGGGGGCGGTCAAGAATAC -3′), and 10 kb upstream of the BIK gene (Forward primer 5′-GTGTTGGGGCTGATAGACCA -3′ and reverse primer 5′-TCAGGGCACTCTGGGAAAGA -3′ as negative control.The primers used for the ChIP assays are listed in Supplementary Table 2.

Luminex Cytokine Assay:
The cytokines in cell culture media were quantified by Luminex instrument (Luminex Corp.) using Multiplex Fluorescent Bead-Based Luminex Cytokine Assays (EMD Millipore).
Transmission Electron Microscopy: To visualize perinuclear region of cells with higher magnification, mouse lungs were fixed overnight in a mixture of 1.25% formaldehyde, 2.5 % glutaraldehyde, and 0.03% picric acid in 0.1 M sodium cacodylate buffer, pH 7.4.The fixed tissues were washed with 0.1 M sodium cacodylate buffer and post-fixed with 1% osmium tetroxide/1.5% potassium ferrocyanide for 2 h.Samples were washed in a maleate buffer and post fixed in 1% uranyl acetate in maleate buffer for 1 h and rinsed in ddH 20 and dehydrated through a series of ethanol (50%, 70%, 95%, (2x)100%) in water for 15 minutes in each solution.
Dehydrated tissues were placed in propylene oxide for 5 min before they were infiltrated in epon mixed 1:1 with propylene oxide overnight at 4°C.The epon resin was polymerized in a 60°C oven for 48 h.Tissues were then sectioned into 80 nm thin sections and imaged using a 1200EX Transmission Electron Microscope (JEOL, Peabody, MA).
Statistics: Data from at least 6 mice per group were presented as the mean ± standard error from the mean (SEM).Statistical analyses were performed using GraphPad Prism Software 5.0 (GraphPad Software, Inc., San Diego, CA).Two-tailed student t-test was used to compare between 2 groups.For all experiments, grouped results were analyzed using one-way or twoway analysis of variance (ANOVA).When significant main effects were detected (p < 0.05), a post test of multiple comparisons was performed (Tukey) to determine differences between treatment groups.
The association between BIK genotype (rs738276) and longitudinal measure of FEV1 in LSC, / GA / AA of BIK and using GG as reference group.
We combined results using a random-effects meta-analysis, evaluating the effect of BIK genotype  were plated in the presence of ROCK inhibitor medium and serum.ROCK inhibitor medium and serum were removed, and mRNA was analyzed 6 or 24 h later by qRT-PCR.n=3/group.N=number of repeats; n=sample size in a single experiment., experimental replicates N=2.n=sample size in a single experiment; N=number of esperimental repeats.Two-tailed student t-test was used to compare between 2 groups and grouped results were analyzed using two-way analysis of variance.Data reported as mean ± SE; * p < 0.05, **p < 0.01, ***p < 0.001.included as an internal control.Cell lysates harvested at 24 h and RF and FF luciferase activity assessed using a dual-luciferase reporter assay system.The RF normalized pPRO, p-78 and p+203 values were further normalized to those of the pGL3 basic vector to indicate the strength of promoter activity.Bars denote mean±SEM (n = 3 independent experiments).The SNP rs738276 is located within a 750bp conserved region of intron 1 of the BIK gene.(E) The pPRO construct was transfected into AALEB, N1, H1299, and H292 cells and analyzed for luciferase activity.Significant luciferase activity was detected only in all four cell lines.(F, G) Promoterluciferase constructs in the pGL3 basic vector were constructed with the BIK promoter (pPRO) and the 750 bp region encompassing rs738276 with the two variants downstream of the BIK promoter (p750A/G), and the p750A/G containing a splice acceptor (p750A/G-SA).Nuclear extracts from H1299 cells were prepared, and 10 µg of each was subjected to EMSA by using biotin-labeled p750A or p750G oligonucleotide probes or the IRF-1 oligo.Competition assays with (F) p750A or p750G and (G) IRF-1 oligo DNA were performed by adding a 0, 10-, 100-, and 1000-fold molar excess of unlabeled specific oligonucleotide probes to the reaction mixture.(H) NHBEs from people with AA or GG allele of Bik SNP were plated on 6-well plates.Cells were treated with 10 ng/ml TNF-alpha for 8 h and the mRNA expression levels of IL-6 and IL-8 were analyzed using qRT-PCR.Two-tailed student t-test was used to compare between 2 groups and grouped results were analyzed using two-way analysis of variance.diet during this time.n=9/group, experimental replicates N=2.Two-tailed student t-test was used to compare between 2 groups and grouped results were analyzed using two-way analysis of variance.Data reported as mean ± SE; *p < 0.05, ** p < 0.01, *** p < 0.001.

FHS
, ECLIPSE, and COPDGene Study was assessed using linear mixed effects model with random intercept with adjustment for age, sex, pack-years, smoking status, and height at baseline visit, time since enrollment at each PFT test and principal components (6): The model included: Fev1 ~ sex + Pack years +Smoking_status+Height +Age +Follow-up years + Recessive/additive model + Follow-up years * recessive/additive genotypel + PCs, where FEV 1, Pack years, Smoking_status, and Height are time-dependent.The additive model is comparing between GG on lung function decline by years of follow up.All statistical tests and plots for LSC, ECLIPSE and COPDGene studies were performed in R (version 3.6.3).The analyses for the FHS were performed in R (version 3.5.1)using the pedigreemm package (version 0.3.3) to account for familial correlation.Study Approval: All animal studies were approved by the Institutional Animal Care and Use Committee and were performed at both the Lovelace Respiratory Research Institute, Albuquerque, NM and Brigham and Women's Hospital, Boston, MA facilities approved by the Association for the Assessment and Accreditation for Laboratory Animal Care International.All human studies and the use of the primary HAECs were approved by the respective institutional review boards or ethics committee and by the Institutional Review Board of Mass General Brigham (IRB Protocol # 2020P000254).All participants have provided a written informed consent.