Linking lipid metabolism to the innate immune response in macrophages through sterol regulatory element binding protein-1a

Abstract

We show that mice with a targeted deficiency in the gene encoding the lipogenic transcription factor SREBP-1a are resistant to endotoxic shock and systemic inflammatory response syndrome induced by cecal ligation and puncture (CLP). When macrophages from the mutant mice were challenged with bacterial lipopolysaccharide, they failed to activate lipogenesis as well as two hallmark inflammasome functions, activation of caspase-1 and secretion of IL-1β. We show that SREBP-1a activates not only genes required for lipogenesis in macrophages but also the gene encoding Nlrp1a, which is a core inflammasome component. Thus, SREBP-1a links lipid metabolism to the innate immune response, which supports our hypothesis that SREBPs evolved to regulate cellular reactions to external challenges that range from nutrient limitation and hypoxia to toxins and pathogens.

Figure 1. Tissue patterns for SREBP mRNA expression

Tissue survey for SREBP-1 and SREBP-1c: total RNA from the indicated tissues was used for qPCR with an internal control DNA standard for quantitation. Values are presented as fg of RNA/µg total RNA. Data are from pooled RNA samples from WT and SREBP-1aDF (n=9/group). SR-1a and SR-1c correspond to SREBP-1a and SREBP-1c respectively. Abbreviations for different tissues are as follows: BAT, brown adipose; ING, inguinal white fat; GON, gonadal white fat; DUOD, duodenum; BMDM, bone marrow derived macrophage; PTM, peritoneal derived thioglycholate elicited macrophage; BMDC, bone marrow dendritic cell.

Figure 2. Role of SREBP-1a in Inflammatory signaling in mice

(A) Survival curves for WT or SREBP-1aDF mice after i.p injection with LPS (10 mg/kg). Animals were monitored over 7 days after LPS injection, WT vs. 1aDF p=0.0308. (B) Serum IL-1β levels were analyzed by ELISA, *WT vs 1aDF after LPS treatment, p=0.0007. (C–D) WT or SREBP-1aDF mice (n=10) underwent CLP using one puncture from a 23-gauge needle. (C) Survival curves for WT or SREBP-1aDF mice after CLP surgery, WT-CLP vs 1aDF-CLP, p=0.0015 by Mantel-Haenszel log rank test. (D) Blood was collected after 24 h and serum IL-1β was measured **WT-CLP vs 1aDF-CLP after 24 h p=0.0082. WT-sham, Sham laparotomy with WT mice; WT-1p, one puncture in cecum of WT; SREBP-1aDF-sham, SREBP-1aDF sham laparotomy; SREBP-1aDF-1p, one puncture in cecum of SREBP-1aDF mice. (E) H and E staining of cecal sections from S. typhimurium infected mice, arrows denote areas of neutrophil infiltration that is shown at a higher magnification for the inset region as noted. (F) Pathology assessment and inflammation scores: color code for bar symbols is as follows: blue-neutrophils, maroon-mononuclear infiltrate, green-submucosal edema, purple-surface erosions, light blue-inflammatory exudate, orange-cryptitis. (G) Cecal S. typhimurium colonization (colony forming units/mg of tissue, CFU/mg) at 72 hrs post inoculation, p=6.5 × e⁻⁶. (H) CXCL1 mRNA in S. typhimurium infected cecum measured by qPCR. #WT vs 1aDF p=0.0006.

Figure 3. SREBP expression levels in BMDM of WT and SREBP-1aDF mice

Total RNA was isolated from BMDM and equal amounts of RNA were used for the quantitative RT-PCR assay. RNA expression level for SREBP-1a (A), SREBP-1c (B), and SREBP-2 (C) are shown as the quantitative expression levels calculated by comparison to qPCR values obtained with a standard curve of serially diluted linear plasmid cDNA. The data are plotted as femtograms of RNA normalized to ribosomal L32 samples analyzed in parallel. Bars represent the standard deviations. (D) nuclear SREBP-1 protein level. Nuclear protein was prepared from BMDM of WT and SREBP-1aDF mice. Aliquots (30 µg) were analyzed by immunoblotting for SREBP-1. *, p = 0.0031, indicating a statistically significant difference between BMDM from the two lines of mice.

Figure 4. SREBP-1a regulates Nlrp1a expression level in BMDM

(A) DNA microarray analysis of gene expression in BMDM of WT and SREBP-1aDF mice (n=9, pooled three mice for each group). The “heatmap” represents genes showing a statistically significant 2-fold or greater induction in WT as well as a 2-fold or difference in expression from SREBP-1aDF compared to WT mice. (B) Representative values from the microarray, NC indicates no change. Expression of Nlrp1a (C) or Nlrp1c (D) was confirmed by qPCR. Data are mean ± SD; n=3 for 3 separate experiments. * WT vs SREBP-1aDF, p=0.0052, ** WT vs SREBP-1aDF, p=0.0049, # ad-GFP vs ad-1a in SREBP-1aDF BMDM, p=0.0391.

Figure 5. LPS mediated IL-1β production is decreased in SREBP-1aDF macrophages

BMDM were treated with 50 ng/ml LPS for the indicated times and collected for analysis. (A) Caspase-1 activity was measured on cell extracts and normalized for zero time for each sample. (B) Macrophages were stimulated with LPS (50 ng/ml) for 6 h, followed by incubation with or without 1 mM ATP for 30 min. IL-1 released into the medium was released into the medium was measured by ELISA *WT vs SREBP-1aDF, p=0.038, **WT vs SREBP-1aDF, p=0.007. (C) IL-1β release from BMDM after infections with S. typhimurium. BMDM from SREBP-1aDF or WT mice were infected with S. typhimurium (MOI=10) for 24 h. Data are shown as geometric means (bars) from three experiments ± the standard errors. #MOI=10; p= 0.004. (D) 100 pmol of si-RNA targeting Nlrp1a, Nlrp3 or a control siRNA (Nci) were added to macrophages that were subsequently treated with LPS (200 ng/ml) for 6 h and then pulsed with 1 mM ATP for 30 min. IL-1 was measured as above. (E–F) Nlrp1a or Nlrp3 RNA levels were measured in samples from D. (G) SREBP-1aDF macrophages were electroporated with vector (pcDNA) or an expression construct encoding SREBP-1a (SR-1a) as described in Experimental Procedures, incubated with LPS +/− ATP and secreted levels of IL-1 were measured as above.

Figure 6. SREBP-1a activates and binds to Nlrp1a promoter

BMDMs in 100-mm dishes were infected with Ad-GFP or Ad-SREBP-1a (10 MOI) for 24 h and RNA was extracted and Nlrp1a or Nlrp1c expression was analyzed by qPCR and normalized using L32. (C) There is a SREBP element in the Nlrp1a promoter between −358 bp and −351 bp. (D–E) Deletion constructs for the Nlrp1a promoter were f analyzed for promoter activity, an internal control expression plasmid for β-galactosidase driven by a CMV promoter was used for normalization. Letters refer to diagrams in C, *pGLM vs p-1208 after transfected SREBP-1a, p=0.0018, **p-1208 vs p-208 after transfected SREBP-1a, p=0.0006. (F) Chromatin from BMDM was analyzed for recruitment of SREBP-1 to the SRE region of Nlrp1a promoter by ChIP as described in Methods (filled arrows in Figure 3C with). (G) As a negative control, we used primers for a non-SRE region for PCR (dashed arrows in Figure 3C). DNA in the precipitation with SREBP-1 antibody was normalized to input and plotted relative to the IgG background. #anti-IgG vs anti-SR1, p=0.02 Data are mean ± SD; n=3 for 3 separate experiments. ‡ad-GFP vs ad-1a in SREBP-1aDF BMDM, p=0.0391.

Figure 7. SREBP-1a activates lipogenesis and is a proinflammatory gene in BMDM

(A) de novo fatty acid biosynthesis was measured before or after LPS stimulation. (B) Nuclear protein was analyzed for SREBP-1 by immunoblotting and α-tubulin was used as normalization control; nSREBP-1 denotes nuclear SREBP-1. (C) Schematic diagram for SREBP-1a promoter (arrows denote location of primers used for ChIP). An NF-κB binding site is located between two Sp1 sites in the SREBP-1a promoter. BMDMs were cultured in control medium or treated with LPS (100 ng/ml) for 24 h. (D) RNA was analyzed by qPCR for SRBEP-1a or 1c as indicated. L32 were used as normalization control. RNA analyses were performed in triplicate and are expressed as the mean ± SD. # LPS treated vs untreated group in WT, p=0.05. (D) ChIP analysis. *LPS treated vs untreated group in NF κB; p=0.026, **LPS treated vs untreated group in Sp1; p=0.003.