Fig 2: Novel inflammatory promoter of Aim2 is regulated by IRF3 and p65.(A) UpSet plot showing the number of alternative first exon (AFE) events out of 77 total that have differential transcription factor (TF) binding and differential chromatin accessibility and all combinations of these sets. (B) Pie chart representing the gene ontology of TF motifs identified through analysis motif enrichment (AME) tool of all AFE promoter sequences. (C) Venn diagram of all motifs defined using HOMER analysis within the annotated and unannotated promoter regions. (D, E) DAVID analysis examining the gene ontology of TFs at the annotated and unannotated promoters of Aim2.

Fig 3: Unannotated 5' untranslated region (UTR) of Aim2 negatively regulates translation through a single iron-responsive element (IRE).(A) Schematic of annotated and unannotated 5'UTR of most prevalent Aim2 isoforms in mouse macrophages. (B) Diagram of cloning strategy of Aim2’s 5'UTR in GFP plasmid. (C) Transfection strategy of 5'UTR-GFP plasmids co-transfected with an mCherry control plasmid at a 1–1 ratio in 293T cells. (D, E) Flow cytometry of 293T cells 72 hr post-transfection with control annotated and unannotated 5'UTR of Aim2 to measure GFP and mCherry (Ctl) protein fluorescence. (F) Using RegRNA2.0, a single IRE was found in the alternative 5'UTR, in addition to multiple Musashi binding elements (MBEs). (G) Diagram of how an IRE functions in the cytoplasm of a cell within a 5'UTR. With low or normal levels of iron, iron binding proteins (IRP1 or IRP2) bind to IRE and block translation. During high levels of iron, within a cell, IRP1 is sequestered by iron-sulfur (Fe-S) clusters and IRP2 is degraded, therefore allowing translation of the protein. (H, I) Flow cytometry of 293T cells 72 hr post-transfection of mCherry (Ctl), along with an annotated 5'UTR-GFP plasmid, unannotated 5'UTR-GFP plasmid, or a GFP plasmid containing the unannotated 5'UTR without the defined IRE. (J, K) Flow cytometry of 293T cells ± 100 µM ferric ammonium citrate (FAC) 72 hr post-transfection of mCherry (Ctl), along with an annotated 5'UTR-GFP plasmid or unannotated 5'UTR-GFP plasmid. (L) Overview of the polysome profiling protocol to analyze translation activity. (M) Cytoplasmic lysates from ±lipopolysaccharide (LPS)-treated cells were fractionated through sucrose gradients. Global RNA polysome profiles generated by the density gradient fractionation system are shown. A representative plot from stimulated primary bone marrow-derived macrophage (BMDM)-fractionated samples is shown. The experiment was performed three times. (N–Q) The relative distribution of Gapdh mRNA, encoding a housekeeping protein, Neat1, long non-coding RNA (lncRNA), annotated and unannotated Aim2 mRNA, was measured by RT-qPCR analysis of RNA. Each of the gradient fractions are calculated as relative enrichment when compared to unfractionated input mRNA; standard deviation represents technical triplicate. (R) Protein lysates of time-course LPS stimulation of 0 hr, 6 hr, 24 hr, 48 hr, and 72 hr without and with 100 µM of FAC (iron) added to immortalized WT BMDMs. Western blot performed on AIM2 and B-ACTIN. (S) Western blot quantification performed in FIJI, standard deviation represents biological triplicates, and p-value assessed using Student’s t-test. Figure 4—source data 1.Supplemental WB Uncrop Primary BMDM +/- Iron.

Fig 4: Global profiling of the cellular alternative splicing landscape in human and mouse macrophages post-inflammatory.(A) Diagram of RNA-seq library generation. (B) Categorization of significant splicing events in human and mouse macrophage. (C) Categorization of significant splicing events found in mouse bone marrow-derived macrophages (BMDM) ±6 hr lipopolysaccharide by using either the Gencode annotation or the GENCODE+ de novo annotation. (D) Venn diagram representing unique and common genes with alternative first exon (AFE) events found in RNA-seq of primary BMDMs post-inflammatory stimulation using the Gencode annotation or the GENCODE+ de novo annotation. Volcano plots of all differentially expressed genes from RNA-seq of either human (E) or mouse (F) macrophages. Genes highlighted in red undergo significant AFE changes following inflammation. Schematic of AFE inclusion and exclusion isoforms, followed by RT-PCR gel results and percent spliced in calculation for Argehf7 (G) Denr (H), and Aim2 (I), was performed in biological triplicates, p-value assessed using Student’s t-test.

Fig 5: GSDMD cleavage after TBI was mainly driven by the NLRP3 inflammasome rather than the NLRP1, NLRC4, and AIM2 inflammasomes. (A) Representative immunoblots images of NLRP1, NLRP3, NLRC4, AIM2, caspase 1, caspase 1 p20, and total protein. (B–G) Quantitative analysis of NLRP1, NLRC4, AIM2, NLRP3 caspase 1, and caspase 1 p20 levels (n = 6). All data are presented as the mean ± SEM and were analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test for significance. *p < 0.05, **p < 0.01, and ***p < 0.001.