Fig 2: Macrophages lacking AUF1 functions display minimal changes in their activation programs and properly acquire M1- and M2a- like phenotypes in vitro. (A) Volcano plot depicting the distribution of the adjusted P values (-Log10 (adjusted P value) and the fold changes (Log2 FC) of mRNA transcripts differing between control and M-Δx3,4 BMDMs treated with LPS for 4hrs and analyzed via 3’end sequencing. Significantly upregulated and downregulated transcripts are indicated by color. (B) Heatmap depicting the extend of significant changes in the levels of transcripts differing between 3 samples of LPS treated control and M-Δx3,4 BMDMs. (C) Quantitation of hnRNPD RNA isoforms as derived from sequencing analyses of activated control and M-Δx3,4 BMDMs. Bar graphs depict mean values of Transcripts Per Kilobase Million (TPKM) of ENSEMBL annotated RNAs for the p37,40,42 & 45 isoforms of AUF1; other protein coding fragments (PF); retained intron transcripts (RI); or RNAs yielded via NMD. (*) asterisks denote statistical significance as per the bioinformatic analyses of the datasets. (D) Functional classification of differential expressed transcripts as identified via Webgestalt (2019). Bubble plots indicate the enrichment scores and overlap of the functional categories. (E) Quantitation of total hnRNPD mRNA in Control and M-Δx3,4 BMDMs (n=5) in a resting state (M0), following exposure to LPS+INFγ, or IL4. Bar graphs denote mean fold changes (FC ± SD) in exon 3 containing mRNAs relative to resting control values (CM0) as assessed via qRT-PCR. (F) Quantitation of factors marking the classical or the alternative polarization of macrophages in control and M-Δx3,4 BMDMs (n=10) either in a resting state (M0), following exposure to LPS+IFNγ, or IL4. Bar graphs denote mean values ( ± SD) of secreted proteins assessed via Cytometric bead arrays; or mean fold changes (FC) in mRNAs relative to resting control values (CM0) as assessed via qRT-PCR. In all bar graphs, (*, ****) denote p values ≤0.05 or ≤0.00001 respectively as determined via unpaired Student’s t-test.

Fig 3: PD-1high ILC2s expressed higher levels of IL-4 and IL-13. PD-1high ILC2s and PD-1low ILC2s were marked as CD45+Lin-CD127+CRTH2+PD-1high and CD45+Lin-CD127+CRTH2+ PD-1low, respectively, and sorted by FACS. (A, B) The gene expressions of PDCD1 (encoding PD-1) (A), IL4, and IL13 (B) were upregulated in the PD-1high group compared with the matched PD-1low group (n=26). (C–F) The representative results and statistical results of IL-4 (C, D) and IL-13 (E, F) protein expression according to the different PD-1 levels assessed by flow cytometry (for each group, n=26). Each dot represents one sample. ***P<0.001, ****P<0.0001. P values were calculated using a paired two-tailed student’s t-test.

Fig 4: Genome-wide transcriptomic analysis of av-deficient TH cells(a) Principal component analysis of the transcriptomes of control versus av-deficient TH cells. (b) Volcano plot depiction of differentially expressed genes in control versus av-deficient TH2 cells. (c) & (d) RNA expression of type 2 genes by control or av-deficient TH cells; Il13 ***P=0.0002, **P=0.0012; Il5 ***P=0.0006, **P=0.0025; Il4 **P=0.0046, *P=0.0167, Gata3 ****P<0.0001. (e) RNA expression of type-1 signalling genes by control or av-deficient TH cells; Statl ****P<0.0001, *P=0.0438; Isg2O *P=0.0148; Runx3 **P=0.0052, *P=0.0220. (c) - (e) mean ± SEM; one-way ANOVA with Tukey’s post-hoc test. (f) Flow cytometric analysis of Runx3 expression by av-deficient TH cells. Data are representative of 2 independent experiments; unpaired two-sided t-test; ***P=0.0003. (g) KEGG pathway analysis of genes downregulated in av-deficient versus control TH2 cells.