Fig 2: The S513A mutation destabilizes Regnase-1 protein but does not affect target mRNA abundance.(A–C) Immunoblot analysis of Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) (A), BMDMs stimulated with LPS (100 ng/ml) (B), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) (C) for indicated time. PECs were pretreated with MG-132 (5 μM) 2 hr before the stimulation. (D)-(F) mRNA expression of Zc3h12a and Il6 in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml) for 4 hr (D), BMDMs stimulated with LPS (100 ng/ml) for 4 hr (E), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml) for indicated time (F). (G)-(I) IL-6 secretion in Zc3h12aWT/WT and Zc3h12aS513A/S513A MEFs stimulated with IL-1β (10 ng/ml), IL-17A (50 ng/ml), or TNF (10 ng/ml) for 24 hr (G), BMDMs stimulated with Pam3CSK4 (1 or 10 ng/ml), poly I:C (10 or 100 μg/ml), LPS (10 or 100 ng/ml), R848 (10 or 100 nM), or CpG DNA (0.1 or 1 μM) for 24 hr (H), and thioglycollate-elicited PECs stimulated with LPS (100 ng/ml), R848 (100 nM), or IL-1β (10 ng/ml) for 24 hr (I). (J) Schematic representation of Model 1 in which 14-3-3-bound Regnase-1 does not have the function of degrading its target mRNAs. This model could explain the experimental observations. (K) Schematic representation of Model 2 in which 14-3-3-bound Regnase-1 maintains some ability to degrade its target mRNAs. This model is not consistent with the experimental observations. In (D)-(I), bars represent mean values of biological replicates (n = 3), and error bars represent standard deviation. Data is representative of two independent experiments, each with three biological replicates.

Fig 3: Nasal and Sinus Mucosa, Related to Figures 4 and 5(A). Expression of ACE2 and TMPRSS2 across donors.(B). Enhanced capture of ACE2 mRNA with second strand synthesis protocol employed in Seq-Well S3. Dot size represents fraction of cells expressing.(C). Cultured human primary basal epithelial cells at confluence were treated with increasing doses (0.1 to 10ng/mL) of IFNa2, IFN?, IL-4, IL-13, IL-17A, and IL-1B for 12 h and bulk RNA-seq analysis was performed (Replicate experiment using Human Donor 1 as in Figure 5)(D). ACE2 expression by stimulation condition. Wilcoxon test between each cytokine (combined doses) versus rest: IFNa Bonferroni-adjusted p = 4.1E-07; IFN? Bonferroni-adjusted p = 9.3E-03; all else n.s. *** p < 0.001.(E). ACE2 expression by IFNa2 dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: *** p < 0.001, * p < 0.05.(F). ACE2 expression by IFN? dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: *** p < 0.001, * p < 0.05.(G). IFITM1 expression by IFNa2 dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: *** p < 0.001.(H). IFITM1 expression by IFN? dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: *** p < 0.001.(I). GBP5 expression among cultured human primary basal epithelial cells. Wilcoxon test: IFNa versus IFN? Bonferroni-adjusted p = 2.94E-07; IFN? Bonferroni-adjusted p = 9.3E-03. TP10K: transcripts per 10,000 reads. *** p < 0.001.(J). GBP5 expression by IFNa2 dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: *** p < 0.001.(K). GBP5 expression by IFN? dose. Bonferroni-corrected t-test compared to 0 ng/mL condition: *** p < 0.001.

Fig 4: SLPI Is Regulated by Colonization and Mediates Protection from AAI(A) Heatmap of immune genes regulated by Bph colonization in mice undergoing AAI. Average normalized read counts for each group are shown in gray, and log2-fold change in mice that received a live, compared to an HK, inoculum for each gene is shown in blue, white, and red. n = 3–5 mice/group.(B) Volcano plot of whole-lung transcriptomic data from mice that received either an HK or live Bph inoculum and then underwent OSC (as shown in Figure 4A). Genes depicted as triangles were significantly enriched in colonized mice not undergoing AAI. Genes involved in an immune system process (defined by the Gene Ontogeny [GO] pathway, GO: 0002376) are shown in light blue and are also depicted in the heatmap shown in (A). n = 3–5 mice/group.(C) ELISA showing SLPI protein expression in the lungs of mice inoculated with live Bph followed by AAI compared to those inoculated with HK. n = 7–8 mice/group.(D) Correlation between SLPI protein expression and fold change in Il17a measured by qRT-PCR in the lungs of mice inoculated with HK or live Bph, followed by AAI. n = 7–8 mice/group(E) qRT-PCR of SLPI from the whole lungs from germ-free, RAG1-/-, and conventionally raised WT mice. n = 9–10 mice/group.(F) Transcription of Slpi in human alveolar epithelial cell line A549 in response to cytokine stimulation. Cells were treated with 1 ng/ml IL-1ß, 100 ng/ml IL-17A, and/or 10 ng/ml TNF-a as shown. Experiment performed in n = 5 biological replicates, each representing the average of 3 technical replicates.Statistical significance: Wald test with BH correction as implemented in DESeq2 in (A) and (B); Wilcoxon rank-sum test for (C); Spearman’s rank test in (D); or Kruskal-Wallis test followed by post hoc two-tailed paired Wilcoxon rank-sum test with adjustment for multiple hypotheses using BH correction in (E) and (F). Boxes indicate 25th and 75th percentiles and whiskers are 1.5 × interquartile range. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 5: The Th17 Response to Bph Is Antigen Specific and Aids Controlling Colonization(A) Left panel: Concatenated flow cytometry plots of cultures of splenocytes of five mice taken 52 days after inoculation with either HK or live Bph. Splenocytes were loaded with either no protein or Bph proteins from a HK culture. Cells were gated on CD4+TCRß+ cells using CD25 as an activation marker and CFSE as a proliferation marker. Representative of 2 independent experiments. Right panel: Quantification of CD25+CFSElo T cells from splenocyte cultures as shown in (A). n = 9–10 mice/group, combined from 2 independent experiments.(B) IL-17A ELISA of culture supernatants from lung CD4+ T cells co-cultured with antigen-loaded CD11c+ dendritic cells (DCs). CD4+ T cells were isolated from the lungs of five mice taken 43 days after inoculation with either HK or live Bph.(C) CFU of Bph recovered from BALs of WT or RAG1-/- mice 14 days after colonization. n = 10 mice/group, combined from 2 independent experiments.(D) Top: Schematic of the experimental approach to test the role of IL-17A during Bph colonization. Bottom: CFU of Bph recovered from lung homogenates (CFU/g), BAL, or nasal lavage (CFU/ml).(E) ELISA showing SLPI protein expression in the lungs of mice inoculated with Bph and treated with anti-IL-17A monoclonal antibody compared to the isotype control.Statistical significance: Kruskal-Wallis followed by post hoc one-tailed paired Wilcoxon rank-sum test with adjustment for multiple hypotheses using BH correction for (A) and (B) or two-tailed Wilcoxon rank-sum test for (C)–(E). Horizontal lines indicate median values. **p < 0.01; ***p < 0.001.