Fig 1: 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 2: ACE2 is an Interferon-Stimulated Gene in Primary Human Barrier Tissue Epithelial Cells(A–D) Basal epithelial cells from distinct sources were cultured to confluence and treated with increasing doses (0.1–10 ng/mL) of IFN-a2, IFN-?, IL-4, IL-17A, and/or IFN-ß for 12 h and bulk RNA-seq analysis was performed. Expression of ACE2 (human) or Ace2 (mouse) by cell type and stimulation condition. (A) Primary mouse basal cells from tracheal epithelium are shown. (B) BEAS-2B human bronchial cell line is shown. (C) Primary human basal cells from nasal scraping, Donor 1, is shown. (D) Primary human basal cells from nasal scraping, Donor 2. Abbreviation is as follows: TP10K, transcripts per 10,000 reads. ***p < 0.001, **p < 0.01, *p < 0.05, Bonferroni-corrected t test compared with untreated condition.(E–H) Co-expression of STAT1/Stat1 and ACE2/Ace2 by cell type. (E) Primary mouse basal cells from tracheal epithelium are shown. (F) BEAS-2B human bronchial cell line is shown. (G) Primary human basal cells from nasal scraping, Donor 1, are shown. (H) Primary human basal cells from nasal scraping, Donor 2 are shown. Abbreviation is as follows: TP10K, transcripts per 10,000 reads. Statistical significance assessed by Spearman’s rank correlation.(I–L) Expression of ACE2 in primary human basal cells from nasal scrapings across a range of concentrations of IFN-? or IFN-a2. (I) IFN-a2 dose response in Donor 1 (p < 0.001 by one-way ANOVA) is shown. (J) IFN-? dose response in Donor 1 (p < 0.01 by one-way ANOVA) is shown. (K) IFN-a2 dose response in Donor 2 (p < 0.001 by one-way ANOVA) is shown. (L) IFN-? dose response in Donor 2 (p < 0.001 by one-way ANOVA). Abbreviation is as follows: TP10K, transcripts per 10,000 reads. ***p < 0.001, **p < 0.01, *p < 0.05, Bonferroni-corrected post hoc testing compared with 0 ng/mL condition.See also Figures S3 and S4 and Table S7.
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: Bordetella pseudohinzii (Bph) Induces a Th17 Response in the Lungs of Colonized Mice(A) Comparison of the abundance of virulence factor classes encoded in the genomes of members in the genus Bordetella. Virulence factors were identified by sequence alignment to the Virulence Factor Database (VFDB) and binned into functional groups defined by VFDB (Chen et al., 2016). Assemblies of Bph isolates described in this study (2–1 and 5–5) are also shown.(B) Recovery of Bph from respiratory tract samples over a 184-day period. CFUs per section of tissue or mL of lavage fluid are shown. Box indicates 25th and 75th percentiles and whiskers are 1.5 × interquartile range. n = 2–6 mice per time point.(C) Representative hematoxylin and eosin staining of mice that received either HK (top panel) or live (bottom panel) Bph taken 60 days after inoculation.(D–F) Flow cytometry of lung tissue digests from mice 30 days after that received HK (blue) or live Bph (red) inoculation. n = 9–10 mice/group, combined from two independent experiments.(D) Neutrophils as a percentage of live cells from lung. Neutrophils were defined as CD11b+Ly6G+.(E) Percentage of Teff (CD4+TCRß+FoxP3-CD44hiCD62Llo) cells from the lungs as a percentage of total T-helper cells.(F) Percentages of IL-17A+IFN?--secreting T-helper 17 (Th17) cells from the lungs.Statistical significance: Mann-Whitney U test. Horizontal lines indicate median values. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 5: 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.
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