Article Figures & Data
Figures
- FIG 1
Type I interferon is necessary for and promotes high-level shedding of S. pneumoniae and IAV. WT and Ifnar1−/− pups were infected i.n. with 103 CFU S. pneumoniae (Spn), 250 PFU IAV-x31, or S. pneumoniae plus IAVx31. (A and B) Ifnar1−/− pups shed significantly fewer bacteria than WT pups (A), and Ifnar1−/− pups shed significantly less IAV than WT pups (B). (C) WT and Ifnar1−/− pups first received IAV and then S. pneumoniae. Ifnar1−/− pups shed significantly fewer bacteria than WT pups. (D and E) Exogenous recombinant IFN-α2 or IFN-β increase pneumococcal shedding from WT but not Ifnar1−/− mice. WT and Ifnar1−/− pups were infected i.n. with 103 CFU S. pneumoniae and daily received either 1,000 IU of recombinant mouse IFN-α2 or 1,000 to 5,000 IU of recombinant mouse IFN-β or vehicle control (0.1% BSA-PBS) by i.n. instillation. Treatment of WT pups with rIFN-α2 (D) or rIFN-β (E) was sufficient to increase pneumococcal shedding over the baseline. Shedding data are shown as a Tukey box-and-whisker plot, with outliers shown as symbols. Each symbol represents the value from an individual pup on a single day. n ≥ 8 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney test).
- FIG 2
Pneumococci are shed and stimulate IFN-I at 21 days p.i. in a pneumolysin-dependent manner. (A and B) WT pups were infected i.n. with 103 CFU S. pneumoniae; mock-treated mice received PBS. RNA was isolated from URT lavages on day 21 p.i. and analyzed by RNA-seq. Shown is a heat map of 543 genes identified as the interferome, comparing S. pneumoniae to mock infection (A) or S. pneumoniae to the S. pneumoniae ply mutant strain (B), demonstrating relative gene expression as log2 fold change, with increased expression in red and decreased expression in blue. n = 5 mice per group. (C) RNA was isolated from URT lavages on day 21 p.i. and analyzed by qRT-PCR. At 21 days p.i., S. pneumoniae-infected mice showed significantly increased expression of the ISGs Ifit2, Mx1, and Oasl2 over mock-infected mice. This increased expression was seen only in WT and not Ifnar1−/− mice. (D to F) WT pups were infected i.n. with 103 CFU S. pneumoniae T4 or T23F (D), or WT and Ifnar1−/− pups were infected i.n. with 103 CFU S. pneumoniae T4 or an isogenic T4 ply mutant (E and F). (D) T4 and T23F are shed 21 days p.i., which correlates with gene expression data. (E) At 21 days p.i., there are more high-shedding events (>200) in S. pneumoniae-infected mice than mice infected with the isogenic S. pneumoniae ply mutant. (F) At 21 days p.i., there is no difference in the number of high-shedding events between S. pneumoniae and S. pneumoniae ply mutant strains in Ifnar1−/− mice. Gene expression data are log10 transformed; each symbol represents the value from an individual pup, and the line represents the mean. Comparisons (Mann-Whitney test) are to mock-infected mice of the respective genotypes. Shedding data are shown as Tukey box-and-whisker plots, with outliers shown as symbols. Each symbol represents the value from an individual pup on a single day. n ≥ 8 pups/group. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (Mann-Whitney test); #, P = 0.03 (one-tailed Fisher’s exact test).
- FIG 3
S. pneumoniae and IAV stimulate IFN-I signaling at 48 h p.i. WT and Ifnar1−/− pups were infected i.n. with 103 CFU the S. pneumoniae T4 or T4Δply mutant, 250 PFU IAV, or T4 plus IAV; mock-infected mice received PBS. RNA was isolated from URT lavages 48 h p.i. and analyzed by qRT-PCR. S. pneumoniae-infected mice showed significantly increased expression of the ISGs Ifit2, Mx1, and Oasl2 compared to that of mock-infected mice. This increased expression was seen only in WT and not Ifnar1−/− mice. The stimulation of ISGs by S. pneumoniae was ply dependent. Gene expression data are log10 transformed; each symbol represents the value from an individual pup. Comparisons (Mann-Whitney test) are to mock-infected mice of the respective genotype. n ≥ 5 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
- FIG 4
S. pneumoniae colonization increases nasal secretions of pups. (A and B) WT and Ifnar1−/− pups were infected i.n. with 103 CFU S. pneumoniae or 250 PFU IAV; mock-infected mice received PBS. RNA was isolated from URT lavages 48 h (A) or 21 days (B) p.i. and analyzed by qRT-PCR. S. pneumoniae-infected mice showed significantly increased expression of the ISG sialyltransferase St3gal1 over mock-infected mice. This increased expression was seen only in WT and not Ifnar1−/− mice. The stimulation of ISGs by S. pneumoniae was also ply dependent. Gene expression data are log10 transformed; each symbol represents the value from an individual pup, and the line represents the mean. Comparisons (Mann-Whitney test) are to mock-infected mice of the respective genotype. n ≥ 5 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Pups infected with IAV or S. pneumoniae secreted more sialic acid from their nares than pups that received PBS. IAV comparisons are days 14 to 18 p.i. to matched PBS controls; S. pneumoniae comparisons are of days 9 to 12 p.i. The increase in nasal secretions from S. pneumoniae-infected pups is IFN-I dependent, as there was no difference detected in sialic acid shed from infected Ifnar1−/− pups compared to PBS controls (days 9 to 12 p.i.). WT and Ifnar1−/− pups were infected i.n. with either 103 CFU S. pneumoniae T23F P2636 (neuraminidase mutant) or 250 PFU IAV-x31 or received PBS. Band integrated pixel density data are for individual pups, with the bar indicating the median. Each symbol represents the value from an individual pup on a single day. n ≥ 3 pups/group. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Mann-Whitney test).
- FIG 5
Lack of IFN-I signaling reduces transmission of IAV and S. pneumoniae. (A) WT and Ifnar1−/− index pups were infected i.n. with 103 CFU S. pneumoniae at a ratio of 6 index to 20 contact in WT litters or 5 index to 18 contact in Ifnar1−/− mice in three separate litters. The S. pneumoniae transmission rate and bacterial burden were higher in WT than Ifnar1−/− contact mice. (B) WT and Ifnar1−/− index pups were infected i.n. with 250 PFU IAV at a ratio of 12 index to 22 contact in WT litters or 10 index to 28 contact in Ifnar1−/− mice in five separate litters. The IAV titer was higher in WT than Ifnar1−/− contact mice. Colonization and titer data are for individual pups, with the line indicating the geometric mean. Each symbol represents the value from an individual pup on a single day. n ≥ 5 pups/group. *, P < 0.05; ***, P < 0.001 (Mann-Whitney test). The dotted line shows the limit of detection.
Supplemental Material
FIG S1
S. pneumoniae colonization and IAV titer do not differ in WT and Ifnar1−/− pups. Pups were infected i.n. with either 103 CFU S. pneumoniae or 250 PFU IAV-x31. (A) S. pneumoniae URT colonization was not different between WT and Ifnar1−/− pups. (B) There was no difference in viral titer in the URT of WT and Ifnar1−/− pups. (C) WT and Ifnar1−/− pups first received IAV and then S. pneumoniae; bacterial colonization was not different between pups. (D and E) WT and Ifnar1−/− pups were infected i.n. with 103 CFU S. pneumoniae and daily received 1,000 IU of recombinant mouse IFN-α2 or 1,000 to 5,000 IU of recombinant mouse IFN-β or vehicle control (0.1% BSA-PBS) by i.n. instillation. Treatment of WT pups with rIFN-α2 (D) or rIFN-β (E) did not affect bacterial colonization. Colonization and titer data are for individual pups, with the line indicating the geometric mean. Each symbol represents the value from an individual pup on a single day. n ≥ 8 pups/group. ns, not significant (Mann-Whitney test). The dotted line shows the limit of detection. Download FIG S1, PDF file, 0.2 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
S. pneumoniae T4 and T23F colonization at 21 days p.i. does not differ in WT and Ifnar1−/− mice. (A to C) WT pups were infected i.n. with 103 CFU S. pneumoniae T4 or T23F (A), or WT and Ifnar1−/− pups were infected i.n. with 103 CFU S. pneumoniae T4 or an isogenic T4 ply mutant; colonization was assessed 21 days p.i. (B and C). (A) The mice are still highly colonized with S. pneumoniae of both T4 and T23F serotypes. (B) Colonization of T4 and the isogenic T4 ply mutant strain is not different. (C) At 21 days p.i., there is no difference in the colonization between T4 and T4 ply mutant strains in Ifnar1−/− mice. Colonization data are for individual pups, with the line indicating the geometric mean. Each symbol represents the value from an individual pup on a single day. n ≥ 8 pups/group. ns, not significant by Mann-Whitney test. Download FIG S2, PDF file, 0.2 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S1
Genes upregulated in S. pneumoniae-infected mice over PBS mice at 21 days p.i. Mice received S. pneumoniae T23F or PBS, i.n., on day 4 of life; on day 21 p.i., the mice were euthanized and RNA was collected by URT lavage. Download Table S1, XLSX file, 0.1 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S2
Interferome analysis of genes upregulated in S. pneumoniae-infected mice at 21 days p.i. The 915 genes identified in the RNA-seq were determined to be ISGs (n = 543) by use of the Interferome database, v2.01, at http://www.interferome.org/interferome/home.jspx (18). Download Table S2, XLSX file, 0.03 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Immunoblot of S. pneumoniae strains for pneumolysin. S. pneumoniae serotypes T4 and T23F used in this study do not produce different levels of pneumolysin. Download FIG S3, PDF file, 0.4 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
Increased expression of Ifna and Ifnb was not detected at 48 h p.i. in mice that had received S. pneumoniae. WT pups were infected i.n. with 103 CFU S. pneumoniae T4 or 250 PFU IAV; mock-infected mice received PBS. RNA was isolated from URT lavages 48 h p.i. and analyzed by qRT-PCR. S. pneumoniae-infected mice showed no increased expression of Ifna or Ifnb over mock-infected mice. This increased expression was seen only in IAV-infected mice. Gene expression data are log10 transformed; each symbol represents the value from an individual pup. Comparisons (Mann-Whitney test) are to mock-infected mice. n ≥ 5 pups/group. ns, not significant; **, P < 0.01. Download FIG S4, PDF file, 0.2 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
TABLE S3
Primers (forward and reverse) used in RT-PCR analysis of the expression of interferon stimulated genes (plus Gapdh control) from the mouse upper respiratory tract. Download Table S3, DOCX file, 0.01 MB.
Copyright © 2021 Zangari et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
