Regulatory T cell-derived IL-1Ra suppresses the innate response to respiratory viral infection

Abstract

Regulatory T (T_reg) cell modulation of adaptive immunity and tissue homeostasis is well described; however, less is known about T_reg cell-mediated regulation of the innate immune response. Here we show that deletion of ST2, the receptor for interleukin (IL)-33, on T_reg cells increased granulocyte influx into the lung and increased cytokine production by innate lymphoid and γδ T cells without alteration of adaptive immunity to influenza. IL-33 induced high levels of the interleukin-1 receptor antagonist (IL-1Ra) in ST2⁺ T_reg cells and deletion of IL-1Ra in T_reg cells increased granulocyte influx into the lung. T_reg cell-specific deletion of ST2 or IL-1Ra improved survival to influenza, which was dependent on IL-1. Adventitial fibroblasts in the lung expressed high levels of the IL-1 receptor and their chemokine production was suppressed by T_reg cell-produced IL-1Ra. Thus, we define a new pathway where IL-33-induced IL-1Ra production by tissue T_reg cells suppresses IL-1-mediated innate immune responses to respiratory viral infection.

Extended Data Figure 1.. Deletion of T_reg cells in Foxp3^DTR mice.

(a-j) Wild-type (WT) C57BL/6J controls (white circles), Foxp3^ERT2Cre controls (grey circles) and Foxp3^DTR mice (green circles) were infected with 0.3LD₅₀ PR8 influenza and all strains were treated with DT on day 2 post infection to deplete T_reg cells in the Foxp3^DTR mice, and harvested on day 5 (WT n=9, Foxp3^ERT2Cre n=10, Foxp3^DTR n=16), 7 (WT n=11, Foxp3^ERT2Cre n=11, Foxp3^DTR n=9), and 10 (WT n=10, Foxp3^ERT2Cre n=9, Foxp3^DTR n=5) post-infection. Uninfected mice treated with DT and harvested 5 days later were indicated on the X axis as (WT n=7, Foxp3^ERT2Cre n=4, Foxp3^DTR n=4). (a) Representative gating strategy to determine the number of i.v. Ab^– alveolar macrophages (SiglecF⁺CD11c^hi), eosinophils (SiglecF⁺CD11c^lo-int), DCs (SiglecF^–CD11c^hi, MHCII^hi), inflammatory monocytes (SiglecF^-CD11c^lo-intCD11b⁺CD64⁺F4/80^int-hiLy6C⁺) and neutrophils (CD11c^–MHCII^–F480^–CD64^–CD11b⁺Ly6G⁺) in the lung during influenza infection. BAL was assessed in a similar manner except that all cells in the BAL were negative for the i.v. Ab. The ratio of Ly6G⁺CD11b⁺ neutrophils compared to total viable i.v. Ab^– parenchymal cells was also assessed. (b) Total number of viable cells that were defined as lung parenchymal cells by i.v. Ab^— staining. (c) Total number of viable BAL cells. (d) Total number of Ly6C⁺ monocytes in the BAL. Total number of IFNγ⁺ (e) and IL-17⁺ (f) CD4⁺ T cells as well as IFNγ⁺ CD8⁺ T cells (g) and IL-17-producing γδ T cells (h) as determined by intracellular cytokine staining. IFNγ Foxp3^ERT2Cre n=10, Foxp3^DTR n=10) (i) and CXCL5 (Foxp3^ERT2Cre n=6, Foxp3^DTR n=6) (j) levels in the BAL on day 5 as measured by ELISA and normalized to total protein (DT in these experiments was given on days 2 and 4 post infection). (k) Survival in the WT controls (black) and Foxp3^DTR (green) mice after infection of an LD₅₀ dose of PR8 influenza and administration of one dose of DT on day 9 of infection. Uninfected Foxp3^DTR mice treated with DT were also included (grey line). Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. Statistics for b-h by two-way ANOVA with Šídák's multiple comparisons test and for i,j Two-tailed unpaired students T-test. Statistical significance for the survival experiment (k) was assessed by Log-Rank (Mantel-Cox) test. Data presented in box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values indicated in the figure.

Extended Data Figure 2.. ST2⁺ T_reg cells expand in the lung and BAL early during influenza.

(a-i) Foxp3^YFPCre mice were infected with influenza and harvested on the indicated day of infection. (a) Representative gating strategy used to analyze lung parenchymal (i.v. Ab^–) T_reg cells and the expression of ST2 and CXCR3. (b-d) Representative flow cytometry, number and percentage of T_reg cells to CD4⁺ T cells in the lung i.v. Ab⁺ blood compartment (b), the lung i.v. Ab^– parenchyma (c) and the BAL (d) (Representative experiment of 2 performed, day 0: n=3, day 4: n=4, lung day 7: n=5, BAL day 7 n=6, lung day 11: n=4, BAL day 11: n=6, day 14: n=3). (e) Representative flow cytometry (left) and total number (right) of ST2⁺ (blue) and CXCR3⁺ (red) and ST2^–CXCR3^– CD3⁺CD4⁺Foxp3⁺ T_reg cells isolated from the BAL on the indicated day post influenza infection (Representative experiment of 2 performed, day 0: n=5, day 4: n=3, day 7: n=6, day 11: n=5). (f) Representative flow cytometry comparing ST2 and CXCR3 expression on parenchymal and blood T_reg cells isolated from lungs harvested on indicated day post influenza infection. (g) Representative flow cytometry (top) and % positive cells (bottom) for CD69 expression on parenchymal and blood T_reg cells isolated from the lung of influenza infected mice on day 5 of infection (Representative experiment of 2 performed, n=4). (h) Representative flow cytometry of ST2 and Helios expression on parenchymal CD3⁺CD4⁺Foxp3⁺ T_reg cells isolated from the lung from mice on the indicated day of infection. (i) Representative flow cytometry of ST2 and KLRG1 expression (top) or CXCR3 and KLRG1 expression (bottom) on parenchymal CD3⁺CD4⁺Foxp3⁺ T_reg cells isolated from the lung from mice on the indicated day of infection. All statistics were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons except for (g) which used an unpaired two-tailed t-test. For line graphs: Error bars, mean ± SD. For box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values are indicated in plots.

Extended Data Figure 3.. Conditional deletion of ST2 on T_reg cells altered T_reg cell activation and the innate immune response to infection.

(a-h) Foxp3^YFPCre controls (white) and Il1rl1^fl/fl littermate controls (grey) and Il1rl1^fl/flFoxp3^YFPCre mice (blue) were infected with 0.3LD₅₀ PR8 influenza and harvested on the indicated day post infection. (a) T_reg cells (left) and Foxp3^–CD4⁺ T cells (right) were sorted from the lungs on day 7 of infection and subjected to RT-qPCR for analysis of Il1rl1 mRNA levels (Each dot represents the mean of 1 experiment, Foxp3^YFPCre n=4, Il1rl1^fl/flFoxp3^YFPCre n=3). (b) MFI of CD69 expression (left; Foxp3^YFPCre n=5, Il1rl1^fl/flFoxp3^YFPCre n=5) and % cells positive for Ki67(right; Foxp3^YFPCre n=6, Il1rl1^fl/flFoxp3^YFPCre n=5) on CD3⁺CD4⁺Foxp3⁺ T_reg cells in the lung parenchyma on day 4 of infection. (c) MFI of KLRG1 (left) and Foxp3 (right) expression of CD3⁺CD4⁺Foxp3⁺ T_reg cells on day 7 of infection in the indicated mouse strains (Il1rl1^fl/fl n=4, Foxp3^YFPCre n=6, Il1rl1^fl/flFoxp3^YFPCre n=4). (d) Representative flow cytometry and total number of CD3⁺CD4⁺Foxp3⁺ T_reg cells in the lung that stained positive for the indicated cytokines on day 9 of influenza infection in Foxp3^YFPCre (white, n=8) and Il1rl1^fl/fl (grey, n=3) littermate controls and Il1rl1^fl/flFoxp3^YFPCre (blue, n=6) mice. (e) Flow cytometry was used to quantify the number of SiglecF^–CD11c^loCD11b⁺CD64^–F4/80^-Ly6G⁺ neutrophils in the BAL (far left), SiglecF⁺CD11c^lo eosinophils in the BAL (left), SiglecF^–CD11c^–CD11b⁺CD64⁺F4/80^int-hiLy6C⁺ inflammatory monocytes in the BAL (right) and, SiglecF⁺CD11c^hi alveolar macrophages in the BAL (far right) in the lung on the indicated day post infection in Foxp3^YFPCre (white, day 0 n=3, day 4 n=6, day 7 n=13, day 9 n=14, day 11 n=14) and Il1rl1^fl/fl(grey, day 7 n=8, day 9 n=3, day 11 n=2) controls and Il1rl1^fl/flFoxp3^YFPCre (blue, day 0 n=4, day 4 n=9, day 7 n=12, day 9 n=11, day 11 n=12) mice. (f) Cytokine and chemokine levels in the BAL of Foxp3^YFPCre control (white, n=16) and Il1rl1^fl/flFoxp3^YFPCre (blue, n=11) mice on day 7 of influenza infection as assessed by Luminex. (g) Representative flow cytometry of ST2 expression of CD45⁺Lineage^-Thy1.2⁺ innate lymphoid cells (left, numbers indicate percentage of ST2⁺ cells with STD) and quantification of the total number of CD45⁺Lineage^-Thy1.2⁺ST2⁺ ILC2s (right). Foxp3^YFPCre (white; n=5) and Il1rl1^fl/fl(grey; n=6) controls and Il1rl1^fl/flFoxp3^YFPCre (blue; n=4) mice. (h) Total number of CD3^-NK1.1⁺ NK cells in the lung on the indicated day of infection. Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. All statistics were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons except for (a,b) which used an unpaired two-tailed t-test and (h) which was calculated by unpaired t-test with Holm-Sidak adjustment for multiple comparisons. For box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values as indicated in plots.

Extended Data Figure 4.. Deletion of ST2 in T_reg cells increased the innate immune response to influenza and improved viral control.

(a-f) Foxp3^ERT2Cre control (light grey) treated with tamoxifen and Il1rl1^fl/flFoxp3^ERT2Cre (black/blue) treated with oil as another control were compared with Il1rl1^fl/flFoxp3^ERT2Cre mice (light blue) treated with tamoxifen to specifically delete the Il1rl1 gene in T_reg cells. After a 14-day tamoxifen washout, mice were infected with influenza and analyzed on day 7. (a) Representative flow cytometry of ST2 and CXCR3 expression on lung (i.v. Ab^-) T_reg cells. (b) MFI of ST2 expression on lung T_reg cells (i.v. Ab^-, CD3⁺CD4⁺Foxp3⁺) (Foxp3^ERT2Cre +TAM n=13, Il1rl1^fl/flFoxp3^ERT2Cre +oil n=12, Il1rl1^fl/flFoxp3^ERT2Cre +TAM n=13). (c) MFI of ST2 expression on lung eosinophils (i.v.Ab^-, SiglecF⁺, CD11c^lo) (Foxp3^ERT2Cre +TAM n=4, Il1rl1^fl/flFoxp3^ERT2Cre +oil n=4, Il1rl1^fl/flFoxp3^ERT2Cre +TAM n=5). (d) MFI of ST2 expression on innate lymphoid cells (CD45⁺, Lineage^-, Thy1.2⁺)(Foxp3^ERT2Cre +TAM n=8, Il1rl1^fl/flFoxp3^ERT2Cre +oil n=9, Il1rl1^fl/flFoxp3^ERT2Cre +TAM n=9). (e) The number of neutrophils and eosinophils in the BAL (Foxp3^ERT2Cre +TAM n=13, Il1rl1^fl/flFoxp3^ERT2Cre +oil n=12, Il1rl1^fl/flFoxp3^ERT2Cre +TAM n=13). (f) The number of lung IL-17⁺γδ T cells and lung IL-5⁺ ILC2s as assessed by flow cytometry (Foxp3^ERT2Cre +TAM n=9, Il1rl1^fl/flFoxp3^ERT2Cre +oil n=8, Il1rl1^fl/flFoxp3^ERT2Cre +TAM n=8). (g) Influenza Polymerase levels as determined by RT-qPCR in the lungs of Foxp3^YFPCre controls (white; day 0 n=9, day 4 n=23, day 7 n=24, day 11 n=31), Il1rl1^fl/fl (grey; day 7 n=7, day 11 n=4) littermate controls and Il1rl1^fl/flFoxp3^YFPCre mice (blue; day 0 n=4, day 4 n=16, day 7 n=21, day 11 n=19) infected with 0.3LD₅₀ PR8 influenza. (h) Matrix Protein 1 RNA levels as determined by RT-qPCR in the lungs of Foxp3^YFPCre controls (white; day 0 n=3, day 4 n=16, day 7 n=16, day 11 n=23) and Il1rl1^fl/flFoxp3^YFPCre mice (blue; day 0 n=4, day 4 n=12, day 7 n=12, day 11 n=11) infected with 0.3LD₅₀ PR8 influenza. (i) Weight loss in Foxp3^YFPCre (white) controls and Il1rl1^fl/flFoxp3^YFPCre mice (blue) after influenza infection (Error bars, mean± SEM). Unless otherwise indicated, data are pooled from 2–4 experiments and each dot represents one mouse. Statistics for b-f were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons. Statistics for g and h were calculated by unpaired t-tests with Holm-Sidak adjustment for multiple comparisons. Statistics for i were calculated by two-way ANOVA with Šídák's multiple comparisons test. For box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values are indicated in plots.

Extended Data Figure 5.. Deletion of Myd88 in T_reg cells phenocopied the immune response to influenza in the Il1rl1^fl/flFoxp3^YFPCre mice.

(a-i) Foxp3^YFPCre controls (white) and myd88^fl/flFoxp3^YFPCre mice (purple) were infected with 0.3LD₅₀ dose PR8 influenza and harvested on the indicated day post infection. (a) T_reg cells and Foxp3^– CD4⁺ T cells were sorted from the lungs on day 7 of infection and Myd88 expression was analyzed by RT-qPCR (Each dot represents the pooled mean of 1 experiment: Foxp3^YFPCre n=4, myd88^fl/flFoxp3^YFPCre n=3). (b) The indicated cell types were sorted from the lungs on day 7 of infection and Myd88 expression was analyzed by RT-qPCR (Each dot represents the pooled mean of 1 experiment: Foxp3^YFPCre n=3, myd88^fl/flFoxp3^YFPCre n=2). (c) Representative flow cytometry of ST2 and CXCR3 expression on lung parenchymal (i.v. Ab^–) CD3⁺CD4⁺Foxp3⁺ T_reg cells on day 7 of influenza infection. (d) Total number of lung parenchymal (i.v. Ab^–) CD3⁺CD4⁺Foxp3⁺ T_reg cells in the indicated strains on the indicated day of influenza infection (left) and the percentage of T_reg cells expressing ST2 in the same mice (right) (Foxp3^YFPCre, day 0 n=3, day 4 n=11, day 7 n=10, day 9 n=6; myd88^fl/flFoxp3^YFPCre, day 0 n=6, day 4 n=11, day 7 n=8, day 9 n=5). (e-f) Representative flow cytometry (left) and number (right) of CD11c^–SiglecF^–CD11b⁺CD64^–LygG⁺ neutrophils (e) and SiglecF⁺CD11c^lo eosinophils (f) in the BAL on the indicated day of infection (Foxp3^YFPCre, day 0 n=5, day 4 n=8, day 7 n=5, day 9 n=4, day 11 n=6; myd88^fl/flFoxp3^YFPCre, day 0 n=6, day 4 n=11, day 7 n=9, day 9 n=4, day 11 n=6). (g-i) Representative flow cytometry at day 7 (left) and total number on the indicated day of infection (right) of IL-17⁺ γδ T cells in the lungs (g) on the indicated day of infection. (h-i) Number of IFNγ⁺CD4⁺ T cells (h) and IFNγ⁺CD8⁺ T cells (i) in the lungs of the indicated Foxp3^YFPCre control and Myd88^fl/flFoxp3^YFPCre mice on the indicated day of influenza infection(Foxp3^YFPCre, day 0 n=4, day 4 n=11, day 7 n=5, day 9 n=10; myd88^fl/flFoxp3^YFPCre, day 0 n=6, day 4 n=11, day 7 n=5, day 9 n=10). Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse, except for a,b where each dot represents the mean value from an individual experiment. Statistics for (a) were calculated by an unpaired two-tailed t-test. Statistics for (b-i) were calculated by unpaired t-test with Holm-Sidak adjustment for multiple comparisons. For box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values are indicated in plots.

Extended Data 6.. Distinct gene expression patterns in ST2⁺ and CXCR3⁺ T_reg cells in the lung of influenza infected mice.

(a-g) Bulk RNAseq was performed on spleen T_reg cells from uninfected mice (black) and on sorted ST2⁺ (blue) and CXCR3⁺ (red) T_reg cells isolated from the pooled lungs of Foxp3^YFPCre mice on day 7 of influenza infection and performed in 3 independent experiments (n=3). (a) Principal component analysis of the different T_reg cell populations. (b) Venn diagram of differentially expressed genes in the ST2⁺ and CXCR3⁺ T_reg subsets compared to each other and to naïve total spleen T_reg cells. (c) Heat map of selected transcription factor expression in the indicated T_reg cell populations performed in triplicate. (d) Transcription factor RNA levels in the indicated cell populations: As above, spleen T_reg cells from uninfected mice (spleen), sorted ST2⁺ T_reg cells (ST2⁺) and CXCR3⁺ T_reg cells (CXCR3⁺) isolated from the lungs of pooled Foxp3^YFPCre mice on day 7 of influenza infection. (e) Heat map of selected trafficking gene expression in the indicated T_reg cell populations. (f) Selected trafficking gene RNA expression levels in the indicated T_reg cell populations. (g) Selected T_reg cell effector gene RNA expression levels in the indicated T_reg cell populations. Floating bars: min to max, line median. All statistics were calculated by unpaired two tailed t-test. P values indicated in figure.

Extended Data 7.. IL-1Ra is a critical mediator of inflammatory responses to influenza.

(a-k) Il1rn^+/+ littermate controls (white circles, n=8) and Il1rn^–/– mice (yellow circles, n=7) were infected with influenza and harvested on day 7. (a-b) The percentage (left) and number (right) of CD11b⁺Ly6G⁺ neutrophils in the lung parenchyma (a) and BAL (b). (c) RNA levels of Cxcl5 relative to β2M in the lungs on day 7 of infection as determined by RT-qPCR. (d-e) The percentage (left) and number (right) of SiglecF⁺CD11c^lo eosinophils in the lung parenchyma (d) and BAL (e). (f) Percentage (left) and number (right) of IL-17-producing γδT cells in the lung as assessed by ICS staining on day 7 of infection. (g-h) Percentage and number of CD4⁺ T cells (g) and CD8⁺ T cells (h) in the lung that were positive for IFNγ and IL-17 expression as assessed by ICS. (i-k) Percentage and number of CD4⁺Foxp3⁺ T_reg cells in the lung parenchyma was determined (i) as well as the percentage and number of T_reg cells that expressed ST2 (j). (k) Influenza Nucleoprotein (left) and Polymerase (right) RNA levels in the lungs on day 7 of influenza infection. (l) Survival of the indicated strains after 0.3LD₅₀ dose influenza. Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. All statistics were calculated by unpaired two-tailed t-test except the survival experiment (l) which was assessed by Log-Rank (Mantel-Cox) test. Box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values are indicated in plots.

Extended Data 8.. Deletion of Il1rn in T_reg cells affected the innate but not adaptive response to influenza.

(a-g) Foxp3^YFPCre controls (white) and Il1rn^fl/fl (grey) littermate controls and Il1rn^fl/flFoxp3^YFPCre (orange) mice were infected with 0.3LD₅₀ PR8 influenza and harvested on the indicated day post infection. (a-b) Representative flow cytometry of the percentage of CD11b⁺Ly6G⁺ neutrophils (a) and SiglecF⁺CD11c^lo eosinophils (b) compared to total viable cells (left) and total number of neutrophils (right) in the BAL in Foxp3^YFPCre controls (white, day 0 n=6, day 5 n=7, day 7 n=20, day 9 n=16) and Il1rn^fl/fl (grey, day 0 n=8, day 5 n=10, day 7 n=14, day 9 n=6) littermate controls and Il1rn^fl/flFoxp3^YFPCre (orange, day 0 n=7, day 5 n=9, day 7 n=12, day 9 n=11) mice on day 7 of infection. (c) Representative flow cytometry of the percentage of IL-17⁺ γδ T cells (right) and total number of IL-17⁺ γδ T cells (left) in the lung of Foxp3^YFPCre controls (n=7) and Il1rn^fl/fl (grey, n=10) littermate controls and Il1rn^fl/flFoxp3^YFPCre (orange, n=9) mice on day 5 of influenza as determined by ICS. (d) Number of IFNγ⁺CD4⁺ T cells (left) and IFNγ⁺CD8⁺ T cells (right) in the lung as assessed by ICS in Foxp3^YFPCre controls (white, n=8) and Il1rn^fl/flFoxp3^YFPCre (orange, n=8) mice on day 7 of infection. (e-f) Percentage (left) and number (right) of total CD4⁺Foxp3⁺ T_reg cells (e) and ST2⁺ T_reg cells (f) in the lung parenchyma assessed by flow cytometry in Foxp3^YFPCre controls (white, n=14) and Il1rn^fl/flFoxp3^YFPCre (orange, n=11) mice on day 7 of infection. (g) Influenza NP RNA levels in the lungs determined by RT-qPCR on day 7 of influenza infection in Foxp3^YFPCre controls (white, n=10) and Il1rn^fl/flFoxp3^YFPCre (orange, n=12) mice were infected with 0.3LD₅₀ PR8 influenza. (h) The number of neutrophils (left) and eosinophils (right) in the BAL on day 7 of 0.3LD₅₀ dose PR8 influenza infection of Il1rn^fl/flFoxp3^ERT2Cre mice treated with either tamoxifen (maroon, n=10) or oil as indicated (grey, n=9). (i) mRNA levels of Il1rn in the indicated cell types sorted from the lungs of Il1rn^fl/fl littermate controls (grey, n=3) and Il1rn^fl/flFoxp3^YFPCre (orange, n=2) infected with 0.3LD₅₀ dose PR8 influenza. (j) mRNA levels of Il1rn in the indicated cell types sorted from the lungs of day 7 infected Il1rn^fl/fl littermate controls (grey, n=3) and Il1rn^fl/flLysM^Cre (purple, n=3) infected with 0.3LD₅₀ dose PR8 influenza. (k) Foxp3^YFPCre controls (white, n=14), Il1rl1^fl/fl littermate controls (gray, n=9) and Il1rl1l^fl/flFoxp3^YFPCre (blue n=7) mice were infected with 0.3LD₅₀ dose PR8 influenza and administered i.p. and i.n. PBS diluent and another group of Il1rl1l^fl/flFoxp3^YFPCre mice were treated with i.p. and i.n. IL-1Ra (blue/orange, n=5) and harvested on day 5 post infection to determine the total number (top) and percentage (bottom) of neutrophils in the BAL. Unless otherwise indicated, data are pooled from 2–3 experiments. Each dot represents one mouse, except for experiments i and j where each dot represents the pooled mean from an experiment. Statistics for (a-b) were calculated by unpaired t-test with Holm-Sidak adjustment for multiple comparisons. Statistics for (d-f, h, j) were calculated by unpaired Student’s t-test. Statistics for (c, g, k) were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons. i was calculated by two-way ANOVA with Šídák's multiple comparisons test. Column Plots: mean with Standard Deviation. Box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values are indicated in plots.

Extended Data 9.. IL-1 may mediate innate inflammatory responses to influenza via IL-1R1-expressing fibroblasts.

(a-b) Il1rl1^fl/flFoxp3^YFPCre (blue circles day 5 n=7, day 7 n=9, day 9 n=13, day 11 n=3), Il1rl1^fl/flFoxp3^YFPCreIl1a/b^-/- (blue and olive circles, day 5 n=11, day 7 n=7, day 9 n=9, day 11 n=4), and Il1a/b^-/- (olive circles, day 5 n=6, day 7 n=5, day 9 n=3, day 11 n=3) and Foxp3^YFPCre control (white circles, day 5 n=7, day 7 n=14, day 9 n=16, day 11 n=5) mice were infected with 0.3LD₅₀ dose PR8 influenza. (a) Total number of CD11b⁺Ly6G⁺ neutrophils in the BAL on the indicated day. (b) Total number of SiglecF⁺CD11c^lo eosinophils in the BAL on the indicated day. (c) Total number of IL-17⁺ γδ T cells in the lungs on day 5 of influenza infection (Foxp3^YFPCre n=7, Il1rl1^fl/flFoxp3^YFPCre n=7, Il1rl1^fl/flFoxp3^YFPCreIl1a/b^-/- n=9). (d) Representative flow cytometry (left) and total number (right) of IL-5⁺- and IL 13-producing ILC2s (CD45⁺Thy1.2⁺Lin^-CD3^-CD4^-ST2⁺) in the lungs on day 7 of influenza infection (Foxp3^YFPCre n=6, Il1rl1^fl/fl n=8, Il1rl1^fl/flFoxp3^YFPCre n=5, Il1rl1^fl/flFoxp3^YFPCreIl1a/b^-/- n=6, and Il1a/b^-/- n=5). (e) Influenza NP RNA levels (copies per ng RNA) in the lungs on day 7 of influenza infection (n=5–20 mice per group as represented by dots). (f) Bubble plot of the indicated gene expression of reanalyzed scRNA-Seq dataset clusters from CD45^- lung mesenchymal cells derived from Dalghren et al. (GEO accession GSE125492) and Tsukui et al. (GEO accession GSE132771) from the indicated cell clusters shown in Figure 8d. (g) Cell sorting strategy to isolate lung CD45^-CD31^-EpCAM⁺ epithelial cells, CD45^-EpCAM^-CD31⁺ endothelial cells, CD45^-PDGFRa⁺PDPN⁺Ly6C-Sca1^- fibroblasts and CD45^-PDGFRa⁺PDPN⁺Ly6C⁺Sca1⁺ adventitial fibroblasts. (h) Representative expression and Geometric mean MFI of IL-1R1 expression on CD45^-EpCAM^-CD31⁺ endothelial cells, CD45^-CD31^-EpCAM⁺ epithelial cells and CD45^-PDGFRa⁺PDPN⁺ fibroblasts isolated from the lungs of naïve mice (n=4 in this representative experiment of 2 performed). (i) Fibroblasts were isolated from the lungs of naïve mice and stimulated with either control media or 10ng/ml IL-1βfor 6hrs and then the expression of the indicated genes were analyzed using RT-qPCR (replicates, n=4). Unless otherwise indicated, data are pooled from 2–3 experiments. For a-e and h each dot represents one mouse. Statistics for (a-b) were calculated by unpaired t-test with Holm-Sidak adjustment for multiple comparisons. Statistics for (c-e, h) were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons. (i) was calculated by unpaired students t-test. Column Plots: mean with Standard Deviation. Box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values are indicated in plots.

Figure 1.. Early deletion of T_reg cells during influenza increased lung granulocytes and improved survival.

(a-f) WT (C57BL/6J) control (white circles), Foxp3^ERT2Cre control (grey circles) and Foxp3^DTR mice (green circles) were infected with influenza. All strains were treated with diphtheria toxin (DT) on day 2 post-infection to deplete T_reg cells in the Foxp3^DTR mice and were harvested on day 5 (WT n=9, Foxp3^ERT2Cre n=10, Foxp3^DTR n=16), 7 (WT n=11, Foxp3^ERT2Cre n=11, Foxp3^DTR n=8), and 10 (WT n=10, Foxp3^ERT2Cre n=9, Foxp3^DTR n=5) post-infection. DT treated mice without infection (indicated by ) and harvested on day 5 were included as additional controls (WT n=7, Foxp3^ERT2Cre n=4, Foxp3^DTR n=4). (a) Schematic of experimental outline. (b) Representative flow cytometry (left) and total number (right) of i.v. Ab^– CD4⁺ Foxp3⁺ T_reg cells in the lung parenchyma on the indicated day of infection. (c-d) Representative flow cytometry of the percentage of neutrophils (i.v. Ab^– CD11b⁺Ly6G⁺) (left) and the total number of neutrophils (i.v. Ab^– CD11c^-SiglecF^-CD64^-F4/80^-CD11b⁺Ly6G⁺) (right) in the lung parenchyma (c) and BAL (d). The mean percentage ± SD of cells for all mice studied is indicated within the flow plots. (e-f) Representative flow cytometry of the percentage and total number of eosinophils (i.v. Ab^–SiglecF⁺CD11c^lo) in the lung parenchyma (e) and BAL (f). The mean percentage ± SD of cells for all mice studied is indicated within the flow plots. (g) Survival of WT control (black) and Foxp3^DTR (green) mice infected with an LD₅₀ dose of PR8 influenza and given one dose of DT on the day of infection. (h) Total number of neutrophils (CD11c^-SiglecF^-CD64^-F4/80^-CD11b⁺GR-1⁺) in the lung parenchyma and BAL on day 5 in Foxp3^ERT2Cre control mice treated with 100μg isotype control IgG (grey, n=8) and Foxp3^DTR mice treated with either 100μg of 1A8 antibody (green, black hash, n=7) or IgG control (green, n=6) on days -1, +1, and +3. All mice were infected with an LD₅₀ dose of PR8 influenza and treated with DT on day 2. (i) Survival after an LD₅₀ dose of PR8 influenza. All groups (Foxp3^ERT2Cre controls and Foxp3^DTR mice) received DT on day 2 of infection. On days -1,+1, +3, +5 of infection a group of Foxp3^DTR mice were treated with 100μg 1A8 antibody while the remaining mice were treated with 100μg isotype IgG control. Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. Statistics for (b-f) and (h) were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test for each time point. Statistical significance for the survival experiments (g, i) was assessed by Log-Rank (Mantel-Cox) test. Data presented in box and whiskers plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 2.. ST2 is expressed on a population of T_reg cells in the lung early during influenza infection.

(a-d) Foxp3^YFPCre mice were infected with influenza and lungs were harvested for analysis at the indicated time points post infection. (a) Representative flow cytometry of ST2 (blue) and CXCR3 (red) expression on intraparenchymal i.v. Ab^–,CD3⁺CD4⁺Foxp3⁺ T_reg cells (left). Quantification of the total number (top) and percentage (bottom) of T_reg cells expressing ST2 (blue) and CXCR3 (red) and neither marker (ST2^-CXCR3^-, grey) or both markers (ST2⁺CXCR3⁺, white) in the lung parenchyma (Representative experiment of 2 performed, day 0: n=6, day 4: n=4, day 7: n=5, day 11: n=5). (b) Representative histogram of GATA3 and T-bet expression as assessed by flow cytometry in ST2⁺ (blue), CXCR3⁺ (red), or ST2^-CXCR3^- (black) T_reg cells, FMO staining control is in grey. (c) Levels of Gata3, Tbx21, Rorc mRNA determined by RT-qPCR in the indicated ST2⁺ (blue), CXCR3⁺ (red), or ST2^-CXCR3^- (grey) T_reg cell populations sorted from the lungs of day 7 infected mice. Experiment was performed 3 times with pooled samples and each dot represents the mean data from one experiment. (d) Representative histogram (left) and percent cells positive for CD69 expression (right) as assessed by flow cytometry in ST2⁺ (blue), CXCR3⁺ (red), and ST2^-CXCR3^- (grey) T_reg cells isolated from the lung on day 5 of influenza infection (Representative experiment of 2 performed, n=5). (e) Representative histogram (left) and percent cells positive for Ki-67 expression (right) as assessed by flow cytometry in ST2⁺ (blue), CXCR3⁺ (red), and ST2^-CXCR3^- (grey) T_reg cells isolated from the lung of day 5 influenza infection (Representative experiment of 2 performed, n=4). (f) Representative flow cytometry (left) and cell numbers (right) of ST2⁺ (blue), CXCR3⁺ (red), and ST2^-CXCR3^- (grey) T_reg cells that are positive for I-A(b) NP311–325⁺ tetramer staining (n=7). Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. All statistics were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons. Column plots: Error bars, mean ± SD, Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 3.. Deletion of ST2 on T_reg cells increased innate immune responses in the lung to influenza.

(a-d) Foxp3^YFPCre controls (white) and Il1rl1^fl/fl littermate controls (grey) and Il1rl1^fl/flFoxp3^YFPCre mice (blue) were infected influenza and harvested on the indicated day post infection. (a) Representative flow cytometry of Foxp3 and ST2 on i.v.Ab^-CD3⁺CD4⁺ T cells in the lung parenchyma, demonstrating specific ST2 deletion on Foxp3⁺ T_reg cells in Il1rl1^fl/flFoxp3^YFPCre mice on day 7 of infection. (b) Total number of CD3⁺CD4⁺Foxp3⁺ T_reg cells in the lung parenchyma on day 0 (Foxp3^YFPCre n=3; Il1rl1^fl/flFoxp3^YFPCre n =4), 4 (Foxp3^YFPCre n=7; Il1rl1^fl/flFoxp3^YFPCre n =9), 7 (Il1rl1^fl/fl n= 5; Foxp3^YFPCre n=14; Il1rl1^fl/flFoxp3^YFPCre n =12), 9 (Il1rl1^fl/fl n= 3; Foxp3^YFPCre n=16; Il1rl1^fl/flFoxp3^YFPCre n =13) and 11 (Il1rl1^fl/fl n= 3; Foxp3^YFPCre n=13; Il1rl1^fl/flFoxp3^YFPCre n =12) post-infection. (c-d) Total number of SiglecF^-CD11c^-CD11b⁺CD64^-F4/80^-Ly6G⁺ neutrophils (c)and SiglecF⁺CD11c^lo eosinophils (d) in the lung parenchyma on day 0 (Foxp3^YFPCre n=3; Il1rl1^fl/flFoxp3^YFPCre n =4), 4 (Foxp3^YFPCre n=7; Il1rl1^fl/flFoxp3^YFPCre n =9), 7 (Il1rl1^fl/fl n= 7; Foxp3^YFPCre n=13; Il1rl1^fl/flFoxp3^YFPCre n =12), 9 (Il1rl1^fl/fl n= 2; Foxp3^YFPCre n=14; Il1rl1^fl/flFoxp3^YFPCre n =11) and 11 (Il1rl1^fl/fl n= 2; Foxp3^YFPCre n=15; Il1rl1^fl/flFoxp3^YFPCre n =12) post-infection. (e) Foxp3^ERT2Cre control mice treated with tamoxifen (light grey, n=13), Il1rl1^fl/fl Foxp3^ERT2Cre mice treated with oil (black and blue, n= 12) and Il1rl1^fl/fl Foxp3^ERT2Cre mice treated with tamoxifen (light blue, n=12) were infected with influenza 2 weeks after tamoxifen washout and the number of neutrophils (left) and eosinophils (right) in the lung parenchyma was assessed on day 7 of infection. (f) Levels of the indicated cytokines and chemokines were assessed in the BAL on day 7 of influenza infection by Luminex (Foxp3^YFPcre n=19, Il1rl1^fl/flFoxp3^YFPCre n=14) or ELISA for CXCL5 (Foxp3^YFPcre n=10, Il1rl1^fl/flFoxp3^YFPCre n=7). (g) Representative flow cytometry (left) and quantification of the total number (right) of IL-17-producing γδ T cells and CD4⁺ T cells in the lung on day 5 of influenza infection in Foxp3^YFPcre (n=7) and Il1rl1^fl/flFoxp3^YFPCre (n=5) mice. (h) Representative flow cytometry (left) and total number (right) of IL-5 and IL-13 producing ILC2s (CD45⁺lineage^-CD3^-CD4^-Thy1.2⁺ST2⁺) and CD4⁺ T cells in the lungs of day 7 influenza infected mice (Il1rl1^fl/fl n= 6; Il1rl1^fl/flFoxp3^YFPCre n =4). (i-j) Representative flow plots for IFNγ and IL-17 ICS and total number of IFNγ positive CD3⁺CD4⁺ T cells (i) and IFNγ positive CD3⁺CD8⁺ T cells (j), on day 0 (Foxp3^YFPCre n=3; Il1rl1^fl/flFoxp3^YFPCre n =4), 4 (Foxp3^YFPCre n=17; Il1rl1^fl/flFoxp3^YFPCre n =14), 7 (Foxp3^YFPCre n=10; Il1rl1^fl/flFoxp3^YFPCre n =11), and 11 (Foxp3^YFPCre n=9; Il1rl1^fl/flFoxp3^YFPCre n =9) post-infection. Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. Statistical significance assessed by unpaired Student’s t-tests with Holm-Sidak adjustment for multiple comparisons for (b, c, d, i, and j). Ordinary one-way ANOVA with Tukey’s multiple comparisons was performed for (e, g, and h). Students unpaired t-test was performed on (f). Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 4.. Deletion of ST2 on T_reg cells improved viral control and survival to influenza.

(a) Influenza nucleoprotein (NP) RNA levels determined by RT-qPCR in the lungs on the indicated day of influenza infection in Foxp3^YFPCre control (white circles; day 0 n=7, day 4 n=23, day 7 n=14, day 11 n=19) and Il1rl1^fl/fl (grey circles; day 7 n=7, day 11 n=4) control mice compared with Il1rl1^fl/flFoxp3^YFPCre (blue circles; day 0 n=4, day 4 n=15, day 7 n=16, day 11 n=19) mice. (b) Influenza NP and NS1 RNA levels as determined by RT-qPCR in the lungs on day 7 of infection in Foxp3^ERT2Cre control (n=12) and Il1rl1^fl/fl Foxp3^ERT2Cre mice (n=14) treated with tamoxifen 2 weeks prior to infection. (c) Survival of Foxp3^YFPCre controls (black) and Il1rl1^fl/fl littermate controls (gray, dashed) and Il1rl1^fl/flFoxp3^YFPCre (blue, dashed) mice infected with LD₅₀ dose of PR8 influenza. (d) Foxp3⁺ T_reg cells were sorted from the lungs of day 7 infected Foxp3^YFPCre (white circles, n=4) and Il1rl1^fl/flFoxp3^YFPCre (blue circles, n=3) mice and analyzed for Areg RNA expression by RT-qPCR (n=mean of 1 independent experiment). (e) RNA was harvested from the lung of Foxp3^YFPCre control (white circles, n=13) and Il1rl1^fl/flFoxp3^YFPCre (blue circles, n=13) mice infected with influenza and analyzed for total lung mRNA expression of Areg. (f) Representative H&E stains of lungs from control mice and Il1rl1^fl/flFoxp3^YFPCre mice infected with influenza. (g) Blinded pathologist review of the extent of inflammation scores in the lungs of control mice (Il1rl1^fl/fl and Foxp3^YFPCre, white circles; day 0 n=5, day 4 n=9, day 7 n=6, n=11) and Il1rl1^fl/flFoxp3^YFPCre mice (blue circles; day 0 n=6, day 4 n=4, day 7 n=3, n=6) infected with influenza. Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. Statistical significance assessed by unpaired Student’s t-tests with Holm-Sidak adjustment for multiple comparisons for (a). Unpaired students t-test was performed on (b, d, e and g). Survival (c) was assessed by Log-Rank (Mantel-Cox) test. Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 5.. Neutrophils mediated improved survival in influenza-infected Il1rl1^fl/flFoxp3^YFPCre mice.

(a-d) Foxp3^YFPCre control (white), Il1rl1^fl/flFoxp3^YFPCre mice (blue), Il1rl1^fl/flFoxp3^YFPCreTcrd^-/- (blue/pink) and Tcrd^-/- (pink) mice were infected with influenza and harvested on the indicated day post infection. (a) Representative flow cytometry (left) and total number (right) of CD3⁺ γδ T cells in the lung on day 7 of infection in Foxp3^YFPCre (n=15), Il1rl1^fl/flFoxp3^YFPCre (n=16), Il1rl1^fl/flFoxp3^YFPCreTcrd^-/- (n=17) and Tcrd^-/- (n=4) mice. (b) Cytokine levels for IL-17a, Il-5, IL-13, IL-33, and IL-1β in the BAL on day 7 of infection in Foxp3^YFPCre (n=15), Il1rl1^fl/flFoxp3^YFPCre (n=11), and Il1rl1^fl/flFoxp3^YFPCreTcrd^-/- (n=8) mice. (c) Total number of neutrophils (left) and eosinophils (right) in the lung on day 7 of infection in the indicated strains in Foxp3^YFPCre (n=15), Il1rl1^fl/flFoxp3^YFPCre (n=16), Il1rl1^fl/flFoxp3^YFPCreTcrd^-/- (n=17) and Tcrd^-/- (n=4) mice. (d) RT-qPCR for the influenza NP gene on day 7 of influenza infection in in Foxp3^YFPCre (n=10), Il1rl1^fl/flFoxp3^YFPCre (n=8), and Il1rl1^fl/flFoxp3^YFPCreTcrd^-/- (n=12). I Survival after LD₅₀ PR8 influenza infection in the indicated strains. (f) Il1rl1^fl/flFoxp3^YFPCre mice were infected with influenza and treated with 100μg IgG control (blue, n=3) or 100μg of the TRFK5 anti-IL-5 antibody (forest green, n=4) on days 4–8 of infection and the number of eosinophils in the lung and BAL were assessed by flow cytometry on day 8. (g) Il1rl1^fl/flFoxp3^YFPCre and Il1rl1^fl/fl control mice were infected with LD₅₀ dose PR8 influenza and treated with either anti-IL-5 antibody or IgG control on days 4–10 as indicated and survival was monitored. (h) The number of neutrophils (CD11c^-SiglecF^-CD64^-F4/80^-CD11b⁺GR-1⁺) was enumerated in the lung parenchyma and BAL on day 4 of LD₅₀ PR8 infection of Il1rl1^fl/fl control mice treated with 100μg of isotype control IgG (grey, n=5) or Il1rl1^fl/flFoxp3^YFPCre treated with either 100μg of anti-Ly6G antibody 1A8 (blue, n=5) or IgG control (teal, n=5) on day -1, +1, +3. (i) Il1rl1^fl/flFoxp3^YFPCre and Il1rl1^fl/fl control mice were infected with LD₅₀ dose PR8 influenza and treated with either 100μg of the anti-Ly6G antibody 1A8 or isotype IgG control on day -1, +1, +3, +5 of infection and survival was monitored. Unless otherwise indicated, data are pooled from 2–3 experiments and each dot represents one mouse. Statistical significance assessed by ordinary one-way ANOVA with Tukey’s multiple comparisons except for (f), which was assessed by Student’s unpaired t-test, and (e, g, i), which was assessed by Log-Rank (Mantel-Cox) test. Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 6.. T_reg cells produce IL-1Ra in response to IL-33-induced ST2 signaling.

(a-c) CD3⁺CD4⁺Foxp3^YFP+ T_reg cells were sorted from spleens of naïve Foxp3^YFPCre mice and CD3⁺CD4⁺Foxp3^YFP+ T_reg cells expressing ST2 (blue) or CXCR3 (red) were sorted from the lungs of Foxp3^YFPCre mice (pooled from up to 10 mice) on day 7 of 0.3LD₅₀ PR8 influenza infection for transcriptome analysis by RNAseq. (a) Volcano plot comparing gene expression in splenic T_reg cells from naïve mice versus lung ST2⁺ T_reg cells from day 7 influenza infected mice. Significant differential gene expression [<0.05 log2 false discovery rate (FDR) cutoff] between ST2⁺ [positive log₂ fold change (FC)] and splenic T_reg cells (negative log₂ FC) T_regs are depicted in blue and aqua, respectively. Highlighted genes have been associated with T_reg or lymphocyte function. Data are pooled from 3 independent experiments. (b) Volcano plot comparing gene expression in splenic T_reg cells from naïve mice versus lung CXCR3⁺ T_reg cells from day 7 influenza infected mice. Significant differential gene expression [<0.05 log2 false discovery rate (FDR) cutoff] between ST2⁺ [positive log₂ fold change (FC)] and splenic T_reg cells (negative log₂ FC) T_regs are depicted in blue and aqua, respectively. Data are pooled from 3 independent experiments. (c) Volcano plot comparing gene expression in ST2⁺ T_reg cells and CXCR3⁺ T_reg cells from the lungs of day 7 infected mice. Significant differential gene expression [<0.05 log2 false discovery rate (FDR) cutoff] between ST2⁺ [positive log₂ fold change (FC)] and CXCR3⁺ (negative log₂ FC) T_regs are depicted in blue and red, respectively. Data are pooled from 3 independent experiments. (d) Il1rn mRNA levels measured by RT-qPCR in the indicated sorted T cell population from the lungs and spleens of day 7 influenza infected Foxp3^YFPCre control mice (white) and Myd88^fl/flFoxp3^YFPCre mice (purple). Each dot represents the mean of one experiment performed in triplicate from a pooled sample of up to 10 mice (n=2–3 independent experiments). (e) Il1rn mRNA levels measured by RT-qPCR in sort total T_reg cells isolated from the lungs of Foxp3^YFPCre control (white), Il1rl1^fl/flFoxp3^YFPCre (blue), and Myd88^fl/flFoxp3^YFPCre mice (purple) on the indicated day post infection with 0.3LD₅₀ influenza. Each dot represents the mean of one experiment performed in triplicate from a pooled sample of 2 to 4 mice (n=2–3 independent experiments). (f) Time course of IL-33 levels in the BAL as measure by Luminex assay (left, n=3–4 per day) and IL-1Ra in the BAL as measured by ELISA (right, day 0 n=4, day 3 n=3, day 5 n=4, day 7 n=9, day 9 n=11) on the indicated day of influenza infection in control Foxp3^YFPcre mice. (g) IL-1ra protein secretion from Treg cells. ST2⁺, CXCR3⁺ and ST2^-CXCR3^- T_reg cells (CD4⁺Foxp3^YFP+) were sorted from pooled spleens of 10–15 naïve Foxp3^YFPCre mice that had been treated with IL-33 to expand the number of ST2⁺ T_reg cells. Sorted cells were then stimulated ex vivo with IL-2/IL-7 alone or with IL-33 and IL-2/IL-7 and the production of IL-1Ra in the supernatant was measured by ELISA after 72 hrs. Each dot represents the mean of one experiment, n=3. ND = none detected. (h) Il1rn mRNA levels measured by RT-qPCR in the lung on day 7 of influenza infection in Il1rl1^fl/fl control (grey, n=7), Foxp3^YFPCre control (white, n=18) and Il1rl1^fl/flFoxp3^YFPCre mice (blue, n=13). Unless otherwise indicated, data are pooled from 2–3 experiments. In (d, e and g) each dot represents the mean of an experiment. Statistical significance for (d, e, h) assessed by one-way ANOVA with Tukey’s multiple comparisons. Column Plots: mean with Standard Deviation. Line Plots: mean with Standard Deviation. Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 7.. Deletion of IL-1Ra in T_reg cells increased innate immunity to influenza.

(a-e) Il1rn^fl/flFoxp3^YFPCre mice (orange circles) with a conditional deletion of Il1rn in T_reg cells were infected with 0.3LD₅₀ PR8 influenza and compared to both Foxp3^YFPCre control mice (white circles) and Il1rn^fl/fl littermate control mice (gray circles). (a) Il1rn mRNA levels in T_reg cells sorted from day 7 infected Il1rn^fl/flFoxp3^YFPCre mice (orange circles, n=2) compared to Foxp3^YFPCre control mice (white circles, n=3) as assessed by RT-qPCR. (b) Total lung Il1rn mRNA levels measured by RT-qPCR on the indicated day of influenza infection (Il1rn^fl/fl day 0 n=8, day 5 n=10, day 7 n=6, day 9 n=6; Foxp3^YFPCre, day 0 n=14, day 5 n=5, day 7 n=15, day 9 n=10; Il1rn^fl/flFoxp3^YFPCre, day 0 n=7, day 5 n=8, day 7 n=12, day 9 n=6). (c) IL-1Ra BAL protein levels measured by ELISA on day 5 of infection (Il1rn^fl/fl n=5; Foxp3^YFPCre n=10; Il1rn^fl/flFoxp3^YFPCre n=9). (d-e) Number of SiglecF^-CD11c^-CD11b⁺CD64^-F4/80^-Ly6G⁺ neutrophils (D) and ⁺CD11c^lo eosinophils (E) in the lung parenchyma (Il1rn^fl/fl day 0 n=8, day 5 n=10, day 7 n=11, day 9 n=5; Foxp3^YFPCre, day 0 n=6, day 5 n=7, day 7 n=20, day 9 n=15; Il1rn^fl/flFoxp3^YFPCre, day 0 n=7, day 5 n=9, day 7 n=11, day 9 n=11). (f) Survival of Foxp3^YFPCre controls (black), Il1rn^fl/fl littermate controls (gray, dashed) and Il1rn^fl/flFoxp3^YFPCre (orange) mice infected with LD₅₀ dose PR8 influenza. (g) Il1rn^fl/flFoxp3^ERT2Cre mice were treated with tamoxifen (maroon, n=10) for 5 days to delete Il1rn in T_reg cells or oil (grey, n=9) as a control and after14 day tamoxifen washout were infected with influenza. On day 7 post-infection the number of neutrophils (left) and eosinophils (right) in the lung was quantified by flow cytometry. (h-i) The indicated cell populations were sorted from the lungs of naïve mice or on day 7 of infection and analyzed by RT-qPCR for il1rn expression (h) and fold upregulation between day 0 (n=2–3) and day 7 (n=3–4) indicated (i). ST2⁺ T_reg cell data was previously shown in Figure 6d and was included as a comparison. (j) Lung (left) and BAL (right) neutrophil numbers after infection of Il1rn^fl/fl littermate control mice (gray circles, day 0 n=5, day 4 n=27, day 7 n=14, day 11 n=8), Il1rn^fl/flLysM^Cre mice (pink circles, day 0 n=4, day 4 n=12, day 7 n=10, day 11 n=12), and Il1rn^fl/flFoxp3^YFPCre mice (orange circles, day 4 n=9, day 7 n=11, day 11 n=3) with influenza. Unless otherwise indicated, data are pooled from 2–3 experiments. For experiments b-e, g and j and each dot represents one mouse, while for a, h and i each dot represents the mean of one experiment. For statistical significance: (a, g) were assessed by unpaired Student’s t-test. (b,d,e, j) were assessed by unpaired t-tests with Holm-Sidak adjustment for multiple comparisons, (c, i) were calculated by Ordinary one-way ANOVA with Tukey’s multiple comparisons, (h) was calculated by two-way ANOVA with Šídák's multiple comparisons test and the survival experiment in (f) was assessed by Log-Rank (Mantel-Cox) test. Column Plots: mean with Standard Deviation. Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.

Figure 8.. Adventitial fibroblasts express high levels of IL-1R1 and are suppressed by T_reg cell produced IL-1Ra.

(a-b) Il1rl1^fl/flFoxp3^YFPCre (blue circles, day 5 n=7, day 7 n=7, day 9 n=11, day 11 n=3), Il1rl1^fl/flFoxp3^YFPCreIl1a/b^-/- (blue and olive circles, day 5 n=9, day 7 n=5, day 9 n=8, day 11 n=4), and Il1a/b^-/- (olive circles, day 5 n=4, day 7 n=4, day 9 n=3, day 11 n=3) as well as control Foxp3^YFPCre (white circles, day 5 n=7, day 7 n=11, day 9 n=16, day 11 n=5) mice were infected with 0.3LD₅₀ dose PR8 influenza and harvested for analysis. (a) Total number of CD11b⁺Ly6G⁺ neutrophils in the lung. (b) Total number of SiglecF⁺CD11c^lo eosinophils in the lung. (c) Survival of Foxp3^YFPCre controls (black), Il1r1l^fl/flFoxp3^YFPCre (blue) and Il1r1l^fl/flFoxp3^YFPCreIl1a/b^-/- (blue and olive hashes) mice infected with LD₅₀ dose of PR8 influenza. (d,e) UMAP visualization (d) of reanalyzed scRNA-Seq dataset clusters from CD45^- lung mesenchymal cells derived from naïve mice in Dalghren et al. (GEO accession GSE125492) and Tsukui et al. (GEO accession GSE132771) and bubble plot of Il1rl1, Il33 and Ccl11 expression in the different cell clusters (E). (f-h) mRNA expression of Il33 (f), Ccl11 (g) and Il1r1 (h) as assessed by RT-qPCR in the indicated cell populations sorted from the lungs of naïve mice (each dot is the mean of 1 sort experiment n=3–8 per cell type). (i) mRNA expression of Cxcl5 in the indicated cell populations sorted from naive or day 7 PR8 influenza infected mice (each dot is the mean of 1 sort experiment n=3–6 per cell type). (j) Lung fibroblasts were stimulated with 10ng/ml of IL-1βfor 24hrs and the production of indicated chemokine or cytokine (n=4 for each group). (k) Ly6C⁺Sca1⁺ Fibroblasts were isolated from Il1rn^fl/fl littermate control mice (gray circles) and Il1rn^fl/flFoxp3^YFPCre mice (orange circles) on day 5 after infection and analyzed for Ccl11 (n=3 each group), Cxcl5 (n=4 each group) and Il33 (n=5 for Il1rn^fl/fl, n=3 for Il1rn^fl/flFoxp3^YFPCre mice) mRNA expression levels using RT-qPCR. Unless otherwise indicated, data are pooled from 2–3 experiments. For (a,b) each dot represents one mouse, while for (f-i) and (k) each dot represents the mean pool result of one experiment and for (j) each dot represents a replicate and is representative of one of the two experiments performed. For statistical significance: (a,b) were assessed by unpaired t-tests with Holm-Sidak adjustment for multiple comparisons, (i) was calculated by two-way ANOVA with Šídák's multiple comparisons test (j-k) were calculated by unpaired Student’s t-test and the survival experiment in (f) was assessed by Log-Rank (Mantel-Cox) test. Column Plots: mean with Standard Deviation. Box and whisker plots: whiskers are max and min, box 25–75 percentile and line is median. P values shown in plots.