Fig 2: Infection of mouse pulmonary stem/progenitor cells (mPSCs) by influenza A virus. (A) The expression of α2,3-linked sialic acid (α2,3 SA) and α2,6-linked sialic acid (α2,6 SA) on mPSCs. The expression of α2,3 SA and α2,6 SA in mPSCs was determined by immunofluorescence assay (IFA) and fluorescence-activated cell sorting (FACS). The expression of CAR and CD54, which were previously demonstrated to be expressed by mPSCs, was also shown. The histograms of α2,3 SA, α2,6 SA, CAR, and CD54 expression of mPSCs were shown in purple, and the negative-staining cells were labeled as green lines. Scale bar of IFA image was 100 μm. (B) Development of cytopathic effects (CPEs) in mPSCs after influenza virus infection. mPSCs were infected with PR8 at a multiplicity of infection (MOI) of 10. The sequential changes in morphology of the same colony were recorded by microscope with a scale bar of 100 μm. (C) Colocalization of Oct4 and viral M1 proteins in influenza virus-infected mPSCs. The expression of Oct4 and viral M1 proteins in mPSCs at 24 h post infection (hpi) was determined by IFA. Scale bar was 50 μm.

Fig 3: Local sympathetic activation attenuates endothelial MAdCAM-1 levelsChR2/TH mice and their controls (transgene negative littermates exposed to light stimulation) underwent daily optogenetic activation of their colon (as described in Figure 1E) and were supplemented with 3% DSS for 7 days.(A) Relative mRNA expression levels of CCR9, CCR7, α4, β7, CCL20, CCL25, E-selectin, and P-selectin in the colon tissue of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group.(B) Expression of the markers CD31 (blue) and TH (red) in the colon of C57BL/6 mice. Scale bar, 200 μm.(C) Expression of the markers CD31 (blue) and the ChR2 fluorescent marker (green) in the colon of ChR2/TH mice. Scale bar, 20 μm.(D) Relative mRNA expression levels of VCAM-1, MAdCAM-1 and ICAM-1 in the colon tissue of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. VCAM-1: N = 9, 11; MAdCAM-1: N = 10, 9; ICAM-1: N = 10, 10.(E) Left: flow cytometry analysis of MAdCAM-1 and ICAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) in the colons of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 8, 8; ICAM-1: N = 9, 8. Right: representative flow cytometry histogram demonstrating the MAdCAM-1 and ICAM-1 levels on CD31+ cells.(F) Relative mRNA expression levels of MAdCAM-1 and ICAM-1 in the small intestine tissue of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 7, 9; ICAM-1: N = 5, 8.(G) Left: flow cytometry analysis of MAdCAM-1 and ICAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) in the colons of ChR2/TH mice and their controls following 7 days of 3% DSS and daily optogenetic activation. The mice received daily IP injections of adrenergic blockers 15 min before each optogenetic stimulation (Phentolamine 10 mg/kg and Nadolol 5 mg/kg). Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 10, 10; ICAM-1: N = 7, 8. Right: representative flow cytometry histogram demonstrating MAdCAM-1 and ICAM-1 levels on CD31+ cells.(H) Left: flow cytometry analysis of MAdCAM-1 and ICAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) in the colons of ChR2/TH mice and their controls. The mice were injected IP with 6-OHDA, and 5 days afterward received 7 days of 3% DSS and daily optogenetic activation. Fold change between the ChR2/TH mice and their controls relative to the mean of the control group. MAdCAM-1: N = 16, 13; ICAM-1: N = 16, 13. Right: representative flow cytometry histogram demonstrating MAdCAM-1 and ICAM-1 levels on CD31+ cells.(I) Experimental design: cells were enzymatically dissociated from colons and incubated for one hour with LPS at 37°C. Afterward, NA was added (0-1000 μM) and the cells were incubated overnight at 37°C. Then MAdCAM-1 levels were analyzed on endothelial cells (CD31+) by flow cytometry.(J) Left: MAdCAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) following exposure to increasing concentrations of NA (as described in Figure 4I). N = 6. Right: representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells.(K) Left: MAdCAM-1 levels on colon-derived enriched endothelial cells (CD31+) following exposure to 100 μM NA overnight at 37°C. N = 6. Right: Representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells.(L) Left: MAdCAM-1 levels on endothelial cells (CD31+; indicated by the fold change in MFI) that were incubated at 37°C with α- or β-adrenergic blockers (Phentolamine or Nadolol respectively) followed by exposure to 100 μM NA overnight. N = 5. Right: representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells.(M) Left: pCREB levels on endothelial cells (CD31+) following exposure to 100 μM NA for 30 min at 37°C. N = 10. Right: representative flow cytometry histogram demonstrating pCREB levels on CD31+ cells.(N) Left: MAdCAM-1 levels on endothelial cells (CD31+) following exposure to 100 μM NA overnight at 37°C. Prior to the administration of NA, the cells were incubated with a CREB inhibitor (666-15). N = 9. Right: representative flow cytometry histogram demonstrating MAdCAM-1 levels on CD31+ cells. Mean ± SEM, as well as data from individual mice, are presented for each group. Students paired t- test for figures 4K, 4M and 4N and Student’s unpaired t test for the rest of the figures. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001. Data represent at least two independent repeats.

Fig 4: Infection of the mouse pulmonary stem/progenitor cells (mPSCs)Oct4+ E3L clone by influenza A virus. (A) The expression of α2,3-linked sialic acid (α2,3 SA) and α2,6-linked sialic acid (α2,6 SA) on the mPSCsOct4+ E3L clone. The expression of α2,3 SA and α2,6 SA in mPSCsOct4+ E3L clone was determined by immunofluorescence assay (IFA) and fluorescence-activated cell sorting (FACS). The expression of CAR and CD54, which was previously demonstrated to be expressed by mPSCs, was also shown. The histograms of α2,3 SA, α2,6 SA, CAR, and CD54 expression were shown in purple, and the negative-staining cells were labeled as green lines. Scale bar of IFA image was 50 μm. (B) Development of cytopathic effect (CPE) in the mPSCsOct4+ E3L clone after influenza virus infection. The mPSCsOct4+ E3L clone was infected with PR8 at a multiplicity of infection (MOI) of 10. The sequential changes in morphology were recorded by microscope with a scale bar of 100 μm. (C) Expression of viral M1 proteins in influenza virus-infected mPSCsOct4+ E3L clone. The expression of viral M1 proteins in the mPSCsOct4+ E3L clone at different time points after virus infection was determined by IFA. Scale bar was 50 μm.

Fig 5: Suppression of NF-κB and the inflammatory circuit by c-Myb depends on IL1α. IL1α and NF-κB inhibition in IL1α up cells reversed the up-regulation of inflammatory genes: Cxcl1 (A), Icam1 (B), Tnfrsf9 (C) upon treatment with IRAK1/4, IL1-Ra and JSH-23. An average from 3 independent experiments is shown. Indicated concentration of inhibitors were added to E0771.LMB IL1α up 28 cells (IL1α up 28), after 24 h Cxcl1 concentrations were determined by CBA and Icam1 and Tnfrsf9 surface expression was measured by flow cytometry and expressed as frequency of positive cells, comparison to vehicle (DMSO)-treated cells. (D) qPCR detection of signature genes in JSH-23-treated (10 µM, 24 h) E0771.LMB IL1α up cells (IL1α up 28). Values were normalized to Gapdh and expressed as a fold change of DMSO-treated cells. An average of 3 independent experiments is shown. E0771.LMB (E) and 4T1 (F) cells overexpressing wt c-Myb (E0071.LMB Mybhigh C2, 4T1 Mybhigh 7) supplemented with recombinant mIL1α (1 ng/mL, 24 h) induces the expression of the signature genes. mRNA levels of indicated genes are determined by qPCR relative to vehicle-treated cells (ctrl) and normalized to Gapdh. An average of 3 independent experiments is shown. (G) Dose-dependent increase in surface Icam1 upon IL1α stimulation (24 h) of E0771.LMB cells overexpressing wt c-Myb (Mybhigh C2) as determined by flow cytometry. (H) Surface Icam1 expression is restored by recombinant IL1α in 4T1 cells overexpressing wt c-Myb (Mybhigh 7) concomitant with loss of IκBα-miRFP703. Mybhigh 7 cells cotransfected with pEGFP-C1 (GFP) and pIκBα−miRFP703 were treated with 1 ng/mL IL1α for 24h, Icam1 was stained and determined in GFP+ cells by flow-cytometry in parallel with IκBα levels. (I) Two 4T1 IL1α KO clones (IL1α KO CD34 and AB20), and scrambled control cells, were cotransfected with pcDNA3.c-Myb (c-Myb)/pcDNA3 (mock) and the NF-κB reporter. Luciferase activity (RLU) was measured 18 h later and normalized to β-galactosidase activity. An average of 3 independent experiments is shown. (J) Putative MYB-binding sites (MBSs, red) in the murine Il1a promoter sequence (980 bp upstream TSS, green). Primer pair used for cloning Il1a-luc reporter construct is indicated in pink. (K) Transactivation assay using 4T1 cells cotransfected with c-Myb or empty vector and Il1a-luc reporter. Il1a promoter activity is expressed as luciferase relative light units normalized to total protein levels. An average of 3 independent experiments is shown. Significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) are indicated.