Table 1.
Information on primary tissue-derived human bronchial epithelial cells.
Fig 1.
Methods for macrophage differentiation, co-culture, and RSV infection.
(A) The timeline of monocyte differentiation into macrophages, then into M0, M1-like, or M2-like macrophages. Human blood-derived monocytes were isolated from enriched leukapheresis product and cultured in IMM. Monocytes were differentiated into naive (M0-like) macrophages using IMM with 50 ng/mL M-CSF for 3–4 days, and further differentiated for 2–3 days into M1- or M2-like macrophages through addition of 50 ng/mL IFNγ and 10 ng/mL LPS, or 10 ng/mL IL-4. Cells were then placed in co-culture with human HBECs for 72 hours. In some instances, the epithelial cells were infected with RSV immediately after being placed in co-culture. (B) A schematic of the macrophages and HBECs being placed in co-culture. The HBECs were maintained at air-liquid interface and the macrophages were in submerged culture at the bottom of the dish. The macrophages and epithelia were not in direct contact. (C) Illustration of co-culture with RSV infection. After cells were placed in co-culture the epithelia were infected by the apical addition of medium containing RSV and incubated for 2 hours; then the inoculum was removed and the epithelia returned to air-liquid interface. The cultures were collected after 72 hours.
Table 2.
Materials used for flow cytometry of macrophages.
Table 3.
Predesigned primer sets for PrimeTime™ standard qPCR assay.
Table 4.
RSV primer sequence and probe sequence for PrimeTime™ standard qPCR assay.
Fig 2.
Morphology of M0, M1, or M2 macrophages after culture.
(A) M0, M1, or M2 macrophages were placed in either M0, M1, or M2 medium (respectively) or ALI medium as either monoculture or co-culture with HBECs for 72 hours and white light images taken to assess morphology. (B) M0, M1, or M2 macrophages were placed in either M0, M1, or M2 medium (respectively), ALI medium, or IMM as co-culture with RSV-infected HBECs. Images were taken after 72 hours of co-culture with infected cells and are a representative image of n = 5. Scale bar represents 0.1 mm.
Fig 3.
Assessment of phenotypic changes after co-culture of macrophages with HBECs.
(A) TER of HBECs was measured after co-culture with macrophages or as monoculture in different medium types (M0, M1, or M2 medium, or ALI medium) for 72 hours. In this figure and the subsequent figures, each medium type is represented by a different colored bar, and if they are in co-culture the bar outline color corresponds to the macrophage type. No significant differences in TER were found as determined by ANOVA, n = 5. (B) Macrophage viability after monoculture in respective media (M0 medium, M1 medium, M2 medium for the corresponding macrophage type) or in ALI medium, or in co-culture (co) with HBECs in either medium. Viability was determined by flow cytometry by exclusion of DRAQ7™ dye. ** = p<0.01 ANOVA with Tukey’s post hoc test compared to M0 in M0 medium, n = 4–5. (C-F). Flow cytometric analysis of cell surface marker expression for M1 markers (CD80, CCR7) or M2 markers (CD206, CD209) in macrophages after monoculture or co-culture in M0, M1, or M2 medium, or in ALI medium. P<0.05 as determined by ANOVA with Tukey’s post hoc tests (n = 4–5) where *p = <0.05, ** = p<0.01 as determined by ANOVA followed by Tukey’s post hoc test comparing M0 cultures, or where indicated, n = 4–5.
Fig 4.
Assessment of phenotypic changes after co-culture of macrophages with HBECs by ELISA.
Media was collected after 72 hours of co-culture of macrophages and HBECs and ELISAs for IL-12p70, IL-10, or TNFα were performed. For statistical analysis, ANOVA was performed within each group and compared to monoculture in the medium appropriate for each macrophage (n = 4–5) but no significant differences were found.
Fig 5.
Images of infected HBECs with or without co-culture with macrophages and in different media.
HBEC were placed in different types of media as indicated with or without co-culture with M0, M1, or M2 macrophages and infected with 0.15 MOI RSV. 2x images of approximately half of the Transwell area were taken 72 hours post infection. Representative images equal to the average fluorescence as quantified in Fig 5 (n = 3–5). Scale bar represents 1 mm. (A) HBECs were uninfected, or infected with RSV when the HBECs were in ALI medium or IMM. (B) Media bathing the HBECs was changed to IMM containing either LPS (10 ng/mL), IFNγ (50 ng/mL), or IL-4 (10 ng/mL). (C) Media bathing HBECs was changed to M0 medium (IMM with 50 ng/mL M-CSF), M1 medium (IMM with 50 ng/mL M-CSF, 50ng/mL IFNγ and 10 ng/mL LPS), or M2 medium (IMM with 50 ng/mL M-CSF and 10 ng/mL IL-4). (D, E, F) HBECs were placed in co-culture with M0, M1, or M2 macrophages and the media changed to M0, M1, or M2 medium, respectively, ALI medium, or IMM.
Fig 6.
Co-culture with RSV infection.
HBECs were placed in different media with or without co-culture with M0, M1, or M2 macrophages and infected with 0.15 MOI RSV. Cells were assessed 72 hours later for (A) Average fluorescence of images taken of epithelia, (B) Transepithelial electrical resistance (TER) of epithelia, (C), Fold change in RSV NS1 by qPCR (GAPDH as housekeeping gene), (D) Macrophage viability as determined by flow cytometry and DRAQ7™ exclusion, or (E, F) Macrophage cell surface expression of CD80 or CD206 (M1 or M2 markers, respectively). In B and C, * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001 compared to IMM, or # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001 compared to ALI as assessed by ANOVA with Dunnett’s post hoc test, n = 3–5. In E and F, * p<0.05, ** p< 0.01 as assessed by ANOVA with Tukey’s post hoc test between each macrophage group, n = 3–5.
Fig 7.
Meso Scale Discovery cytokine assay on media of macrophages and HBECs infected with RSV in co-culture.
HBEC were placed in different media with or without co-culture with M0, M1, or M2 macrophages and infected with 0.15 MOI RSV. Media was collected from the basolateral side of the epithelia at 24 and 72 hours post infection and collected. Data for 24 hours is shown, as results at 72 hours were the same. Basolateral media or apical wash were assessed for the release of IL-10, IL-6, IL-12p70, or TNFα by Meso Scale Discovery (MSD) cytokine assay. * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001 compared to IMM or where indicated, or # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001 compared to ALI as assessed by ANOVA with Dunnett’s post hoc test, †† p<0.01 as assessed by ANOVA with Tukey’s post hoc test comparing samples with M1 medium or M1 macrophages, n = 3–4.