Upregulation of cell-surface mucin MUC15 in human nasal epithelial cells upon influenza A virus infection

Abstract

Background: Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract, and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1, the roles of other mucins are still poorly understood, especially in viral infections.

Methods: To further identify mucins significant in influenza infection, we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus.

Results: We found that the expression of MUC15 was significantly upregulated upon infection, and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly, positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines, chemokines, EGFR and phosphorylated ERK) started to peak and plateau, MUC15 may potentially serve an immunomodulatory function later during influenza viral infection.

Conclusions: Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus, we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection.

Keywords: H3N2; Immunomodulation; MUC15; Mucin.

Fig. 1

MUC15 was upregulated after H3N2 infection in hNECs a: Fold changes of MUC mRNA expression in hNECs infected by H3N2 at 48 h post infection (hpi) with uninfected subjects (Uni) as baseline, detected by RNA sequencing (black column, n = 3) and mRNA array (white column, n = 5). b: Quantitative real-time RT-PCR analysis of MUC genes in hNECs infected with H3N2 with uninfected control subjects as baseline (n = 13), using PGK1 as the internal control. Relative mRNA expression levels were calculated using 2^ ^(−ΔΔCt) method; the fold change was calculated compared against uninfected control subjects. c: Western blot analysis of MUC15 protein expression on hNECs infected with H3N2. Cells were harvested at various times as indicated. d: The western blot band intensities were measured using Image J, and graph is presented as the ratio of MUC15-to-GAPDH and then normalized to each uninfected control (n = 5) (left y axis and bars). Plaque assay show viral replication in hNECs infected by H3N2 (n = 8) (right y axis and spots). e: IF co-staining of MUC15 (red) and HA (green) on fully differentiated hNECs with or without H3N2 infection (Cell nuclei are stained in blue with DAPI, 49–6-Diamidino-2-phenylindole dihydrochloride). */§, **/^##, ***/^###, **** denotes P value of less than 0.05, 0.01, 0.001, < 0.0001 compared with uninfected control, respectively. Median values with 25th and 75th percentiles are indicated by error bar. Uni: uninfected control

Fig. 2

Active viral replication induced MUC15 upregulation but MUC15 did not interfere with viral replication a: Plaque assay showed viral replication in hNECs with or without H3N2 infection or UV-inactivated H3N2 (UV) (n = 2). b: Quantitative real-time RT-PCR analysis of MUC15 mRNA expression in hNECs treated with or without H3N2, UV-inactivated H3N2 (UV) or 25 μg/mL poly (I:C) for 24 and 48 h with uninfected control subjects as baseline (n = 2), using PGK1 as the internal control. Relative mRNA expression levels were calculated using 2^ ^(−ΔΔCt) method; the fold change was compared against uninfected control subjects. c: Western blot analysis of MUC15 protein expression on hNECs treated with or without H3N2, UV-inactivated H3N2 (UV) or 25 μg/mL poly (I:C). Cells were harvested at various times as indicated (n = 2). d: Viral replication in hNECs transfected with non-targeting siRNA (NT-siRNA) or MUC15-siRNA and normal hNECs (n = 1). e-f: Viral replication in MDCK cells (E, n = 3) and hNECs (F, n = 1) treated with 0, 2 or 30 ng recombinant MUC15 protein (rMUC15) before or after H3N2 infection. g: Immunoprecipitation with MUC15 protein followed by Western blotting for hemagglutinin (HA), matrix protein 1 (M1), neuraminidase (NA) and MUC15 (n = 2). Median values with 25th and 75th percentiles are indicated by error bar. * denotes P value of less than 0.05 compared with uninfected control. Uni: uninfected control; Unt: untreated control

Fig. 3

Expression of MUC15 was positively correlated with inflammatory factors a: Quantitative real-time RT-PCR analysis of IL1B, IL6, IL8, TNF, CCL2, IFNB1, CXCL10, (solid line, left y axis) and MUC15 (dotted line, right y axis) mRNA expression in hNECs infected with H3N2 (n = 11), using PGK1 as the internal control. Relative mRNA expression levels were calculated using 2^ ^(−ΔΔCt) method; the fold change was compared against uninfected control subjects. b: The correlations between mRNA level (2^ ^(−ΔΔCt)) of MUC15 versus IL1B, IL6, IL8, TNF, CCL2, IFNB1, CXCL10 in hNECs infected with H3N2 were analyzed (n = 11). The timepoints post-infection that were included in the graphs are 8, 16, 24, 48 and 72 hpi. *, **, ***, **** denotes P value of less than 0.05, 0.01, 0.001, < 0.0001 compared with uninfected control, respectively. Uni: uninfected control

Fig. 4

EGFR-pERK1/2-AP1 pathway was triggered by viral infection. a: Quantitative real-time RT-PCR analysis of EGFR (solid line, left y axis) and MUC15 (dotted line, right y axis) mRNA expression in hNECs infected with H3N2 (n = 11), using PGK1 as the internal control. Relative mRNA expression levels were calculated using 2^ ^(−ΔΔCt) method; the fold change was compared against uninfected control subjects. b: The correlations between protein level (blot band intensities) of MUC15 versus EGFR in hNECs with or without H3N2 infection were analyzed (n = 5). c: Western blot analysis of EGFR, pERK, total ERK and GAPDH protein expression on hNECs with or without H3N2infection. Cells were harvested at various times as indicated (n = 5). d: Quantitative real-time RT-PCR analysis of JUN and FOS mRNA expressions in hNECs with or without H3N2 infection (n = 11), using PGK1 as the internal control. Relative mRNA expression levels were calculated using 2^ ^(−ΔΔCt) method; the fold change was compared against uninfected control subjects. e: The correlations between mRNA level (2^ ^(−ΔΔCt)) of MUC15 versus JUN and FOS in hNECs infected with H3N2 were analyzed (n = 11). f: The hypothesis of MUC15’s potential role in hNECs infected with H3N2: The increase of MUC15 expression triggered by H3N2 viral replication inhibits the activation of epithelial growth factor receptor (EGFR), which initiates the downstream ERK1/2-AP1 pathway. Thus, MUC15 may downregulate the host immune response at the later phase of viral infection. *, **, ***, **** denotes P value of less than 0.05, 0.01, 0.001, < 0.0001 compared with uninfected control, respectively. Uni: uninfected control