Modulation of cytokeratin and cytokine/chemokine expression following influenza virus infection of differentiated human tonsillar epithelial cells

Abstract

The tonsils have been identified as a site of replication for Epstein-Barr virus, adenovirus, human papillomavirus, and other respiratory viruses. Human tonsil epithelial cells (HTECs) are a heterogeneous group of actively differentiating cells. Here, we investigated the cellular features and susceptibility of differentiated HTECs to specific influenza viruses, including expression of avian-type and mammalian-type sialic acid (SA) receptors, viral replication dynamics, and the associated cytokine secretion profiles. We found that differentiated HTECs possess more abundant α2,3-linked SA (preferentially bound by avian influenza viruses) than α2,6-linked SA (preferentially bound by mammalian strains). This dual receptor expression suggests a role in influenza virus adaptation and tropism within the tonsils by facilitating the binding and entry of multiple influenza virus strains. Our results indicated the susceptibility of differentiated HTECs to a wide range of influenza viruses from human, swine, and avian hosts. Virus production for most strains was detected as early as 1 day post-infection (dpi), and typically peaked by 3 dpi. However, pandemic H1N1 virus showed remarkably delayed replication kinetics that did not peak until at least 7 dpi. Notably, influenza virus infection impacted the expression of cytokeratins in HTEC cultures, which correlated with altered cytokine secretion patterns. These patterns varied within the strains but were most distinct in swine H3N2 infection. In conclusion, differentiated HTECs exhibited a strain-specific pattern of influenza virus replication and innate immune responses that included changes in cytokeratin and cytokine expression. These studies shed light on the complex interplay between influenza viruses and host cells in the tonsils.

Importance: To develop effective interventions against influenza, it is important to identify host factors affecting pathogenesis and immune responses. Tonsils are lymphoepithelial organs characterized by infiltration of B and T lymphocytes into the squamous epithelium of tonsillar crypts, beneath which germinal centers play key roles in antigen processing and the immune response. Influenza virus tropism in the human upper respiratory tract is a key determinant of host-range, pathogenesis, and transmission. Accordingly, experimental models using primary cells from the human respiratory tract are relevant for assessing virus tropism and replication competence. Our study addresses the dynamics of influenza virus replication in HTECs, including cellular tropism, infectivity, and cytokeratin and cytokine expression. The results of this study highlight the complex interplay between structural proteins and immune signaling pathways, all of which provide valuable insights into host-virus interactions.

Keywords: chemokines; crypts; cytokeratins, keratin; cytokines; influenza viruses; reticular epithelial cells; sialic acid receptors; squamous epithelial cells; tonsils; tropism.

Fig 1

Characteristics of well-differentiated HTECs. (A) Phase-contrast microscopy images of primary HTEC cultures at different time points during the differentiation process (20× magnification): (A.a) day 0 at the ALI, (A.b) day 5 at the ALI, (A.c) day 10 at the ALI, and (A.d) day 30 at the ALI, revealing well-differentiated epithelial cells of different sizes, types, and cytoplasm-to-nucleus ratios. (B) SEM provided details of differentiated HTEC characteristics. (B.a) The apical surface of the HTECs. Note the heterogeneous cell sizes and shapes, representing squamous surface epithelial cells disrupted by reticulated crypts (detailed in insets). (B.b) Increased magnification showing structures resembling cilia (arrows). (B.c–B.i) Areas of higher magnification (color-matched to larger figure) highlighting tight junctions between cells (B.c; arrow) and crypts rich in microvilli (d–i; scale bar: 2, 5, or 10 µm, as indicated). Note the mesh-like arrangement of crypts. (C) Confocal immunofluorescence imaging of the HTEC culture apical surface. HTECs were fixed and stained at 15 days in culture with monoclonal antibodies against villin (Alexafluor594; red) as a marker for microvilli, and intracellular β-tubulin (Alexafluor488; green) associated with cytoskeletal microtubules and sparse cilia. The isotype background is shown in Fig. S1. Scale bars represent 50 µm in panels (C.a; 20× magnification), 20 µm in (C.b; 40×), 10 µm in (C.c; 63×), and 5 µm in (C.d; 68×). Additional fields are given in Fig. S1. Panel (C.d) displays a Z-stack (10 layers) orthogonal view, highlighting the surface localization of cilia (green) and microvilli (red). CK8/18 (Alexafluor647; magenta) is included as a cell marker. The complete Z-stack is provided as Video S1 and at a higher resolution in Video S2.

Fig 2

Distribution of CK and lectin staining in HTECs as determined by flow cytometry. (A) HTECs were fixed and permeabilized, then stained with monoclonal antibodies specific for CK5, CK14, CK8/18, and CK19 for flow cytometric analysis. Percentages give fraction positive for the indicated CK when gated against matched isotype controls. (B) HTECs were stained with MAA I-FITC lectin for avian-adapted α2,3-linked SA receptors or with SNA-BV786 lectin for mammalian-adapted α2,6-linked SA receptors. (B.a) MAA I lectin staining was nearly universal in more than five replicate experiments. (B.b) SNA lectin staining was more limited over the same experiments. (B.c) Dual staining showed that coincident expression of avian with mammalian-adapted SA in HTECs was robust. Gating is based on unstained and fluorescence-minus-one controls as shown in Fig. S2. Inset statistics give means ± SEM of the indicated populations from three replicate experiments.

Fig 3

Influenza virus growth kinetics in HTECs. Well-differentiated HTECs were inoculated at a multiplicity of Infection (MOI) of 0.1 with the designated influenza viruses. (A) Human viruses A/TN/1–560/2009 (pH1N1) and A/HK4801/2014 (H3N2). (B) Human viruses A/WSN/1933 (H1N1) and B/Brisbane/60/2008 (IB). (C) Swine viruses A/Swine/NC/1816/2002 (H1N1) and A/Swine/OH/15TOSU4783/2016 (H3N2). (D) Strains A/Vietnam/1203/2004 (H5N1) and Avian A/scaup/GA/W22-145E/22 (H5N1). Viruses released from the cells were harvested with supernatants at the indicated time points and titrated to determine the TCID50 levels. The area under the curve (AUC) means ± SDs of three independent experiments, each using three HTEC cultures, are presented.

Fig 4

Longitudinal immune response profile of virus-infected HTECs. At the indicated times post viral infection, HTEC supernatants were collected and analyzed using a MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel. (A) Heat map showing the cytokine response in HTEC cultures to influenza virus infection over time. The colors shown represent the mean log₂ fold changes in cytokine/chemokine concentration relative to mock-infected cells from two independent experiments that included three internal replicates, each. Analytes are grouped hierarchically based on similar expression patterns, as indicated by the clustering diagram at left. The complete linear regression on the log₂ fold changes for each cytokine, with the virus and time as independent variables, including P-values, is given in Table S1. (B) Principal component analysis of log₁₀-transformed cytokine immune responses in HTEC cultures by strain and time post-infection. Strains are denoted by color, and the time post-infection (0–5 days) is indicated by shape. Note the unique clustering of Swine H3N2 data points. (C) Detail of selected cytokines with distinctly elevated levels of expression in HTEC cultures infected with Swine H3N2 relative to the other viral strains tested.

Fig 5

CK expression in pH1N1 influenza-infected HTECs. HTECs were infected with A/TN/1–560/2009 (pH1N1) at an MOI of 0.1. Infected HTECs were collected at 5, 7, and 10 dpi, then fixed, permeabilized, and stained with anti-influenza nucleoprotein monoclonal antibody (FluNP), as well as with monoclonal antibodies against either CK 5, CK14, CK8/18, or CK19, then analyzed by flow cytometry. (A) Representative flow plots showing the trend of decreased CK expression at early times (day 5) post-infection, and the subsequent return to nearly uniform CK expression as time of infection progressed. CK5 gives the most dramatic example of CK expression dynamics, while CK14 and CK19 show nearly complete return to mock-infection levels of CK expression. Gates were based on unstained, isotype, and fluorescence-minus-one controls (Fig. S5). (B) Heatmap summary of the data shown in (A). Percentages are the fraction of viable HTECs from either the FluNP(+) or FluNP(−) cohorts within the culture that expressed each CK at the time point assayed. While both cohorts experienced decreased CK expression, note the pronounced decrease in CK expression in uninfected cells, especially for CK5 and CK8/18 (n = 6 inserts/each virus/each time point).

Fig 6

CK expression in HTECs infected with three different strains of influenza. (A–C) HTECs were infected with A/Swine/NC/1816/2002 (sH1N1), A/HONG KONG/ 4801/2014 (H3N2), or A/Swine/OH/ 15TOSU4783/2016 (sH3N2), at an MOI of 0.1. Infected HTECs were collected at 1 or 3 dpi and assessed by flow cytometry, as described for Fig. 5. Specific CK expression is given vertically, and FluNP expression is shown horizontally. Fluorescence-minus-one gating plots are given in Fig. S6. (D) Heatmap summary of data from A–C showing the fraction of total viable HTECs from either the FluNP(+) or FluNP(−) cohorts within the culture that expressed each CK at the time point assayed. Notably, CK expression decreased in both cohorts but was more pronounced in uninfected HTECs (n = 6 inserts/each virus/each time point).

Fig 7

Correlations between cytokine production and influenza vs CK populations after influenza infection of HTECs. Data from Fig. 4 to 6 were combined to determine correlations between cytokine expressions, influenza infection (FLU+ or FLU−), and specific CK expression in HTEC cultures. The data include all influenza strains and time points after infection. Colors represent the correlation coefficient as determined by a Spearman correlation, and numbers mark those with statistical significance (P < 0.1). Cytokines are grouped by similar overall correlation patterns. Gray and red bars at the right highlight different general immunological characteristics.