Fig 1: Exogenous and endogenous SPINK6 affect virus maturation and viral replication in human airway organoids. Confocal imaging (left) shows intrinsic SPINK6 (green) expression in the epithelial cells in 2D human airway organoids. Nuclei and actin filaments are counterstained and shown as blue and purple, respectively. Scale bar, 10 µm. Flow cytometry analysis demonstrates HAT and SPINK6 expression in human airway organoids. The plots (middle) show the representative result of one experiment performed three times. The percentages of HAT and SPINK6 positive cells (right) are shown as mean and SD of three independent experiments.At 24 h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times.2D human airway organoids were incubated with the fluorogenic substrate in triplicate for 1 h in the presence of wtSPINK6 or mutSPINK6, and applied to fluorescence assay. The fluorescence intensity of mutSPINK6‐treated organoids is arbitrarily set as 1. Data represent the mean and SD of a representative experiment performed three times.After inoculation of H1N1/pdm, 2D human airway organoids were incubated with wtSPINK6 or PBS in triplicate for 24 h (left). Cell‐free culture media were harvested for viral titration (right) and examination of HA cleavage by WB after 10‐fold concentration (middle). Viral titer data present the mean and SD of triplicated samples.2D airway organoids were applied to fluorescence assay after incubation with α‐SPINK6 or isotypic IgG in triplicate for 4 h (left). Data present the mean and SD of triplicated samples.After inoculation of H1N1/pdm, 2D human airway organoids were incubated with an α‐SPINK6 or isotype IgG in triplicate for 24 h (left). Cell‐free culture media were harvested for viral load detection (right) and examination of HA cleavage by WB after 10‐fold concentration (middle). Viral load data present the mean and SD of triplicated samples. Data information: *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t test. Source data are available online for this figure.
Fig 2: SPINK6 constrains proteolytic activity of trypsin, and trypsin‐mediated HA cleavage and viral growth. TPCK trypsin is premixed with wtSPINK6 protein or mutSPINK6 protein or PBS in triplicate, incubated with a fluorogenic substrate for 30 min, and then applied to fluorescence assay. Data are presented as mean and SD in a representative experiment performed three times. The fluorescence intensity of TPCK trypsin/PBS mixture is arbitrarily set as 1. ***P < 0.001.At 48 h post‐transfection of wtSPINK6 plasmid in 293T cells, cell lysate and concentrated supernatant (50×) were applied to the detection of SPINK6 protein and β‐actin by WB.At 48 h after co‐transfection of H7 plasmid with wtSPINK6 plasmid or blank vector, the transfectants were incubated with or without TPCK trypsin for 30 min, and then applied to examine HA cleavage by WB. Intensities of HA2 and HA0 bands are quantified with ImageJ. HA2/HA0 ratio of each sample is shown at the bottom.At 48 h after transfection of H7 plasmid, the transfectants were incubated with TPCK trypsin in the presence of recombinant wtSPINK6 protein or PBS or a protease inhibitor AEBSF for 30 min and then applied to examine HA cleavage.At 24 h after transfection of wtSPINK6 plasmid or blank vector in triplicate, A549 cells were inoculated with H7N9/ah virus. At the indicated hours post‐infection (hpi), culture media (supernatant) and cell lysate were harvested for viral load detection. Data are presented as mean and SD in a representative experiment performed three times. **P < 0.01; ***P < 0.001. Student’s t‐test. Source data are available online for this figure.
Fig 3: The association SNP genotypes are correlated with differential SPINK6 mRNA expression in human tissues. The association SNP rs1432689 genotypes are correlated with differential SPINK6 mRNA expression in over 1,000 human lung tissues from three centers. The box denotes interquartile range; the thick line and diamond within the box are the median and mean, respectively; whiskers are the minimum and maximum and open dots are outliers. Linear regression was used for eQTL data analysis. Data of three cohorts were applied to meta‐analysis and resulted in the P‐value of the association SNP.rs1432689 genotypes are correlated with SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database. Sample sizes for each genotype are shown in the x‐axis. The y‐axis shows the normalized expression of SPINK6 (Norm. expression) in the indicated trusses. Median and interquartile range are represented by a white line and dark box, respectively. Data distribution is colored with light blue; a wider section of the violin plot indicates the data on the section have a higher frequency. Linear regression was used for data analysis.
Fig 4: SPINK6 inhibits virus growth and maturation‐mediated HAT and KLK5. At 36 h after transfection of the indicted protease and wtSPINK6 plasmids or blank vector in triplicate, BHK21 cells were inoculated with H7N9/ah at a MOI of 0.5. Cell‐free culture media were harvested at 24 hpi for viral titration. Data represent the mean and SD of the triplicated wells in a representative experiment performed three times. **P < 0.01. Student’s t‐test.At 24 h after transfection of HAT or KLK5 plasmid in sextuplicate, A549 cells were inoculated with H1N1/pdm at MOI of 0.25. The infected cells were incubated with recombinant wtSPINK6 or mutSPINK6 protein in triplicate for 24 h. Culture media in each well were stored in two aliquots, one was applied to viral titration with conventional plaque assay (black bars) and the other was pre‐treated with TPCK trypsin for 1 h prior to plaque assay (grey bars). Data represent the mean and SD of the triplicated wells in a representative experiment performed three times. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t‐test.
Fig 5: SPINK6 treatment suppresses viral growth and improves survival in mouse influenza infection. Regime of the mouse experiment. Balb/c mice were a mouse‐adapted strain of pandemic H1N1 virus. At 24 h before inoculation, and 8, 24, and 36 h post‐inoculation, two groups of mice were intranasally administered with wtSPINK6 protein or mutSPINK6 protein. Ten mice treated with wtSPINK6 or mutSPINK6 were monitored daily for disease signs, body weight, and survival for 14 days. Five mice treated with wtSPINK6 or mutSPINK6 were sacrificed at 3 and 4 days after the viral challenge. Lung tissues were collected for the quantification of viral growth and immunofluorescence staining.Survival rates of mice treated with wtSPINK6 and mutSPINK6 were analyzed with Mantel–Cox test.Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6. Data represent mean ± SD. **P < 0.01; ***P < 0.001. Student’s t‐test.Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP‐positive cells (green). Nuclei are counterstained with DAPI (blue). Representative confocal images of virus‐infected cells in the indicated mice. Scale bar, 10 µm.
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