Figure 1. TGF-β response following rhinovirus infection in PBMCs. (a) Experimental design of the sample collection from healthy and asthmatic individuals. (b) Graphical abstract of the data collection. (c) Gene expression analysis of TGF-βRI expression on untouched PBMCs, normalized on the control group and the housekeeping gene HPRT (n = 19, 28). (d) Gene expression analysis of TGF-βRII expression on untouched PBMCs, normalized on the control group and the housekeeping gene HPRT (n = 19, 28). (e) Detection of RV1b load of RV-infected PBMCs via qPCR, normalized on HPRT mRNA expression (n = 19, 20). (f) Gene expression analysis of TGF-βRII expression in PBMCs, with control medium or infected with rhinovirus, and normalized on the control group and the house-keeping gene HPRT (n = 15, 18, 17, 16). (g) Gene expression analysis of TGF-β1 expression on PBMCs with control medium or infected with rhinovirus, normalized on the control group and the housekeeping gene HPRT (n = 15, 18, 17, 16). (h) ELISA assay of TGF-β1 production in the supernatant of control medium and rhinovirus-infected PBMCs, activated in acidic condition before measurement, according to the supplier’s instructions (n = 19, 28). Data are shown as Mean+SEM. * p < 0.05, **** p < 0.0001.

Figure 1. TGF-β response following rhinovirus infection in PBMCs. (a) Experimental design of the sample collection from healthy and asthmatic individuals. (b) Graphical abstract of the data collection. (c) Gene expression analysis of TGF-βRI expression on untouched PBMCs, normalized on the control group and the housekeeping gene HPRT (n = 19, 28). (d) Gene expression analysis of TGF-βRII expression on untouched PBMCs, normalized on the control group and the housekeeping gene HPRT (n = 19, 28). (e) Detection of RV1b load of RV-infected PBMCs via qPCR, normalized on HPRT mRNA expression (n = 19, 20). (f) Gene expression analysis of TGF-βRII expression in PBMCs, with control medium or infected with rhinovirus, and normalized on the control group and the house-keeping gene HPRT (n = 15, 18, 17, 16). (g) Gene expression analysis of TGF-β1 expression on PBMCs with control medium or infected with rhinovirus, normalized on the control group and the housekeeping gene HPRT (n = 15, 18, 17, 16). (h) ELISA assay of TGF-β1 production in the supernatant of control medium and rhinovirus-infected PBMCs, activated in acidic condition before measurement, according to the supplier’s instructions (n = 19, 28). Data are shown as Mean+SEM. * p < 0.05, **** p < 0.0001.

Figure 2. High rhinovirus load was associated with more TGF-βRII and induced Treg immune response. (a) Experimental design. (b) Correlation of TGF-βRII/HPRT expression in RV-infected PBMCs with the RV1b/HPRT mRNA expression in controls. (c) Correlation of TGF-βRII/HPRT expression in RV-infected PBMCs with the RV1b/HPRT mRNA expression in asthmatics. (d) Flow cytometry analysis of Foxp3+ CD25+ CD4+ Tregs in CN and RV condition. A representative dot plot of each group and the FMO control is shown (n = 10, 9, 10, 9). Data are shown as Mean+SEM. * p < 0.05, ** p < 0.01, *** p < 0,001.

Figure 2. High rhinovirus load was associated with more TGF-βRII and induced Treg immune response. (a) Experimental design. (b) Correlation of TGF-βRII/HPRT expression in RV-infected PBMCs with the RV1b/HPRT mRNA expression in controls. (c) Correlation of TGF-βRII/HPRT expression in RV-infected PBMCs with the RV1b/HPRT mRNA expression in asthmatics. (d) Flow cytometry analysis of Foxp3+ CD25+ CD4+ Tregs in CN and RV condition. A representative dot plot of each group and the FMO control is shown (n = 10, 9, 10, 9). Data are shown as Mean+SEM. * p < 0.05, ** p < 0.01, *** p < 0,001.

Figure 3. NK cells produce less TGF-β upon rhinovirus infection. (a) Experimental design. (b) Flow cytometry analysis of GARP-positive NK cells (n = 10, 9, 10, 9). (c) Flow cytometry analysis of intracellular TGF-β+ GARP+ NK cells. A representative dot plot for each group is shown (n = 10, 9, 10, 9). (d) Flow cytometry analysis of CD56 bright NK cells (n = 15, 24, 15, 24). (e) IFNγ ELISA of supernatants from CN and RV conditions (n = 19, 22, 19, 22). Data are shown as Mean+SEM. * p < 0.05, *** p < 0.001.

Figure 3. NK cells produce less TGF-β upon rhinovirus infection. (a) Experimental design. (b) Flow cytometry analysis of GARP-positive NK cells (n = 10, 9, 10, 9). (c) Flow cytometry analysis of intracellular TGF-β+ GARP+ NK cells. A representative dot plot for each group is shown (n = 10, 9, 10, 9). (d) Flow cytometry analysis of CD56 bright NK cells (n = 15, 24, 15, 24). (e) IFNγ ELISA of supernatants from CN and RV conditions (n = 19, 22, 19, 22). Data are shown as Mean+SEM. * p < 0.05, *** p < 0.001.

Table 1. Clinical characteristics of the control and asthmatic individuals in the human AZCRA study.

Table 1. Clinical characteristics of the control and asthmatic individuals in the human AZCRA study.

IDGenderAgeAsthma Severity 1Treatment Asthma Symptom Control (GINA 2020)C01F34///C02F33///C03F29///C04F44///C05M62///C06M64///C07M26///C08F23///C09F63///C10F53///C11M55///C12M21///C13F22///C14M31///C15M25///C16M43///C17M30///C18F28///C19M24///A01M28Mild persistentSteroid + non-steroidWell controlledA02F33Mod. PersistentSteroid + non-steroidPartly controlledA03M63Mild persistentSteroid + non-steroidWell controlledA04M53Mild persistentSteroid + non-steroidPartly controlledA05M57Mild persistentSteroid + non-steroidWell controlledA06F47Mild persistentSteroid + non-steroidWell controlledA07F40Mod. PersistentSteroid + non-steroidPartly controlledA08M60IntermittentNoneWell controlledA09F44Mild persistentSteroid + non-steroidWell controlledA10F51Mild persistentSteroid + non-steroidPartly controlledA11M26IntermittentNonePartly controlledA12F56IntermittentNoneWell controlledA13M31IntermittentSteroid + non-steroidPartly controlledA14M24Mild persistentSteroidPartly controlledA15F47IntermittentSteroid + non-steroidWell controlledA16F26IntermittentNonePartly controlledA17F54IntermittentSteroid + non-steroidWell controlledA18F47Mod. PersistentSteroid + non-steroidPartly controlledA19F24Mod. PersistentSteroid + non-steroidUncontrolledA20M23IntermittentNoneWell controlledA21M41Mod. PersistentSteroid + non-steroidPartly controlledA22F64Mod. PersistentSteroid + non-steroidPartly controlledA23F62UnknownNoneUnknownA24F52IntermittentSteroid + non-steroidPartly controlledA25M48IntermittentSteroid + non-steroidPartly controlledA26M35IntermittentSteroid + non-steroidWell controlledA27F52UnknownSteroid + non-steroidWell controlledA28F54Mod. PersistentSteroid + non-steroidPartly controlled

Table 2. Flow cytometry antibodies.

Table 2. Flow cytometry antibodies.

AntibodyCompanyCatalog NumberRRIDBV421 anti-human CD56BD Biosciences562752AB_2732054APC-Fire anti-human CD3Biolegend300470AB_2629689PerCP/Cyanine 5.5 anti-human GARPBiolegend352513AB_2734371PE/Cyanine7 anti-human CD25 antibodyBiolegend302612AB_314282Alexa Fluor 647 anti-human FOXP3 antibodyBiolegend320214AB_492984PE anti-human LAP recombinantBiolegend364403AB_2910407PE anti-mouse IgG1, κBiolegend400113AB_326435Zombie Aqua Fixable Viability KitBiolegend423101