Respiratory Syncytial Virus Inhibits Lung Epithelial Na+ Channels by Up-regulating Inducible Nitric-oxide Synthase

Respiratory syncytial virus (RSV) infection has been shown to reduce Na+-driven alveolar fluid clearance in BALB/c mice in vivo. To investigate the cellular mechanisms by which RSV inhibits amiloride-sensitive epithelial Na+ channels (ENaC), the main pathways through which Na+ ions enter lung epithelial cells, we infected human Clara-like lung (H441) cells with RSV that expresses green fluorescent protein (rRA2). 3-6 days later patch clamp recordings showed that infected cells (i.e. cells expressing green fluorescence; GFP(+)) had significantly lower whole-cell amiloride-sensitive currents and single channel activity (NPo) as compared with non-infected (GFP(-)), non-inoculated, or cells infected with UV-inactivated RSV. Both α and β ENaC mRNA levels were significantly reduced in GFP(+) cells as measured by real-time reverse transcription-PCR. Infection with RSV increased expression of the inducible nitric-oxide synthase (iNOS) and nitrite concentration in the culture medium; nuclear translocation of NF-κB p65 subunit and NF-κB activation were also up-regulated. iNOS up-regulation in GFP(+) cells was prevented by knocking down IκB kinase γ before infection. Furthermore, pretreatment of H441 cells with the specific iNOS inhibitor 1400W (1 μm) resulted in a doubling of the amiloride-sensitive Na+ current in GFP(+) cells. Additionally, preincubation of H441 cells with A77-1726 (20 μm), a de novo UTP synthesis inhibitor, and 1400W completely reversed the RSV inhibition of amiloride-sensitive currents in GFP(+) cells. Thus, both UTP- and iNOS-generated reactive species contribute to ENaC down-regulation in RSV-infected airway epithelial cells. Respiratory syncytial virus (RSV) infection has been shown to reduce Na+-driven alveolar fluid clearance in BALB/c mice in vivo. To investigate the cellular mechanisms by which RSV inhibits amiloride-sensitive epithelial Na+ channels (ENaC), the main pathways through which Na+ ions enter lung epithelial cells, we infected human Clara-like lung (H441) cells with RSV that expresses green fluorescent protein (rRA2). 3-6 days later patch clamp recordings showed that infected cells (i.e. cells expressing green fluorescence; GFP(+)) had significantly lower whole-cell amiloride-sensitive currents and single channel activity (NPo) as compared with non-infected (GFP(-)), non-inoculated, or cells infected with UV-inactivated RSV. Both α and β ENaC mRNA levels were significantly reduced in GFP(+) cells as measured by real-time reverse transcription-PCR. Infection with RSV increased expression of the inducible nitric-oxide synthase (iNOS) and nitrite concentration in the culture medium; nuclear translocation of NF-κB p65 subunit and NF-κB activation were also up-regulated. iNOS up-regulation in GFP(+) cells was prevented by knocking down IκB kinase γ before infection. Furthermore, pretreatment of H441 cells with the specific iNOS inhibitor 1400W (1 μm) resulted in a doubling of the amiloride-sensitive Na+ current in GFP(+) cells. Additionally, preincubation of H441 cells with A77-1726 (20 μm), a de novo UTP synthesis inhibitor, and 1400W completely reversed the RSV inhibition of amiloride-sensitive currents in GFP(+) cells. Thus, both UTP- and iNOS-generated reactive species contribute to ENaC down-regulation in RSV-infected airway epithelial cells. Respiratory syncytial virus (RSV) 2The abbreviations used are: RSV, respiratory syncytial virus; IKK, IκB kinase; AFC, alveolar fluid clearance; ENaC, epithelial Na+ channels; RT, reverse transcription; iNOS, inducible nitric-oxide synthase; PBS, phosphate-buffered saline; siRNA, small interfering RNA; pF, picofarads; GFP, green fluorescent protein; m.o.i., multiplicity of infection; HPRT, hypoxanthine phosphoribosyltransferase; LPS, lipopolysaccharide; IFN-γ, interferon γ; PE, phycoerythrin; SH, small hydrophobic. is a member of the Pneumovirus genus within the family of the Paramyxoviridae, which is characterized by a linear, negative-sense, single-stranded RNA genome (1Sullender W.M. Clin. Microbiol. Rev. 2000; 13: 1-15Crossref PubMed Scopus (226) Google Scholar). It is the most common cause of lower respiratory tract disease in infants and children worldwide (2Smyth R.L. Openshaw P.J. Lancet. 2006; 368: 312-322Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar) and may also be under-diagnosed as a cause of community-acquired lower respiratory tract infections among adults (3Dowell S.F. Anderson L.J. Gary Jr., H.E. Erdman D.D. Plouffe J.F. File Jr., T.M. Marston B.J. Breiman R.F. J. Infect. Dis. 1996; 174: 456-462Crossref PubMed Scopus (357) Google Scholar). The primary targets of RSV infection are respiratory epithelial cells (4Zhang L. Peeples M.E. Boucher R.C. Collins P.L. Pickles R.J. J. Virol. 2002; 76: 5654-5666Crossref PubMed Scopus (420) Google Scholar). Clinical manifestations may range from a mild cold syndrome to severe respiratory distress and failure (5Hammer J. Numa A. Newth C.J. Pediatr. Pulmonol. 1997; 23: 176-183Crossref PubMed Scopus (134) Google Scholar). Respiratory tract fluid accumulation and rhinorrhea are common findings in most cases. Our previous studies showed that infection of BALB/c mice with RSV impairs Na+-driven alveolar fluid clearance (AFC) across the distal lung and upper airways, respectively, resulting in increased levels of lung water and mild hypoxemia (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. 2006; PubMed Scopus Google Scholar). Na+ across the of both Na+ and ions through as amiloride-sensitive and and and a of as the and S. Rev. Physiol. PubMed Scopus Google S. A. L. J. Physiol. 2002; PubMed Scopus Google Scholar). the activity of of may Na+ Furthermore, in in Na+ among infected and non-infected cells and the of infection the Thus, the cellular mechanisms by which RSV epithelial Na+ be in vivo. To RSV infection of airway epithelial cells ENaC levels and activity and mechanisms we infected H441 cells, a human expressing ENaC, with a RSV that expresses green fluorescent protein 3-6 days later we measured the activity and mRNA levels of and γ ENaC in infected (i.e. H441 cells expressing green fluorescence; non-infected as as cells whole-cell and single channel patch clamp recordings and real-time of previous that RSV infection of epithelial cells iNOS S. J. PubMed Scopus Google Scholar) and the inhibition of ENaC by and reactive and mechanisms Matalon S. Am. J. Physiol. Google L. Am. J. Physiol. Google we the that increased levels of from iNOS were the of ENaC To we measured ENaC NF-κB iNOS and levels of nitrite in the in RSV-infected cells with 1400W iNOS or Our that or reactive species by the of with reactive species are the down-regulation of ENaC activity in RSV-infected cells. Furthermore, in that RSV inhibition of was by UTP (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. 2006; PubMed Scopus Google we also measured whole-cell amiloride-sensitive currents in RSV-infected cells with inhibitor of de novo UTP or Our showed that down-regulation of ENaC activity in RSV-infected cells is to increased levels of UTP as Thus, RSV infection ENaC by cells were from and in with and as A. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. PubMed Scopus Google Scholar). were in and in a of and cells and were used in were from the and a of in and in with to channel A. A. Matalon S. Am. J. Physiol. Physiol. 2000; Google Scholar). The was of respiratory syncytial virus that green fluorescent protein in infected cells was as The of RSV the RSV with the of the was with that of J. Virol. PubMed Scopus Google Cell. Full Text PDF PubMed Scopus Google Scholar). of a by a RSV by a virus which by and a The expressing RSV and and also been J. Virol. PubMed Google J. Virol. PubMed Google Scholar). cells cells were infected with virus a multiplicity of infection of and with the RSV and the J. Virol. PubMed Google Scholar). The cells were and the was with days the were and used to cells. The was with and cells were days in the was by in cells. were by and in cells W.M. Anderson PubMed Scopus Google Scholar). were in RSV virus was by through as before A. J. J. Virol. PubMed Scopus Google Scholar). of of were by to of in a the of and (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google Scholar). Infection of H441 H441 cells were infected with to a multiplicity of infection of or were to to the cells the was cells were and with RSV which the cells had to cells were to UV-inactivated or RSV cells were to of was to infection of H441 cells with or RSV resulted in H441 cells were infected with UV-inactivated or RSV as and days infection cells were from by to and were and were to a of in were and cells were in the The of to H441 cells was by with a to with as cells were by to green to H441 that were infected by (i.e. GFP(+) The GFP(+) cells were with as H441 cells with as the of previous in (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google we that the inhibition of alveolar fluid clearance in mice infected with RSV infection. expression of we to patch clamp recordings infection. H441 cells were with phosphate-buffered and a the of fluorescent cells were by the of green whole-cell patch clamp cells were with and The of measured with a was were from with a were with the and The from to with The was before of a the and the The was with patch clamp were with and through and whole-cell the cells were and whole-cell currents were by a from to in were by the current and from the of the the currents to were cells with the currents were by the currents in the of from from cells with currents or were single channel Na+ currents from H441 cells the were with a and were with the were as in the previous were measured a of currents were and with The and of single channel currents were from from of recordings as A. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. PubMed Scopus Google A. A. Matalon S. Am. J. Physiol. Physiol. 2000; Google Scholar). were or to to The of the of channels in a patch by the which the activity of was from recordings as before A. A. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000; PubMed Google Scholar). of of ENaC days H441 cells were from by and and in of were green with a to of and cells were RNA was from of cells with was reverse of and γ ENaC were with real-time a to of human hypoxanthine were used as The of the used ENaC α and reverse ENaC β and reverse ENaC γ and reverse and reverse were from The levels of or γ ENaC were as which was by of the of the from of or γ The in expression of or γ ENaC mRNA infection was by of in which was by of the of cells of from the of or GFP(+) cells as J. PubMed Scopus Google Scholar). iNOS with with H441 were in cold with in by the of cellular with in cells were with a in in as the with cells were with by with to in the were and with were and with the was from from H441 cells with and human interferon γ as the of iNOS days H441 cells were a to of non-inoculated, GFP(+) and levels of iNOS were with real-time a The of the used iNOS and reverse J. PubMed Scopus Google Scholar) and and reverse were from The levels of iNOS mRNA was as which was by of the of the HPRT, from that of The in expression of iNOS mRNA infection was by of in which was by of the of cells of the from the of or GFP(+) cells as J. PubMed Scopus Google Scholar). of in the culture of cells as as that of the cells or UV-inactivated infections were measured as of with and human as the was by to the of S. J. 2002; PubMed Scopus Google Scholar) with from with before the the that as or contribute to the nitrite To protein in culture H441 cells were with PBS, and of cold and with inhibitor was were from the and The was and protein concentration was by the as a as (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google concentration was by the protein in of NF-κB p65 were as before J. PubMed Scopus Google Scholar). The nuclear were by and to and with a and and with a The levels as were as J. PubMed Scopus Google Scholar). H441 cells were with cold PBS, and the cells were in and with The cells were and the were in The were and nuclear in were in of and with specific the NF-κB and of The were with were with from and H441 cells were the by cells to were with or a of human specific H441 cells were infected with The cells were and by days RNA was and iNOS mRNA levels were measured with real-time as To the of and from cells were and the of expression was by are as the were by of by the of or the as with a were significantly from H441 with RSV of (i.e. H441 cells with and expressing green fluorescence; GFP(+)) cells were infection of H441 cells with UV-inactivated to that green were as cells. The of infected cells cells in the and of infection; the infection were and were and and days infection in H441 in cells was compared with cells in culture are with the of of RSV in cells (4Zhang L. Peeples M.E. Boucher R.C. Collins P.L. Pickles R.J. J. Virol. 2002; 76: 5654-5666Crossref PubMed Scopus (420) Google J. Virol. PubMed Google Scholar). the has been that the protein of RSV in J. J. Virol. PubMed Scopus Google Scholar). we to by infection. shown in is a of H441 cells in was among cells, cells infected with RSV and UV-inactivated Furthermore, in of we infected H441 cells with GFP(+) cells, and with The of cells was significantly from of the cells and cells days infection from showed that the of in H441 cells. 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PubMed Scopus Google Scholar) infected with A77-1726 was the whole-cell patch clamp days GFP(+) cells a Na+ current which was by A77-1726 had and amiloride-sensitive currents of H441 both the and amiloride-sensitive currents in GFP(+) cells were significantly of the GFP(+) cells and and and the real-time showed that the of α and β ENaC mRNA levels in GFP(+) cells were by with A77-1726 and Infection through of that inhibition of UTP amiloride-sensitive currents of cells to we the of to of H441 cells with a iNOS by a fluorescent showed levels of and in infected GFP(+) and cells as compared with cells. fluorescent of cells was as compared with cells days infection as in levels of UV-inactivated cells were to cells. as in GFP(+) H441 cells had significantly levels of of levels of iNOS protein as compared with cells and cells. with the up-regulation of iNOS protein nitrite levels in the culture of H441 cells infected with were as compared with the levels of cells the nitrite levels in the of H441 cells infected with UV-inactivated RSV levels H441 cells with (1 and human showed a of nitrite concentration compared with of H441 cells with nitrite in the in culture of cells or cells infected with or UV-inactivated of H441 cells were with (1 and human were measured by and to protein as and are by the nitrite of the by the of the are the from compared with the of the of GFP(+) cells, of nitrite were in both GFP(+) and cells. Infection a of iNOS up-regulation has been to activation of NF-κB J. Full Text PDF PubMed Google Scholar). NF-κB has also been shown to a in the up-regulation of to RSV A. J. Infect. Dis. 2002; PubMed Scopus Google Scholar). Our in nuclear translocation of the NF-κB p65 subunit in H441 cells and infection as compared with cells and NF-κB activity was also significantly increased infection as shown by in nuclear translocation of p65 was in cells to UV-inactivated of NF-κB the of the of we the activation of NF-κB and up-regulation of iNOS in cells. H441 cells were with and infected with as and later cells were by of and and cells were and iNOS mRNA was measured by real-time by the specific was by with shown in GFP(+) cells with showed a of iNOS mRNA compared with cells, with the findings of iNOS up-regulation shown in the the iNOS mRNA levels of GFP(+) cells with were from iNOS mRNA levels of or cells. that infection to in iNOS expression and of iNOS the investigate the up-regulation of iNOS and to the of ENaC activity RSV we H441 cells with a specific iNOS inhibitor, or infected the cells with and and amiloride-sensitive Na+ currents from GFP(+) cells shown in the nitrite concentration in the culture from infected cells was lower that of with from that 1400W by Additionally, in the whole-cell and amiloride-sensitive current were and GFP(+) cells of the GFP(+) cells were and The amiloride-sensitive currents of the GFP(+) cells were as as compared with GFP(+) cells that the of by up-regulation of iNOS infection in to the ENaC activity the the of α and β ENaC mRNA levels of GFP(+) cells was by with 1400W and to the with A77-1726 preincubation and of UTP and Na+ of RSV-infected H441 the of we H441 cells with both A77-1726 and 1400W infected with and whole-cell currents shown in and amiloride-sensitive currents of GFP(+) cells with both A77-1726 and 1400W were and and respectively, Both and amiloride-sensitive currents of GFP(+) cells with A77-1726 and 1400W were to the levels of of the cells and respectively, the we that RSV infection inhibits amiloride-sensitive Na+ across human airway cells both the whole-cell and single channel Infection of H441 cells with RSV resulted in a inhibition of the whole-cell Na+ currents and of the amiloride-sensitive Furthermore, RSV infection ENaC mRNA levels of infected cells. findings are with in that RSV infection alveolar fluid clearance and the of I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. 2006; PubMed Scopus Google I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. Mol. PubMed Scopus Google Scholar). Infection with RSV or γ mRNA in lung of mice (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google Scholar). a small of epithelial cells infected with RSV, studies in may infected cells. To is the that virus Na+ in human cells the single cells to of the epithelial of the distal and and are the of RSV infection. in the of epithelial in A. A. S. A. 2002; PubMed Scopus Google Scholar) and protein which is to in and in the respiratory tract S. 2000; PubMed Scopus Google Scholar). H441 cells amiloride-sensitive Na+ and currents and been used as a epithelial channel A. J. L. C.J. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2002; Scopus Google L. I.C. Sullender W.M. Matalon S. Am. J. Mol. 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PubMed Scopus Google is that infection of cells with RSV the protein may cause of in studies cells levels of as compared with or RSV-infected cells the inhibition of ENaC were completely by the inhibition of iNOS and de novo UTP which were to in cells. of the that the down-regulation of ENaC in H441 cells was to the of to the of expression was in as the of infected cells and of H441 cells with expressing or amiloride-sensitive currents in H441 cells is that most of were which may and to The of virus and of has a of to RSV in a of J. PubMed Scopus Google and J. J. Openshaw P.J. J. Virol. PubMed Scopus Google Scholar). The that UV-inactivated iNOS or ENaC that were the to we infected H441 cells with and that whole-cell ENaC currents in H441 cells to the as the and amiloride-sensitive current of GFP(+) cells infected by and and of GFP(+) cells infected by virus we that in ENaC down-regulation was the of by infection we shown that the of and of BALB/c mice were both prevented and reversed by or of of the de novo of synthesis as or Furthermore, the of and A77-1726 were to a by of which and UTP synthesis the I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. 2006; PubMed Scopus Google I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. Mol. PubMed Scopus Google Scholar). in H441 cells with A77-1726 before infection amiloride-sensitive whole-cell the of UTP ENaC down-regulation in RSV infection. Our previous studies showed that RSV infection increased both UTP and levels in the of BALB/c mice and of reduced both UTP and to levels I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. Mol. PubMed Scopus Google Scholar). findings are with the that de novo and synthesis pathways are the of was reversed by of that UTP (6Davis I.C. Sullender W.M. Hickman-Davis J.M. Lindsey J.R. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: 112-120Crossref PubMed Scopus (75) Google I.C. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. 2006; PubMed Scopus Google Scholar). The mechanisms by which RSV infection UTP and UTP the inhibition of Na+ channel currents been main targets of are and the to from also protein kinase J. 2000; Google Scholar). of protein kinase has been shown to reduce ENaC activity and subunit Am. J. Physiol. Google J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Our previous that infection of mice with RSV in activation of protein kinase as by and translocation from the to the I.C. A. Hickman-Davis J.M. Sullender W.M. Matalon S. Am. J. Physiol. Lung Mol. Physiol. 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Google L. Am. J. Physiol. Google A. A. Matalon S. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000; PubMed Google Scholar). It is to that is that are to ENaC as reactive been in the of the of of C.J. L. J.F. Matalon S. J. 2002; Full Text Full Text PDF PubMed Scopus Google L. J.F. Jr., J.R. Matalon S. Am. J. Mol. PubMed Scopus Google L. Jr., J.R. Matalon S. J. 2006; Full Text Full Text PDF PubMed Scopus Google S. J. L. A. Mol. 2006; PubMed Scopus Google channels Matalon S. Am. J. Physiol. Physiol. 2000; PubMed Google and L. PubMed Scopus Google J. Physiol. 1996; PubMed Scopus Google Scholar). the that RSV infection inhibits Na+ channel activity in whole-cell and single channel levels in human respiratory epithelial cells. findings be from of the in which the of of epithelial cells to studies and expression within epithelial cells, to the of respiratory disease in infected a of days of findings are the that is in the inhibition of Na+ RSV infection through up-regulation of iNOS by activation of NF-κB in that as a respiratory fluid the of RSV Our to virus with the and with the with and