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• W2023147099 abstract "Nontypeable Haemophilus influenzae(NTHi) is an important human pathogen that causes chronic otitis media with effusion (COME) in children and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms underlying mucin overproduction in NTHi infections still remain unclear. Here, we show that NTHi strongly up-regulatesMUC5AC mucin transcription only after bacterial cell disruption. Maximal up-regulation is induced by heat-stable bacterial cytoplasmic proteins, whereas NTHi surface membrane proteins induce only moderate MUC5AC transcription. These results demonstrate an important role for cytoplasmic molecules from lysed bacteria in the pathogenesis of NTHi infections, and may well explain why many patients still have persistent symptoms such as middle ear effusion in COME after intensive antibiotic treatment. Furthermore, our results indicate that activation of p38 mitogen-activated protein kinase is required for NTHi-induced MUC5AC transcription, whereas activation of phosphoinositide 3-kinase-Akt pathway leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 mitogen-activated protein kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi infections and lead to novel therapeutic intervention for COME and COPD. Nontypeable Haemophilus influenzae(NTHi) is an important human pathogen that causes chronic otitis media with effusion (COME) in children and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms underlying mucin overproduction in NTHi infections still remain unclear. Here, we show that NTHi strongly up-regulatesMUC5AC mucin transcription only after bacterial cell disruption. Maximal up-regulation is induced by heat-stable bacterial cytoplasmic proteins, whereas NTHi surface membrane proteins induce only moderate MUC5AC transcription. These results demonstrate an important role for cytoplasmic molecules from lysed bacteria in the pathogenesis of NTHi infections, and may well explain why many patients still have persistent symptoms such as middle ear effusion in COME after intensive antibiotic treatment. Furthermore, our results indicate that activation of p38 mitogen-activated protein kinase is required for NTHi-induced MUC5AC transcription, whereas activation of phosphoinositide 3-kinase-Akt pathway leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 mitogen-activated protein kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi infections and lead to novel therapeutic intervention for COME and COPD. nontypeableH. influenzae chronic otitis media with effusion chronic obstructive pulmonary disease mitogen-activated protein kinase phosphoinositide 3-kinase soluble cytoplasmic fraction reverse transcription lipopolysaccharide lipooligosaccharide phosphate-buffered saline dominant-negative mutant Nontypeable Haemophilus influenzae(NTHi),1 a Gram-negative bacillus, is an important human pathogen in both children and adults (1Kuklinska D. Killeen M. Eur. J. Clin. Microbiol. 1984; 3: 249-252Google Scholar, 2Moxon E. J. Antimicrob. Chemother. 1986; 18: 17-24Google Scholar). In children, it causes chronic otitis media with effusion (COME), one of the most common childhood infections and the leading cause of conductive hearing loss in the United States (3St. Geme III, J.W. Infect. Agents Dis. 1993; 2: 1-16Google Scholar), whereas in adults, it exacerbates chronic obstructive pulmonary disease (COPD), the fourth leading cause of patient deaths in the United States (4Foxwell A. Kyd J. Cripps A. Microbiol. Mol. Biol. Rev. 1998; 62: 294-308Google Scholar, 5Murphy T. Sethi S. Am. Rev. Respir. Dis. 1992; 146: 1067-1083Google Scholar). Despite the need for prophylactic measures, development of a vaccine for preventing NTHi infections remains a great challenge because of high antigenic variation. Moreover, inappropriate antibiotic treatment contributes to the worldwide emergence of antibiotic-resistant strains. Therefore, there is an urgent need for developing novel therapeutic strategies for the treatment of these diseases based on a full understanding of the molecular pathogenesis of NTHi infections.Although significant progress has been made toward identifying the virulence factors of NTHi, the molecular pathogenesis of NTHi infections is still largely unknown. Interestingly, there is evidence that up-regulation of mucin production induced by bacteria could play an important role. Mucins are high molecular weight glycoproteins that constitute the major component of mucus secretions in the middle ear, trachea, digestive, and reproductive tracts (6Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Google Scholar). They protect and lubricate the epithelial surface and trap particles, including bacteria and viruses, for mucociliary clearance (7Rose M.C. Am. J. Physiol. 1992; 263: L413-L429Google Scholar). In COME and COPD, excessive production of mucin occurs, overwhelming the normal mucociliary clearance mechanisms. As mucus levels increase, they contribute significantly to airway obstruction in COPD (8Larivee P. Levine S.J. Rieves R.D. Shelhamer J.H. Shmura S. Takishima T. Airway Secretion: Physiological Bases for the Control of Mucus Hypersecretion. Marcel Dekker, Inc., New York1994: 469-511Google Scholar, 9Kim W.D. Eur. Respir. J. 1997; 10: 1914-1917Google Scholar) and conductive hearing loss in COME (10Majima Y. Hamaguchi Y. Hirata K. Takeuchi K. Morishita A. Sakakura Y. Ann. Otol. Rhinol. Laryngol. 1998; 97: 272-274Google Scholar). In addition to the obstructive outcome, mucin has been reported to bind to almost all known bacterial pathogens (11Kubiet M. Ramphal R. Weber A. Smith A. Infect. Immun. 2000; 68: 3362-3367Google Scholar, 12Namavar F. Sprrius M. Veerman E.C.I. Appelmelk B.J. Vandenbroucke-Grauls C.M.J.E. Infect. Immun. 1998; 66: 444-447Google Scholar, 13Mntle M. Husar S.D. Infect. Immun. 1994; 62: 1219-1227Google Scholar, 14Scharfman A. Kroczynski H. Carnoy C. Brussel E.V. Lamblin G. Ramphal R. Roussel P. Infect. Immun. 1996; 64: 5417-5420Google Scholar). The combination of defective mucociliary clearance and mucin-bacteria interaction could greatly increase the ability of bacteria to persist in a host. To date, 13 mucin genes have been cloned (6Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Google Scholar, 7Rose M.C. Am. J. Physiol. 1992; 263: L413-L429Google Scholar, 15Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Google Scholar, 16Williams S. McGluckin M. Gotley D. Eyre H. Sutherland G. Antails T. Cancer Res. 1999; 59: 4083-4089Google Scholar, 17Williams S.J. Wreschner D.H. Tran M. Eyre H.J. Sutherland G.R. McGuckin M.A. J. Biol. Chem. 2001; 276: 18327-18336Google Scholar) and one, MUC5AC, has been shown to be highly expressed in airway and middle ear epithelial cells (18Li D Gallup M. Fan N. Szymkowski D.E. Basbaum C.B. J. Biol. Chem. 1998; 273: 6812-6820Google Scholar). Furthermore, recent studies have demonstrated that expression level ofMUC5AC mRNA in the middle ear is higher in patients with COME than in normal individuals (19Smirnova M.G. Kiselev S.L. Birchall J.P. Pearson J.P. Eur. Cytokine Netw. 2001; 12: 119-125Google Scholar). Taken together, these studies strongly suggest that up-regulation of MUC5AC mucin gene plays an important role in the pathogenesis of NTHi infections.Although little is known about how NTHi up-regulates MUC5ACmucin transcription, previous studies have shown that bacteria can activate transcription of host defense genes via activation of specific signal transduction cascades. Among the commonly known signaling events, the mitogen-activated protein kinase (MAP kinase) pathways are thought to be most important in transmitting extracellular signals from the cell surface to the nucleus (20Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Google Scholar). p38, a major MAP kinase superfamily member, has been shown to be involved in NTHi-induced inflammatory responses (23Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T.F. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Google Scholar). In addition to p38 MAP kinase, phosphoinositide 3-kinase (PI 3-kinase) represents another major signaling transducer involved in the regulation of cell proliferation, survival, metabolism, cytoskeleton reorganization, and membrane trafficking (21Duronio V. Scheid M.P. Ettinger S. Cell. Signal. 1998; 10: 233-239Google Scholar), as well as bacterial pathogenesis (22Bierne H. Dramsi S. Gratacap M.P. Randriamampita C. Carpenter G. Payrastre B. Cossart P. Cell. Microbiol. 2000; 2: 465-476Google Scholar). However, the role of both p38 MAP kinase and PI-3 kinase in mucin up-regulation has not yet been explored.Because mucin overproduction plays an important role in the pathogenesis of COME and COPD, and NTHi is a major pathogen of these diseases, we hypothesize that NTHi up-regulates MUC5AC mucin transcription via activation of specific signal transduction pathways. Here, we show that previously unrecognized cytoplasmic protein components of NTHi up-regulate MUC5AC mucin gene transcription via a positive p38 MAP kinase pathway and a negative PI 3-kinase-Akt signaling pathway. These studies provide new insights into the molecular pathogenesis of NTHi infections and may open up novel targets for therapeutic intervention.DISCUSSIONNTHi has now become well established as an important human pathogen in both children and adults. In children, it causes COME, one of the most common childhood infections and the leading cause of conductive hearing loss in children (1Kuklinska D. Killeen M. Eur. J. Clin. Microbiol. 1984; 3: 249-252Google Scholar, 2Moxon E. J. Antimicrob. Chemother. 1986; 18: 17-24Google Scholar, 3St. Geme III, J.W. Infect. Agents Dis. 1993; 2: 1-16Google Scholar, 4Foxwell A. Kyd J. Cripps A. Microbiol. Mol. Biol. Rev. 1998; 62: 294-308Google Scholar). In adults, it causes lower respiratory tract infections in the setting of COPD, the fourth leading cause of patient death in the United States (5Murphy T. Sethi S. Am. Rev. Respir. Dis. 1992; 146: 1067-1083Google Scholar). Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms by which mucin is up-regulated in NTHi infections still remain poorly understood. In the present study, we performed experiments to determine the involvement of NTHi in up-regulation ofMUC5AC mucin gene transcription in human epithelial cells. Here, we show that NTHi cytoplasmic proteins up-regulateMUC5AC transcription via a positive p38 MAP kinase signaling pathway and a negative PI 3-kinase-Akt signaling pathway (Fig.8).A major finding in this study is the experimental evidence for the involvement of bacterial cytoplasmic proteins in MUC5ACinduction. This result, although rather unexpected, may well explain why many patients still have persistent symptoms such as middle ear effusion in COME even after intensive treatment with antibiotics (37Lim D.J. Auris Nasus Larynx. 1985; 12: S8-S10Google Scholar). One of the major characteristics of NTHi is its tendency to autolyze. Its autolysis can be triggered in vitro when the bacteria culture is old, and in vivo under various conditions including antibiotic treatment. Clinical microbiology studies have shown that most effusions from the patients with COME were negative on bacteria culture, whereas bacterial DNA could be detected by PCR in 80% of effusions, often in the absence of viable bacteria on culture (30Kubba H. Pearson J.P. Birchall J.H. Clin. Otolaryngol. 2000; 25: 181-194Google Scholar). In addition, DeMaria et al. (38DeMaria T.F. Prior R.B. Briggs B.R. Lim D.L. Birck H.G. J. Clin. Microbiol. 1984; 20: 15-17Google Scholar) reported that endotoxin was present in 67% of middle ear effusions that were negative as determined by culture for any bacterium. Despite some potential underestimation of the prevalence of viable bacteria by conventional culture, these results clearly indicate that bacterial breakdown products or components released from lysed bacteria persist in the middle ear even after bacteria die and thus may act as long lasting stimuli of mucin production and inflammatory responses (37Lim D.J. Auris Nasus Larynx. 1985; 12: S8-S10Google Scholar). Taken together, our present study and the previous findings suggest that the cytoplasmic proteins released from the lysed NTHi bacteria after treatment with antibiotics may contribute substantially to the pathogenesis of otitis media by directly up-regulatingMUC5AC mucin transcription.Another unexpected finding in this study is the negative effect of NTHi LOS on MUC5AC transcription. We previously showed that LPS from other Gram-negative bacteria such as Pseudomonas aeruginosa and S. typhimurium up-regulatesMUC2 and MUC5AC transcription (31Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Google Scholar, 39Austin D. Miyata S. Gallup M. Li J.-D. Chpelin C. Coste A. Escudier E. Nadel J. Basbaum C. Biochim. Biophys. Acta. 1998; 1406: 251-259Google Scholar). Additionally, induction of proinflammatory cytokines by NTHi LOS has also been reported (24Clemans D.L. Bauer R.J. Hanson J.A. Hobbs M.V. St. Geme III, J.W. Marrs C.F. Gilsdorf J.R. Infect. Immun. 2000; 68: 4430-4440Google Scholar). Based on these studies, we initially expected to observe a stimulating effect of LOS on MUC5AC. The negative effect shown in Fig. 2 (B and C) is unexpected, because it was in sharp contrast to the up-regulation of mucin by LPS from S. typhimurium and P. aeruginosa. In comparison with LPS, LOS lacks an O-specific polysaccharide (33Philips N.J. Apicella M.A. Griffiss M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Google Scholar). Therefore it seems logical that the lack of O-specific polysaccharide may account for the negative effect onMUC5AC induction. However, this notion is not supported by the fact that LPS molecules purified from a polysaccharide-deficient strain and a wild-type strain of P. aeruginosa were equipotent in induction of MUC2 (31Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Google Scholar), suggesting that lipid A and the sugar core region are sufficient for mucin induction. In view of the structure of other regions, LOS also appears to differ from LPS in lipid A (33Philips N.J. Apicella M.A. Griffiss M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Google Scholar). An antigenic analysis of NTHi lipid A by Apicellaet al. (40Apicella M.A. Dudas K.C. Campagnari A. Rice P. Mylotte J.M. Murphy T.F. Infect. Immun. 1985; 50: 9-14Google Scholar) showed that a monoclonal antibody specific for the lipid A portion of NTHi LOS recognized the lipid A determinant on most NTHi strains but did not recognize the lipid A of 39 stains from 14 non-H. influenzae species. Thus, differences in the lipid A region between NTHi LOS and other bacterial LPS may be responsible for the difference in mucin induction. Although no direct up-regulation on MUC5AC by NTHi LOS was shown in vitro, our data do not preclude the possibility that LOS may indirectly up-regulate MUC5AC in vivo by inducing cytokines such as TNF-α, which has been shown to up-regulate mucin (15Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Google Scholar).In the present study, we provided evidence for the first time that activation of p38 MAP kinase is required for up-regulation of MUC5AC by NTHi cytoplasmic protein(s). In addition, we showed that PI 3-kinase-Akt signaling pathway is also activated by NTHi, which, however, leads to down-regulation of p38 MAP kinase activity. Negative cross-talk has been established by previous studies between PI 3-kinase-Akt pathway and MAP kinases including the extracellular signal-regulated kinases and the c-Jun NH2-terminal kinase (41Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Google Scholar). Whether or not there is also negative interaction between PI 3-kinase-Akt and p38 MAP kinase has remained unclear. Recently, a report by Gratton et al. (36Gratton J.P. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; 276: 30359-30365Google Scholar) showed that blockade of PI 3-kinase-Akt led to enhanced vascular endothelial growth factor activation of p38 MAP kinase. However, little is known about the involvement of this negative cross-talk in bacterial pathogenesis as well as in mucin gene regulation. In the present study, we revealed that PI 3-kinase-Akt serves as an inhibitory signaling pathway in NTH-induced MUC5AC transcription via a negative cross-talk with p38 MAP kinase. Although we showed that inhibition of PI 3-kinase-Akt signaling by wortmannin enhanced, whereas activation of PI 3-kinase-Akt by overexpression of an activated form of p110 attenuated, NTHi-induced activation of p38 MAP kinase, we can not rule out the possibility that PI 3-kinase-Akt pathway may interact with the upstream kinases of p38 MAP kinases such as MAP kinase kinases 3 and 6. It is also unclear whether a direct physical interaction between PI 3-kinase-Akt and MAP kinase kinases 3 and 6-p38 MAP kinase is involved in this cross-talk. These questions will be addressed in our future studies.In summary, NTHi cytoplasmic proteins up-regulate MUC5ACmucin gene transcription in human epithelial cells. Activation of p38 MAP kinase is required for NTHi-induced MUC5ACtranscription. In addition to p38, NTHi cytoplasmic proteins also induce activation of PI 3-kinase-Akt, which, however, leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 MAP kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and lead to novel therapeutic intervention for COME and COPD. Nontypeable Haemophilus influenzae(NTHi),1 a Gram-negative bacillus, is an important human pathogen in both children and adults (1Kuklinska D. Killeen M. Eur. J. Clin. Microbiol. 1984; 3: 249-252Google Scholar, 2Moxon E. J. Antimicrob. Chemother. 1986; 18: 17-24Google Scholar). In children, it causes chronic otitis media with effusion (COME), one of the most common childhood infections and the leading cause of conductive hearing loss in the United States (3St. Geme III, J.W. Infect. Agents Dis. 1993; 2: 1-16Google Scholar), whereas in adults, it exacerbates chronic obstructive pulmonary disease (COPD), the fourth leading cause of patient deaths in the United States (4Foxwell A. Kyd J. Cripps A. Microbiol. Mol. Biol. Rev. 1998; 62: 294-308Google Scholar, 5Murphy T. Sethi S. Am. Rev. Respir. Dis. 1992; 146: 1067-1083Google Scholar). Despite the need for prophylactic measures, development of a vaccine for preventing NTHi infections remains a great challenge because of high antigenic variation. Moreover, inappropriate antibiotic treatment contributes to the worldwide emergence of antibiotic-resistant strains. Therefore, there is an urgent need for developing novel therapeutic strategies for the treatment of these diseases based on a full understanding of the molecular pathogenesis of NTHi infections. Although significant progress has been made toward identifying the virulence factors of NTHi, the molecular pathogenesis of NTHi infections is still largely unknown. Interestingly, there is evidence that up-regulation of mucin production induced by bacteria could play an important role. Mucins are high molecular weight glycoproteins that constitute the major component of mucus secretions in the middle ear, trachea, digestive, and reproductive tracts (6Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Google Scholar). They protect and lubricate the epithelial surface and trap particles, including bacteria and viruses, for mucociliary clearance (7Rose M.C. Am. J. Physiol. 1992; 263: L413-L429Google Scholar). In COME and COPD, excessive production of mucin occurs, overwhelming the normal mucociliary clearance mechanisms. As mucus levels increase, they contribute significantly to airway obstruction in COPD (8Larivee P. Levine S.J. Rieves R.D. Shelhamer J.H. Shmura S. Takishima T. Airway Secretion: Physiological Bases for the Control of Mucus Hypersecretion. Marcel Dekker, Inc., New York1994: 469-511Google Scholar, 9Kim W.D. Eur. Respir. J. 1997; 10: 1914-1917Google Scholar) and conductive hearing loss in COME (10Majima Y. Hamaguchi Y. Hirata K. Takeuchi K. Morishita A. Sakakura Y. Ann. Otol. Rhinol. Laryngol. 1998; 97: 272-274Google Scholar). In addition to the obstructive outcome, mucin has been reported to bind to almost all known bacterial pathogens (11Kubiet M. Ramphal R. Weber A. Smith A. Infect. Immun. 2000; 68: 3362-3367Google Scholar, 12Namavar F. Sprrius M. Veerman E.C.I. Appelmelk B.J. Vandenbroucke-Grauls C.M.J.E. Infect. Immun. 1998; 66: 444-447Google Scholar, 13Mntle M. Husar S.D. Infect. Immun. 1994; 62: 1219-1227Google Scholar, 14Scharfman A. Kroczynski H. Carnoy C. Brussel E.V. Lamblin G. Ramphal R. Roussel P. Infect. Immun. 1996; 64: 5417-5420Google Scholar). The combination of defective mucociliary clearance and mucin-bacteria interaction could greatly increase the ability of bacteria to persist in a host. To date, 13 mucin genes have been cloned (6Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Google Scholar, 7Rose M.C. Am. J. Physiol. 1992; 263: L413-L429Google Scholar, 15Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Google Scholar, 16Williams S. McGluckin M. Gotley D. Eyre H. Sutherland G. Antails T. Cancer Res. 1999; 59: 4083-4089Google Scholar, 17Williams S.J. Wreschner D.H. Tran M. Eyre H.J. Sutherland G.R. McGuckin M.A. J. Biol. Chem. 2001; 276: 18327-18336Google Scholar) and one, MUC5AC, has been shown to be highly expressed in airway and middle ear epithelial cells (18Li D Gallup M. Fan N. Szymkowski D.E. Basbaum C.B. J. Biol. Chem. 1998; 273: 6812-6820Google Scholar). Furthermore, recent studies have demonstrated that expression level ofMUC5AC mRNA in the middle ear is higher in patients with COME than in normal individuals (19Smirnova M.G. Kiselev S.L. Birchall J.P. Pearson J.P. Eur. Cytokine Netw. 2001; 12: 119-125Google Scholar). Taken together, these studies strongly suggest that up-regulation of MUC5AC mucin gene plays an important role in the pathogenesis of NTHi infections. Although little is known about how NTHi up-regulates MUC5ACmucin transcription, previous studies have shown that bacteria can activate transcription of host defense genes via activation of specific signal transduction cascades. Among the commonly known signaling events, the mitogen-activated protein kinase (MAP kinase) pathways are thought to be most important in transmitting extracellular signals from the cell surface to the nucleus (20Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Google Scholar). p38, a major MAP kinase superfamily member, has been shown to be involved in NTHi-induced inflammatory responses (23Shuto T. Xu H. Wang B. Han J. Kai H. Gu X.X. Murphy T.F. Lim D.J. Li J.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8774-8779Google Scholar). In addition to p38 MAP kinase, phosphoinositide 3-kinase (PI 3-kinase) represents another major signaling transducer involved in the regulation of cell proliferation, survival, metabolism, cytoskeleton reorganization, and membrane trafficking (21Duronio V. Scheid M.P. Ettinger S. Cell. Signal. 1998; 10: 233-239Google Scholar), as well as bacterial pathogenesis (22Bierne H. Dramsi S. Gratacap M.P. Randriamampita C. Carpenter G. Payrastre B. Cossart P. Cell. Microbiol. 2000; 2: 465-476Google Scholar). However, the role of both p38 MAP kinase and PI-3 kinase in mucin up-regulation has not yet been explored. Because mucin overproduction plays an important role in the pathogenesis of COME and COPD, and NTHi is a major pathogen of these diseases, we hypothesize that NTHi up-regulates MUC5AC mucin transcription via activation of specific signal transduction pathways. Here, we show that previously unrecognized cytoplasmic protein components of NTHi up-regulate MUC5AC mucin gene transcription via a positive p38 MAP kinase pathway and a negative PI 3-kinase-Akt signaling pathway. These studies provide new insights into the molecular pathogenesis of NTHi infections and may open up novel targets for therapeutic intervention. DISCUSSIONNTHi has now become well established as an important human pathogen in both children and adults. In children, it causes COME, one of the most common childhood infections and the leading cause of conductive hearing loss in children (1Kuklinska D. Killeen M. Eur. J. Clin. Microbiol. 1984; 3: 249-252Google Scholar, 2Moxon E. J. Antimicrob. Chemother. 1986; 18: 17-24Google Scholar, 3St. Geme III, J.W. Infect. Agents Dis. 1993; 2: 1-16Google Scholar, 4Foxwell A. Kyd J. Cripps A. Microbiol. Mol. Biol. Rev. 1998; 62: 294-308Google Scholar). In adults, it causes lower respiratory tract infections in the setting of COPD, the fourth leading cause of patient death in the United States (5Murphy T. Sethi S. Am. Rev. Respir. Dis. 1992; 146: 1067-1083Google Scholar). Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms by which mucin is up-regulated in NTHi infections still remain poorly understood. In the present study, we performed experiments to determine the involvement of NTHi in up-regulation ofMUC5AC mucin gene transcription in human epithelial cells. Here, we show that NTHi cytoplasmic proteins up-regulateMUC5AC transcription via a positive p38 MAP kinase signaling pathway and a negative PI 3-kinase-Akt signaling pathway (Fig.8).A major finding in this study is the experimental evidence for the involvement of bacterial cytoplasmic proteins in MUC5ACinduction. This result, although rather unexpected, may well explain why many patients still have persistent symptoms such as middle ear effusion in COME even after intensive treatment with antibiotics (37Lim D.J. Auris Nasus Larynx. 1985; 12: S8-S10Google Scholar). One of the major characteristics of NTHi is its tendency to autolyze. Its autolysis can be triggered in vitro when the bacteria culture is old, and in vivo under various conditions including antibiotic treatment. Clinical microbiology studies have shown that most effusions from the patients with COME were negative on bacteria culture, whereas bacterial DNA could be detected by PCR in 80% of effusions, often in the absence of viable bacteria on culture (30Kubba H. Pearson J.P. Birchall J.H. Clin. Otolaryngol. 2000; 25: 181-194Google Scholar). In addition, DeMaria et al. (38DeMaria T.F. Prior R.B. Briggs B.R. Lim D.L. Birck H.G. J. Clin. Microbiol. 1984; 20: 15-17Google Scholar) reported that endotoxin was present in 67% of middle ear effusions that were negative as determined by culture for any bacterium. Despite some potential underestimation of the prevalence of viable bacteria by conventional culture, these results clearly indicate that bacterial breakdown products or components released from lysed bacteria persist in the middle ear even after bacteria die and thus may act as long lasting stimuli of mucin production and inflammatory responses (37Lim D.J. Auris Nasus Larynx. 1985; 12: S8-S10Google Scholar). Taken together, our present study and the previous findings suggest that the cytoplasmic proteins released from the lysed NTHi bacteria after treatment with antibiotics may contribute substantially to the pathogenesis of otitis media by directly up-regulatingMUC5AC mucin transcription.Another unexpected finding in this study is the negative effect of NTHi LOS on MUC5AC transcription. We previously showed that LPS from other Gram-negative bacteria such as Pseudomonas aeruginosa and S. typhimurium up-regulatesMUC2 and MUC5AC transcription (31Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Google Scholar, 39Austin D. Miyata S. Gallup M. Li J.-D. Chpelin C. Coste A. Escudier E. Nadel J. Basbaum C. Biochim. Biophys. Acta. 1998; 1406: 251-259Google Scholar). Additionally, induction of proinflammatory cytokines by NTHi LOS has also been reported (24Clemans D.L. Bauer R.J. Hanson J.A. Hobbs M.V. St. Geme III, J.W. Marrs C.F. Gilsdorf J.R. Infect. Immun. 2000; 68: 4430-4440Google Scholar). Based on these studies, we initially expected to observe a stimulating effect of LOS on MUC5AC. The negative effect shown in Fig. 2 (B and C) is unexpected, because it was in sharp contrast to the up-regulation of mucin by LPS from S. typhimurium and P. aeruginosa. In comparison with LPS, LOS lacks an O-specific polysaccharide (33Philips N.J. Apicella M.A. Griffiss M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Google Scholar). Therefore it seems logical that the lack of O-specific polysaccharide may account for the negative effect onMUC5AC induction. However, this notion is not supported by the fact that LPS molecules purified from a polysaccharide-deficient strain and a wild-type strain of P. aeruginosa were equipotent in induction of MUC2 (31Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Google Scholar), suggesting that lipid A and the sugar core region are sufficient for mucin induction. In view of the structure of other regions, LOS also appears to differ from LPS in lipid A (33Philips N.J. Apicella M.A. Griffiss M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Google Scholar). An antigenic analysis of NTHi lipid A by Apicellaet al. (40Apicella M.A. Dudas K.C. Campagnari A. Rice P. Mylotte J.M. Murphy T.F. Infect. Immun. 1985; 50: 9-14Google Scholar) showed that a monoclonal antibody specific for the lipid A portion of NTHi LOS recognized the lipid A determinant on most NTHi strains but did not recognize the lipid A of 39 stains from 14 non-H. influenzae species. Thus, differences in the lipid A region between NTHi LOS and other bacterial LPS may be responsible for the difference in mucin induction. Although no direct up-regulation on MUC5AC by NTHi LOS was shown in vitro, our data do not preclude the possibility that LOS may indirectly up-regulate MUC5AC in vivo by inducing cytokines such as TNF-α, which has been shown to up-regulate mucin (15Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Google Scholar).In the present study, we provided evidence for the first time that activation of p38 MAP kinase is required for up-regulation of MUC5AC by NTHi cytoplasmic protein(s). In addition, we showed that PI 3-kinase-Akt signaling pathway is also activated by NTHi, which, however, leads to down-regulation of p38 MAP kinase activity. Negative cross-talk has been established by previous studies between PI 3-kinase-Akt pathway and MAP kinases including the extracellular signal-regulated kinases and the c-Jun NH2-terminal kinase (41Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Google Scholar). Whether or not there is also negative interaction between PI 3-kinase-Akt and p38 MAP kinase has remained unclear. Recently, a report by Gratton et al. (36Gratton J.P. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; 276: 30359-30365Google Scholar) showed that blockade of PI 3-kinase-Akt led to enhanced vascular endothelial growth factor activation of p38 MAP kinase. However, little is known about the involvement of this negative cross-talk in bacterial pathogenesis as well as in mucin gene regulation. In the present study, we revealed that PI 3-kinase-Akt serves as an inhibitory signaling pathway in NTH-induced MUC5AC transcription via a negative cross-talk with p38 MAP kinase. Although we showed that inhibition of PI 3-kinase-Akt signaling by wortmannin enhanced, whereas activation of PI 3-kinase-Akt by overexpression of an activated form of p110 attenuated, NTHi-induced activation of p38 MAP kinase, we can not rule out the possibility that PI 3-kinase-Akt pathway may interact with the upstream kinases of p38 MAP kinases such as MAP kinase kinases 3 and 6. It is also unclear whether a direct physical interaction between PI 3-kinase-Akt and MAP kinase kinases 3 and 6-p38 MAP kinase is involved in this cross-talk. These questions will be addressed in our future studies.In summary, NTHi cytoplasmic proteins up-regulate MUC5ACmucin gene transcription in human epithelial cells. Activation of p38 MAP kinase is required for NTHi-induced MUC5ACtranscription. In addition to p38, NTHi cytoplasmic proteins also induce activation of PI 3-kinase-Akt, which, however, leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 MAP kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and lead to novel therapeutic intervention for COME and COPD. NTHi has now become well established as an important human pathogen in both children and adults. In children, it causes COME, one of the most common childhood infections and the leading cause of conductive hearing loss in children (1Kuklinska D. Killeen M. Eur. J. Clin. Microbiol. 1984; 3: 249-252Google Scholar, 2Moxon E. J. Antimicrob. Chemother. 1986; 18: 17-24Google Scholar, 3St. Geme III, J.W. Infect. Agents Dis. 1993; 2: 1-16Google Scholar, 4Foxwell A. Kyd J. Cripps A. Microbiol. Mol. Biol. Rev. 1998; 62: 294-308Google Scholar). In adults, it causes lower respiratory tract infections in the setting of COPD, the fourth leading cause of patient death in the United States (5Murphy T. Sethi S. Am. Rev. Respir. Dis. 1992; 146: 1067-1083Google Scholar). Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms by which mucin is up-regulated in NTHi infections still remain poorly understood. In the present study, we performed experiments to determine the involvement of NTHi in up-regulation ofMUC5AC mucin gene transcription in human epithelial cells. Here, we show that NTHi cytoplasmic proteins up-regulateMUC5AC transcription via a positive p38 MAP kinase signaling pathway and a negative PI 3-kinase-Akt signaling pathway (Fig.8). A major finding in this study is the experimental evidence for the involvement of bacterial cytoplasmic proteins in MUC5ACinduction. This result, although rather unexpected, may well explain why many patients still have persistent symptoms such as middle ear effusion in COME even after intensive treatment with antibiotics (37Lim D.J. Auris Nasus Larynx. 1985; 12: S8-S10Google Scholar). One of the major characteristics of NTHi is its tendency to autolyze. Its autolysis can be triggered in vitro when the bacteria culture is old, and in vivo under various conditions including antibiotic treatment. Clinical microbiology studies have shown that most effusions from the patients with COME were negative on bacteria culture, whereas bacterial DNA could be detected by PCR in 80% of effusions, often in the absence of viable bacteria on culture (30Kubba H. Pearson J.P. Birchall J.H. Clin. Otolaryngol. 2000; 25: 181-194Google Scholar). In addition, DeMaria et al. (38DeMaria T.F. Prior R.B. Briggs B.R. Lim D.L. Birck H.G. J. Clin. Microbiol. 1984; 20: 15-17Google Scholar) reported that endotoxin was present in 67% of middle ear effusions that were negative as determined by culture for any bacterium. Despite some potential underestimation of the prevalence of viable bacteria by conventional culture, these results clearly indicate that bacterial breakdown products or components released from lysed bacteria persist in the middle ear even after bacteria die and thus may act as long lasting stimuli of mucin production and inflammatory responses (37Lim D.J. Auris Nasus Larynx. 1985; 12: S8-S10Google Scholar). Taken together, our present study and the previous findings suggest that the cytoplasmic proteins released from the lysed NTHi bacteria after treatment with antibiotics may contribute substantially to the pathogenesis of otitis media by directly up-regulatingMUC5AC mucin transcription. Another unexpected finding in this study is the negative effect of NTHi LOS on MUC5AC transcription. We previously showed that LPS from other Gram-negative bacteria such as Pseudomonas aeruginosa and S. typhimurium up-regulatesMUC2 and MUC5AC transcription (31Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Google Scholar, 39Austin D. Miyata S. Gallup M. Li J.-D. Chpelin C. Coste A. Escudier E. Nadel J. Basbaum C. Biochim. Biophys. Acta. 1998; 1406: 251-259Google Scholar). Additionally, induction of proinflammatory cytokines by NTHi LOS has also been reported (24Clemans D.L. Bauer R.J. Hanson J.A. Hobbs M.V. St. Geme III, J.W. Marrs C.F. Gilsdorf J.R. Infect. Immun. 2000; 68: 4430-4440Google Scholar). Based on these studies, we initially expected to observe a stimulating effect of LOS on MUC5AC. The negative effect shown in Fig. 2 (B and C) is unexpected, because it was in sharp contrast to the up-regulation of mucin by LPS from S. typhimurium and P. aeruginosa. In comparison with LPS, LOS lacks an O-specific polysaccharide (33Philips N.J. Apicella M.A. Griffiss M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Google Scholar). Therefore it seems logical that the lack of O-specific polysaccharide may account for the negative effect onMUC5AC induction. However, this notion is not supported by the fact that LPS molecules purified from a polysaccharide-deficient strain and a wild-type strain of P. aeruginosa were equipotent in induction of MUC2 (31Li J.D. Dohrman A.F. Gallup M. Miyata S. Gum J.R. Kim Y.S. Nadel J.A. Prince A. Basbaum C.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 967-972Google Scholar), suggesting that lipid A and the sugar core region are sufficient for mucin induction. In view of the structure of other regions, LOS also appears to differ from LPS in lipid A (33Philips N.J. Apicella M.A. Griffiss M. Gibson B.W. Biochemistry. 1992; 31: 4515-4526Google Scholar). An antigenic analysis of NTHi lipid A by Apicellaet al. (40Apicella M.A. Dudas K.C. Campagnari A. Rice P. Mylotte J.M. Murphy T.F. Infect. Immun. 1985; 50: 9-14Google Scholar) showed that a monoclonal antibody specific for the lipid A portion of NTHi LOS recognized the lipid A determinant on most NTHi strains but did not recognize the lipid A of 39 stains from 14 non-H. influenzae species. Thus, differences in the lipid A region between NTHi LOS and other bacterial LPS may be responsible for the difference in mucin induction. Although no direct up-regulation on MUC5AC by NTHi LOS was shown in vitro, our data do not preclude the possibility that LOS may indirectly up-regulate MUC5AC in vivo by inducing cytokines such as TNF-α, which has been shown to up-regulate mucin (15Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Google Scholar). In the present study, we provided evidence for the first time that activation of p38 MAP kinase is required for up-regulation of MUC5AC by NTHi cytoplasmic protein(s). In addition, we showed that PI 3-kinase-Akt signaling pathway is also activated by NTHi, which, however, leads to down-regulation of p38 MAP kinase activity. Negative cross-talk has been established by previous studies between PI 3-kinase-Akt pathway and MAP kinases including the extracellular signal-regulated kinases and the c-Jun NH2-terminal kinase (41Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Google Scholar). Whether or not there is also negative interaction between PI 3-kinase-Akt and p38 MAP kinase has remained unclear. Recently, a report by Gratton et al. (36Gratton J.P. Morales-Ruiz M. Kureishi Y. Fulton D. Walsh K. Sessa W.C. J. Biol. Chem. 2001; 276: 30359-30365Google Scholar) showed that blockade of PI 3-kinase-Akt led to enhanced vascular endothelial growth factor activation of p38 MAP kinase. However, little is known about the involvement of this negative cross-talk in bacterial pathogenesis as well as in mucin gene regulation. In the present study, we revealed that PI 3-kinase-Akt serves as an inhibitory signaling pathway in NTH-induced MUC5AC transcription via a negative cross-talk with p38 MAP kinase. Although we showed that inhibition of PI 3-kinase-Akt signaling by wortmannin enhanced, whereas activation of PI 3-kinase-Akt by overexpression of an activated form of p110 attenuated, NTHi-induced activation of p38 MAP kinase, we can not rule out the possibility that PI 3-kinase-Akt pathway may interact with the upstream kinases of p38 MAP kinases such as MAP kinase kinases 3 and 6. It is also unclear whether a direct physical interaction between PI 3-kinase-Akt and MAP kinase kinases 3 and 6-p38 MAP kinase is involved in this cross-talk. These questions will be addressed in our future studies. In summary, NTHi cytoplasmic proteins up-regulate MUC5ACmucin gene transcription in human epithelial cells. Activation of p38 MAP kinase is required for NTHi-induced MUC5ACtranscription. In addition to p38, NTHi cytoplasmic proteins also induce activation of PI 3-kinase-Akt, which, however, leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 MAP kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and lead to novel therapeutic intervention for COME and COPD. We thank David Kolodrubetz, Paul Patrick Cleary, and Davida Rixter for critically reading this manuscript. We thank David Stokoe for providing PI 3-kinase and Akt expression plasmids. We also thank Akira Imasato for help with the graphics." @default.
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• W2023147099 title "Novel Cytoplasmic Proteins of Nontypeable Haemophilus influenzae Up-regulate Human MUC5AC Mucin Transcription via a Positive p38 Mitogen-activated Protein Kinase Pathway and a Negative Phosphoinositide 3-Kinase-Akt Pathway" @default.
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