Why three non-governmental organizations are launching an alternative to the World Health Report

At the World Health Assembly in May 2003, three civil society groups—the People's Health Movement, the Global Equity Gauge Alliance, and Medact—discussed the need for civil society to produce its own alternative to the World Health Organisation's World Health Report. We felt strongly that we needed to produce a global health report that had equity and the right to health at its heart. We also needed a way to monitor the performance of global health institutions themselves. The idea of an alternative to the World Health Report has developed into an initiative called the Global Health Watch, which we are launching next year.The Three Key Players Medact ( http://www.medact.org ) is a United Kingdom–based global health charity, undertaking education, research, and advocacy on conflict, poverty, and the environment. The Global Equity Gauge Alliance ( http://www.gega.org.za ) was created to participate in and support an active approach to monitoring health inequalities and promoting equity within and between societies. The Alliance currently includes 11 member-teams, called Equity Gauges, located in ten countries in the Americas, Africa, and Asia. The People's Health Movement ( http://www.phmovement.org ) is a global network of activists, organisations, and social movements. Its goal is to re-establish health and equitable development as top priorities in local, national, and international policy-making, with comprehensive primary health care as the strategy to achieve these priorities.
The Three Key Players
Medact ( http://www.medact.org ) is a United Kingdom–based global health charity, undertaking education, research, and advocacy on conflict, poverty, and the environment.The Global Equity Gauge Alliance ( http://www.gega.org.za ) was created to participate in and support an active approach to monitoring health inequalities and promoting equity within and between societies. The Alliance currently includes 11 member-teams, called Equity Gauges, located in ten countries in the Americas, Africa, and Asia.The People's Health Movement ( http://www.phmovement.org ) is a global network of activists, organisations, and social movements. Its goal is to re-establish health and equitable development as top priorities in local, national, and international policy-making, with comprehensive primary health care as the strategy to achieve these priorities.Why an Alternative Is Needed Concerted action by civil society has had tremendous success in the field of international health—global grassroots campaigns on infant feeding, smoking, and drug prices have changed policies and people's lives.
 The Watch: Examining the world's health from an alternative perspective (Illustration: Giovanni Maki, Public Library of Science) But over the last two decades—at the same time as these campaigns have scored victories—there has, in some parts of the world, been a stagnation and even reversal of the dramatic gains in life expectancy witnessed by many others for much of the 20th century. These reversals, unprecedented outside times of war and famine since the early 1800s and a scandal in a world of enormous wealth and technological prowess, have once more thrown the spotlight on how underlying social and economic problems affect health and health services. The setbacks have also underlined appalling failures of health development policy. Ambitious targets to achieve ‘Health for All’ agreed to at the end of the 1970s by health ministers from around the world have failed miserably; a similar fate seems likely for the targets set out in the Millennium Development Goals for 2015. As a result, there are large question marks hanging over the effectiveness of international health policy. These are the reasons why we have decided to produce Global Health Watch, which we hope will become a regular report on international health issues ( Box 1 ). We believe that civil society campaigners need to look at the broader health agenda—beyond single-issue advocacy. Major concerns about health systems such as poor pay and working conditions for health professionals, creeping commercialisation, and plummeting public investment have not had the attention they deserve. Likewise, broader determinants of health—such as education, water, food, and the environment—are often insufficiently regarded when health policies are formulated. The Watch attempts to focus minds on the need for more integrated planning across sectors and on the creation of health systems that promote social justice rather undermine it. Box 1. Global Health Watch—2005 Report Contents
 Section A: The Politics and Economics of Health in the 21st Century

 Section B: The Health Care Sector
 Health systems that promote social justice Responding to the commercialisation of health care The pharmaceutical industry, access to medicines, and intellectual property rights Human resources: the lifeblood of health systems Responding to HIV/AIDS Gene technology and the attainment of health for all Sexual and reproductive health

 Section C: Beyond Health Care
 Environmental challenges Militarism and conflict Water The right to food: land, agriculture, and household food security Education

 Section D: Marginalised Groups
 Indigenous peoples People with disabilities

 Section E: Monitoring of Institutions and Resource Flows
 World Health Organisation World Bank World Trade Organisation and trade agreements Global Fund and Pepfar (United States President's Emergency Plan for AIDS Relief) Monitoring of international promises on aid and debt relief

 Section F: Summary and Strategies for Action
Why an Alternative Is Needed
Concerted action by civil society has had tremendous success in the field of international health—global grassroots campaigns on infant feeding, smoking, and drug prices have changed policies and people's lives.But over the last two decades—at the same time as these campaigns have scored victories—there has, in some parts of the world, been a stagnation and even reversal of the dramatic gains in life expectancy witnessed by many others for much of the 20th century. These reversals, unprecedented outside times of war and famine since the early 1800s and a scandal in a world of enormous wealth and technological prowess, have once more thrown the spotlight on how underlying social and economic problems affect health and health services.The setbacks have also underlined appalling failures of health development policy. Ambitious targets to achieve ‘Health for All’ agreed to at the end of the 1970s by health ministers from around the world have failed miserably; a similar fate seems likely for the targets set out in the Millennium Development Goals for 2015. As a result, there are large question marks hanging over the effectiveness of international health policy.These are the reasons why we have decided to produce Global Health Watch, which we hope will become a regular report on international health issues ( Box 1 ). We believe that civil society campaigners need to look at the broader health agenda—beyond single-issue advocacy. Major concerns about health systems such as poor pay and working conditions for health professionals, creeping commercialisation, and plummeting public investment have not had the attention they deserve. Likewise, broader determinants of health—such as education, water, food, and the environment—are often insufficiently regarded when health policies are formulated. The Watch attempts to focus minds on the need for more integrated planning across sectors and on the creation of health systems that promote social justice rather undermine it.Box 1. Global Health Watch—2005 Report Contents
Section A: The Politics and Economics of Health in the 21st CenturySection A: The Politics and Economics of Health in the 21st CenturySection B: The Health Care Sector
 Health systems that promote social justice Responding to the commercialisation of health care The pharmaceutical industry, access to medicines, and intellectual property rights Human resources: the lifeblood of health systems Responding to HIV/AIDS Gene technology and the attainment of health for all Sexual and reproductive healthSection B: The Health Care SectorHealth systems that promote social justiceResponding to the commercialisation of health careThe pharmaceutical industry, access to medicines, and intellectual property rightsHuman resources: the lifeblood of health systemsResponding to HIV/AIDSGene technology and the attainment of health for allSexual and reproductive healthSection C: Beyond Health Care
 Environmental challenges Militarism and conflict Water The right to food: land, agriculture, and household food security EducationSection C: Beyond Health CareEnvironmental challengesMilitarism and conflictWaterThe right to food: land, agriculture, and household food securityEducationSection D: Marginalised Groups
 Indigenous peoples People with disabilitiesSection D: Marginalised GroupsIndigenous peoplesPeople with disabilitiesSection E: Monitoring of Institutions and Resource Flows
 World Health Organisation World Bank World Trade Organisation and trade agreements Global Fund and Pepfar (United States President's Emergency Plan for AIDS Relief) Monitoring of international promises on aid and debt reliefSection E: Monitoring of Institutions and Resource FlowsWorld Health OrganisationWorld BankWorld Trade Organisation and trade agreementsGlobal Fund and Pepfar (United States President's Emergency Plan for AIDS Relief)Monitoring of international promises on aid and debt reliefSection F: Summary and Strategies for ActionSection F: Summary and Strategies for ActionHow Will the Watch Be Different? This is how the Watch will be alternative: it will present options for health policy-makers that question the dominant reform agenda that emphasises market-driven and diseased-based approaches to health care. A policy bias against government action and a lack of creative thinking about how governments can shape health care markets to work in favour of equity and social inclusion are unfortunate features of global health debates. More recently, the emphasis has been placed once again on campaigns against specific diseases such as HIV/AIDS and tuberculosis, despite the universally acknowledged importance of building and maintaining health systems that can respond to the broader needs of patients. We hope the Watch will present some alternative and imaginative thinking about how health services can respond creatively to the many challenges they face, with a strong focus on basic principles of equity and universality and avoiding top-down disease-focussed programmes that neglect the broader determinants of health. We have invited some of the most interesting and innovative thinkers in health policy—from both developing and developed countries and from academia and civil society—to help us achieve these objectives. The Watch will also be ‘alternative’ in another sense—it will act as a regular monitor of the policies, governance, and funding of the institutions affecting global health, including the World Health Organisation and World Bank, something no other health report undertakes. We hope to offer proposals for reform, as well as to stimulate further action by civil society to make these institutions more accountable and responsive to the needs of the poor and vulnerable.
How Will the Watch Be Different?
This is how the Watch will be alternative: it will present options for health policy-makers that question the dominant reform agenda that emphasises market-driven and diseased-based approaches to health care. A policy bias against government action and a lack of creative thinking about how governments can shape health care markets to work in favour of equity and social inclusion are unfortunate features of global health debates. More recently, the emphasis has been placed once again on campaigns against specific diseases such as HIV/AIDS and tuberculosis, despite the universally acknowledged importance of building and maintaining health systems that can respond to the broader needs of patients.We hope the Watch will present some alternative and imaginative thinking about how health services can respond creatively to the many challenges they face, with a strong focus on basic principles of equity and universality and avoiding top-down disease-focussed programmes that neglect the broader determinants of health. We have invited some of the most interesting and innovative thinkers in health policy—from both developing and developed countries and from academia and civil society—to help us achieve these objectives.The Watch will also be ‘alternative’ in another sense—it will act as a regular monitor of the policies, governance, and funding of the institutions affecting global health, including the World Health Organisation and World Bank, something no other health report undertakes. We hope to offer proposals for reform, as well as to stimulate further action by civil society to make these institutions more accountable and responsive to the needs of the poor and vulnerable.Linking Civil Society Groups It is important to say that the three networks and organisations that have convened the Watch are really just its initiators. In the end we hope the Watch will be backed by as many individuals, organisations, and social movements as possible, strengthening the links between civil society organisations across countries and across health-related sectors, and increasing the power and influence of the report itself. Already, many have expressed their interest in the project, and their willingness to contribute: through writing chapters, contributing case studies, and launching the Watch and promoting it in their country when it is finished. Groups from India and Brazil are planning parallel national Watches. We plan to launch the Watch at the second People's Health Assembly, which will be held in Ecuador in July 2005. We don't want this report to be addressed just to health activists or health policy-makers or academics. If we are going to create change we need to capture the imagination of the broader health professional community and the public at large. That is why we encourage readers to get involved and tell others about the Watch and to use it to throw down a challenge to those who call the shots at national and international levels.
Linking Civil Society Groups
It is important to say that the three networks and organisations that have convened the Watch are really just its initiators. In the end we hope the Watch will be backed by as many individuals, organisations, and social movements as possible, strengthening the links between civil society organisations across countries and across health-related sectors, and increasing the power and influence of the report itself. Already, many have expressed their interest in the project, and their willingness to contribute: through writing chapters, contributing case studies, and launching the Watch and promoting it in their country when it is finished. Groups from India and Brazil are planning parallel national Watches.We plan to launch the Watch at the second People's Health Assembly, which will be held in Ecuador in July 2005. We don't want this report to be addressed just to health activists or health policy-makers or academics. If we are going to create change we need to capture the imagination of the broader health professional community and the public at large. That is why we encourage readers to get involved and tell others about the Watch and to use it to throw down a challenge to those who call the shots at national and international levels.


Background The intratracheal instillation of  Pseudomonas aeruginosa  entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na + -K + -ATPase.
Background
The intratracheal instillation of  Pseudomonas aeruginosa  entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na + -K + -ATPase.Pseudomonas aeruginosaMethods Pseudomonas aeruginosa  entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na + -K + -ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.
Methods
Pseudomonas aeruginosa  entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na + -K + -ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.Pseudomonas aeruginosaResults The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α 1 Na + -K + -ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.
Results
The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α 1 Na + -K + -ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.Conclusions These results show that  Pseudomonas  infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.
Conclusions
These results show that  Pseudomonas  infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.Pseudomonas

Background The epithelial sodium channel (ENaC) is expressed in epithelial cells of several tissues involved in salt and water reabsorption. The channel is composed of three related subunits (α, β, γ) that are able to reconstitute a functional channel when expressed in  Xenopus laevis  oocytes [ 1 , 2 ]. ENaC is expressed in a wide range of tissues, including the kidney [ 1 , 3 - 5 ], distal colon [ 1 , 3 , 5 ], lung [ 6 - 8 ], ear epithelium [ 9 , 10 ], papilla of the tongue [ 11 - 13 ], eyes [ 14 ], chondrocytes [ 15 ] and differentiating epithelia [ 16 ]. ENaC synthesis and activity are highly regulated by hormones, such as aldosterone, vasopressin and catecholamines, by intracellular pH, feedback inhibition and extracellular proteases [ 17 , 18 ]. In the lung, vectorial Na +  transport from the alveoli to the interstitium is the main force that drives water out of the alveoli [ 19 , 20 ]. This transport mechanism plays a crucial role late in gestation and at birth when sodium transport is involved in lung liquid clearance [ 21 ]. Its importance at birth has been shown unambiguously in αENaC gene knockout mice, where the inability to clear lung water rapidly leads to hypoxemia and death [ 22 ]. Na +  transport is also important in adults for lung liquid clearance [ 19 , 23 ]. Increased ENaC expression has been detected in the lung and in alveolar epithelial cells  in vitro , following stimulation with steroids, β-agonists, catecholamines, and agents that increase cAMP concentration [ 24 - 27 ]. αENaC expression in the lung is modulated at birth when considerable liquid clearance is required [ 3 , 6 , 27 ]. It is also upregulated during hyperoxia [ 28 , 29 ] and downregulated during hypoxia, which could explain high altitude lung edema (HALE) [ 30 , 31 ]. Several lines of evidence suggest that up-regulation or downregulation of ENaC activity in the lung could be associated with lung infection. In type I pseudohypoaldosteronism, a recessive genetic disease leading to a non-functional ENaC, susceptibility to lung infection has been reported [ 32 - 34 ]. Although ENaC is not the primary defect associated with cystic fibrosis (CF), airway cells from CF patients show a 2–3-fold increase in Na +  transport compared to normal cells [ 35 , 36 ]. This sodium hyperabsorption results from the inability of cystic fibrosis transmembrane regulator (CFTR) in CF cells to downregulate ENaC activity [ 37 , 38 ]. Pseudomonas aeruginosa  is a bacterium occuring naturally in a wide range of environments such as in soil, fresh and seawater, plants and decomposing organic matter [ 39 ]. Although not usually pathogenic, this common bacterium can evoke opportunistic infections in immunodeficient persons, such as patients with severe burns [ 39 ].  Pseudomonas  can promote nosocomial lung infection after artificial ventilation [ 40 ] and is also present in patients with bronchiectasis [ 39 ]. Chronic lung infections are the major cause of morbidity and mortality in CF patients [ 41 ] where  Pseudomonas aeruginosa  is the main source of chronic lung infection in CF patients [ 42 ]. Instillation of  Pseudomonas aeruginosa  in the lung of anaesthetised rabbits has been reported to promote acute pneumonia, resulting in alveolar epithelial injury, loss of epithelial barrier integrity, lung edema, pleural empyema and pleural effusions within 8 h of infection [ 43 ]. A more chronic pneumonia model has been developed in the mouse by the intratracheal instillation of  P. aeruginosa  entrapped in agar beads. In this model, the lungs of susceptible mouse strains develop severe lung infection with a strong inflammatory response and some lung edema [ 44 , 45 ].  Pseudomonas  by itself has been shown to inhibit active sodium absorption in cultured airway epithelial cells [ 46 ]. Here, we studied its impact on the expression of genes involved in the modulation of liquid absorption in alveolar and airway epithelium, namely the three ENaC subunits and the catalytic subunit of Na + -K + -ATPase. Pseudomonas  entrapped in agar beads was instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains, and the expression of α, β, γENaC and α 1  Na + -K + -ATPase mRNA was studied by Northern blotting in lungs infected between 3 hours and 14 days.
Background
The epithelial sodium channel (ENaC) is expressed in epithelial cells of several tissues involved in salt and water reabsorption. The channel is composed of three related subunits (α, β, γ) that are able to reconstitute a functional channel when expressed in  Xenopus laevis  oocytes [ 1 , 2 ]. ENaC is expressed in a wide range of tissues, including the kidney [ 1 , 3 - 5 ], distal colon [ 1 , 3 , 5 ], lung [ 6 - 8 ], ear epithelium [ 9 , 10 ], papilla of the tongue [ 11 - 13 ], eyes [ 14 ], chondrocytes [ 15 ] and differentiating epithelia [ 16 ]. ENaC synthesis and activity are highly regulated by hormones, such as aldosterone, vasopressin and catecholamines, by intracellular pH, feedback inhibition and extracellular proteases [ 17 , 18 ]. In the lung, vectorial Na +  transport from the alveoli to the interstitium is the main force that drives water out of the alveoli [ 19 , 20 ]. This transport mechanism plays a crucial role late in gestation and at birth when sodium transport is involved in lung liquid clearance [ 21 ]. Its importance at birth has been shown unambiguously in αENaC gene knockout mice, where the inability to clear lung water rapidly leads to hypoxemia and death [ 22 ]. Na +  transport is also important in adults for lung liquid clearance [ 19 , 23 ].Xenopus laevisIncreased ENaC expression has been detected in the lung and in alveolar epithelial cells  in vitro , following stimulation with steroids, β-agonists, catecholamines, and agents that increase cAMP concentration [ 24 - 27 ]. αENaC expression in the lung is modulated at birth when considerable liquid clearance is required [ 3 , 6 , 27 ]. It is also upregulated during hyperoxia [ 28 , 29 ] and downregulated during hypoxia, which could explain high altitude lung edema (HALE) [ 30 , 31 ]. Several lines of evidence suggest that up-regulation or downregulation of ENaC activity in the lung could be associated with lung infection. In type I pseudohypoaldosteronism, a recessive genetic disease leading to a non-functional ENaC, susceptibility to lung infection has been reported [ 32 - 34 ]. Although ENaC is not the primary defect associated with cystic fibrosis (CF), airway cells from CF patients show a 2–3-fold increase in Na +  transport compared to normal cells [ 35 , 36 ]. This sodium hyperabsorption results from the inability of cystic fibrosis transmembrane regulator (CFTR) in CF cells to downregulate ENaC activity [ 37 , 38 ].in vitroPseudomonas aeruginosa  is a bacterium occuring naturally in a wide range of environments such as in soil, fresh and seawater, plants and decomposing organic matter [ 39 ]. Although not usually pathogenic, this common bacterium can evoke opportunistic infections in immunodeficient persons, such as patients with severe burns [ 39 ].  Pseudomonas  can promote nosocomial lung infection after artificial ventilation [ 40 ] and is also present in patients with bronchiectasis [ 39 ]. Chronic lung infections are the major cause of morbidity and mortality in CF patients [ 41 ] where  Pseudomonas aeruginosa  is the main source of chronic lung infection in CF patients [ 42 ].Pseudomonas aeruginosaPseudomonasPseudomonas aeruginosaInstillation of  Pseudomonas aeruginosa  in the lung of anaesthetised rabbits has been reported to promote acute pneumonia, resulting in alveolar epithelial injury, loss of epithelial barrier integrity, lung edema, pleural empyema and pleural effusions within 8 h of infection [ 43 ]. A more chronic pneumonia model has been developed in the mouse by the intratracheal instillation of  P. aeruginosa  entrapped in agar beads. In this model, the lungs of susceptible mouse strains develop severe lung infection with a strong inflammatory response and some lung edema [ 44 , 45 ].  Pseudomonas  by itself has been shown to inhibit active sodium absorption in cultured airway epithelial cells [ 46 ]. Here, we studied its impact on the expression of genes involved in the modulation of liquid absorption in alveolar and airway epithelium, namely the three ENaC subunits and the catalytic subunit of Na + -K + -ATPase.Pseudomonas aeruginosaP. aeruginosaPseudomonasPseudomonas  entrapped in agar beads was instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains, and the expression of α, β, γENaC and α 1  Na + -K + -ATPase mRNA was studied by Northern blotting in lungs infected between 3 hours and 14 days.PseudomonasMethods Infection of mice with  P. aeruginosa Clinical strain 508 of  P. aeruginosa  (provided by Dr. Jacqueline Lagacé, Université de Montréal, Montreal, Canada) was entrapped in agar beads, and 50-μl suspensions containing 2 × 10 5  to 1 × 10 6  CFU/ml were instilled intratracheally in male mice of resistant (BALB/c) or susceptible (DBA/2, C57BL/6 and A/J) strains as described previously [ 44 , 45 ]. Macrophage and polymorphonuclear (PMN) counts in bronchoalveolar lavage (BAL) BAL were performed as described elsewhere with a few modifications [ 44 ]. The infected mice were sacrificed by CO 2  inhalation at different time points after  P. aeruginosa  instillation in the lungs. The trachea was cannulated, and the lungs were washed seven times with 1 ml PBS. Total cell counts were conducted in a hemacytometer. Differential cell counts were made by Diff-Quick staining (American Scientific Products) of Cytospin preparations. Number of animals: day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3. Northern blotting The lungs from infected mice were harvested between 3 h to 14 days after infection, homogenized in 5 ml of 4 M guanidine isothiocyanate, and centrifuged on a cesium chloride gradient [ 44 ]. Fifteen to 20 μg of total RNA purified from the lungs were electrophoresed on 1% agarose-formaldehyde gel and transferred to Nytran membranes (Schleicher & Schuell, Keene, NH, USA) by overnight blotting with 10 X SSC. Hybridization was performed, as reported previously [ 3 ], in Church buffer (0.5 M Na phosphate, pH 7.2, 7% SDS (w/v), 1 mM EDTA, pH 8) [ 47 ]. The nylon membranes were hybridized successively with different cDNA probes. (αENaC, βENaC and γENaC, α 1 Na + -K + -ATPase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or 18S rRNA). To detect αENaC mRNA, the blots were hybridized with 764-bp mouse αENaC cDNA (His-445 to stop codon) [ 3 ]. The probes for rat β and γENaC cDNA were gifts from Dr. B.C. Rossier (Institut de pharmacologie et de toxicologie de l'Université de Lausanne, Lausanne, Switzerland) and coded for the entire cDNA [ 2 ]. The α 1 Na + -K + -ATPase probe was a gift from Dr. J. Orlowski (Physiology Department, McGill University, Montreal, Quebec, Canada) and consisted of a NarI-StuI 332-bp fragment coding from nucleotide 89 to 421 (from the 5'UTR to Arg-61) of the rat kidney and brain α isoform [ 48 ]. For quantitative study, αENaC mRNA expression was normalized to murine GADPH with a 455 bp cDNA probe cloned between nucleotide 146 and 601 [ 44 ] or with 18S rRNA, using a 640-bp cDNA probe between nucleotidet 852 and 1492 of the rat 18S rRNA sequence [ 26 ]. The blots were exposed to Kodak Xar-film with an intensifying screen, or to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) for densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control from the same strain. The data from the different strains were pooled and subjected to statistical analysis. Between each round of hybridization, the membranes were stripped by treatment with 0.1 X SSC, 1% SDS and 2.5 mM EDTA at 95°C. The blots were allowed to cool gradually with agitation for 30 min at room temperature. The membranes were then rinsed with 5 X SSC and rehybridized. Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied. Statistics For the BAL cell count, the data are presented as means ± SE (standard error). For ENaC and Na + , K + -ATPase mRNA expresion, the comparisons between groups were analyzed by Wilcoxon signed rank non-parametric test using Statsview software (SAS Institute, Inc., Cary, NC, USA). Probability  p  values < 0.05 were considered to be significant.
Methods
Infection of mice with  P. aeruginosa Clinical strain 508 of  P. aeruginosa  (provided by Dr. Jacqueline Lagacé, Université de Montréal, Montreal, Canada) was entrapped in agar beads, and 50-μl suspensions containing 2 × 10 5  to 1 × 10 6  CFU/ml were instilled intratracheally in male mice of resistant (BALB/c) or susceptible (DBA/2, C57BL/6 and A/J) strains as described previously [ 44 , 45 ].
Infection of mice with  P. aeruginosa
P. aeruginosaClinical strain 508 of  P. aeruginosa  (provided by Dr. Jacqueline Lagacé, Université de Montréal, Montreal, Canada) was entrapped in agar beads, and 50-μl suspensions containing 2 × 10 5  to 1 × 10 6  CFU/ml were instilled intratracheally in male mice of resistant (BALB/c) or susceptible (DBA/2, C57BL/6 and A/J) strains as described previously [ 44 , 45 ].P. aeruginosaMacrophage and polymorphonuclear (PMN) counts in bronchoalveolar lavage (BAL) BAL were performed as described elsewhere with a few modifications [ 44 ]. The infected mice were sacrificed by CO 2  inhalation at different time points after  P. aeruginosa  instillation in the lungs. The trachea was cannulated, and the lungs were washed seven times with 1 ml PBS. Total cell counts were conducted in a hemacytometer. Differential cell counts were made by Diff-Quick staining (American Scientific Products) of Cytospin preparations. Number of animals: day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3.
Macrophage and polymorphonuclear (PMN) counts in bronchoalveolar lavage (BAL)
BAL were performed as described elsewhere with a few modifications [ 44 ]. The infected mice were sacrificed by CO 2  inhalation at different time points after  P. aeruginosa  instillation in the lungs. The trachea was cannulated, and the lungs were washed seven times with 1 ml PBS. Total cell counts were conducted in a hemacytometer. Differential cell counts were made by Diff-Quick staining (American Scientific Products) of Cytospin preparations. Number of animals: day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3.P. aeruginosaNorthern blotting The lungs from infected mice were harvested between 3 h to 14 days after infection, homogenized in 5 ml of 4 M guanidine isothiocyanate, and centrifuged on a cesium chloride gradient [ 44 ]. Fifteen to 20 μg of total RNA purified from the lungs were electrophoresed on 1% agarose-formaldehyde gel and transferred to Nytran membranes (Schleicher & Schuell, Keene, NH, USA) by overnight blotting with 10 X SSC. Hybridization was performed, as reported previously [ 3 ], in Church buffer (0.5 M Na phosphate, pH 7.2, 7% SDS (w/v), 1 mM EDTA, pH 8) [ 47 ]. The nylon membranes were hybridized successively with different cDNA probes. (αENaC, βENaC and γENaC, α 1 Na + -K + -ATPase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or 18S rRNA). To detect αENaC mRNA, the blots were hybridized with 764-bp mouse αENaC cDNA (His-445 to stop codon) [ 3 ]. The probes for rat β and γENaC cDNA were gifts from Dr. B.C. Rossier (Institut de pharmacologie et de toxicologie de l'Université de Lausanne, Lausanne, Switzerland) and coded for the entire cDNA [ 2 ]. The α 1 Na + -K + -ATPase probe was a gift from Dr. J. Orlowski (Physiology Department, McGill University, Montreal, Quebec, Canada) and consisted of a NarI-StuI 332-bp fragment coding from nucleotide 89 to 421 (from the 5'UTR to Arg-61) of the rat kidney and brain α isoform [ 48 ]. For quantitative study, αENaC mRNA expression was normalized to murine GADPH with a 455 bp cDNA probe cloned between nucleotide 146 and 601 [ 44 ] or with 18S rRNA, using a 640-bp cDNA probe between nucleotidet 852 and 1492 of the rat 18S rRNA sequence [ 26 ]. The blots were exposed to Kodak Xar-film with an intensifying screen, or to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) for densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control from the same strain. The data from the different strains were pooled and subjected to statistical analysis. Between each round of hybridization, the membranes were stripped by treatment with 0.1 X SSC, 1% SDS and 2.5 mM EDTA at 95°C. The blots were allowed to cool gradually with agitation for 30 min at room temperature. The membranes were then rinsed with 5 X SSC and rehybridized. Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.
Northern blotting
The lungs from infected mice were harvested between 3 h to 14 days after infection, homogenized in 5 ml of 4 M guanidine isothiocyanate, and centrifuged on a cesium chloride gradient [ 44 ]. Fifteen to 20 μg of total RNA purified from the lungs were electrophoresed on 1% agarose-formaldehyde gel and transferred to Nytran membranes (Schleicher & Schuell, Keene, NH, USA) by overnight blotting with 10 X SSC. Hybridization was performed, as reported previously [ 3 ], in Church buffer (0.5 M Na phosphate, pH 7.2, 7% SDS (w/v), 1 mM EDTA, pH 8) [ 47 ]. The nylon membranes were hybridized successively with different cDNA probes. (αENaC, βENaC and γENaC, α 1 Na + -K + -ATPase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or 18S rRNA). To detect αENaC mRNA, the blots were hybridized with 764-bp mouse αENaC cDNA (His-445 to stop codon) [ 3 ]. The probes for rat β and γENaC cDNA were gifts from Dr. B.C. Rossier (Institut de pharmacologie et de toxicologie de l'Université de Lausanne, Lausanne, Switzerland) and coded for the entire cDNA [ 2 ]. The α 1 Na + -K + -ATPase probe was a gift from Dr. J. Orlowski (Physiology Department, McGill University, Montreal, Quebec, Canada) and consisted of a NarI-StuI 332-bp fragment coding from nucleotide 89 to 421 (from the 5'UTR to Arg-61) of the rat kidney and brain α isoform [ 48 ]. For quantitative study, αENaC mRNA expression was normalized to murine GADPH with a 455 bp cDNA probe cloned between nucleotide 146 and 601 [ 44 ] or with 18S rRNA, using a 640-bp cDNA probe between nucleotidet 852 and 1492 of the rat 18S rRNA sequence [ 26 ]. The blots were exposed to Kodak Xar-film with an intensifying screen, or to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) for densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control from the same strain. The data from the different strains were pooled and subjected to statistical analysis.Between each round of hybridization, the membranes were stripped by treatment with 0.1 X SSC, 1% SDS and 2.5 mM EDTA at 95°C. The blots were allowed to cool gradually with agitation for 30 min at room temperature. The membranes were then rinsed with 5 X SSC and rehybridized. Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.Statistics For the BAL cell count, the data are presented as means ± SE (standard error). For ENaC and Na + , K + -ATPase mRNA expresion, the comparisons between groups were analyzed by Wilcoxon signed rank non-parametric test using Statsview software (SAS Institute, Inc., Cary, NC, USA). Probability  p  values < 0.05 were considered to be significant.
Statistics
For the BAL cell count, the data are presented as means ± SE (standard error). For ENaC and Na + , K + -ATPase mRNA expresion, the comparisons between groups were analyzed by Wilcoxon signed rank non-parametric test using Statsview software (SAS Institute, Inc., Cary, NC, USA). Probability  p  values < 0.05 were considered to be significant.pResults Inflammation in mice infected with P. aeruginosa The inflammation process evoked by  Pseudomonas  instillation in the lung of C57BL/6 mice was monitored by studying the number of total cells in BAL at different times after infection. As shown in Figure  1 , the inflammation process was more pronounced on days 1 and 4 post-infection. Significant PMN recruitment was noted on day 1 after infection since these cells constituted 90% of the cell population in BAL at that time (Fig.  1 ). The proportion of PMN decreased gradually over time. On days 6 and 14, there was a significant reduction of PMN in BAL (p < 0.05) compared to day 1. PMN still constituted 18% of the cells in BAL on day 14. The infection also led to modulation in the number of macrophages with a significant increase (p < 0.05) on day 4 post-infection (Fig.  1 ). Figure 1 Differential cell counting in C57BL/6 mice infected with 1–2 × 10 5  Pseudomonas aeruginosa  embedded in agar beads.  Pseudomonas  infection leads to strong inflammation with recruitment of PMN and macrophages in bronchoalveolar lavage on days 1 and 4 post-infection. Day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3. Differential cell counting: PMN in light grey, macrophages in dark grey. Modulation of α, β and γENaC expression following lung infection with P. aeruginosa Pseudomonas  embedded in agar beads was administered intratracheally in resistant (BALB/c) and susceptible strains of mice (DBA/2, C57BL/6, A/J) as described previously [ 44 , 49 ]. α, β and γENaC expression in infected lungs was measured by Northern blot hybridization (Fig.  2 ). Expression of the three subunits was highly modulated in time after lung infection, but showed a similar pattern between the four mouse strains tested. The BALB/c strain that is resistant to  Pseudomonas  infection [ 45 ], as well as the DBA/2, C57BL/6 and A/J susceptible strains, showed increased α, β and γENaC expression at 24 h, followed by a decrease on day 3 post-infection. The GADPH standard gene did not manifest any modulation of its expression. Densitometric quantitative analyses of the Northern blots were performed for the four mouse strains. The relative expression at each time point was determined relative to uninfected animals of the same strain and the data from the 4 strains were pooled for analysis (Fig.  3 ). αENaC mRNA expression presented a significant decline to a median of 43% on days 3 and 7 post-infection, and was still decreased to a median of 45% on day 14 post-infection compared to uninfected controls (p < 0.05, Fig.  3 ). βENaC mRNA expression was increased to a median of 241% of uninfected control values, 24 h post-infection (p < 0.05), and was followed by a decrease to medians of 42% and 54% on day 3 and 7 post-infection (p < 0.05) (Fig.  3 ). Although the expression of γ ENaC mRNA showed an expression pattern very similar to the α and βENaC subunits, with an increased expression at 24 h (median of 171%) followed by a decreased expression on day 3 (median of 53%) and 7 (median of 66%) of infection, these changes failed however to reach significance (Fig.  3 ). No modulation of α, β or γENaC mRNA was detected when the lungs were instilled with agarose beads only (data not shown). We also investigated the expression of α 1  Na + -K + -ATPase mRNA coding for the catalytic domain of the sodium pump, but could not find any significant change during infection (Fig.  3 ). Figure 2 Expression of α, β and γENaC mRNA in the lung following infection with  Pseudomonas aeruginosa .  Representative Northern blot of α, β and γENaC mRNA expression following infection with  Pseudomonas  in resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) strains of mice. There is a characteristic modulation of the three ENaC subunits that is not different between strains. Figure 3 Densitometric analysis of the modulation of αENaC, βENaC, γENaC and α 1 Na + -K + -ATPase mRNA following  Pseudomonas  infection.  The modulation of αENaC, βENaC, γENaC and α 1 Na + -K + -ATPase mRNA by Northern blots hybridization was subjected to a densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control coming from the same strain. The α and βENaC mRNA were modulated at some time point by Pseudomonas infection compared to uninfected animals. There was no modulation for γENaC or α 1 Na + -K + -ATPase mRNA. αENaC mRNA was downregulated compared to uninfected controls on days 3, 7 and 14 post-infection (*, p < 0.05). βENaC mRNA was elevated at 24 h post-infection (*, p < 0.05) compared to uninfected controls and was downregulated thereafter on day 3 and 7 post-infection (*, p < 0.05). Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.
Results
Inflammation in mice infected with P. aeruginosa The inflammation process evoked by  Pseudomonas  instillation in the lung of C57BL/6 mice was monitored by studying the number of total cells in BAL at different times after infection. As shown in Figure  1 , the inflammation process was more pronounced on days 1 and 4 post-infection. Significant PMN recruitment was noted on day 1 after infection since these cells constituted 90% of the cell population in BAL at that time (Fig.  1 ). The proportion of PMN decreased gradually over time. On days 6 and 14, there was a significant reduction of PMN in BAL (p < 0.05) compared to day 1. PMN still constituted 18% of the cells in BAL on day 14. The infection also led to modulation in the number of macrophages with a significant increase (p < 0.05) on day 4 post-infection (Fig.  1 ). Figure 1 Differential cell counting in C57BL/6 mice infected with 1–2 × 10 5  Pseudomonas aeruginosa  embedded in agar beads.  Pseudomonas  infection leads to strong inflammation with recruitment of PMN and macrophages in bronchoalveolar lavage on days 1 and 4 post-infection. Day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3. Differential cell counting: PMN in light grey, macrophages in dark grey.
Inflammation in mice infected with P. aeruginosa
The inflammation process evoked by  Pseudomonas  instillation in the lung of C57BL/6 mice was monitored by studying the number of total cells in BAL at different times after infection. As shown in Figure  1 , the inflammation process was more pronounced on days 1 and 4 post-infection. Significant PMN recruitment was noted on day 1 after infection since these cells constituted 90% of the cell population in BAL at that time (Fig.  1 ). The proportion of PMN decreased gradually over time. On days 6 and 14, there was a significant reduction of PMN in BAL (p < 0.05) compared to day 1. PMN still constituted 18% of the cells in BAL on day 14. The infection also led to modulation in the number of macrophages with a significant increase (p < 0.05) on day 4 post-infection (Fig.  1 ).PseudomonasModulation of α, β and γENaC expression following lung infection with P. aeruginosa Pseudomonas  embedded in agar beads was administered intratracheally in resistant (BALB/c) and susceptible strains of mice (DBA/2, C57BL/6, A/J) as described previously [ 44 , 49 ]. α, β and γENaC expression in infected lungs was measured by Northern blot hybridization (Fig.  2 ). Expression of the three subunits was highly modulated in time after lung infection, but showed a similar pattern between the four mouse strains tested. The BALB/c strain that is resistant to  Pseudomonas  infection [ 45 ], as well as the DBA/2, C57BL/6 and A/J susceptible strains, showed increased α, β and γENaC expression at 24 h, followed by a decrease on day 3 post-infection. The GADPH standard gene did not manifest any modulation of its expression. Densitometric quantitative analyses of the Northern blots were performed for the four mouse strains. The relative expression at each time point was determined relative to uninfected animals of the same strain and the data from the 4 strains were pooled for analysis (Fig.  3 ). αENaC mRNA expression presented a significant decline to a median of 43% on days 3 and 7 post-infection, and was still decreased to a median of 45% on day 14 post-infection compared to uninfected controls (p < 0.05, Fig.  3 ). βENaC mRNA expression was increased to a median of 241% of uninfected control values, 24 h post-infection (p < 0.05), and was followed by a decrease to medians of 42% and 54% on day 3 and 7 post-infection (p < 0.05) (Fig.  3 ). Although the expression of γ ENaC mRNA showed an expression pattern very similar to the α and βENaC subunits, with an increased expression at 24 h (median of 171%) followed by a decreased expression on day 3 (median of 53%) and 7 (median of 66%) of infection, these changes failed however to reach significance (Fig.  3 ). No modulation of α, β or γENaC mRNA was detected when the lungs were instilled with agarose beads only (data not shown). We also investigated the expression of α 1  Na + -K + -ATPase mRNA coding for the catalytic domain of the sodium pump, but could not find any significant change during infection (Fig.  3 ). Figure 2 Expression of α, β and γENaC mRNA in the lung following infection with  Pseudomonas aeruginosa .  Representative Northern blot of α, β and γENaC mRNA expression following infection with  Pseudomonas  in resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) strains of mice. There is a characteristic modulation of the three ENaC subunits that is not different between strains. Figure 3 Densitometric analysis of the modulation of αENaC, βENaC, γENaC and α 1 Na + -K + -ATPase mRNA following  Pseudomonas  infection.  The modulation of αENaC, βENaC, γENaC and α 1 Na + -K + -ATPase mRNA by Northern blots hybridization was subjected to a densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control coming from the same strain. The α and βENaC mRNA were modulated at some time point by Pseudomonas infection compared to uninfected animals. There was no modulation for γENaC or α 1 Na + -K + -ATPase mRNA. αENaC mRNA was downregulated compared to uninfected controls on days 3, 7 and 14 post-infection (*, p < 0.05). βENaC mRNA was elevated at 24 h post-infection (*, p < 0.05) compared to uninfected controls and was downregulated thereafter on day 3 and 7 post-infection (*, p < 0.05). Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.
Modulation of α, β and γENaC expression following lung infection with P. aeruginosa
Pseudomonas  embedded in agar beads was administered intratracheally in resistant (BALB/c) and susceptible strains of mice (DBA/2, C57BL/6, A/J) as described previously [ 44 , 49 ]. α, β and γENaC expression in infected lungs was measured by Northern blot hybridization (Fig.  2 ). Expression of the three subunits was highly modulated in time after lung infection, but showed a similar pattern between the four mouse strains tested. The BALB/c strain that is resistant to  Pseudomonas  infection [ 45 ], as well as the DBA/2, C57BL/6 and A/J susceptible strains, showed increased α, β and γENaC expression at 24 h, followed by a decrease on day 3 post-infection. The GADPH standard gene did not manifest any modulation of its expression. Densitometric quantitative analyses of the Northern blots were performed for the four mouse strains. The relative expression at each time point was determined relative to uninfected animals of the same strain and the data from the 4 strains were pooled for analysis (Fig.  3 ). αENaC mRNA expression presented a significant decline to a median of 43% on days 3 and 7 post-infection, and was still decreased to a median of 45% on day 14 post-infection compared to uninfected controls (p < 0.05, Fig.  3 ). βENaC mRNA expression was increased to a median of 241% of uninfected control values, 24 h post-infection (p < 0.05), and was followed by a decrease to medians of 42% and 54% on day 3 and 7 post-infection (p < 0.05) (Fig.  3 ). Although the expression of γ ENaC mRNA showed an expression pattern very similar to the α and βENaC subunits, with an increased expression at 24 h (median of 171%) followed by a decreased expression on day 3 (median of 53%) and 7 (median of 66%) of infection, these changes failed however to reach significance (Fig.  3 ). No modulation of α, β or γENaC mRNA was detected when the lungs were instilled with agarose beads only (data not shown). We also investigated the expression of α 1  Na + -K + -ATPase mRNA coding for the catalytic domain of the sodium pump, but could not find any significant change during infection (Fig.  3 ).PseudomonasPseudomonasDiscussion The instillation of  Pseudomonas  enmeshed in agarose beads in the lung is a good model to study lung inflammation [ 44 , 45 ] and lung injury [ 43 ] secondary to an infection. For this study,  P. aeruginosa  enmeshed in agarose beads was instilled into the mouse lung because the model allows the development of chronic lung infection in susceptible mouse strains [ 44 , 45 ]. The infection leads to cellular infiltration and alveolar edema that stand on day 3 post-infection and that can be still demonstrated on day 14 post-infection in  Pseudomonas -susceptible mouse strains [ 45 ]. Because the lung inflammation associated with  Pseudomonas  infection is accompanied by lung injury [ 43 ], and because we have shown recently that ENaC expression can be modulated under conditions that promote lung injury [ 50 ], we tested here if  Pseudomonas  was affecting the mRNA expression level of the three ENaC subunits as well as the catalytic subunit of the Na +  pump since these elements are involved in lung liquid balance across the alveolar epithelium [ 19 , 23 ]. The results reported here indicate that  Pseudomonas  infection modulated the expression of the three ENaC mRNA with a characteristic pattern. There was no significant difference, however, in the expression profile of ENaC mRNA between the  Pseudomonas -resistant (Balb/C) and -susceptible (DBA/2, C57BL/6, A/J) mouse strains. Modulation of ENaC expression is therefore most likely not a genetic marker linked to the susceptibility of mouse strains to establishment of a chronic infection with  Pseudomonas . Pseudomonas  infection affected ENaC mRNA with a pattern consisting of increased expression at 24 h, followed by a marked decrease on day 3 post-infection. The change in ENaC mRNA was related to bacterial infection, since agarose beads alone failed to evoke any modulation of these RNA. The three ENaC subunits were modulated with a similar profile, with some noticeable differences, however. Although αENaC mRNA expression tends to increase by day 1, the most noticeable feature brought by  Pseudomonas  infection to αENaC mRNA was the significant decreases after 3 days, 7 days and 14 days post-infection. To the best of our knowledge, this is the first report demonstrating that bacterial infection  in vivo  can lead to modulation of ENaC mRNA expression. Recently, αENaC mRNA expression was found to be downregulated in the mouse lung after 7 and 14 days of adenoviral infection [ 51 ]. Furthermore, there is some evidence that αENaC expression is also decreased in other models of lung injury. Folkesson et al. [ 52 ] reported a decline in ENaC expression following subacute lung injury, 10 days after intratracheal administration of bleomycin. More recently, we recorded a decrease in ENaC expression after ischemia-reperfusion lung injury [ 50 ]. All these results, and the results reported in the present report, suggest that the modulation of ENaC expression associated with lung infection could be a widespread mechanism, not specific to a given pathogen or injury process, but a general response of the lung to inflammation and injury. The β ENaC subunit was also modulated by  Pseudomonas  infection. There was a significant increase in the mRNA expression on day 1 post-infection, followed, as for αENaC, by a decreased expression on day 3 and day 7 post-infection. Different stoichiometries have been proposed for ENaC. One model suggests a 2α, β, γ ratio [ 53 , 54 ] whereas others postulate an octomeric [ 55 ] or nonameric structure [ 55 , 56 ]. Although the expression of the α subunit alone is sufficient to allow ENaC activity [ 1 ], the three subunits are needed to get a fully functional channel [ 2 ]. The expression of the three subunits in  Xenopus laevis  oocytes increases amiloride-sensitive Na +  current by 100% compared to αENaC alone [ 2 ]. The α, β and α, γ channels are 20 times less effective in driving amiloride-sensitive current than the native channels and show differences in their biophysical properties [ 57 , 58 ]. Gene inactivation or over-expression of the different ENaC subunits has revealed important differences in the role each subunit plays in lung liquid management. αENaC knockout mice develop respiratory distress and die within 40 h from birth because of their inability to clear lung liquid [ 22 ]. Lung liquid clearance at birth is also slower in γENaC knockout mice [ 59 ], but is not affected in βENaC knockouts [ 60 ]. Increased transgenic expression of βENaC targeted in the airway epithelia, but not α or γ subunits, showed an increase Na +  transport across the airway epithelium and a reduced height of the airway surface liquid [ 61 ]. For all these reasons, it is difficult to predict how the modulation of ENaC mRNA expression and its effect on the ratio of the three subunits, would have an impact on ENaC activity. In addition, ENaC mRNA content also does not necessarily reflect the amount of active channel at the membrane. One thing seems clear however, because of its prominence in the lung, the diminution of αENaC expression that we detected in the lung following  Pseudomonas  infection, could certainly influence amiloride-sensitive current and lung liquid clearance as in αENaC KO mice rescued by transgenic expression of αENaC that has a lower expression of ENaC in the lung [ 62 , 63 ]. In such model, there is a reduced ENaC current in tracheal cells [ 63 ], and a much slower lung liquid clearance following thiourea or hyperoxia-induced lung edema [ 64 ]. The general biphasic modulation of ENaC mRNA expression with an increase at 24 h followed by a decrease thereafter is an interesting finding that could explain some contradictory reports concerning ENaC expression in lung following  Pseudomonas  infection. Acute bacterial pneumonia in rats has been shown to increase alveolar epithelial fluid clearance [ 65 , 66 ] when in late pneumonia, there is a decrease in the lung liquid clearance ability of the lung [ 66 ]. These contradictory results could be well explained by the modulation of ENaC expression reported here. The long term ENaC downregulation by  Pseudomonas  infection could be of potential clinical significance to understand the slow improvement in some ARDS patients. In contrast to α and β ENaC, α 1  Na + -K + -ATPase mRNA was unaffected in the course of lung infection. This is similar to what has been reported during adenovirus lung infection [ 51 ] where αENaC, aquaporin 1 (AQP1) and AQP5 mRNA show decreased expression, but not α 1  Na + -K + -ATPase. In ischemia reperfusion injury, there was also no modulation of α 1  Na + -K + -ATPase expression despite significant ENaC downregulation [ 50 ]. These results, as well as the data reported here, suggest that the inflammatory process seems to selectively affect, and not in a non-specific way, some elements of lung liquid clearance. It would be difficult at this time to speculate on the reasons for this modulation. Na + -K + -ATPase is an important element in lung liquid clearance, however, by being one of the key generator of membrane potential, the enzyme also affects other channels and ion transport process. It is possible that by modulating ENaC expression and not α 1  Na + -K + -ATPase,  Pseudomonas  infection alters the Na +  transport system but does not change other important cell functions meditated by Na + -K + -ATPase. Furthermore, despite a similar mRNA expression level, there could be a fall in protein content or activity of the sodium pump. Additional experiments are necessary to answer this question. Several studies report that in lung epithelial cells, viral infection [ 67 , 68 ], mycoplasma [ 69 ], bacterial infection [ 70 , 71 ], and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) [ 70 , 72 ], interleudin-1β (IL-1β) [ 73 ], or TGF-β [ 74 ] decrease the expression of water channels, such as AQP1 and AQP5 and reduce the short circuit current generated by cells. Adenoviral lung infection in mice results in pulmonary inflammation and lung edema with lowered expression of AQP1, AQP5 and αENaC [ 51 ]. All these data, including the results presented here, suggest that lung inflammation, by decreasing the expression of αENaC and water channels, could hamper the liquid clearance ability of the lungs and favour edema formation.
Discussion
The instillation of  Pseudomonas  enmeshed in agarose beads in the lung is a good model to study lung inflammation [ 44 , 45 ] and lung injury [ 43 ] secondary to an infection. For this study,  P. aeruginosa  enmeshed in agarose beads was instilled into the mouse lung because the model allows the development of chronic lung infection in susceptible mouse strains [ 44 , 45 ]. The infection leads to cellular infiltration and alveolar edema that stand on day 3 post-infection and that can be still demonstrated on day 14 post-infection in  Pseudomonas -susceptible mouse strains [ 45 ]. Because the lung inflammation associated with  Pseudomonas  infection is accompanied by lung injury [ 43 ], and because we have shown recently that ENaC expression can be modulated under conditions that promote lung injury [ 50 ], we tested here if  Pseudomonas  was affecting the mRNA expression level of the three ENaC subunits as well as the catalytic subunit of the Na +  pump since these elements are involved in lung liquid balance across the alveolar epithelium [ 19 , 23 ]. The results reported here indicate that  Pseudomonas  infection modulated the expression of the three ENaC mRNA with a characteristic pattern. There was no significant difference, however, in the expression profile of ENaC mRNA between the  Pseudomonas -resistant (Balb/C) and -susceptible (DBA/2, C57BL/6, A/J) mouse strains. Modulation of ENaC expression is therefore most likely not a genetic marker linked to the susceptibility of mouse strains to establishment of a chronic infection with  Pseudomonas .PseudomonasP. aeruginosaPseudomonasPseudomonasPseudomonasPseudomonasPseudomonasPseudomonasPseudomonas  infection affected ENaC mRNA with a pattern consisting of increased expression at 24 h, followed by a marked decrease on day 3 post-infection. The change in ENaC mRNA was related to bacterial infection, since agarose beads alone failed to evoke any modulation of these RNA. The three ENaC subunits were modulated with a similar profile, with some noticeable differences, however. Although αENaC mRNA expression tends to increase by day 1, the most noticeable feature brought by  Pseudomonas  infection to αENaC mRNA was the significant decreases after 3 days, 7 days and 14 days post-infection. To the best of our knowledge, this is the first report demonstrating that bacterial infection  in vivo  can lead to modulation of ENaC mRNA expression. Recently, αENaC mRNA expression was found to be downregulated in the mouse lung after 7 and 14 days of adenoviral infection [ 51 ]. Furthermore, there is some evidence that αENaC expression is also decreased in other models of lung injury. Folkesson et al. [ 52 ] reported a decline in ENaC expression following subacute lung injury, 10 days after intratracheal administration of bleomycin. More recently, we recorded a decrease in ENaC expression after ischemia-reperfusion lung injury [ 50 ]. All these results, and the results reported in the present report, suggest that the modulation of ENaC expression associated with lung infection could be a widespread mechanism, not specific to a given pathogen or injury process, but a general response of the lung to inflammation and injury.PseudomonasPseudomonasin vivoThe β ENaC subunit was also modulated by  Pseudomonas  infection. There was a significant increase in the mRNA expression on day 1 post-infection, followed, as for αENaC, by a decreased expression on day 3 and day 7 post-infection. Different stoichiometries have been proposed for ENaC. One model suggests a 2α, β, γ ratio [ 53 , 54 ] whereas others postulate an octomeric [ 55 ] or nonameric structure [ 55 , 56 ]. Although the expression of the α subunit alone is sufficient to allow ENaC activity [ 1 ], the three subunits are needed to get a fully functional channel [ 2 ]. The expression of the three subunits in  Xenopus laevis  oocytes increases amiloride-sensitive Na +  current by 100% compared to αENaC alone [ 2 ]. The α, β and α, γ channels are 20 times less effective in driving amiloride-sensitive current than the native channels and show differences in their biophysical properties [ 57 , 58 ]. Gene inactivation or over-expression of the different ENaC subunits has revealed important differences in the role each subunit plays in lung liquid management. αENaC knockout mice develop respiratory distress and die within 40 h from birth because of their inability to clear lung liquid [ 22 ]. Lung liquid clearance at birth is also slower in γENaC knockout mice [ 59 ], but is not affected in βENaC knockouts [ 60 ]. Increased transgenic expression of βENaC targeted in the airway epithelia, but not α or γ subunits, showed an increase Na +  transport across the airway epithelium and a reduced height of the airway surface liquid [ 61 ]. For all these reasons, it is difficult to predict how the modulation of ENaC mRNA expression and its effect on the ratio of the three subunits, would have an impact on ENaC activity. In addition, ENaC mRNA content also does not necessarily reflect the amount of active channel at the membrane. One thing seems clear however, because of its prominence in the lung, the diminution of αENaC expression that we detected in the lung following  Pseudomonas  infection, could certainly influence amiloride-sensitive current and lung liquid clearance as in αENaC KO mice rescued by transgenic expression of αENaC that has a lower expression of ENaC in the lung [ 62 , 63 ]. In such model, there is a reduced ENaC current in tracheal cells [ 63 ], and a much slower lung liquid clearance following thiourea or hyperoxia-induced lung edema [ 64 ].PseudomonasXenopus laevisPseudomonasThe general biphasic modulation of ENaC mRNA expression with an increase at 24 h followed by a decrease thereafter is an interesting finding that could explain some contradictory reports concerning ENaC expression in lung following  Pseudomonas  infection. Acute bacterial pneumonia in rats has been shown to increase alveolar epithelial fluid clearance [ 65 , 66 ] when in late pneumonia, there is a decrease in the lung liquid clearance ability of the lung [ 66 ]. These contradictory results could be well explained by the modulation of ENaC expression reported here. The long term ENaC downregulation by  Pseudomonas  infection could be of potential clinical significance to understand the slow improvement in some ARDS patients.PseudomonasPseudomonasIn contrast to α and β ENaC, α 1  Na + -K + -ATPase mRNA was unaffected in the course of lung infection. This is similar to what has been reported during adenovirus lung infection [ 51 ] where αENaC, aquaporin 1 (AQP1) and AQP5 mRNA show decreased expression, but not α 1  Na + -K + -ATPase. In ischemia reperfusion injury, there was also no modulation of α 1  Na + -K + -ATPase expression despite significant ENaC downregulation [ 50 ]. These results, as well as the data reported here, suggest that the inflammatory process seems to selectively affect, and not in a non-specific way, some elements of lung liquid clearance. It would be difficult at this time to speculate on the reasons for this modulation. Na + -K + -ATPase is an important element in lung liquid clearance, however, by being one of the key generator of membrane potential, the enzyme also affects other channels and ion transport process. It is possible that by modulating ENaC expression and not α 1  Na + -K + -ATPase,  Pseudomonas  infection alters the Na +  transport system but does not change other important cell functions meditated by Na + -K + -ATPase. Furthermore, despite a similar mRNA expression level, there could be a fall in protein content or activity of the sodium pump. Additional experiments are necessary to answer this question.PseudomonasSeveral studies report that in lung epithelial cells, viral infection [ 67 , 68 ], mycoplasma [ 69 ], bacterial infection [ 70 , 71 ], and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) [ 70 , 72 ], interleudin-1β (IL-1β) [ 73 ], or TGF-β [ 74 ] decrease the expression of water channels, such as AQP1 and AQP5 and reduce the short circuit current generated by cells. Adenoviral lung infection in mice results in pulmonary inflammation and lung edema with lowered expression of AQP1, AQP5 and αENaC [ 51 ]. All these data, including the results presented here, suggest that lung inflammation, by decreasing the expression of αENaC and water channels, could hamper the liquid clearance ability of the lungs and favour edema formation.Conclusions We have shown in this report that  Pseudomonas  infection modulates ENaC mRNA expression. This modulation is independent of mouse strain susceptibility to establishment of chronic infection with  Pseudomonas . Although there is an elevation of ENaC expression after 24 h, the most important feature is probably the long-lasting decrease of αENaC transcripts on days 3 and 7 post-infection. The lung inflammation induced by  Pseudomonas  infection therefore seems to favour a reduction in the expression of an essential element involved in lung liquid clearance as well as the regulation of airway surface liquid volume.
Conclusions
We have shown in this report that  Pseudomonas  infection modulates ENaC mRNA expression. This modulation is independent of mouse strain susceptibility to establishment of chronic infection with  Pseudomonas . Although there is an elevation of ENaC expression after 24 h, the most important feature is probably the long-lasting decrease of αENaC transcripts on days 3 and 7 post-infection. The lung inflammation induced by  Pseudomonas  infection therefore seems to favour a reduction in the expression of an essential element involved in lung liquid clearance as well as the regulation of airway surface liquid volume.PseudomonasPseudomonasPseudomonasAuthors' contributions AD performed the hybridization, the statistical analysis of the blots and wrote the manuscript. DG performed the  Pseudomonas  instillation, RNA extraction and Northern blotting of RNA sample. The BAL recovery as well as PMN and macrophage counting was performed by CG. YB and DR designed and co-ordinated the study. All authors read and approved the final manuscript.
Authors' contributions
AD performed the hybridization, the statistical analysis of the blots and wrote the manuscript. DG performed the  Pseudomonas  instillation, RNA extraction and Northern blotting of RNA sample. The BAL recovery as well as PMN and macrophage counting was performed by CG. YB and DR designed and co-ordinated the study. All authors read and approved the final manuscript.Pseudomonas