CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14^−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.     

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.     

α1-antitrypsin promotes SPLUNC1-mediated lung defense against Pseudomonas aeruginosa infection in mice

Background

Pseudomonas aeruginosa (PA) infection is involved in various lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. However, treatment of PA infection is not very effective in part due to antibiotic resistance. α1-antitrypsin (A1AT) has been shown to reduce PA infection in humans and animals, but the underlying mechanisms remain unclear. The goal of our study is to test whether a novel endogenous host defense protein, short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is involved in the therapeutic effect of A1AT during lung PA infection.

Method

SPLUNC1 knockout (KO) and littermate wild-type (WT) mice on the C57BL/6 background were intranasally infected with PA to determine the therapeutic effects of A1AT. A1AT was aerosolized to mice 2 hrs after the PA infection, and mice were sacrificed 24 hrs later. PA load and inflammation were quantified in the lung, and SPLUNC1 protein in bronchoalveolar lavage (BAL) fluid was examined by Western blot.

Results

In WT mice, PA infection significantly increased neutrophil elastase (NE) activity, but reduced SPLUNC1 protein in BAL fluid. Notably, PA-infected mice treated with A1AT versus bovine serum albumin (BSA) demonstrated higher levels of SPLUNC1 protein expression, which are accompanied by lower levels of NE activity, lung bacterial load, and pro-inflammatory cytokine production. To determine whether A1AT therapeutic effects are dependent on SPLUNC1, lung PA load in A1AT- or BSA-treated SPLUNC1 KO mice was examined. Unlike the WT mice, A1AT treatment in SPLUNC1 KO mice had no significant impact on lung PA load and pro-inflammatory cytokine production.

Conclusion

A1AT reduces lung bacterial infection in mice in part by preventing NE-mediated SPLUNC1 degradation.     

Affecting Pseudomonas aeruginosa Phenotypic Plasticity by Quorum Sensing Dysregulation Hampers Pathogenicity in Murine Chronic Lung Infection

In Pseudomonas aeruginosa quorum sensing (QS) activates the production of virulence factors, playing a critical role in pathogenesis. Multiple negative regulators modulate the timing and the extent of the QS response either in the pre-quorum or post-quorum phases of growth. This regulation likely increases P. aeruginosa phenotypic plasticity and population fitness, facilitating colonization of challenging environments such as higher organisms. Accordingly, in addition to the factors required for QS signals synthesis and response, also QS regulators have been proposed as targets for anti-virulence therapies. However, while it is known that P. aeruginosa mutants impaired in QS are attenuated in their pathogenic potential, the effect of mutations causing a dysregulated timing and/or magnitude of the QS response has been poorly investigated so far in animal models of infection. In order to investigate the impact of QS dysregulation on P. aeruginosa pathogenesis in a murine model of lung infection, the QteE and RsaL proteins have been selected as representatives of negative regulators controlling P. aeruginosa QS in the pre- and post-quorum periods, respectively. Results showed that the qteE mutation does not affect P. aeruginosa lethality and ability to establish chronic infection in mice, despite causing a premature QS response and enhanced virulence factors production in test tube cultures compared to the wild type. Conversely, the post-quorum dysregulation caused by the rsaL mutation hampers the establishment of P. aeruginosa chronic lung infection in mice without affecting the mortality rate. On the whole, this study contributes to a better understanding of the impact of QS regulation on P. aeruginosa phenotypic plasticity during the infection process. Possible fallouts of these findings in the anti-virulence therapy field are also discussed.     

Inhibition of Macrophage Migration Inhibitory Factor Ameliorates Ocular Pseudomonas aeruginosa-Induced Keratitis

Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.     

Topical flagellin protects the injured corneas from Pseudomonas aeruginosa infection

Among bacterial pathogens, Pseudomonas (P.) aeruginosa infection is the most sight threatening. The corneal innate immune responses are key mediators of the host’s defense to P. aeruginosa. Using a mouse model of Pseudomonas keratitis, we evaluated the protective effects of topical application of flagellin, a ligand for Toll-Like receptor 5 (TLR5), on the development of Pseudomonas keratitis and elucidated the underlying mechanisms. Topical application of purified flagellin 6 and 24 h prior to P. aeruginosa inoculation on injured mouse corneas significantly attenuated clinical symptoms of P. aeruginosa keratitis, decreased bacterial burden, and suppressed infection induced inflammation in the B6 mouse cornea. Topical application of flagellin on wounded cornea induced PMN infiltration and markedly upregulated cathelicidin-related antimicrobial peptide (CRAMP) expression. In PMN depleted mice, flagellin promoted bacterial clearance in the cornea compared to that of the PBS treated mice, but was unable to prevent corneal perforation and systemic bacterial dissemination and sepses. Deletion of CRAMP increased corneal susceptibility to P. aeruginosa and abolished flagellin-induced protection in B6 mice. Our findings illustrate the profound protective effect of flagellin on the cornea innate defense, a response that can be exploited for prophylactic purposes to prevent contact lens associated Pseudomonas keratitis.     

Homotrimeric Macrophage Migration Inhibitory Factor (MIF) Drives Inflammatory Responses in the Corneal Epithelium by Promoting Caveolin-rich Platform Assembly in Response to Infection

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa–induced IL-8 responses via the formation of caveolin-1-rich “signaling hubs” in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.     

Host Genetic Background Influences the Response to the Opportunistic Pseudomonas aeruginosa Infection Altering Cell-Mediated Immunity and Bacterial Replication

Pseudomonas aeruginosa is a common cause of healthcare-associated infections including pneumonia, bloodstream, urinary tract, and surgical site infections. The clinical outcome of P. aeruginosa infections may be extremely variable among individuals at risk and patients affected by cystic fibrosis. However, risk factors for P. aeruginosa infection remain largely unknown. To identify and track the host factors influencing P. aeruginosa lung infections, inbred immunocompetent mouse strains were screened in a pneumonia model system. A/J, BALB/cJ, BALB/cAnNCrl, BALB/cByJ, C3H/HeOuJ, C57BL/6J, C57BL/6NCrl, DBA/2J, and 129S2/SvPasCRL mice were infected with P. aeruginosa clinical strain and monitored for body weight and mortality up to seven days. The most deviant survival phenotypes were observed for A/J, 129S2/SvPasCRL and DBA/2J showing high susceptibility while BALB/cAnNCrl and C3H/HeOuJ showing more resistance to P. aeruginosa infection. Next, one of the most susceptible and resistant mouse strains were characterized for their deviant clinical and immunological phenotype by scoring bacterial count, cell-mediated immunity, cytokines and chemokines profile and lung pathology in an early time course. Susceptible A/J mice showed significantly higher bacterial burden, higher cytokines and chemokines levels but lower leukocyte recruitment, particularly neutrophils, when compared to C3H/HeOuJ resistant mice. Pathologic scores showed lower inflammatory severity, reduced intraluminal and interstitial inflammation extent, bronchial and parenchymal involvement and diminished alveolar damage in the lungs of A/J when compared to C3H/HeOuJ. Our findings indicate that during an early phase of infection a prompt inflammatory response in the airways set the conditions for a non-permissive environment to P. aeruginosa replication and lock the spread to other organs. Host gene(s) may have a role in the reduction of cell-mediated immunity playing a critical role in the control of P. aeruginosa infection. These results now provide a basis for mapping genomic regions underlying host susceptibility to P. aeruginosa infection.     

The pattern of methacholine responsiveness in mice is dependent on antigen challenge dose

Background

The relative contributions of the cytotoxic phenotype of P. aeruginosa expressing type III secretory toxins and an immunocompromised condition lacking normal Toll-like receptor 4 (TLR4) signaling in the pathogenesis of acute lung injury and sepsis were evaluated in a mouse model for Pseudomonas aeruginosa pneumonia. By using lipopolysaccharide-resistant C3H/HeJ mice missing normal TLR4 signaling due to a mutation on the tlr4 gene, we evaluated how TLR4 signaling modulates the pneumonia caused by cytotoxic P. aeruginosa expressing type III secretory toxins.

Methods

We infected C3H/HeJ or C3H/FeJ mice with three different doses of either a cytotoxic P. aeruginosa strain (wild type PA103) or its non-cytotoxic isogenic mutant missing the type III secretory toxins (PA103ΔUT). Survival of the infected mice was evaluated, and the severity of acute lung injury quantified by measuring alveolar epithelial permeability as an index of acute epithelial injury and the water to dry weight ratios of lung homogenates as an index of lung edema. Bacteriological analysis and cytokine assays were performed in the infected mice.

Results

Development of acute lung injury and sepsis was observed in all mouse strains when the cytotoxic P. aeruginosa strain but not the non-cytotoxic strain was instilled in the airspaces of the mice. Only C3H/HeJ mice had severe bacteremia and high mortality when a low dose of the cytotoxic P. aeruginosa strain was instilled in their lungs.

Conclusion

The cytotoxic phenotype of P. aeruginosa is the critical factor causing acute lung injury and sepsis in infected hosts. When the P. aeruginosa is a cytotoxic strain, the TLR4 signaling system is essential to clear the batcteria to prevent lethal lung injury and bacteremia.     

Crystal structures of respiratory pathogen neuraminidases

Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 Å resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.     

The Ability of Virulence Factor Expression by Pseudomonas aeruginosa to Predict Clinical Disease in Hospitalized Patients

Background

Pseudomonas aeruginosa is an opportunistic pathogen that frequently causes hospital acquired colonization and infection. Accurate identification of host and bacterial factors associated with infection could aid treatment decisions for patients with P. aeruginosa cultured from clinical sites.

Methods

We identified a prospective cohort of 248 hospitalized patients with positive P. aeruginosa cultures. Clinical data were analyzed to determine whether an individual met predefined criteria for infection versus colonization. P. aeruginosa isolates were tested for the expression of multiple phenotypes previously associated with virulence in animal models and humans. Logistic regression models were constructed to determine the degree of association between host and bacterial factors with P. aeruginosa infection of the bloodstream, lung, soft tissue and urinary tract.

Results

One host factor (i.e. diabetes mellitus), and one bacterial factor, a Type 3 secretion system positive phenotype, were significantly associated with P. aeruginosa infection in our cohort. Subgroup analysis of patients with P. aeruginosa isolated from the urinary tract revealed that the presence of a urinary tract catheter or stent was an additional factor for P. aeruginosa infection.

Conclusions

Among hospitalized patients with culture-documented P. aeruginosa, infection is more likely to be present in those with diabetes mellitus and those harboring a Type 3 secretion positive bacterial strain.     

Plasmacytoid Dendritic Cells Mediate the Regulation of Inflammatory Type T Cell Response for Optimal Immunity against Respiratory Chlamydia Pneumoniae Infection

Chlamydia pneumoniae (Cpn) infection is a leading cause for a variety of respiratory diseases and has been implicated in the pathogenesis of chronic inflammatory diseases. The regulatory mechanisms in host defense against Cpn infection are less understood. In this study, we investigated the role of plasmacytoid dendritic cells (pDCs) in immune regulation in Cpn respiratory tract infection. We found that in vivo depletion of pDCs increased the severity of infection and lung pathology. Mice depleted of pDC had greater body weight loss, higher lung bacterial burden and excessive tissue inflammation compared to the control mice. Analysis of specific T cell cytokine production pattern in the lung following Cpn infection revealed that pDC depleted mice produced significantly higher amounts of inflammatory cytokines, especially TNF-α, but lower IL-10 compared to the controls. In particular, pDC depleted mice showed pathogenic T cell responses characterized by inflammatory type-1 (CD8 and CD4) and inflammatory Th2 cell responses. Moreover, pDC depletion dramatically reduced CD4 regulatory T cells (Tregs) in the lungs and draining lymph nodes. Furthermore, pDC-T cell co-culture experiments showed that pDCs isolated from Cpn infected mice were potent in inducing IL-10 producing CD4 Tregs. Together, these findings provide in vivo evidence for a critical role of pDCs in homeostatic regulation of immunity during Cpn infection. Our findings highlight the importance of a ‘balanced’ immune response for host protective immunity and preventing detrimental immunopathology during microbial infections.     

Effect of Long-Term Voluntary Exercise Wheel Running on Susceptibility to Bacterial Pulmonary Infections in a Mouse Model

Regular moderate exercise has been suggested to exert anti-inflammatory effects and improve immune effector functions, resulting in reduced disease incidence and viral infection susceptibility. Whether regular exercise also affects bacterial infection susceptibility is unknown. The aim of this study was to investigate whether regular voluntary exercise wheel running prior to a pulmonary infection with bacteria (P. aeruginosa) affects lung bacteriology, sickness severity and phagocyte immune function in mice. Balb/c mice were randomly placed in a cage with or without a running wheel. After 28 days, mice were intranasally infected with P. aeruginosa. Our study showed that regular exercise resulted in a higher sickness severity score and bacterial (P. aeruginosa) loads in the lungs. The phagocytic capacity of monocytes and neutrophils from spleen and lungs was not affected. Although regular moderate exercise has many health benefits, healthy mice showed increased bacterial (P. aeruginosa) load and symptoms, after regular voluntary exercise, with perseverance of the phagocytic capacity of monocytes and neutrophils. Whether patients, suffering from bacterial infectious diseases, should be encouraged to engage in exercise and physical activities with caution requires further research.     

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background

We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE.

Methodology/Principal Findings

Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice.

Conclusions

Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.     

Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion

The diaminopimelic acid pathway of lysine biosynthesis has been suggested to provide attractive targets for the development of novel antibacterial drugs. Here we report the characterization of two enzymes from this pathway in the human pathogen Pseudomonas aeruginosa, utilizing structural biology, biochemistry and genetics. We show that tetrahydrodipicolinate N-succinyltransferase (DapD) from P. aeruginosa is specific for the L-stereoisomer of the amino substrate L-2-aminopimelate, and its D-enantiomer acts as a weak inhibitor. The crystal structures of this enzyme with L-2-aminopimelate and D-2-aminopimelate, respectively, reveal that both compounds bind at the same site of the enzyme. Comparison of the binding interactions of these ligands in the enzyme active site suggests misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition. P. aeruginosa mutants where the dapA gene had been deleted were viable and able to grow in a mouse lung infection model, suggesting that DapA is not an optimal target for drug development against this organism. Structure-based sequence alignments, based on the DapA crystal structure determined to 1.6 Å resolution revealed the presence of two homologues, PA0223 and PA4188, in P. aeruginosa that could substitute for DapA in the P. aeruginosa PAO1ΔdapA mutant. In vitro experiments using recombinant PA0223 protein could however not detect any DapA activity.     

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.     

Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.     

Autophagy Enhances Bacterial Clearance during P. aeruginosa Lung Infection

Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.     

Impairment in inflammasome signaling by the chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients results in an increase in inflammatory response

Pseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1β by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1β expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.Subject terms: Cell death, Immune cell death