Isolation and characterization of quorum‐sensing signalling molecules in Pseudomonas aeruginosa isolates recovered from nosocomial infections

Pseudomonas aeruginosa is one of the most common pathogens in nosocomial infections. Many studies have documented the role of quorum‐sensing (QS) systems in antibiotic tolerance of P. aeruginosa. N‐acyl homoserine lactones (AHLs) serve as QS signalling molecules and can be a target for modulating bacterial pathogenicity. In this study, nosocomial isolates of P. aeruginosa were characterized for the presence of different types of QS signalling molecules. AHLs were solvent extracted and quantified by determination of β‐galactosidase activity using the Escherichia coli MG4 reporter strain. Further characterization was performed by analytical thin layer chromatography coupled with detection using the Agrobacterium tumefaciens A136 biosensor strain. All P. aeruginosa isolates produced AHLs, but there were differences in the quantity and nature of AHLs. We identified AHLs belonging to C4‐homoserine lactone (HSL), C6‐HSL, C8‐HSL, C10‐HSL and C12‐HSL. AHL profiling of P. aeruginosa isolates showed differences in the amounts and types of AHLs, suggesting differences in the virulence factors and the potential for infection. Our results may be investigated further using animal model systems.     

Effect of plant phenolic compounds on biofilm formation by Pseudomonas aeruginosa

In the natural environment, bacteria predominantly exist in matrix‐enclosed multicellular communities associated with various surfaces, referred to as biofilms. Bacteria in biofilms are extremely resistant to antibacterial agents thus causing serious problems for antimicrobial therapy. In this study, we showed that different plant phenolic compounds, at concentrations that did not or weakly suppressed bacterial growth, increased the capacity of Pseudomonas aeruginosa PAO1 to form biofilms. Biofilm formation of P. aeruginosa PAO1 was enhanced 3‐ to 7‐fold under the action of vanillin and epicatechin, and 2‐ to 2.5‐fold in the presence of 4‐hydroxybenzoic, gallic, cinnamic, sinapic, ferulic, and chlorogenic acids. At higher concentrations, these compounds displayed an inhibiting effect. Similar experiments carried out for comparison with Agrobacterium tumefaciens C58 showed the same pattern. Vanillin, 4‐hydroxybenzoic, and gallic acids at concentrations within the range of 40 to 400 μg/mL increased the production of N–3‐oxo‐dodecanoyl‐homoserine lactone in P. aeruginosa PAO1 which suggests a possible relationship between stimulation of biofilm formation and Las Quorum Sensing system of this bacterium. Using biosensors to detect N‐acyl‐homoserine lactones (AHL), we demonstrated that the plant phenolics studied did not mimic AHLs.     

Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant–microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.     

N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa

In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37 degrees C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.     

Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells.

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.     

Effects of bacterial N-acyl homoserine lactones on human Jurkat T lymphocytes-OdDHL induces apoptosis via the mitochondrial pathway

Diverse Gram-negative bacteria communicate with each other by using diffusible N-acyl-homoserine lactone (AHL) signaling molecules to coordinate gene expression with cell population density. This mechanism termed ‘quorum sensing’ is involved in the regulation of physiological functions as well as multiple virulence determinants. It becomes more and more evident, that bacteria communicate not only with each other but also with their host. Up to now, little is known about this interkingdom communication. The AHL quorum sensing molecule N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL) from Pseudomonas aeruginosa has been shown to influence the immune system of the host. The role and potential influence of other AHL molecules from other bacteria have so far not been determined. In this paper, we investigated the role of 7 different AHLs on apoptosis of human Jurkat T lymphocytes. We found, that among all homoserine lactones tested, only OdDHL rapidly induced apoptosis which was accompanied by the breakdown of the mitochondrial transmembrane potential (ΔΨ_m). Since overexpression of anti-apoptotic Bcl-2 completely abrogated the apoptotic effect, we presume that OdDHL induces apoptosis by activation of the intrinsic mitochondrial apoptosis pathway. The reason that bacteria induce apoptosis is largely unknown. We suspect that through apoptosis an anti-inflammatory response is triggered.     

Occurrence of diversified N‐acyl homoserine lactone mediated biofilm‐forming bacteria in rice rhizoplane

Quorum sensing (QS)‐mediated biofilm‐forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere. In the present work, we have reported on the occurrence of diversified bacterial species capable of producing N‐acyl homoserine lactone (AHL) as the QS signal in the roots of a rice plant grown under field conditions. The AHL‐producing bacteria were directly isolated from the rice root by the biosensor reporter (Chromobacterium violaceum CV026) overlay method and characterized for biofilm production by the microtiter plate method. A total of 48 QS‐positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings. The in vitro biofilm production and genetic diversity as revealed by BOX‐PCR fingerprinting showed high variability among the isolates. Most of the best biofilm‐forming isolates produced a N‐butyryl dl ‐homoserine lactone (a C4‐AHL type) signal in the medium. The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7‐4; QS36‐2), A. enteropelongenes (QS20‐8), A. veronii (QS36‐3), Enterobacter sp. (QS20‐11), Klebsiella pneumoniae (QS24‐6), Kosakonia cowanii (QS24‐21), Providentia rettigeri (QS24‐2), Sphingomonas aquatilis (QS24‐17), and Pseudomonas sihuiensis (QS24‐20). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions. Developing inoculants from these strains would ensure competitive colonization on the rhizoplane of the crop through their biofilm and thereby improve plant growth and health.     

Response of leaf-associated bacterial communities to primary acyl-homoserine lactone in the tobacco phyllosphere

The phyllosphere is inhabited by large populations of epiphytic bacteria that are able to modulate their phenotypes and behavior by quorum sensing (QS). However, the impact of acyl-homoserine lactones (AHLs) involved in QS on the ecology of bacteria in their natural habitat remains unclear. Therefore, we used a bioassay and liquid chromatography-mass spectrometry to detect AHLs in the tobacco phyllosphere. Our results identified several AHLs in the tobacco phyllosphere, the majority of which were short-chain AHLs. Furthermore, the addition of an exogenous N-(3-oxohexanoyl) homoserine lactone (3OC6HSL), which is seen in the naturally occurring tobacco phyllosphere, generated variability in the composition of the bacterial community as determined by denaturing gradient gel electrophoresis (DGGE) analysis and phospholipid fatty acid (PLFA) analysis. Notably, the ratio of Gram-positive (GP) bacteria increased in response to treatment with 1 μM AHL, but decreased incipiently when treated with 10 μM AHL. These observations provide insight into the composition of the leaf-colonizing epiphyte community responsible for AHLs, particularly GP bacteria as they do not use AHLs as signaling molecules for QS.     

Effects of various anti-asthmatic agents on mite allergen-pulsed murine bone marrow-derived dendritic cells

Background Dendritic cells (DCs) play an important role in the immune response and are critically involved in asthma. β₂‐agonists could potentially exacerbate type 2 T helper (Th2) cell‐mediated immune response. Objectives To determine the effects of various anti‐asthmatic agents on DCs function both in vitro and in vivo. Methods Murine bone marrow‐derived DCs were pulsed with mite allergen in the presence of pranlukast, salbutamol, salmeterol or fluticasone. These DCs were then inoculated intranasally into naïve mice to induce allergic airway inflammation in vivo. Results Pranlukast reduced IL‐10 and increased IL‐12, while fluticasone reduced both IL‐10 and IL‐12 production by mite allergen‐pulsed DCs. Allergic airway inflammation in pranlukast‐ and fluticasone‐treated and mite allergen pulsed DCs‐harbouring mice was attenuated and such response was associated with inhibition of Th2 response in the airway. Salbutamol did not alter cytokine production, while salmeterol reduced IL‐12 production by mite allergen‐pulsed DCs. Lung pathology in β₂‐agonist‐harbouring mice was comparable with those of mite allergen‐pulsed DCs‐harbouring mice. Conclusions Our results indicate that leukotriene receptor antagonists and corticosteroids inhibit DCs‐induced Th2 skewed immune response, and that short‐ and long‐acting β₂‐agonists do not modify DCs‐induced allergic airway inflammation.     

Exogenous Yersinia pestis quorum sensing molecules N-octanoyl-homoserine lactone and N-(3-oxooctanoyl)-homoserine lactone regulate the LcrV virulence factor

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y. pestis cultures down-regulates LcrV protein expression. DNA microarray analysis shows 10 additional T3SS genes are consistently down-regulated by C8 or oxo-C8. This is the first report demonstrating that AHLs regulate Y. pestis virulence factor expression.     

Mucosal B cells: phenotypic characteristics, transcriptional regulation, and homing properties

Summary: Mucosal antibody defense depends on a complex cooperation between local B cells and secretory epithelia. Mucosa‐associated lymphoid tissue gives rise to B cells with striking J‐chain expression that are seeded to secretory effector sites. Such preferential homing constitutes the biological basis for local production of polymeric immunoglobulin A (pIgA) and pentameric IgM with high affinity to the epithelial pIg receptor that readily can export these antibodies to the mucosal surface. This ultimate functional goal of mucosal B‐cell differentiation appears to explain why the J chain is also expressed by IgG‐ and IgD‐producing plasma cells (PCs) occurring at secretory tissue sites; these immunocytes may be considered as ‘spin‐offs’ from early effector clones that through class switch are on their way to pIgA production. Abundant evidence supports the notion that intestinal PCs are largely derived from B cells initially activated in gut‐associated lymphoid tissue (GALT). Nevertheless, insufficient knowledge exists concerning the relative importance of M cells, major histocompatibility complex class II‐expressing epithelial cells, and professional antigen‐presenting cells for the uptake, processing, and presentation of luminal antigens in GALT to accomplish the extensive and sustained priming and expansion of mucosal B cells. Likewise, it is unclear how the germinal center reaction in GALT so strikingly can promote class switch to IgA and expression of J chain. Although B‐cell migration from GALT to the intestinal lamina propria is guided by rather well‐defined adhesion molecules and chemokines/chemokine receptors, the cues directing preferential homing to different segments of the gut require better definition. This is even more so for the molecules involved in homing of mucosal B cells to secretory effector sites beyond the gut, and in this respect, the role of Waldever's ring (including the palatine tonsils and adenoids) as a regional inductive tissue needs further characterization. Data suggest a remarkable compartmentalization of the mucosal immune system that must be taken into account in the development of effective local vaccines to protect specifically the airways, eyes, oral cavity, small and large intestines, and urogenital tract.     

Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1

The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3' position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.     

Effects of topical platelet activating factor on the guinea-pig tracheobronchial mucosa in vivo

Platelet activating factor (PAF) has been reported to produce a variety of airway effects including epithelial damage and increased airway-lung absorption of hydrophilic tracers. The present study examines effects of PAF on the guinea-pig tracheobronchial mucosa in vivo. Vehicle with and without PAF (4.0 and 8.0 nmol) was superfused onto the tracheobronchial mucosa. The levels of ¹²⁵I-albumin, previously given intravenously, were determined in tracheobronchial lavage fluids as an index of mucosal exudation of plasma. The mucosa was also examined by scanning electron microscopy. In separate animals, ^99mTc-DTPA (a low molecular weight, 492 Da, hydrophilic tracer) was superfused onto the mucosal surface through an oro-tracheal catheter, together with vehicle or PAF (8.0 nmol). A gamma camera determined the disappearance rate of ^99mTc-DTPA from the airways as an index of mucosal absorption. PAF produced dose-dependent mucosal exudation of plasma up to 20-fold greater than control (P < 0.001). However, PAF did not damage the epithelium and the absorption ability of the airway mucosa was unaffected. The results, in contrast to previous reports, suggest that PAF may not readily damage the airway mucosa even at large exudative doses of the agent. The present finding support the view that the plasticity of the epithelial junctions allows the creation of valve-like paracellular pathways for unidirectional clearance of extravasated plasma into the airway lumen. We suggest that endogenous PAF may participate in first line respiratory defence reactions by causing lumenal entry of bulk plasma without harming the epithelium.     

TPI ASM8 reduces eosinophil progenitors in sputum after allergen challenge

Background TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides (AON), one targeting the common beta chain (βc) of the IL‐3/IL‐5/GM‐CSF receptors and the other targeting the chemokine receptor CCR3. Inhalation of TPI ASM8 significantly improves lung function and sputum eosinophilia after allergen inhalation challenge in asthmatics. Objective This study assessed whether TPI ASM8 reduces airway levels of haemopoietic progenitor cells. Methods This open‐label study was conducted in 14 stable, allergic mild asthmatic subjects with early‐ and late‐phase allergen‐induced bronchoconstriction. Subjects underwent allergen challenges after 4‐day treatment with placebo, 4 mg b.i.d. and 8 mg o.d. of TPI ASM8. Sputum was induced before, 7 and 24 h after allergen challenges for progenitor measurements. Treatments were separated by 2–3 weeks. Results TPI ASM8 reduced allergen‐induced sputum eosinophils, and the early and late asthmatic responses (P<0.05). TPI ASM8 also reduced the number of CD34⁺CCR3⁺ cells (P=0.004) and CD34⁺IL‐5Rα⁺ cells (P=0.016), and the proportion of CD34⁺ cells expressing IL‐5Rα (P=0.036). Conclusions and Clinical Relevance TPI ASM8 was safe and well tolerated. The results of this study demonstrate blocking of CCR3 and βc expression by TPI ASM8 significantly inhibits the accumulation of eosinophils and eosinophil progenitors in the airways after allergen challenge. Inhibition of airway progenitor cell accumulation presents a novel therapeutic target. Cite this as: H. Imaoka, H. Campbell, I. Babirad, R. M. Watson, M. Mistry, R. Sehmi and G. M. Gauvreau, Clinical & Experimental Allergy, 2011 (41) 1740–1746.     

Effects of hydrogen peroxide on the guinea-pig tracheobronchial mucosa in vivo

Lumenal entry of plasma (mucosal exudation) is a key feature of airway inflammation. In airways challenged with histamine-type mediators and allergen the mucosal exudation response occurs without causing epithelial derangement and without increased airway absorption. In contrast, reactive oxygen metabolites may cause mucosal damage. In this study, involving guinea-pig airways, we have examined effects of H₂O₂ on airway exudation and absorption in vivo. Vehicle or H₂O₂ (0.1 and 0.5 M ) was superfused onto the tracheobronchial mucosal surface through an oro-tracheal catheter. ¹²⁵I-albumin, given intravenously, was determined in tracheobronchial tissue and in lavage fluids 10 min after challenge as an index of mucosal exudation of plasma. The tracheobronchial mucosa was also examined by scanning electron microscopy. In separate animals, ^99mTc-DTPA was superfused 20 min after vehicle or H₂O₂ (0.1 and 0.5 M ) had been given. A gamma camera determined the disappearance rate of ^99mTc-DTPA from the airways as an index of airway absorption. The high dose of H₂O₂ (0.5 M ) produced epithelial damage, increased the absorption of ^99mTc-DTPA (P < 0.001), and increased the exudation of plasma (P < 0.001). Notably, it appeared that all extravasated plasma had entered the airway lumen within 10 min. These data demonstrate that H₂O₂ differs from exudative autacoids such as histamine by causing both epithelial damage and plasma exudation responses. These data also agree with the view that the epithelial lining determines the rate of absorption and is responsible for the valve-like function that allows lumenal entry of extravasated bulk plasma without any increased inward perviousness.     

Expression of toll‐like receptors in T lymphocytes stimulated with N‐(3‐oxododecanoyl)‐L‐homoserine lactone from Pseudomonas aeruginosa

The establishment of chronic Pseudomonas aeruginosa infections is correlated with the disturbance of the host immune system. The P. aeruginosa quorum‐sensing molecule N‐3‐(oxododecanoyl)‐L‐homoserine lactone (3‐O‐C12‐HSL) has the potential to modulate the host immune system. The immune system recognizes pathogens via toll‐like receptors (TLRs). We found that 3‐O‐C12‐HSL induced TLR changes in monocytes. However, the role of T cells in P. aeruginosa infection has not been delineated. In order to understand this activity, we examined whether 3‐O‐C12‐HSL has an effect on the immune function and the expression of TLRs in T lymphocytes. Human peripheral blood mononuclear cells (PBMCs) cells were cultured with 0, 1, 10, 50, or 100 μM 3‐O‐C12‐HSL for 12 h. TLR2/TLR4 expression and T‐lymphocyte proliferation were increased in a dose‐dependent manner, and 100 μM 3‐O‐C12‐HSL significantly increased TLR2 expression. Moreover, tumor necrosis factor‐α production of these PBMCs was inhibited. To conclude, 3‐O‐C12‐HSL can induce lymphocyte cell proliferation. These findings provide a new perspective on our understanding of the persistence of the chronic inflammation that accompanies P. aeruginosa infection.     

Airway remodelling and inflammation in asthma are dependent on the extracellular matrix protein fibulin‐1c

Asthma is a chronic inflammatory disease of the airways. It is characterized by allergic airway inflammation, airway remodelling, and airway hyperresponsiveness (AHR). Asthma patients, in particular those with chronic or severe asthma, have airway remodelling that is associated with the accumulation of extracellular matrix (ECM) proteins, such as collagens. Fibulin‐1 (Fbln1) is an important ECM protein that stabilizes collagen and other ECM proteins. The level of Fbln1c, one of the four Fbln1 variants, which predominates in both humans and mice, is increased in the serum and airways fluids in asthma but its function is unclear. We show that the level of Fbln1c was increased in the lungs of mice with house dust mite (HDM)‐induced chronic allergic airway disease (AAD). Genetic deletion of Fbln1c and therapeutic inhibition of Fbln1c in mice with chronic AAD reduced airway collagen deposition, and protected against AHR. Fbln1c‐deficient (Fbln1c^–/–) mice had reduced mucin (MUC) 5 AC levels, but not MUC5B levels, in the airways as compared with wild‐type (WT) mice. Fbln1c interacted with fibronectin and periostin that was linked to collagen deposition around the small airways. Fbln1c^–/– mice with AAD also had reduced numbers of α‐smooth muscle actin‐positive cells around the airways and reduced airway contractility as compared with WT mice. After HDM challenge, these mice also had fewer airway inflammatory cells, reduced interleukin (IL)‐5, IL‐13, IL‐33, tumour necrosis factor (TNF) and CXCL1 levels in the lungs, and reduced IL‐5, IL‐33 and TNF levels in lung‐draining lymph nodes. Therapeutic targeting of Fbln1c reduced the numbers of GATA3‐positive Th2 cells in the lymph nodes and lungs after chronic HDM challenge. Treatment also reduced the secretion of IL‐5 and IL‐13 from co‐cultured dendritic cells and T cells restimulated with HDM extract. Human epithelial cells cultured with Fbln1c peptide produced more CXCL1 mRNA than medium‐treated controls. Our data show that Fbln1c may be a therapeutic target in chronic asthma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunomodulatory and protective roles of quorum-sensing signaling molecules N-acyl homoserine lactones during infection of mice with Aeromonas hydrophila

Aeromonas hydrophila leads to both intestinal and extraintestinal infections in animals and humans, and the underlying mechanisms leading to mortality are largely unknown. By using a septicemic mouse model of infection, we showed that animals challenged with A. hydrophila die because of kidney and liver damage, hypoglycemia, and thrombocytopenia. Pretreatment of animals with quorum-sensing-associated signaling molecules N-acyl homoserine lactones (AHLs), such as butanoyl and hexanoyl homoserine lactones (C(4)- and C(6)-HSLs), as well as N-3-oxododecanoyl (3-oxo-C(12))-HSL, prevented clinical sequelae, resulting in increased survivability of mice. Since little is known as to how different AHLs modulate the immune response during infection, we treated mice with the above AHLs prior to lethal A. hydrophila infection. When we compared results in such animals to those in controls, the treated animals exhibited a significantly reduced bacterial load in the blood and other mouse organs, as well as various levels of cytokines/chemokines. Importantly, neutrophil numbers were significantly elevated in the blood of C(6)-HSL-treated mice compared to those in animals given phosphate-buffered saline and then infected with the bacteria. These findings coincided with the fact that neutropenic animals were more susceptible to A. hydrophila infection than normal mice. Our data suggested that neutrophils quickly cleared bacteria by either phagocytosis or possibly another mechanism(s) during infection. In a parallel study, we indeed showed that other predominant immune cells inflicted during A. hydrophila infections, such as murine macrophages, when they were pretreated with AHLs, rapidly phagocytosed bacteria, whereas untreated cells phagocytosed fewer bacteria. This study is the first to report that AHL pretreatment modulates the innate immune response in mice and enhances their survivability during A. hydrophila infection.     

SP‐D and regulation of the pulmonary innate immune system in allergic airway changes

The airway mucosal surfaces are constantly exposed to inhaled particles that can be potentially toxic, infectious or allergenic and should elicit inflammatory changes. The proximal and distal air spaces, however, are normally infection and inflammation free due to a specialized interplay between cellular and molecular components of the pulmonary innate immune system. Surfactant protein D (SP‐D) is an epithelial‐cell‐derived immune modulator that belongs to the small family of structurally related Ca²⁺‐dependent C‐type collagen‐like lectins. While collectins can be detected in mucosal surfaces of various organs, SP‐A and SP‐D (the ‘lung collectins’) are constitutively expressed in the lung at high concentrations. Both proteins are considered important players of the pulmonary immune responses. Under normal conditions however, SP‐A‐/‐ mice display no pathological features in the lung. SP‐D‐/‐ mice, on the other hand, show chronic inflammatory alterations indicating a special importance of this molecule in regulating immune homeostasis and the function of the innate immune cells. Recent studies in our laboratory and others implied significant associations between changes in SP‐D levels and the presence of airway inflammation both in animal models and patients raising a potential usefulness of this molecule as a disease biomarker. Research on wild‐type and mutant recombinant molecules in vivo and in vitro showed that SP‐D binds carbohydrates, lipids and nucleic acids with a broad spectrum specificity and initiates phagocytosis of inhaled pathogens as well as apoptotic cells. Investigations on gene‐deficient and conditional over expressor mice in addition, provided evidence that SP‐D directly modulates macrophage and dendritic cell function as well as T cell‐dependent inflammatory events. Thus, SP‐D has a unique, dual functional capacity to induce pathogen elimination on the one hand and control of pro‐inflammatory mechanisms on the other, suggesting a potential suitability for therapeutic prevention and treatment of chronic airway inflammation without compromising the host defence function of the airways. This paper will review recent findings on the mechanisms of immune‐protective function of SP‐D in the lung. Cite this as: L. R. Forbes and A. Haczku, Clinical & Experimental Allergy, 2010 (40) 547–562.