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Pseudomonas aeruginosa(P.a) infections are a major public health issue in ventilator-associated pneumoniae, cystic fibrosis, and chronic obstructive pulmonary disease exacerbations. P.ais multidrug resistant, and there is an urgent need to develop new therapeutic approaches. Here, we evaluated the effect of direct pulmonary transplantation of gene-modified (elafin and interleukin [IL]-6) syngeneic macrophages in a mouse model of acute P.ainfection. Wild-type (WT) or Elafin-transgenic (eTg) alveolar macrophages (AMs) or bone marrow-derived macrophages (BMDMs) were recovered from bronchoalveolar lavage or generated from WT or eTg mouse bone marrow. Cells were modified with adenovirus IL-6 (Ad-IL-6), characterized in vitro, and transferred by oropharyngeal instillation in the lungs of naive mice. The protective effect was assessed during P.aacute infection (survival studies, mechanistic studies of the inflammatory response). We show that a single bolus of genetically modified syngeneic AMs or BMDMs provided protection in our P.a-induced model. Mechanistically, Elafin-modified AMs had an IL-6-IL-10-IL-4R-IL-22-antimicrobial molecular signature that, in synergy with IL-6, enhanced epithelial cell proliferation and tissue repair in the alveolar unit. We believe that this innovative cell therapy strategy could be of value in acute bacterial infections in the lung.

Silver nanoparticles (AgNPs) are microbicidal agents which could be potentially used as an alternative to antivirals to treat human infectious diseases, especially influenza virus infections where antivirals have generally proven unsuccessful. However, concerns about the use of AgNPs on humans arise from their potential toxicity, although mechanisms are not well-understood. We show here, in the context of an influenza virus infection of lung epithelial cells, that AgNPs down-regulated influenza induced CCL-5 and -IFN-β release (two cytokines important in antiviral immunity) through RIG-I inhibition, while enhancing IL-8 production, a cytokine important for mobilizing host antibacterial responses. AgNPs activity was independent of coating and was not observed with gold nanoparticles. Down-stream analysis indicated that AgNPs disorganized the mitochondrial network and prevented the antiviral IRF-7 transcription factor influx into the nucleus. Importantly, we showed that the modulation of RIG-I-IRF-7 pathway was concomitant with inhibition of either classical or alternative autophagy (ATG-5- and Rab-9 dependent, respectively), depending on the epithelial cell type used. Altogether, this demonstration of a AgNPs-mediated functional dichotomy (down-regulation of IFN-dependent antiviral responses and up-regulation of IL-8-dependent antibacterial responses) may have practical implications for their use in the clinic.

BackgroundPseudomonas aeruginosalung infections are a huge problem in ventilator-associated pneumonia, cystic fibrosis (CF) and in chronic obstructive pulmonary disease (COPD) exacerbations. This bacterium secretes virulence factors that may subvert host innate immunity.ObjectiveWe evaluated the effect of P. aeruginosaelastase LasB, an important virulence factor secreted by the type II secretion system, on ion transport, innate immune responses and epithelial repair, both in vitro and in vivo.MethodsWild-type (WT) or cystic fibrosis transmembrane conductance regulator (CFTR)-mutated epithelial cells (cell lines and primary cells from patients) were treated with WT or ΔLasB pseudomonas aeruginosa O1 (PAO1) secretomes. The effect of LasB and PAO1 infection was also assessed in vivo in murine models.ResultsWe showed that LasB was the most abundant protein in WT PAO1 secretomes and that it decreased epithelial CFTR expression and activity. In airway epithelial cell lines and primary bronchial epithelial cells, LasB degraded the immune mediators interleukin (IL)-6 and trappin-2, an important epithelial-derived antimicrobial molecule. We further showed that an IL-6/STAT3 signalling pathway was downregulated by LasB, resulting in inhibition of epithelial cell repair. In mice, intranasally instillated LasB induced significant weight loss, inflammation, injury and death. By contrast, we showed that overexpression of IL-6 and trappin-2 protected mice against WT-PAO1-induced death, by upregulating IL-17/IL-22 antimicrobial and repair pathways.ConclusionsOur data demonstrate that PAO1 LasB is a major P. aeruginosasecreted factor that modulates ion transport, immune response and tissue repair. Targeting this virulence factor or upregulating protective factors such as IL-6 or antimicrobial molecules such as trappin-2 could be beneficial in P. aeruginosa-infected individuals.

Surfactant protein C (SP-C) consists of a hydrophobic α-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC(1-13)) were studied. Binding studies showed that SPC(1-13)binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-α by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-α in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.

Surfactant protein A (SP-A) is known to cause bacterial permeabilization. The aim of this work was to gain insight into the mechanism by which SP-A induces permeabilization of rough lipopolysaccharide (Re-LPS) membranes. In the presence of calcium, large interconnected aggregates of fluorescently labeled TR-SP-A were observed on the surface of Re-LPS films by epifluorescence microscopy. Using Re-LPS monolayer relaxation experiments at constant surface pressure, we demonstrated that SP-A induced Re-LPS molecular loss by promoting the formation of three-dimensional lipid-protein aggregates in Re-LPS membranes. This resulted in decreased van der Waals interactions between Re-LPS acyl chains, as determined by differential scanning calorimetry, which rendered the membrane leaky. We also showed that the coexistence of gel and fluid lipid phases within the Re-LPS membrane conferred susceptibility to SP-A-mediated permeabilization. Taken together, our results seem to indicate that the calcium-dependent permeabilization of Re-LPS membranes by SP-A is related to the extraction of LPS molecules from the membrane due to the formation of calcium-mediated protein aggregates that contain LPS.

The present study investigated the ability of surfactant associated protein A1 (SFTPA1), a major component of lung surfactant, to bind and serve as a signal in human cultured myometrial cells. By using ligand blot analysis with 125I-SFTPA1, we consistently identified two myometrial SFTPA1 interacting proteins (55 and 200 kDa). We found that the SFTPA1 immunoreactive protein was present in myometrial cells. We also showed by indirect immunofluorescence the nuclear translocation of RELA (also known as NFkappaB p65 subunit) after activation of myometrial cells by SFTPA1. Neutralization of TLR4 did not reverse this effect. Moreover, SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) and protein kinase C zeta (PRKCZ). The prolonged treatment of myometrial cells with SFTPA1 upregulated PTGS2 (COX2) protein levels. We next evaluated whether SFTPA1 affected the actin dynamic. Stimulation of myometrial cells with SFTPA1 markedly enhanced the intensity of the filamentous-actin pool stained with fluorescein isothiocyanate-phalloidin. Inhibition of PRKC or Rho-associated, coiled-coil containing protein kinase 1 (ROCK) reduced the SFTPA1-mediated stress fiber formation. Our data support the hypothesis that human myometrial cells express functional SFTPA1 binding sites and respond to SFTPA1 to initiate activation of signaling events related to human parturition.

Due to the inhalation of airborne particles containing bacterial lipopolysaccharide (LPS), these molecules might incorporate into the 1,2-dipalmitoylphosphatidylcholine (DPPC)-rich monolayer and interact with surfactant protein A (SP-A), the major surfactant protein component involved in host defense. In this study, epifluorescence microscopy combined with a surface balance was used to examine the interaction of SP-A with mixed monolayers of DPPC/rough LPS (Re-LPS). Binary monolayers of Re-LPS plus DPPC showed negative deviations from ideal behavior of the mean areas in the films consistent with partial miscibility and attractive interaction between the lipids. This interaction resulted in rearrangement and reduction of the size of DPPC-rich solid domains in DPPC/Re-LPS monolayers. The adsorption of SP-A to these monolayers caused expansion in the lipid molecular areas. SP-A interacted strongly with Re-LPS and promoted the formation of DPPC-rich solid domains. Fluorescently labeled Texas red-SP-A accumulated at the fluid-solid boundary regions and formed networks of interconnected filaments in the fluid phase of DPPC/Re-LPS monolayers in a Ca2+-independent manner. These lattice-like structures were also observed when TR-SP-A interacted with lipid A monolayers. These novel results deepen our understanding of the specific interaction of SP-A with the lipid A moiety of bacterial LPS.

Surfactant protein A (SFTPA1), a member of the collagenous lectin (collectin) family, was first described as a major constituent of lung surfactant, but has recently also been found in the female genital tract. Various microorganisms colonize this area and may cause intrauterine infection or trigger preterm labor. We found that SFTPA1 was not produced in the uterus. Instead, it was immunodetected transiently in rat myometrium at the end (Days 19 and 21) of gestation, but not postpartum, and in cultured myometrial cells. Fluorescence microscopy showed that Texas Red-labeled SFTPA1 bound to myometrial cells. This result was confirmed by biochemical approaches. [125I]-SFTPA1 bound to two myometrial cell proteins (55 and 210 kDa). This interaction was dependent on the integrity of the collagenlike domain of SFTPA1. SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) in myometrial cells. Bacterial lipopolysaccharide (LPS), an agent known to trigger uterine contractions and preterm birth, also activated MAPK1/3. The prolonged treatment of myometrial cells with LPS or SFTPA1 upregulated PTGS2 (COX2) protein levels. The addition of rough-type LPS to SFTPA1 blocked the interaction of SFTPA1 with its binding sites and the activation of MAPK1/3 and PTGS2 by SFTPA1. Our data provide the first demonstration of a direct effect of SFTPA1 on rat myometrial cells and inhibitory cross talk between SFTPA1 and LPS signals, providing new insight into the mechanisms of normal and preterm parturition.

SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immunity. SP-A interacts with the bacterial rough LPS (lipopolysaccharide) Re-LPS (Re595 mutant of LPS from Salmonella minnesota), but not with smooth LPS. In the present study, we first examined the characteristics of the interaction of human SP-A with Re-LPS. Fluorescence intensity and anisotropy measurements of FITC-labelled Re-LPS in the presence and absence of SP-A indicated that SP-A bound to Re-LPS in solution in a Ca2+-independent manner, with a dissociation constant of 2.8×10−8 M. In the presence of calcium, a high-mobility complex of SP-A and [3H]Rb-LPS (Rb mutant of LPS from Escherichia coli strain LCD 25) micelles was formed, as detected by sucrose density gradients. Re-LPS aggregation induced by SP-A was further characterized by light scattering. On the other hand, human SP-A inhibited TNF-α (tumour necrosis factor-α) secretion by human macrophage-like U937 cells stimulated with either Re-LPS or smooth LPS. We further examined the effects of human SP-A on the binding of Re-LPS to LBP (LPS-binding protein) and CD14. SP-A decreased the binding of Re-LPS to CD14, but not to LBP, as detected by cross-linking experiments with 125I-ASD-Re-LPS [125I-labelled sulphosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Re-LPS] and fluorescence analysis with FITC-Re-LPS. When SP-A, LBP and CD14 were incubated together, SP-A reduced the ability of LBP to transfer 125I-ASD-Re-LPS to CD14. These SP-A effects were not due to the ability of SP-A to aggregate Re-LPS in the presence of calcium, since they were observed in both the absence and the presence of calcium. These studies suggest that SP-A could contribute to modulate Re-LPS responses by altering the competence of the LBP–CD14 receptor complex.

Influenza virus infection leads to severe and complicated disease, particularly in patients with lung cancer. It alters the tumor microenvironment (TME), which may potentiate lung cancer progression and disrupt responses to antitumoral treatments. Consequently, influenza vaccination and antiviral treatments should be recommended to all patients with lung cancer.