COVID-19 PBMCs are doubly harmful, through LDN-mediated lung epithelial damage and monocytic impaired responsiveness to live Pseudomonas aeruginosa exposure.

Introduction: Although many studies have underscored the importance of T cells, phenotypically and functionally, fewer have studied the functions of myeloid cells in COVID disease. In particular, the potential role of myeloid cells such as monocytes and low-density neutrophils (LDNs) in innate responses and particular in the defense against secondary bacterial infections has been much less documented. Methods: Here, we compared, in a longitudinal study, healthy subjects, idiopathic fibrosis patients, COVID patients who were either hospitalized/moderate (M-) or admitted to ICU (COV-ICU) and patients in ICU hospitalized for other reasons (non-COV-ICU). Results: We show that COVID patients have an increased proportion of low-density neutrophils (LDNs), which produce high levels of proteases (particularly, NE, MMP-8 and MMP-9) (unlike non-COV-ICU patients), which are partly responsible for causing type II alveolar cell damage in co-culture experiments. In addition, we showed that M- and ICU-COVID monocytes had reduced responsiveness towards further live Pseudomonas aeruginosa (PAO1 strain) infection, an important pathogen colonizing COVID patients in ICU, as assessed by an impaired secretion of myeloid cytokines (IL-1, TNF, IL-8,…). By contrast, lymphoid cytokines (in particular type 2/type 3) levels remained high, both basally and post PAO1 infection, as reflected by the unimpaired capacity of T cells to proliferate, when stimulated with anti-CD3/CD28 beads. Discussion: Overall, our results demonstrate that COVID circulatory T cells have a biased type 2/3 phenotype, unconducive to proper anti-viral responses and that myeloid cells have a dual deleterious phenotype, through their LDN-mediated damaging effect on alveolar cells and their impaired responsiveness (monocyte-mediated) towards bacterial pathogens such as P. aeruginosa.

A specific innate immune response silences the virulence of Pseudomonas aeruginosa in a latent infection model in the Drosophila melanogaster host.

Microbial pathogenicity often depends on the route of infection. For instance, P. aeruginosa or S. marcescens cause acute systemic infections when low numbers of bacteria are injected into D. melanogaster flies whereas flies succumb much slower to the continuous ingestion of these pathogens, even though both manage to escape from the gut compartment and reach the hemocoel. Here, we have developed a latent P. aeruginosa infection model by feeding flies on the bacteria for a short period. The bacteria stably colonize internal tissues yet hardly cause any damage since latently-infected flies live almost as long as noninfected control flies. The apparently dormant bacteria display particular characteristics in terms of bacterial colony morphology, composition of the outer cell wall, and motility. The virulence of these bacteria can however be reactivated upon wounding the host. We show that melanization but not the cellular or the systemic humoral response is the predominant host defense that establishes latency and may coerce the bacteria to a dormant state. In addition, the lasting activation of the melanization responses in latently-infected flies provides a degree of protection to the host against a secondary fungal infection. Latent infection by an ingested pathogen protects against a variety of homologous or heterologous systemic secondary infectious challenges, a situation previously described for the endosymbiotic Wolbachia bacteria, a guard against viral infections.

Neutrophils and galectin-3 defend mice from lethal bacterial infection and humans from acute respiratory failure.

Respiratory infection by Pseudomonas aeruginosa, common in hospitalized immunocompromised and immunocompetent ventilated patients, can be life-threatening because of antibiotic resistance. This raises the question of whether the host's immune system can be educated to combat this bacterium. Here we show that prior exposure to a single low dose of lipopolysaccharide (LPS) protects mice from a lethal infection by P. aeruginosa. LPS exposure trained the innate immune system by promoting expansion of neutrophil and interstitial macrophage populations distinguishable from other immune cells with enrichment of gene sets for phagocytosis- and cell-killing-associated genes. The cell-killing gene set in the neutrophil population uniquely expressed Lgals3, which encodes the multifunctional antibacterial protein, galectin-3. Intravital imaging for bacterial phagocytosis, assessment of bacterial killing and neutrophil-associated galectin-3 protein levels together with use of galectin-3-deficient mice collectively highlight neutrophils and galectin-3 as central players in LPS-mediated protection. Patients with acute respiratory failure revealed significantly higher galectin-3 levels in endotracheal aspirates (ETAs) of survivors compared to non-survivors, galectin-3 levels strongly correlating with a neutrophil signature in the ETAs and a prognostically favorable hypoinflammatory plasma biomarker subphenotype. Taken together, our study provides impetus for harnessing the potential of galectin-3-expressing neutrophils to protect from lethal infections and respiratory failure.

Extracellular vesicles released by host epithelial cells during Pseudomonas aeruginosa infection function as homing beacons for neutrophils.

BACKGROUND: Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause sight threatening infections in the eye and fatal infections in the cystic fibrosis airway. Extracellular vesicles (EVs) are released by host cells during infection and by the bacteria themselves; however, there are no studies on the composition and functional role of host-derived EVs during PA infection of the eye or lung. Here we investigated the composition and capacity of EVs released by PA infected epithelial cells to modulate innate immune responses in host cells. METHODS: Human telomerase immortalized corneal epithelial cells (hTCEpi) cells and human telomerase immortalized bronchial epithelial cells (HBECs) were treated with a standard invasive test strain of Pseudomonas aeruginosa, PAO1, for 6 h. Host derived EVs were isolated by qEV size exclusion chromatography. EV proteomic profiles during infection were compared using mass spectrometry and functional studies were carried out using hTCEpi cells, HBECs, differentiated neutrophil-like HL-60 cells, and primary human neutrophils isolated from peripheral blood. RESULTS: EVs released from PA infected corneal epithelial cells increased pro-inflammatory cytokine production in naïve corneal epithelial cells and induced neutrophil chemotaxis independent of cytokine production. The EVs released from PA infected bronchial epithelial cells were also chemotactic although they failed to induce cytokine secretion from naïve HBECs. At the proteomic level, EVs derived from PA infected corneal epithelial cells exhibited lower complexity compared to bronchial epithelial cells, with the latter having reduced protein expression compared to the non-infected control. CONCLUSIONS: This is the first study to comprehensively profile EVs released by corneal and bronchial epithelial cells during Pseudomonas infection. Together, these findings show that EVs released by PA infected corneal and bronchial epithelial cells function as potent mediators of neutrophil migration, contributing to the exuberant neutrophil response that occurs during infection in these tissues.

Pseudomonas aeruginosa two-component system LadS/PA0034 regulates macrophage phagocytosis via fimbrial protein cupA1.

Pseudomonas aeruginosa is one of the most common nosocomial pathogens worldwide, known for its virulence, drug resistance, and elaborate sensor-response network. The primary challenge encountered by pathogens during the initial stages of infection is the immune clearance arising from the host. The resident macrophages of barrier organs serve as the frontline defense against these pathogens. Central to our understanding is the mechanism by which bacteria modify their behavior to circumvent macrophage-mediated clearance, ensuring their persistence and colonization. To successfully evade macrophage-mediated phagocytosis, bacteria must possess an adaptive response mechanism. Two-component systems provide bacteria the agility to navigate diverse environmental challenges, translating external stimuli into cellular adaptive responses. Here, we report that the well-documented histidine kinase, LadS, coupled to a cognate two-component response regulator, PA0034, governs the expression of a vital adhesin called chaperone-usher pathway pilus cupA. The LadS/PA0034 system is susceptible to interference from the reactive oxygen species likely to be produced by macrophages and further lead to a poor adhesive phenotype with scantily cupA pilus, impairing the phagocytosis efficiency of macrophages during acute infection. This dynamic underscores the intriguing interplay: as macrophages deploy reactive oxygen species to combat bacterial invasion, the bacteria recalibrate their exterior to elude these defenses. IMPORTANCE: The notoriety of Pseudomonas aeruginosa is underscored by its virulence, drug resistance, and elaborate sensor-response network. Yet, the mechanisms by which P. aeruginosa maneuvers to escape phagocytosis during acute infections remain elusive. This study pinpoints a two-component response regulator, PA0034, coupled with the histidine kinase LadS, and responds to macrophage-derived reactive oxygen species. The macrophage-derived reactive oxygen species can impair the LadS/PA0034 system, resulting in reduced expression of cupA pilus in the exterior of P. aeruginosa. Since the cupA pilus is an important adhesin of P. aeruginosa, its deficiency reduces bacterial adhesion and changes their behavior to adopt a planktonic lifestyle, subsequently inhibiting the phagocytosis of macrophages by interfering with bacterial adhesion. Briefly, reactive oxygen species may act as environmental cues for the LadS/PA0034 system. Upon recognition, P. aeruginosa may transition to a poorly adhesive state, efficiently avoiding engulfment by macrophages.

Development of a nano-emulsion based multivalent protein subunit vaccine against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

Palmitic acid-capped MIL-101-Al as a nano-adjuvant to amplify immune responses against Pseudomonas aeruginosa.

As a highly contagious opportunistic pathogen, Pseudomonas aeruginosa (P. aeruginosa) is one of the main causes of healthcare-associated infections. The drug-resistant nature of P. aeruginosa can render antibiotic treatments ineffective, leading to a high morbidity and mortality. Higher specificity and reduced toxicity are features of immunotherapy, which can generate robust immune responses and preserve long-term immunological memory to completely eradicate infections. In this study, we developed a type of P. aeruginosa vaccine based on a metal-organic framework. Specifically, MIL-101-Al nanoparticles were synthesized to encapsulate antigens derived from the bacterial lysate (BL) of PAO1, a drug-resistant P. aeruginosa, and the adjuvant unmethylated cytosine-phosphate-guanine oligonucleotide (CpG), which were then modified with palmitic acid (PAA) to obtain MIL-BC@PAA. The stability and biocompatibility were significantly increased by capping with PAA. Moreover, MIL-BC@PAA showed significantly enhanced uptake by antigen presenting cells (APCs), and promoted their maturation. Importantly, immunity studies revealed the greatly elicited antigen-specific humoral and cellular responses, and a protection rate of about 70% was observed in P. aeruginosa-challenged mice. Overall, these results demonstrate the promising potential of MIL-BC@PAA as an ideal nanovaccine for P. aeruginosa vaccination.

Metabolic Modulation-Mediated Antibiotic and Immune Activation for Treatment of Chronic Lung Infections.

The Pseudomonas aeruginosa biofilm in recalcitrant chronic lung infections not only develops high antimicrobial tolerance but also induces an aberrant host inflammatory response. The metabolic condition plays a vital role in both the antimicrobial susceptibility of bacteria and the inflammatory response of immune cells, thereby offering a potential therapeutic target. Herein, we described a metabolic modulation strategy by using ultrasound-responsive liposomal nanoparticles containing a sonosensitizer and a hypoxia-activated prodrug against biofilm-associated chronic lung infections. Under ultrasound stimulation, the sonosensitizer generates antibacterial reactive oxygen species by oxygen consumption. Subsequently, the oxygen consumption-mediated hypoxia not only induces the anaerobic metabolism of bacteria for antibiotic activation but also triggers the glycolysis pathway of immune cells for inflammatory activation. Such metabolic modulation strategy demonstrated efficient therapeutic efficacy for P. aeruginosa biofilm-induced chronic lung infections in mice models and provides a promising way for combating biofilm-associated chronic infections.

Unraveling the immune functions of large yellow croaker Tmem208 in response to Pseudomonas plecoglossicida: Insights from cloning, expression profiling, and transcriptome analysis.

Pseudomonas plecoglossicida, the causative agent of Visceral White Spot Disease, poses substantial risks to large yellow croaker (Larimichthys crocea) aquaculture. Previous genome-wide association studies (GWAS), directed towards elucidating the resistance mechanisms of large yellow croaker against this affliction, suggested that the transmembrane protein 208 (named Lctmem208) may confer a potential advantage. TMEM proteins, particularly TMEM208 located in the endoplasmic reticulum, plays significant roles in autophagy, ER stress, and dynamics of cancer cell. However, research on TMEM's function in teleost fish immunity remains sparse, highlighting a need for further study. This study embarks on a comprehensive examination of LcTmem208, encompassing cloning, molecular characterization, and its dynamics in immune function in response to Pseudomonas plecoglossicida infection. Our findings reveal that LcTmem208 is highly conserved across teleost species, exhibiting pronounced expression in immune-relevant tissues, which escalates significantly upon pathogenic challenge. Transcriptome analysis subsequent to LcTmem208 overexpression in kidney cells unveiled its pivotal role in modulating immune-responsive processes, notably the p53 signaling pathway and cytokine-mediated interactions. Enhanced phagocytic activity in macrophages overexpressing LcTmem208 underscores its importance in innate immunity. Taken together, this is the first time reported the critical involvement of LcTmem208 in regulating innate immune responses of defensing P. plecoglossicida, thereby offering valuable insights into teleost fish immunity and potential strategies for the selective breeding of disease-resistant strains of large yellow croaker in aquaculture practices.

Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells.

Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

Anti-inflammatory roles of type I interferon signaling in the lung.

Excessive or persistent inflammation may have detrimental effects on lung structure and function. Currently, our understanding of conserved host mechanisms that control the inflammatory response remains incompletely understood. In this study, we investigated the role of type I interferon signaling in the inflammatory response against diverse clinically relevant stimuli. Using mice deficient in type I interferon signaling (IFNAR1-/-), we demonstrate that the absence of interferon signaling resulted in a robust and persistent inflammatory response against Pseudomonas aeruginosa, lipopolysaccharide, and chemotherapeutic agent bleomycin. The elevated inflammatory response in IFNAR1-/- mice was manifested as elevated myeloid cells, such as macrophages and neutrophils, in the bronchoalveolar lavage. The inflammatory cell response in the IFNAR1-/- mice persisted to 14 days and there is impaired recovery and fibrotic remodeling of the lung in IFNAR1-/- mice after bleomycin injury. In the Pseudomonas infection model, the elevated inflammatory cell response led to improved bacterial clearance in IFNAR1-/- mice, although there was similar lung injury and survival. We performed RNA sequencing of lung tissue in wild-type and IFNAR1-/- mice after LPS and bleomycin injury. Our unbiased analysis identified differentially expressed genes between IFNAR1-/- and wild-type mice, including previously unknown regulation of nucleotide-binding oligomerization domain (NOD)-like receptor signaling, retinoic acid-inducible gene-I (RIG-I) signaling, and necroptosis pathway by type I interferon signaling in both models. These data provide novel insights into the conserved anti-inflammatory mechanisms of the type I interferon signaling.NEW & NOTEWORTHY Type I interferons are known for their antiviral activities. In this study, we demonstrate a conserved anti-inflammatory role of type I interferon signaling against diverse stimuli in the lung. We show that exacerbated inflammatory response in the absence of type I interferon signaling has both acute and chronic consequences in the lung including structural changes.

Monoclonal antibodies against lipopolysaccharide protect against Pseudomonas aeruginosa challenge in mice.

Pseudomonas aeruginosa is a common cause of hospital-acquired infections, including central line-associated bloodstream infections and ventilator-associated pneumonia. Unfortunately, effective control of these infections can be difficult, in part due to the prevalence of multi-drug resistant strains of P. aeruginosa. There remains a need for novel therapeutic interventions against P. aeruginosa, and the use of monoclonal antibodies (mAb) is a promising alternative strategy to current standard of care treatments such as antibiotics. To develop mAbs against P. aeruginosa, we utilized ammonium metavanadate, which induces cell envelope stress responses and upregulates polysaccharide expression. Mice were immunized with P. aeruginosa grown with ammonium metavanadate and we developed two IgG2b mAbs, WVDC-0357 and WVDC-0496, directed against the O-antigen lipopolysaccharide of P. aeruginosa. Functional assays revealed that WVDC-0357 and WVDC-0496 directly reduced the viability of P. aeruginosa and mediated bacterial agglutination. In a lethal sepsis model of infection, prophylactic treatment of mice with WVDC-0357 and WVDC-0496 at doses as low as 15 mg/kg conferred 100% survival against challenge. In both sepsis and acute pneumonia models of infection, treatment with WVDC-0357 and WVDC-0496 significantly reduced bacterial burden and inflammatory cytokine production post-challenge. Furthermore, histopathological examination of the lungs revealed that WVDC-0357 and WVDC-0496 reduced inflammatory cell infiltration. Overall, our results indicate that mAbs directed against lipopolysaccharide are a promising therapy for the treatment and prevention of P. aeruginosa infections.

Activation of cannabinoid-2 receptor protects against Pseudomonas aeruginosa induced acute lung injury and inflammation.

BACKGROUND: Bacterial pneumonia is a major risk factor for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pseudomonas aeruginosa (PA), an opportunistic pathogen with an increasing resistance acquired against multiple drugs, is one of the main causative agents of ALI and ARDS in diverse clinical settings. Given the anti-inflammatory role of the cannabinoid-2 receptor (CB2R), the effect of CB2R activation in the regulation of PA-induced ALI and inflammation was tested in a mouse model as an alternative to conventional antibiotic therapy. METHODS: In order to activate CB2R, a selective synthetic agonist, JWH133, was administered intraperitoneally (i.p.) to C57BL/6J mice. Furthermore, SR144528 (a selective CB2R antagonist) was administered in combination with JWH133 to test the specificity of the CB2R-mediated effect. PA was administered intratracheally (i.t.) for induction of pneumonia in mice. At 24 h after PA exposure, lung mechanics were measured using the FlexiVent system. The total cell number, protein content, and neutrophil population in the bronchoalveolar lavage fluid (BALF) were determined. The bacterial load in the whole lung was also measured. Lung injury was evaluated by histological examination and PA-induced inflammation was assessed by measuring the levels of BALF cytokines and chemokines. Neutrophil activation (examined by immunofluorescence and immunoblot) and PA-induced inflammatory signaling (analyzed by immunoblot) were also studied. RESULTS: CB2R activation by JWH133 was found to significantly reduce PA-induced ALI and the bacterial burden. CB2R activation also suppressed the PA-induced increase in immune cell infiltration, neutrophil population, and inflammatory cytokines. These effects were abrogated by a CB2R antagonist, SR144528, further confirming the specificity of the CB2R-mediated effects. CB2R-knock out (CB2RKO) mice had a significantly higher level of PA-induced inflammation as compared to that in WT mice. CB2R activation diminished the excess activation of neutrophils, whereas mice lacking CB2R had elevated neutrophil activation. Pharmacological activation of CB2R significantly reduced the PA-induced NF-κB and NLRP3 inflammasome activation, whereas CB2KO mice had elevated NLRP3 inflammasome. CONCLUSION: Our findings indicate that CB2R activation ameliorates PA-induced lung injury and inflammation, thus paving the path for new therapeutic avenues against PA pneumonia.

Contribution of IL-38 in Lung Immunity during Pseudomonas Aeruginosa-induced Pneumonia.

OBJECTIVE: Interleukin-38 (IL-38), a new type of cytokine, is involved in processes such as tissue repair, inflammatory response, and immune response. However, its function in pneumonia caused by Pseudomonas aeruginosa (P. aeruginosa) is still unclear. METHODS: In this study, we detected circulating IL-38 and cytokines such as IL-1ß, IL-6, IL-17A, TNF-α, IL-8, and IL-10 in adults affected by early stage pneumonia caused by P. aeruginosa. Collected clinical data of these patients, such as the APACHE II score, levels of PCT, and oxygenation index when they entering the ICU. Using P. aeruginosa-induced pneumonia WT murine model to evaluate the effect of IL-38 on Treg differentiation, cell apoptosis, survival, tissue damage, inflammation, and bacterial removal. RESULTS: In clinical research, although IL-38 is significantly increased during the early stages of clinical P. aeruginosa pneumonia, the concentration of IL-38 in the serum of patients who died with P. aeruginosa pneumonia was relatively lower than that of surviving patients. It reveals IL-38 may insufficiently secreted in patients who died with P. aeruginosa pneumonia. Besides, the serum IL-38 level of patients with P. aeruginosa pneumonia on the day of admission to the ICU showed significantly positive correlations with IL-10 and the PaO2/FiO2 ratio but negative correlations with IL-1ß, IL-6, IL-8, IL-17, TNF-α, APACHE II score, and PCT In summary, IL-38 might be a molecule for adjuvant therapy in P. aeruginosa pneumonia. In experimental animal models, first recombinant IL-38 improved survival, whereas anti-IL-38 antibody reduced survival in the experimental pneumonia murine model. Secondly, IL-38 exposure reduced the inflammatory response, as suggested by the lung injury, and reduced cytokine levels (IL-1ß, IL-6, IL- 17A, TNF-α, and IL-8, but not IL-10). It also increased bacterial clearance and reduced cell apoptosis in the lungs. Furthermore, IL-38 was shown to reduce TBK1 expression in vitro when naive CD4+ T lymphocytes were differentiated to Tregs and played a protective role in P. aeruginosa pneumonia. CONCLUSIONS: To summarize, the above findings provide additional insights into the mechanism of IL-38 in the treatment of P. aeruginosa pneumonia.

Destructing biofilms by cationic dextran through phase transition.

Eliminating biofilms from infected tissue presents one of the most challenging issues in clinical treatment of chronic wounds. In biofilms, the extracellular polymeric substances (EPS) form gel structures by electrostatic forces between macromolecules. We hypothesized that cationic polymers could induce the gel-to-sol phase transition of the network, leading to biofilms disruptions. We first validated this assumption by using polyethyleneimine (PEI) as a model molecule, and further synthesized two cationic dextrans with high biodegradability for in vitro and in vivo evaluation. All the cationic polymers could destruct Pseudomonas aeruginosa (P. aeruginosa) biofilms. Treating biofilm with cationic dextrans significantly enhanced the bacterial antibiotic sensitivity. When tested in a biofilm-presenting mouse wound healing model, the cationic dextrans efficiently controlled infection, and accelerated the healing process. Our findings suggest that devising cationic polymers to trigger phase transition of biofilm is an effective, straightforward, and perhaps generic strategy for anti-bacterial therapies.

Vaccination of mice with hybrid protein containing Exotoxin S and PcrV with adjuvants alum and MPL protects Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa as a common pathogen causing urinary tract infections (UTIs) has been resistant to different antibiotics and developing an effective vaccine can be an alternative strategy. In the present study, the immunogenicity and protection efficacy of formulations composed of a hybrid protein composed of P. aeruginosa V-antigen (PcrV) and exoenzyme S (ExoS) with alum and MPL were evaluated. The hybrid protein could increase the specific systemic and mucosal immune responses, as well as cellular responses as compared with control groups. Combining of alum or MPL adjuvant with the hybrid protein significantly improved the levels of IgG1, serum IgA, mucosal IgG, and IL-17 as compared to the ExoS.PcrV alone. After bladder challenge with a P. aeruginosa strain, the bacterial loads of bladder and kidneys were significantly decreased in mice received ExoS.PcrV admixed with alum and ExoS.PcrV admixed with MPL than controls. The present study indicated that immunization of mice with a hybrid protein composed of ExoS and PcrV could induce multifactorial immune responses and opsonize the bacteria and decrease the viable bacterial cells. Because P. aeruginosa have caused therapeutic challenges worldwide, our study proposed ExoS.PcrV + alum and ExoS.PcrV + MPL as promising candidates for the prevention of infections caused by P. aeruginosa.

Sensory nerves promote corneal inflammation resolution via CGRP mediated transformation of macrophages to the M2 phenotype through the PI3K/AKT signaling pathway.

OBJECTIVES: To explore the role of the corneal sensory nerves in Pseudomonas aeruginosa (P. aeruginosa) keratitis, the synergistic effect between the sensory neurons and macrophages in calcitonin gene-related peptide (CGRP) release, and the functional mechanisms of CGRP-mediated transformation of macrophages to the M2 phenotype. METHODS: Corneal nerve loss, macrophage recruitment, and CGRP expression were evaluated. To explore the synergistic effect between the sensory neurons and macrophages, RAW 264.7 cells were challenged with lipopolysaccharide (LPS), then trigeminal ganglion (TG) sensory neurons were isolated and co-incubated with macrophages, and CGRP expression was tested. To investigate the biological function of cornea neuron-initiated immune responses mediated by CGRP, BIBN 4096BS was used to inhibit CGRP in vivo and α-CGRP was used to simulate CGRP in vitro. The expressions of inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IL-10), M1 (CD80/CD86), M2 (CD163/CD206) macrophage markers, and signal transducers (PI3K/AKT) were detected. RESULTS: P. aeruginosa infection induced corneal nerve loss, macrophage recruitment, and CGRP up-expression. CGRP was co-localized with macrophages. Co-culture showed that sensory neurons and macrophages can mediate CGRP release. More CGRP was released when the two types of cells were combined to respond to LPS. BIBN 4096BS promoted pro-inflammatory cytokines and inhibited the anti-inflammatory cytokines and signal transducers, while, α-CGRP inhibited the pro-inflammatory cytokines and M1 markers and promoted the anti-inflammatory cytokine, M2 markers, and signal transducers. CONCLUSIONS: P. aeruginosa infection induces corneal sensory neuron activation, macrophage recruitment, and CGRP up-expression. The synergistic effect between the sensory neurons and macrophages promotes CGRP release. CGRP inhibits corneal inflammation and promotes the transformation of macrophages to the M2 phenotype through the PI3K/AKT signaling pathway.

Therapeutic evaluation of immunomodulators in reducing surgical wound infection.

Despite many advances in infection control practices, including prophylactic antibiotics, surgical site infections (SSIs) remain a significant cause of morbidity, prolonged hospitalization, and death worldwide. Our innate immune system possesses a multitude of powerful antimicrobial strategies which make it highly effective in combating bacterial, fungal, and viral infections. However, pathogens use various stealth mechanisms to avoid the innate immune system, which in turn buy them time to colonize wounds and damage tissues at surgical sites. We hypothesized that immunomodulators that can jumpstart and activate innate immune responses at surgical sites, would likely reduce infection at surgical sites. We used three immunomodulators; fMLP (formyl-Methionine-Lysine-Proline), CCL3 (MIP-1α), and LPS (Lipopolysaccharide), based on their documented ability to elicit strong inflammatory responses; in a surgical wound infection model with Pseudomonas aeruginosa to evaluate our hypothesis. Our data indicate that one-time topical treatment with these immunomodulators at low doses significantly increased proinflammatory responses in infected and uninfected surgical wounds and were as effective, (or even better), than a potent prophylactic antibiotic (Tobramycin) in reducing P. aeruginosa infection in wounds. Our data further show that immunomodulators did not have adverse effects on tissue repair and wound healing processes. Rather, they enhanced healing in both infected and uninfected wounds. Collectively, our data demonstrate that harnessing the power of the innate immune system by immunomodulators can significantly boost infection control and potentially stimulate healing. We propose that topical treatment with these immunomodulators at the time of surgery may have therapeutic potential in combating SSI, alone or in combination with prophylactic antibiotics.

Passive immunization with anti- chimeric protein PilQ/PilA -DSL region IgY does not protect against mortality associated with Pseudomonas aeruginosa sepsis in a rabbit model.

BACKGROUND: Pseudomonas aeruginosa sepsis is associated with unacceptably high mortality and, for many of those who survive, long-term morbidity. The aims of this study were to production of IgY against chimeric protein pilQ-pilA-DSL region and killed- whole cell Pseudomonas aeruginosa O1 (PAO1) strain and their efficacy for immunoprophylaxis of sepsis caused by P. aeruginosa in a rabbit model. METHODS: Specific IgY was obtained by immunization of hens. The purity of IgY was determined by SDS-PAGE analysis. The effect of IgY on growth and hydrophobicity of P. aeruginosa were performed through time-kill assay and microbial adhesion to hydrocarbons test (MATH), respectively. The efficacy of specific IgYs was examined against P. aeruginosa sepsis in a rabbit model. The rabbits were monitored for 72 h to record physiological characters and survival. Hematologic factors, C-reactive protein, pro-inflammatory cytokines, and bacterial count from blood and solid organs were measured, periodically. RESULTS: We found that the growth inhibitory effect of the anti- killed whole cell IgY was higher than anti-pilQ-pilA IgY (P < 0.001). The hydrophobicity effect of PAO1 increased when bacteria were opsonized by anti- killed whole cell IgY while the hydrophobicity activity was decreased following incubation of PAO1 with anti-pilQ-pilA IgY in a broth medium (P < 0.001). Following intravenous (IV) administration of produced IgYs, no significant difference was observed in the survival, decrease in inflammatory mediators and clinical symptoms between the groups 48h post infection (P > 0.05). Moreover, no considerable decrease was observed in the bacterial load of blood, lungs and kidneys in rabbits treated with specific IgYs and control groups (P > 0.05). No bacteria were found in the spleen and liver samples from infected rabbits. CONCLUSION: Although produced IgYs had a good immunoreactivity, IV immunization of IgYs was not protective against P. aeruginosa sepsis in the rabbit model. Further studies are needed to assess the immune response and decreasing mortality rate using the rabbit sepsis model.

The cyclic dinucleotide 2'3'-cGAMP induces a broad antibacterial and antiviral response in the sea anemone Nematostella vectensis.

In mammals, cyclic dinucleotides (CDNs) bind and activate STING to initiate an antiviral type I interferon response. CDNs and STING originated in bacteria and are present in most animals. By contrast, interferons are believed to have emerged in vertebrates; thus, the function of CDN signaling in invertebrates is unclear. Here, we use a CDN, 2'3' cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP), to activate immune responses in a model cnidarian invertebrate, the starlet sea anemone Nematostella vectensis Using RNA sequencing, we found that 2'3'-cGAMP induces robust transcription of both antiviral and antibacterial genes in N. vectensis Many of the antiviral genes induced by 2'3'-cGAMP are homologs of vertebrate interferon-stimulated genes, implying that the interferon response predates the evolution of interferons. Knockdown experiments identified a role for NF-κB in specifically inducing antibacterial genes downstream of 2'3'-cGAMP. Some of these putative antibacterial genes were also found to be induced during Pseudomonas aeruginosa infection. We characterized the protein product of one of the putative antibacterial genes, the N. vectensis homolog of Dae4, and found that it has conserved antibacterial activity. This work suggests that a broad antibacterial and antiviral transcriptional response is an evolutionarily ancestral output of 2'3'-cGAMP signaling in animals.