Exploring the therapeutic potential of Leuconostoc mesenteroides lysates in wound healing and immune modulation on keratinocyte cells.

The skin, being the body's largest organ, primarily functions as a formidable defense mechanism against potential microbial infections. The skin's microbiota, consisting of a complex assembly of microorganisms, exerts a pivotal influence on skin homeostasis by modulating keratinocytes and their cytokine secretion, thereby playing an integral role in promoting optimal cutaneous health. Leuconostoc mesenteroides finds extensive application in the production of fermented foods and bacteriocins. Empirical studies validate the effectiveness of L. mesenteroides treatments in enhancing immune function and demonstrating notable antioxidant characteristics. This study investigates the potential of L. mesenteroides in improving skin health and wound healing. It also aims to comprehend their impact on wound healing markers, cytokine production, and cell cycle regulation compared to ferulic acid, known for its wound healing effects. Our findings indicate that L. mesenteroides lysate possesses antibacterial properties against Staphylococcus aureus and Pseudomonas aeruginosa, along with the ability to mitigate their toxic effects in a pathogen-simulating model employing HaCaT keratinocyte cells. Additionally, the lysate demonstrated noteworthy wound closure after a 24-hour treatment, along with a significant reduction in interleukin-6 levels and oxidative stress index. Modulation of the cell cycle is evident by decreasing G0/G1 phases and increasing S and G2/M phases and enhanced expression of wound healing marker genes and proteins CDH1. In conclusion, L. mesenteroides lysate exhibits immune-modulating and antibacterial properties, offering potential alternatives to conventional treatments for various skin conditions. These findings contribute to the exploration of innovative approaches to enhancing human life through skin health and wound healing.

Pseudomonas aeruginosa in chronic lung disease: untangling the dysregulated host immune response.

Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen capable of exploiting barriers and immune defects to cause chronic lung infections in conditions such as cystic fibrosis. In these contexts, host immune responses are ineffective at clearing persistent bacterial infection, instead driving a cycle of inflammatory lung damage. This review outlines key components of the host immune response to chronic P. aeruginosa infection within the lung, beginning with initial pathogen recognition, followed by a robust yet maladaptive innate immune response, and an ineffective adaptive immune response that propagates lung damage while permitting bacterial persistence. Untangling the interplay between host immunity and chronic P. aeruginosa infection will allow for the development and refinement of strategies to modulate immune-associated lung damage and potentiate the immune system to combat chronic infection more effectively.

The potential of bacterial anti-phagocytic proteins in suppressing the clearance of extracellular vesicles mediated by host phagocytosis.

Extracellular vesicles (EVs), characterized by low immunogenicity, high biocompatibility and targeting specificity along with excellent blood-brain barrier permeability, are increasingly recognized as promising drug delivery vehicles for treating a variety of diseases, such as cancer, inflammation and viral infection. However, recent findings demonstrate that the intracellular delivery efficiency of EVs fall short of expectations due to phagocytic clearance mediated by the host mononuclear phagocyte system through Fcγ receptors, complement receptors as well as non-opsonic phagocytic receptors. In this text, we investigate a range of bacterial virulence proteins that antagonize host phagocytic machinery, aiming to explore their potential in engineering EVs to counteract phagocytosis. Special emphasis is placed on IdeS secreted by Group A Streptococcus and ImpA secreted by Pseudomonas aeruginosa, as they not only counteract phagocytosis but also bind to highly upregulated surface biomarkers αVß3 on cancer cells or cleave the tumor growth and metastasis-promoting factor CD44, respectively. This suggests that bacterial anti-phagocytic proteins, after decorated onto EVs using pre-loading or post-loading strategies, can not only improve EV-based drug delivery efficiency by evading host phagocytosis and thus achieve better therapeutic outcomes but also further enable an innovative synergistic EV-based cancer therapy approach by integrating both phagocytosis antagonism and cancer targeting or deactivation.

Neutralizing antibodies after nebulized phage therapy in cystic fibrosis patients.

BACKGROUND: Cystic fibrosis (CF) patients are prone to recurrent multi-drug-resistant (MDR) bacterial lung infections. Under this scenario, phage therapy has been proposed as a promising tool. However, the limited number of reported cases hampers the understanding of clinical outcomes. Anti-phage immune responses have often been overlooked and only described following invasive routes of administration. METHODS: Three monophage treatments against Staphylococcus aureus and/or Pseudomonas aeruginosa lung infections were conducted in cystic fibrosis patients. In-house phage preparations were nebulized over 10 days with standard-of-care antibiotics. Clinical indicators, bacterial counts, phage and antibiotic susceptibility, phage detection, and immune responses were monitored. FINDINGS: Bacterial load was reduced by 3-6 log in two of the treatments. No adverse events were described. Phages remained in sputum up to 33 days after completion of the treatment. In all cases, phage-neutralizing antibodies were detected in serum from 10 to 42 days post treatment, with this being the first report of anti-phage antibodies after nebulized therapy. CONCLUSIONS: Nebulized phage therapy reduced bacterial load, improving quality of life even without bacterial eradication. The emergence of antibodies emphasizes the importance of long-term monitoring to better understand clinical outcomes. These findings encourage the use of personalized monophage therapies in contrast to ready-to-use cocktails, which might induce undesirable antibody generation. FUNDING: This study was supported by the Spanish Ministry of Science, Innovation and Universities; Generalitat Valenciana; and a crowdfunding in collaboration with the Spanish Cystic Fibrosis Foundation.

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.

Biofilm-derived membrane vesicles exhibit potent immunomodulatory activity in Pseudomonas aeruginosa PAO1.

Pathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesicles in Gram-negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic-derived MVs (p-MVs) and biofilm-derived MVs (b-MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b-MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p-MVs. The mRNA expression levels of Toll-like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b-MV-mediated cytokine induction, suggesting that lipopolysaccharide of native b-MVs is the origin of the immune response. In addition, heat-treated or homogenized b-MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen-associated molecular patterns in lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.

A Bispecific Monoclonal Antibody Targeting Psl and PcrV Enhances Neutrophil-Mediated Killing of Pseudomonas aeruginosa in Patients with Bronchiectasis.

Rationale: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. Objectives: This study evaluated the efficacy of gremubamab to enhance killing of P. aeruginosa by neutrophils from patients with bronchiectasis and to prevent P. aeruginosa-associated cytotoxicity. Methods: P. aeruginosa isolates from a global bronchiectasis cohort (n = 100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in vitro and in vivo. Patients with bronchiectasis (n = 11) and control subjects (n = 10) were enrolled, and the effect of gremubamab in peripheral blood neutrophil opsonophagocytic killing (OPK) assays against P. aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n = 30; 19 chronic P. aeruginosa infection, 11 no known P. aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. Measurements and Main Results: Psl and PcrV were conserved in isolates from chronically infected patients with bronchiectasis. Seventy-three of 100 isolates had a full psl locus, and 99 of 100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the monoclonal antibody-mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6 ± 8.1%) and phagocytosis (+70.0 ± 48.8%), similar to effects observed in neutrophils from control subjects (OPK, +30.1 ± 7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P. aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. Conclusions: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P. aeruginosa and reduced virulence.

The Hippo kinases control inflammatory Hippo signaling and restrict bacterial infection in phagocytes.

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells.

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa. Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

AAV-vectored expression of monospecific or bispecific monoclonal antibodies protects mice from lethal Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.

Septin-dependent defense mechanisms against Pseudomonas aeruginosa are stalled in cystic fibrosis bronchial epithelial cells.

Airway epithelial cells form a physical barrier against inhaled pathogens and coordinate innate immune responses in the lungs. Bronchial cells in people with cystic fibrosis (pwCF) are colonized by Pseudomonas aeruginosa because of the accumulation of mucus in the lower airways and an altered immune response. This leads to chronic inflammation, lung tissue damage, and accelerated decline in lung function. Thus, identifying the molecular factors involved in the host response in the airways is crucial for developing new therapeutic strategies. The septin (SEPT) cytoskeleton is involved in tissue barrier integrity and anti-infective responses. SEPT7 is critical for maintaining SEPT complexes and for sensing pathogenic microbes. In the lungs, SEPT7 may be involved in the epithelial barrier resistance to infection; however, its role in cystic fibrosis (CF) P. aeruginosa infection is unknown. This study aimed to investigate the role of SEPT7 in controlling P. aeruginosa infection in bronchial epithelial cells, particularly in CF. The study findings showed that SEPT7 encages P. aeruginosa in bronchial epithelial cells and its inhibition downregulates the expression of other SEPTs. In addition, P. aeruginosa does not regulate SEPT7 expression. Finally, we found that inhibiting SEPT7 expression in bronchial epithelial cells (BEAS-2B 16HBE14o- and primary cells) resulted in higher levels of internalized P. aeruginosa and decreased IL-6 production during infection, suggesting a crucial role of SEPT7 in the host response against this bacterium. However, these effects were not observed in the CF cells (16HBE14o-/F508del and primary cells) which may explain the persistence of infection in pwCF. The study findings suggest the modification of SEPT7 expression as a potential approach for the anti-infective control of P. aeruginosa, particularly in CF.

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Immunometabolic Regulation of Bacterial Infection, Biofilms, and Antibiotic Susceptibility.

BACKGROUND: Upon infection, mucosal tissues activate a brisk inflammatory response to clear the pathogen, i.e., resistance to disease. Resistance to disease is orchestrated by tissue-resident macrophages, which undergo profound metabolic reprogramming after sensing the pathogen. These metabolically activated macrophages release many inflammatory factors, which promote their bactericidal function. However, in immunocompetent individuals, pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella evade this type of immunity, generating communities that thrive for the long term. SUMMARY: These organisms develop features that render them less susceptible to eradication, such as biofilms and increased tolerance to antibiotics. Furthermore, after antibiotic therapy withdrawal, "persister" cells rapidly upsurge, triggering inflammatory relapses that worsen host health. How these pathogens persisted in inflamed tissues replete with activated macrophages remains poorly understood. KEY MESSAGES: In this review, we discuss recent findings indicating that the ability of P. aeruginosa, S. aureus, and Salmonella to evolve biofilms and antibiotic tolerance is promoted by the similar metabolic routes that regulate macrophage metabolic reprogramming.

Targeting HuR-Vav3 mRNA interaction prevents Pseudomonas aeruginosa adhesion to the cystic fibrosis airway epithelium.

Cystic fibrosis (CF) is characterized by chronic bacterial infections leading to progressive bronchiectasis and respiratory failure. Pseudomonas aeruginosa (Pa) is the predominant opportunistic pathogen infecting the CF airways. The guanine nucleotide exchange factor Vav3 plays a critical role in Pa adhesion to the CF airways by inducing luminal fibronectin deposition that favors bacteria trapping. Here we report that Vav3 overexpression in CF is caused by upregulation of the mRNA-stabilizing protein HuR. We found that HuR accumulates in the cytoplasm of CF airway epithelial cells and that it binds to and stabilizes Vav3 mRNA. Interestingly, disruption of the HuR-Vav3 mRNA interaction improved the CF epithelial integrity, inhibited the formation of the fibronectin-made bacterial docking platforms, and prevented Pa adhesion to the CF airway epithelium. These findings indicate that targeting HuR represents a promising antiadhesive approach in CF that can prevent initial stages of Pa infection in a context of emergence of multidrug-resistant pathogens.

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.

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.

Outer Membrane Vesicles Displaying a Heterologous PcrV-HitA Fusion Antigen Promote Protection against Pulmonary Pseudomonas aeruginosa Infection.

Along with surging threats and antibiotic resistance of Pseudomonas aeruginosa in health care settings, it is imperative to develop effective vaccines against P. aeruginosa infection. In this study, we used an Asd (aspartate-semialdehyde dehydrogenase)-based balanced-lethal host-vector system of a recombinant Yersinia pseudotuberculosis mutant to produce self-adjuvanting outer membrane vesicles (OMVs). The OMVs were used as a carrier to deliver the heterologous PcrV-HitAT (PH) fusion antigen of P. aeruginosa for vaccine evaluation. Intramuscular vaccination with OMVs carrying the PH antigen (referred to rOMV-PH) afforded 73% protection against intranasal challenge with 5 × 106 (25 50% lethal doses) of the cytotoxic PA103 strain and complete protection against a noncytotoxic PAO1 strain. In contrast, vaccination with the PH-deficient OMVs or PH antigen alone failed to offer effective protection against the same challenge. Immune analysis showed that the rOMV-PH vaccination induced potent humoral and Th1/Th17 responses compared to the PH vaccination. The rOMV-PH vaccination rapidly cleared P. aeruginosa burdens with coordinated production of proinflammatory cytokines in mice. Moreover, antigen-specific CD4+ and CD8+ T cells and their producing cytokines (tumor necrosis factor alpha and interleukin-17A), rather than antibodies, were essential for protection against pneumonic P. aeruginosa infection. Our studies demonstrated that the recombinant Y. pseudotuberculosis OMVs delivering heterologous P. aeruginosa antigens could be a new promising vaccine candidate for preventing the spread of drug-resistant P. aeruginosa. IMPORTANCE Hospital- and community-acquired infections with Pseudomonas aeruginosa cause a high rate of morbidity and mortality in patients who have underlying medical conditions. The spread of multidrug-resistant P. aeruginosa strains is becoming a great challenge for treatment using antibiotics. Thus, a vaccine as one of the alternative strategies is urgently required to prevent P. aeruginosa infection.

Granulocytic Myeloid-Derived Suppressor Cells in Cystic Fibrosis.

Cystic Fibrosis (CF) is a genetic disease that causes chronic and severe lung inflammation and infection associated with high rates of mortality. In CF, disrupted ion exchange in the epithelium results in excessive mucus production and reduced mucociliary clearance, leading to immune system exacerbation and chronic infections with pathogens such as P. aeruginosa and S. aureus. Constant immune stimulation leads to altered immune responses including T cell impairment and neutrophil dysfunction. Specifically, CF is considered a Th17-mediated disease, and it has been proposed that both P. aeruginosa and a subset of neutrophils known as granulocytic myeloid suppressor cells (gMDSCs) play a role in T cell suppression. The exact mechanisms behind these interactions are yet to be determined, but recent works demonstrate a role for arginase-1. It is also believed that P. aeruginosa drives gMDSC function as a means of immune evasion, leading to chronic infection. Herein, we review the current literature regarding immune suppression in CF by gMDSCs with an emphasis on T cell impairment and the role of P. aeruginosa in this dynamic interaction.

Vaccination induces rapid protection against bacterial pneumonia via training alveolar macrophage in mice.

Vaccination strategies for rapid protection against multidrug-resistant bacterial infection are very important, especially for hospitalized patients who have high risk of exposure to these bacteria. However, few such vaccination strategies exist due to a shortage of knowledge supporting their rapid effect. Here, we demonstrated that a single intranasal immunization of inactivated whole cell of Acinetobacter baumannii elicits rapid protection against broad A. baumannii-infected pneumonia via training of innate immune response in Rag1-/- mice. Immunization-trained alveolar macrophages (AMs) showed enhanced TNF-α production upon restimulation. Adoptive transfer of immunization-trained AMs into naive mice mediated rapid protection against infection. Elevated TLR4 expression on vaccination-trained AMs contributed to rapid protection. Moreover, immunization-induced rapid protection was also seen in Pseudomonas aeruginosa and Klebsiella pneumoniae pneumonia models, but not in Staphylococcus aureus and Streptococcus pneumoniae model. Our data reveal that a single intranasal immunization induces rapid and efficient protection against certain Gram-negative bacterial pneumonia via training AMs response, which highlights the importance and the possibility of harnessing trained immunity of AMs to design rapid-effecting vaccine.