Intracellular Bacterial Killing Research Articles

RSV enhances Staphylococcus aureus bacterial growth in the lung.

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo. Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV.

Nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection.

Abstract Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading cause of Gram-negative pneumonia and pneumonic sepsis for which limited therapeutic options are available. The lungs are densely innervated by nociceptive sensory neurons (nociceptors), which mediate breathing, cough, and bronchoconstriction. However, the role of these neurons in host defense against CRKP-induced pneumonic sepsis is unknown. We found that nociceptors play a host deleterious role to promote pneumonic sepsis. Nociceptor-ablated mice showed an enhanced survival and bacterial clearance after CRKP lung infection. Furthermore, nociceptors ablation resulted in the increased influx of neutrophils and Ly6Chi monocytes and cytokine induction. Depletion of Ly6Chi monocytes, but not of neutrophils, impaired lung bacterial clearance and bacterial dissemination in nociceptor-ablated mice, indicating that Ly6Chi monocytes are a critical cellular population downstream of nociceptors to regulate pneumonic sepsis. Moreover, nociceptive neuropeptide calcitonin gene-related peptide (CGRP) suppressed induction of reactive oxygen species and intracellular bacterial killing. Collectively, our data suggest that the nociceptor neuronal signaling could be a therapeutic target for treating multidrug-resistant CRKP lung infections and pneumonic sepsis. Supported by grants from NIH/NIGMS P20GM113117, American Lung Association, and JCRC Heart Research Award.

Investigating the effectiveness of liposome-bacteriophage nanocomplex in killing Staphylococcus aureus using epithelial cell coculture models

Host cell invasion with strong antibiotics evading is a major feature of respiratory Staphylococcus aureus infections with severe recurrence. Bacteriophage (phage) therapy and design of liposomal phage to target intracellular pathogens have been described recently. The practicality for pulmonary delivery of liposomal phage, and how formulation compositions affecting the aerosolization and intracellular bacterial killing remain unexplored. In the present study, three commonly used phospholipids (SPC, EPC, and HSPC) were selected to investigate their ability for phage K nebulization and intracellular therapy in the form of liposome-phage nanocomplexes. The three lipid nanocarriers showed protection on phage K upon mesh nebulization and the pulmonary deposition efficiency was influenced by the lipid used. Moreover, the intracellular bacterial killing was strongly depended on the lipid types, where EPC-phage exhibited the best killing performance with no relapsing. Phage K with the aid of EPC liposomes was also observed to manage the tissue infection in a 3D spheroid model more effectively than other groups. Altogether, this novel EPC liposome-phage nanocomplex can be a promising formulation approach that enables inhalable phage to manage respiratory infections caused by bacteria strongly associated with human epithelial cells.

Bosutinib Stimulates Macrophage Survival, Phagocytosis, and Intracellular Killing of Bacteria.

Host-acting compounds are emerging as potential alternatives to combating antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduce the bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodeling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-κB activation, which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, cotreatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infections through multiple mechanisms and could be used to boost the host innate immunity to combat drug-resistant bacterial infections.

Mitoxantrone targets both host and bacteria to overcome vancomycin resistance in Enterococcus faecalis.

Antibiotic resistance critically limits treatment options for infection caused by opportunistic pathogens such as enterococci. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin-resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria through induction of reactive oxygen species and DNA damage. MTX also synergizes with vancomycin against VRE, rendering the resistant strains more permeable to MTX. In a murine wound infection model, single-dose MTX treatment effectively reduces VRE numbers, with further reduction when combined with vancomycin. Multiple MTX treatments accelerate wound closure. MTX also promotes macrophage recruitment and proinflammatory cytokine induction at the wound site and augments intracellular bacterial killing in macrophages by up-regulating the expression of lysosomal enzymes. These results show that MTX represents a promising bacterium- and host-targeted therapeutic for overcoming vancomycin resistance.

Butyrate-induced IL-22 expression in fish macrophages contributes to bacterial clearance

IL-22 has been characterized as a critical cytokine in maintaining barrier integrity and host immunity. So far, it has been known that IL-22 is mainly produced by lymphoid lineage cells. In the present study, we have thoroughly investigated butyrate-induced production and function of IL-22 in fish macrophages. Our results demonstrated that short-chain fatty acids (SCFAs), major microbiota-derived metabolites, promoted the expression of IL-22 in head kidney macrophages (HKMs) of turbot (Scophthalmus maximus L.). Interestingly, butyrate-mediated intracellular bacterial killing in HKMs diminished when IL-22 expression was interfered. Furthermore, the turbot fed the diet containing sodium butyrate (NaB) exhibited significantly lower mortality after bacterial infection, compared to the fish fed a basal diet. At the meantime, a higher level of IL-22 expression and bactericidal activity was detected in HKMs from the turbot fed NaB-supplemented diet. In addition, NaB treatment promoted the expression of antimicrobial peptides (AMPs) β-defensins in zebrafish (Danio rerio). However, butyrate-induced expression of AMPs was reduced in IL-22 mutant zebrafish compared to wild-type (WT) fish. Meanwhile, NaB treatment was incapable to protect IL-22 mutant fish from bacterial infection as it did in WT zebrafish. Importantly, our results demonstrated that IL-22 expression was remarkably suppressed in macrophage-depleted zebrafish, indicating that macrophage might be a cell source of IL-22 production in vivo. In conclusion, all these findings collectively revealed that SCFAs regulated the production and function of IL-22 in fish macrophages, which facilitated host resistance to bacterial invasion.

In vitro and Intracellular Antibacterial Activity of Sudapyridine (WX-081) Against Tuberculosis.

Sudapyridine (WX-081) has exhibited equivalent efficacy than its counterpart parent drug bedaquiline (BDQ) but better safety profile against Mycobacterium tuberculosis (Mtb). Our study was aimed to evaluate in vitro activity of WX-081 against the clinical isolates of Mtb with different drug-resistance profiles and the intracellular bactericidal activity against the reference strain. The minimum inhibitory concentrations (MICs) of WX-081 and BDQ were tested against 114 Mtb clinical isolates. The intracellular activity of WX-081 and BDQ against the Mtb reference strain H37Rv in THP-1 cells was also evaluated in parallel. The MICs for WX-081 of the enrolled isolates ranged from 0.0156 μg/mL to 1 μg/mL. The MIC50 and MIC90 of WX-081 were, respectively, 0.25 μg/mL and 0.5 μg/mL, with 95.6% of the enrolled strains having MICs ≤0.25 μg/mL. For a given strain, the MIC value of WX-081 was generally equivalent to or 2-fold than MIC of BDQ. The intracellular bacterial killing was acquired with the tested drug concentrations that were presumed attainable during clinical usage. WX-081 exhibited potent efficacy against the clinical isolates in vitro. The intracellular killing effect of sudapyridine against the reference strain supports its potential efficacy in treating TB patients.

Elexacaftor/tezacaftor/ivacaftor corrects monocyte microbicidal deficiency in cystic fibrosis.

Cystic fibrosis (CF), which is caused by mutations in the CF transmembrane conductance regulator (CFTR), is characterised by chronic bacterial lung infection and inflammation. In CF, monocytes and monocyte-derived macrophages have been shown to display defective phagocytosis and antimicrobial activity against relevant lung pathogens, including Pseudomonas aeruginosa. Thus, we addressed the effect of CFTR triple modulator therapy (elexacaftor/tezacaftor/ivacaftor (ETI)) on the activity of CF monocytes against P. aeruginosa. Monocytes from people with CF (PWCF) before and after 1 and 6 months of ETI therapy were isolated from blood and infected with P. aeruginosa to assess phagocytic activity and intracellular bacterial killing. The oxidative burst and interleukin-6 secretion were also determined. Monocytes from healthy controls were also included. Longitudinal analysis of the clinical parameters confirmed an improvement of lung function and lung microbiology by ETI. Both the phagocytic and microbicidal deficiencies of CF monocytes also improved significantly, although not completely. Furthermore, we measured an exuberant oxidative burst in CF monocytes before therapy, which was reduced considerably by ETI. This led to an improvement of reactive oxygen species-dependent bactericidal activity. Inflammatory response to bacterial stimuli was also lowered compared with pre-therapy. PWCF on ETI therapy, in a real-life setting, in addition to clinical recovery, showed significant improvement in monocyte activity against P. aeruginosa, which may have contributed to the overall effect of ETI on pulmonary disease. This also suggests that CF monocyte dysfunctions may be specifically targeted to ameliorate lung function in CF.

Bacterial secretions in growth medium stimulate the mouse respiratory innate immune response.

Introduction. Finding a safe innate immune response stimulator is one of the greatest challenges facing immunologists and vaccine manufacturers.Gap statement. The role of sterile bacterial secretions (SBSs) of Pseudomonas aeruginosa in stimulating the innate immune response was not investigated previously.Aim. The comparative effect of SBSs and bacterial cells of P. aeruginosa isolates isolated from freshwater (PAE) and infected wounds (PAC) on the respiratory tract innate immune response.Methodology. Four test mice groups were instilled intranasally (i.n.) with 106 c.f.u of PAC, 106 c.f.u of PAE, SBS of PAC, and SBS of PAE. Two control groups were given i.n. either LB broth or PBS. Time-course changes in IL-1 beta mRNA, TNF-alpha mRNA, IL-1β and TNF-α, leukocyte count, bacterial uptake, and intracellular bacterial killing by mouse alveolar macrophages (AMs) and histological changes were examined. Lung bacterial burdens were counted in first and second test groups.Results. The maximum level of IL-1β was seen as early as 2 h (1360±180 pg ml-1) post-instillation (i.n.) with SBS of PAC and 1 h (1910±244 pgml-1) post-instillation with SBS of PAE. The maximum level of TNF-α was seen as early as 4 h (953±192 pg ml-1) post-instillation with SBS of PAC and (1197±298 pg ml-1) post-instillation with SBS of PAE. These values were almost in line with IL-1β and TNF-α gene expression. Moderate infiltration of leukocytes in bronchoalveolar lavage (BAL) and lung sections and moderate activity of AMs (bacterial uptake and bacterial killing) were observed. The above innate immune response parameters in mice instilled i.n. with PAC and PAE were higher (P<0.05) than in the mice groups instilled i.n. with SBSs. The PAC was persistent in the lungs of mice for up to 72 h (3.5±0.22 log10 of c.f.u. g-1) and up to 48 h (2.05±0.21 log10 of c.f.u. g-1) for PAE.Conclusion. The administration of mice with SBS i.n. stimulates cellular and molecular arms of the innate immune response in the respiratory tract, opening the door to the possibility of using SBS of P. aeruginosa as an adjuvant.

TLR9 Negatively Regulates Intracellular Bacterial Killing by Pyroptosis in Burkholderia pseudomallei-Infected Mouse Macrophage Cell Line (Raw264.7).

ABSTRACTMelioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-β, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention.IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-β, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms.

Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

Collagen – Zinc Oxide Nanoparticles (ZnO NPs) Composites for Wound Healing – A Review

Fish Collagen which is also called as Marine collagen has gained immense attention in the recent years as an appropriate alternative to mammalian collagen. Fish collagen is essentially the superhero of collagen sourced from animals. Fish collagen is made up of mostly Type 1 collagen, which makes up to 70 percent of total skin. Collagen is found to heal wounds by attracting new skin cells to the wound site. It promotes healing and provide platform for new tissue growth. Zinc Oxide Nanoparticles are nanoparticles of ZnO that have diameter less than 100 nanometres. Recently, ZnO Nanoparticles have shown to disrupt bacterial cell membrane integrity, reduce cell surface hydrophobicity and enhancement of intracellular bacterial killing. The ZnO nanoparticles were synthesized from the plant. The paper will describe the potency of fish skin waste, problems in healing burn injuries, collagen extraction, green synthesis of ZnO NPs, collagen and ZnO NPs applications in wound dressing. Since ZnO nanoparticles have much anti-bacterial activity it can be combined with collagen and characterized for the application in wound healing management.

Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen causing invasive disease, including community-acquired bacteraemia, and remains a leading cause of global mortality. Understanding the role of phagocytes in killing bacteria is still limited, especially in vivo. In this study, we established a zebrafish model to study the interaction between intravenously administered pneumococci and professional phagocytes such as macrophages and neutrophils, to unravel bacterial killing mechanisms employed by these immune cells. Our model confirmed the key role of polysaccharide capsule in promoting pneumococcal virulence through inhibition of phagocytosis. Conversely, we show pneumococci lacking a capsule are rapidly internalised by macrophages. Low doses of encapsulated S. pneumoniae cause near 100% mortality within 48 hours postinfection (hpi), while 50 times higher doses of unencapsulated pneumococci are easily cleared. Time course analysis of in vivo bacterial numbers reveals that while encapsulated pneumococcus proliferates to levels exceeding 105 CFU at the time of host death, unencapsulated bacteria are unable to grow and are cleared within 20 hpi. Using genetically induced macrophage depletion, we confirmed an essential role for macrophages in bacterial clearance. Additionally, we show that upon phagocytosis by macrophages, phagosomes undergo rapid acidification. Genetic and chemical inhibition of vacuolar ATPase (v-ATPase) prevents intracellular bacterial killing and induces host death indicating a key role of phagosomal acidification in immunity to invading pneumococci. We also show that our model can be used to study the efficacy of antimicrobials against pneumococci in vivo. Collectively, our data confirm that larval zebrafish can be used to dissect killing mechanisms during pneumococcal infection in vivo and highlight key roles for phagosomal acidification in macrophages for pathogen clearance.

Resistin production does not affect outcomes in a mouse model of acute surgical sepsis.

Because of the strong correlation between the blood concentration of circulating resistin and the illness severity of septic patients, resistin has been proposed as a mediator of sepsis pathophysiology. In vitro data indicate that human resistin directly impairs neutrophil migration and intracellular bacterial killing, although the significance of these findings in vivo remain unclear. The objectives of the present study were: (1) to validate the expression of human resistin in a clinically relevant, murine model of surgical sepsis, (2) to assess how sepsis-induced changes in resistin correlate with markers of infection and organ dysfunction, and (3) to investigate whether the expression of human resistin alters immune function or disease outcomes in vivo. 107 male, C57BL/6 mice transgenic for the human resistin gene and its promoter elements (Retn+/-/-, or Retn+) were generated on a Retn-/- (mouse resistin knockout, or Rko) background. Outcomes were compared between age-matched transgenic and knockout mice. Acute sepsis was defined as the initial 24 h following cecal ligation and puncture (CLP). Physiologic and laboratory parameters correlating to the human Sequential Organ Failure Assessment (SOFA) Score were measured in mice, and innate immune cell number/function in the blood and peritoneal cavity were assessed. CLP significantly increased circulating levels of human resistin. The severity of sepsis-induced leukopenia was comparable between Retn+ and Rko mice. Resistin was associated with increased production of neutrophil reactive oxygen species, a decrease in circulating neutrophils at 6 h and an increase in peritoneal Ly6Chi monocytes at 6 h and 24 h post-sepsis. However, intraperitoneal bacterial growth, organ dysfunction and mouse survival did not differ with resistin production in septic mice. Ex vivo resistin-induced impairment of neutrophil function do not appear to translate to increased sepsis severity or poorer outcomes in vivo following CLP.

Phagocytosis and intracellular killing of Pasteurella multocida B:2 by macrophages: A comparative study between buffalo and cattle.

Background and Aim:Pasteurella multocida B:2 is the causative agent of hemorrhagic septicemia (HS) in buffalo and cattle. Buffaloes are known to be more susceptible to HS than cattle, but the reason for this remains unknown. This study aimed to compare the in vitro efficiency with which buffalo and cattle macrophages can kill P. multocida B:2.Materials and Methods:Monocyte-derived macrophages of buffalo and cattle were used in this study. They were exposed to 1×106 colony-forming unit/mL of live P. multocida B:2 before the cells were harvested at 0, 30, 60, and 120 min post-exposure and viewed under a fluorescence microscope to count viable and non-viable macrophages and the macrophages with phagocytosing P. multocida B:2 cells. The phagocytosis, intracellular bacterial killing, and macrophage death rates were calculated and compared between the two species and sampling points.Results:In general, the rates of phagocytosis, intracellular killing, and macrophage death increased with time of exposure for both animal species. No significant (p>0.05) differences were noted between the phagocytosis rates by the macrophages of buffalo and cattle throughout the experiment. However, the rates of intracellular killing were significantly (p<0.05) higher in cattle macrophages at 30 min and 120 min post-exposure than those of buffalo. The death rates of buffalo macrophages were significantly (p<0.05) higher than those of cattle at 60 min and 120 min post-exposure.ConclusionWith higher bacteria killing ability and lower macrophage death, cattle appeared to be more efficient at handling P. multocida B:2 infection than buffalo.

Roscovitine Worsens Mycobacterium abscessus Infection by Reducing DUOX2-mediated Neutrophil Response.

Persistent neutrophilic inflammation associated with chronic pulmonary infection causes progressive lung injury and, eventually, death in individuals with cystic fibrosis (CF), a genetic disease caused by biallelic mutations in the CF transmembrane conductance regulator (CFTR) gene. Therefore, we examined whether roscovitine, a cyclin-dependent kinase inhibitor that (in other conditions) reduces inflammation while promoting host defense, might provide a beneficial effect in the context of CF. Herein, using CFTR-depleted zebrafish larvae as an innovative vertebrate model of CF immunopathophysiology, combined with murine and human approaches, we sought to determine the effects of roscovitine on innate immune responses to tissue injury and pathogens in the CF condition. We show that roscovitine exerts antiinflammatory and proresolution effects in neutrophilic inflammation induced by infection or tail amputation in zebrafish. Roscovitine reduces overactive epithelial reactive oxygen species (ROS)-mediated neutrophil trafficking by reducing DUOX2/NADPH-oxidase activity and accelerates inflammation resolution by inducing neutrophil apoptosis and reverse migration. It is important to note that, although roscovitine efficiently enhances intracellular bacterial killing of Mycobacterium abscessus in human CF macrophages ex vivo, we found that treatment with roscovitine results in worse infection in mouse and zebrafish models. By interfering with DUOX2/NADPH oxidase-dependent ROS production, roscovitine reduces the number of neutrophils at infection sites and, consequently, compromises granuloma formation and maintenance, favoring extracellular multiplication of M. abscessus and more severe infection. Our findings bring important new understanding of the immune-targeted action of roscovitine and have significant therapeutic implications for safely targeting inflammation in CF.

Metformin strengthens uroepithelial immunity against E. coli infection

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.

Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions

Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.

L-Threoascorbic acid treatment promotes S. aureus-infected primary human endothelial cells survival and function, as well as intracellular bacterial killing, and immunomodulates the release of IL-1β and soluble ICAM-1

BackgroundVitamin C (ascorbic acid, AscH2) has been shown to enhance immunity. Here, we studied its immunomodulatory effect on human endothelial cells (ECs) during S. aureus infection. Materials and methodsThe ex vivo effects of AscH2 were performed on primary human umbilical vein endothelial cells (HUVECs) infected or not with S. aureus. ResultsAscH2 treatment induced a marked downregulation of nitric oxide (NO) production and a moderate upregulation of arginase activity in S. aureus-infected HUVECs (respectively, p < 0.05 and p > 0.05). Although the upregulated release levels of soluble intercellular adhesion molecular 1 (sICAM-1/sCD54) and sE-selectin (sCD62E) molecules were not significantly different between treated and untreated S. aureus-infected HUVECs, AscH2 treatment induced reversing effect on sICAM-1 release when comparing to uninfected control HUVECs. Moreover, AscH2 treatment appears to have a significant effect on preventing HUVEC necrosis induced by S. aureus infection (p < 0.05). Furthermore, AscH2 treatment induced a significant upregulation of cell protective redox biomarker in S. aureus-infected, as shown by superoxide dismutase (SOD) activity (p < 0.05), but not by catalase activity (p > 0.05). Additionally, S. aureus infection markedly downregulated total bound calcium ions (bCa2+) levels as compared to control HUVECs, whereas, AscH2 treatment induced a slight upregulation of bCa2+ levels in infected HUVECs as compared to infected and untreated HUVECs (p > 0.05). On the other hand, AscH2 treatment downregulated increased total cellular cholesterol content (tccCHOL) levels in HUVECs induced by S. aureus infection (p < 0.05). In addition, AscH2 treatment markedly reversed S. aureus effect on upregulation of intracellular glucose (iGLU) levels within infected HUVECs (p < 0.05). Moreover, AscH2 treatment significantly downregulated S. aureus growth (p < 0.05), and significantly upregulated bacterial internalization and intracellular killing by HUVECs (p < 0.05), as well as their cell cycle activation (p < 0.01). Finally, AscH2 treatment has a slight effect on the production of interleukin 6 (IL-6), but induced a marked downregulation of that of IL-1β in S. aureus-infected HUVECs (respectively, p > 0.05, and p < 0.05). ConclusionsOur outcomes demonstrated that, during S. aureus infection, AscH2 treatment promotes human ECs survival and function, as well as prevents inflammatory response exacerbation, while inducing bactericidal activity.

Failed Eradication Therapy of New-Onset Pseudomonas aeruginosa Infections in Children With Cystic Fibrosis Is Associated With Bacterial Resistance to Neutrophil Functions.

BackgroundAntibiotics, such as inhaled tobramycin, are used to eradicate new-onset Pseudomonas aeruginosa (PA) infections in patients with cystic fibrosis (CF) but frequently fail due to reasons poorly understood. We hypothesized that PA isolates’ resistance to neutrophil antibacterial functions was associated with failed eradication in patients harboring those strains.MethodsWe analyzed all PA isolates from a cohort of 39 CF children with new-onset PA infections undergoing tobramycin eradication therapy, where 30 patients had eradicated and 9 patients had persistent infection. We characterized several bacterial phenotypes and measured the isolates’ susceptibility to neutrophil antibacterial functions using in vitro assays of phagocytosis and intracellular bacterial killing.ResultsPA isolates from persistent infections were more resistant to neutrophil functions, with lower phagocytosis and intracellular bacterial killing compared to those from eradicated infections. In multivariable analyses, in vitro neutrophil responses were positively associated with twitching motility, and negatively with mucoidy. In vitro neutrophil phagocytosis was a predictor of persistent infection following tobramycin even after adjustment for clinical risk factors.ConclusionsPA isolates from new-onset CF infection show strain-specific susceptibility to neutrophil antibacterial functions, and infection with PA isolates resistant to neutrophil phagocytosis is an independent risk factor for failed tobramycin eradication.