Bacterial Phagocytosis Research Articles

Alpha-defensins inhibit ERK/STAT3 signaling during monocyte-macrophage differentiation and impede macrophage function

Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder associated with a 5–tenfold decrease in lung levels of alpha-1-antitrypsin (AAT) and an increased risk for obstructive lung disease. α-defensins are cationic broad-spectrum cytotoxic and pro-inflammatory peptides found in the azurophilic granules of neutrophils. The concentration of α-defensins is less than 30 nM in the bronchoalveolar lavage fluid of healthy controls but is up to 6 μM in AATD individuals with significant lung function impairment. Alveolar macrophages are generally classified into pro-inflammatory (M1) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. Therefore, monocyte-macrophage differentiation should be tightly controlled to maintain lung integrity. In this study, we determined the effect of α-defensins on monocyte-macrophage differentiation and identified the molecular mechanism of this effect. The results of this study demonstrate that 2.5 μM of α-defensins inhibit the phosphorylation of ERK1/2 and STAT3 and suppress the expression of M2 macrophage markers, CD163 and CD206. In addition, a scratch assay shows that the high concentration of α-defensins inhibits cell movement by ~ 50%, and the phagocytosis assay using flow cytometry shows that α-defensins significantly reduce the bacterial phagocytosis rate of monocyte-derived macrophages (MDMs). To examine whether exogenous AAT is able to alleviate the inhibitory effect of α-defensins on macrophage function, we incubated MDMs with AAT prior to α-defensin treatment and demonstrate that AAT improves the migratory ability and phagocytic ability of MDMs compared with MDMs incubated only with α-defensins. Taken together, this study suggests that a high concentration of α-defensins inhibits the activation of ERK/STAT3 signaling, negatively regulates the expression of M2 macrophage markers, and impairs innate immune function of macrophages.

Irisflorentin promotes bacterial phagocytosis and inhibits inflammatory responses in macrophages during bacterial infection

Bacterial infection remains a big concern in the patients of ICU, which is the main cause of life-threatening organ dysfunction, or even sepsis. The poor control of bacterial infection caused by antibiotic resistance, etc. or the overwhelming immune response are the most important patho genic factors in intensive care unit (ICU) patients. As main pathogens, antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), impose serious challenges during sepsis and require alternative therapeutic options. Irisflorentin (IFL) is one of the major bioactive compounds isolated from the roots of Belamcanda chinensis (Shegan). In this study, IFL could suppress inflammatory response induced by MRSA or a synthetic mimic of bacterial lipoprotein (Pam3CSK4). IFL treatment enhanced the ability of macrophages to phagocytose bacteria likely through up-regulating the expression of phagocytic receptors SR-A1 and FcγR2a. Furthermore, IFL inhibited Pam3CSK4-induced production of pro-inflammatory cytokines, including IL-6 and TNF-α in Raw 264.7 cells, mouse primary macrophages or dendritic cells. IFL treatment also inhibited heat-killed MRSA-induced secretion of IL-6 and TNF-α in mouse bone marrow-derived macrophages. Moreover, IFL attenuated M1 polarization of macrophages as indicated by the down-regulated expression of its polarization markers CD86 and iNOS. Mechanistically, IFL markedly decreased the Pam3CSK4-induced activation of ERK, JNK or p38 MAPK pathways in macrophages. Taken together, IFL may serve as a promising compound for the therapy of bacterial infection, particularly those caused by antibiotic-resistant bacteria, such as MRSA.

Fei-Yan-Qing-Hua decoction decreases hyperinflammation by inhibiting HMGB1/RAGE signaling and promotes bacterial phagocytosis in the treatment of sepsis

Ethnopharmacological relevanceFei-Yan-Qing-Hua decoction (FYQHD), derived from the renowned formula Ma Xing Shi Gan tang documented in Zhang Zhong Jing's “Treatise on Exogenous Febrile Disease” during the Han Dynasty, has demonstrated notable efficacy in the clinical treatment of pneumonia resulting from bacterial infection. However, its molecular mechanisms underlying the therapeutic effects remains elusive. Aim of the studyThis study aimed to investigate the protective effects of FYQHD against lipopolysaccharide (LPS) and carbapenem-resistant Klebsiella pneumoniae (CRKP)-induced sepsis in mice and to elucidate its specific mechanism of action. Materials and methodsSepsis models were established in mice through intraperitoneal injection of LPS or CRKP. FYQHD was administered via gavage at low and high doses. Serum cytokines, bacterial load, and pathological damage were assessed using enzyme-linked immunosorbent assay (ELISA), minimal inhibitory concentration (MIC) detection, and hematoxylin and eosin staining (H&E), respectively. In vitro, the immunoregulatory effects of FYQHD on macrophages were investigated through ELISA, MIC, quantitative real-time PCR (Q-PCR), immunofluorescence, Western blot, and a network pharmacological approach. ResultsThe application of FYQHD in the treatment of LPS or CRKP-induced septic mouse models revealed significant outcomes. FYQHD increased the survival rate of mice exposed to a lethal dose of LPS to 33.3%, prevented hypothermia (with a rise of 3.58 °C), reduced pro-inflammatory variables (including TNF-α, IL-6, and MCP-1), and mitigated tissue damage in LPS or CRKP-induced septic mice. Additionally, FYQHD decreased bacterial load in CRKP-infected mice. In vitro, FYQHD suppressed the expression of inflammatory cytokines in macrophages activated by LPS or HK-CRKP. Mechanistically, FYQHD inhibited the PI3K/AKT/mTOR/4E-BP1 signaling pathway, thereby suppressing the translational level of inflammatory cytokines. Furthermore, it reduced the expression of HMGB1/RAGE, a positive feedback loop in the inflammatory response. Moreover, FYQHD was found to enhance the phagocytic activity of macrophages by upregulating the expression of phagocytic receptors such as CD169 and SR-A1. ConclusionFYQHD provides protection against bacterial sepsis by concurrently inhibiting the inflammatory response and augmenting the phagocytic ability of immune cells.

Nicotinamide mononucleotide as a therapeutic agent to alleviate multi-organ failure in sepsis

BackgroundSepsis-caused multi-organ failure remains the major cause of morbidity and mortality in intensive care units with limited therapeutics. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD+), has been recently reported to be protective in sepsis; however, its therapeutic effects remain to be determined. This study sought to investigate the therapeutic effects of NMN in septic organ failure and its underlying mechanisms.MethodsSepsis was induced by feces-injection-in-peritoneum in mice. NMN was given after an hour of sepsis onset. Cultured neutrophils, macrophages and endothelial cells were incubated with various agents.ResultsWe demonstrate that administration of NMN elevated NAD+ levels and reduced serum lactate levels, oxidative stress, inflammation, and caspase-3 activity in multiple organs of septic mice, which correlated with the attenuation of heart dysfunction, pulmonary microvascular permeability, liver injury, and kidney dysfunction, leading to lower mortality. The therapeutic effects of NMN were associated with lower bacterial burden in blood, and less ROS production in septic mice. NMN improved bacterial phagocytosis and bactericidal activity of macrophages and neutrophils while reducing the lipopolysaccharides-induced inflammatory response of macrophages. In cultured endothelial cells, NMN mitigated mitochondrial dysfunction, inflammation, apoptosis, and barrier dysfunction induced by septic conditions, all of which were offset by SIRT3 inhibition.ConclusionNAD+ repletion with NMN prevents mitochondrial dysfunction and restrains bacterial dissemination while limiting inflammatory damage through SIRT3 signaling in sepsis. Thus, NMN may represent a therapeutic option for sepsis.

Utilization of Platelet-Rich Plasma (PRP) in the Performance of Blood Cultures

Sepsis is a leading cause of death and morbidity. A precise microbiologic diagnosis in these patients reduces mortality, morbidity, hospital length of stay and costs. Current blood culture technology is woefully inadequate in the detection of circulating bacteria, with detection rates in the range of only 32 to 71%. Platelets play a key role in innate immunity. Platelets interact directly with innate immune cells, exert important immunomodulatory effects, and promote leucocyte migration to sites of infection. During active bacterial infection, platelets release platelet factor -4 which binds onto a variety of bacterial surfaces, thus rendering bacteria more susceptible to opsonization by leucocytes. Platelet surface Toll-like receptors play a key role in regulating bacterial phagocytosis by leucocytes. FcgammaRllA mediates immune complex mediated platelet activation for killing of opsonized bacteria. Platelets also have been observed to “corral” circulating bacteria by forming hollow spheres around them. For these reasons, we performed a prospective pilot study involving 100 patients with suspected sepsis testing the hypothesis that BC performed with PRP is superior to traditional BC performed with whole blood in detecting circulating bacteria. PRP was prepared via centrifugation of blood contained in 3 cc CBC tubes containing EDTA obtained upon admission. PRP and whole blood were injected into separate and identical culture media bottles and cultures were incubated identically in our state-of-the art microbiology laboratory. PRP culture technique detected two patients with circulating bacteria in whom traditional blood cultures were negative: Enterobacter faecalis in an elderly woman with bilateral pneumonia and Burkolderia capecia in an immunocompromised elderly man with pneumonia. In two patients with urosepsis culture results were concordant with identification of E. coli in both techniques. In eight patients, traditional BC technique detected circulating bacteria, wherein PRP-generated cultures were negative: three with Klebsiella pneumoniae, two with Enterococcus faecalis, two with E. Coli, and one with anaerobic Bacteroides Theta-iota group. Three positive cultures generated by traditional methodology were deemed contaminants by I.D. consultation. No contaminants or false positives were detected by the PRP-generated technique. Thus, in this pilot study, evaluating the utility of blood cultures prepared from platelet-rich-plasma, two patients were detected to harbor pathogenic circulating bacteria which was undetected by standard state-of-the-art blood culture techniques. Two distinct disadvantages of BC prepared from PRP relative to traditional BC technique in this study were the time delay in preparing PRP from the patient's CBC tube: these had been stored in the lab for typically 4 to 48 hours before centrifugation and subsequent inoculation, and the dramatic difference in volume represented by PRP vs. traditional BC: typically .5 to 1.1 cc vs. 32 to 40 cc, respectively. An additional disadvantage was the presence of EDTA in CBC tubes, which is confirmed to exert antibacterial properties. The results of this pilot study are intriguing. Further studies comparing PRP-generated BC and traditional BC in the detection of circulating bacteria with emerging identification techniques such as metrogenomic analysis in detecting bacterial DNA, RNA or peptide signatures would be interesting.

Expression of Concern for Meconi et al., "Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis".

Expression of Concern for Meconi et al., "Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis".

Microenvironment-Regulating Drug Delivery Nanoparticles for Treating and Preventing Typical Biofilm-Induced Oral Diseases.

The oral cavity comprises an environment full of microorganisms. Dysregulation of this microbial-cellular microenvironment will lead to a series of oral diseases, such as implant-associated infection caused by Staphylococcus aureus (S. aureus) biofilms and periodontitis initiated by Streptococcus oralis (S. oralis). In this study, a liposome-encapsulated indocyanine green (ICG) and rapamycin drug-delivery nanoparticle (ICG-rapamycin) is designed to treat and prevent two typical biofilm-induced oral diseases by regulating the microbial-cellular microenvironment. ICG-rapamycin elevates the reactive oxygen species (ROS) and temperature levels to facilitate photodynamic and photothermal mechanisms under near-infrared (NIR) laser irradiation for anti-bacteria. In addition, it prevents biofilm formation by promoting bacterial motility with increasing the ATP levels. The nanoparticles modulate the microbial-cellular interaction to reduce cellular inflammation and enhance bacterial clearance, which includes promoting the M2 polarization of macrophages, upregulating the anti-inflammatory factor TGF-β, and enhancing the bacterial phagocytosis of macrophages. Based on these findings, ICG-rapamycin is applied to implant-infected and periodontitis animal models to confirm the effects in vivo. This study demonstrates that ICG-rapamycin can treat and prevent biofilm-induced oral diseases by regulating the microbial-cellular microenvironment, thus providing a promising strategy for future clinical applications.

LncRNA MALAT1/microRNA-30b axis regulates macrophage polarization and function.

Macrophages (Mφ) are long-lived myeloid cells that can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, tissue damage, and regeneration. Noncoding RNA are a class of nonprotein-coding transcriptome with numerous interdependent biological roles; however, their functional interaction in the regulation of Mφ polarization and immune responses remain unclear. Here, we show antagonistic relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. MALAT1 expression displays a time-dependent induction during Mφ differentiation and, upon challenge with TLR4 agonist (E. coli LPS). MALAT1 knockdown promoted the expression of M2Mφ markers without affecting M1Mφ markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 differentiation. Compared to the control, MALAT1 knockdown resulted in reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions in Mφ. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2 Mφ differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. Concurrent MALAT1 knockdown and miR-30b overexpression exhibited the most significant attenuation in both assays. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, M1Mφ markers and downregulation of miR-30b expression in gingival tissues suggesting a pro-inflammatory function of MALAT1 in vivo. Overall, we unraveled the role of MALAT1 in Mφ polarization and delineated the underlying mechanism of its regulation by involving MALAT-1-driven miR-30b sequestration.

Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

Insect-pathogen crosstalk and the cellular-molecular mechanisms of insect immunity: uncovering the underlying signaling pathways and immune regulatory function of non-coding RNAs.

Multicellular organisms are constantly subjected to pathogens that might be harmful. Although insects lack an adaptive immune system, they possess highly effective anti-infective mechanisms. Bacterial phagocytosis and parasite encapsulation are some forms of cellular responses. Insects often defend themselves against infections through a humoral response. This phenomenon includes the secretion of antimicrobial peptides into the hemolymph. Specific receptors for detecting infection are required for the recognition of foreign pathogens such as the proteins that recognize glucans and peptidoglycans, together referred to as PGRPs and βGRPs. Activation of these receptors leads to the stimulation of signaling pathways which further activates the genes encoding for antimicrobial peptides. Some instances of such pathways are the JAK-STAT, Imd, and Toll. The host immune response that frequently accompanies infections has, however, been circumvented by diseases, which may have assisted insects evolve their own complicated immune systems. The role of ncRNAs in insect immunology has been discussed in several notable studies and reviews. This paper examines the most recent research on the immune regulatory function of ncRNAs during insect-pathogen crosstalk, including insect- and pathogen-encoded miRNAs and lncRNAs, and provides an overview of the important insect signaling pathways and effector mechanisms activated by diverse pathogen invaders.

Pyrrolidine dithiocarbamate in combination with L-N-monomethyl arginine alleviates Staphylococcus aureus infection via regulation of CXCL8/CXCR1 axis in peritoneal macrophages in vitro

The CXCL8/CXCR1 axis in conjoint with the free radicals and anti-oxidants dictates the severity of inflammation caused by the bacteria, Staphylococcus aureus. S.aureus mediated inflammatory processes is regulated by NF-κB and its product, iNOS. The objective of this study was to examine the effects of inhibition of NF-κB and iNOS on CXCL8/CXCR1, alteration in M1/M2 polarization of macrophages and associated inflammatory responses during S.aureus infection in vitro. For this, the murine peritoneal macrophages were pretreated with NF-κB inhibitor, Pyrrolidine dithiocarbamate (PDTC) and iNOS inhibitor, L-N-monomethyl arginine (LNMMA), either alone or in combination, followed by time-dependent S.aureus infection. The chemotactic migrations of macrophages were determined by the agarose spot assay. The iNOS, NF-κB and CXCR1 protein expressions were evaluated. The ROS level (superoxide, H2O2, NO) and antioxidant activities (SOD, CAT, GSH, arginase) were measured. The intra-macrophage phagoctyic activity had been analyzed by confocal microscopy. S.aureus activated macrophages showed increased iNOS expression that symbolizes M1 characterization of macrophages. The results suggest that the combination treatment of LNMMA + PDTC was effective in diminution of CXCL8 production and CXCR1 expression through downregulation of NF-κB and iNOS signaling pathway. Consequently, there was decrement in macrophage migration, reduced ROS generation, elevated antioxidant enzyme activity as well as bacterial phagocytosis at 90 min post bacterial infection. The increased arginase activity further proves the switch from pro-inflammatory M1 to anti-inflammatory M2 polarization of macrophages. Concludingly, the combination of PDTC + LNMMA could resolve S.aureus mediated inflammation through mitigation of CXCL8/CXCR1 pathway switching from M1 to M2 polarization.

Bone Marrow Mesenchymal Stem Cell-Derived Dermcidin-Containing Migrasomes enhance LC3-Associated Phagocytosis of Pulmonary Macrophages and Protect against Post-Stroke Pneumonia.

Pneumonia is one of the leading causes of death in patients with acute ischemic stroke (AIS). Antibiotics fail to improve prognosis of patients with post-stroke pneumonia, albeit suppressing infection, due to adverse impacts on the immune system. The current study reports that bone marrow mesenchymal stem cells (BM-MSC) downregulate bacterial load in the lungs of stroke mice models. RNA-sequencing of the lung from BM-MSC-treated stroke models indicates that BM-MSC modulates pulmonary macrophage activities after cerebral ischemia. Mechanistically, BM-MSC promotes the bacterial phagocytosis of pulmonary macrophages through releasing migrasomes, which are migration-dependent extracellular vesicles. With liquid chromatography-tandem mass spectrometry (LC-MS/MS), the result shows that BM-MSC are found to load the antibacterial peptide dermcidin (DCD) in migrasomes upon bacterial stimulation. Besides the antibiotic effect, DCD enhances LC3-associated phagocytosis (LAP) of macrophages, facilitating their bacterial clearance. The data demonstrate that BM-MSC is a promising therapeutic candidate against post-stroke pneumonia, with dual functions of anti-infection and immunol modulation, which is more than a match for antibiotics treatment.

Bioactive compounds in Layacha brown rice (Oryza sativa L.) improve immune responses in mice via activation of transcription factor Nrf2

Dysregulated or weak immunity is caused by ageing, chemo-radiotherapy, COVID-19, infections, steroids, pollutants and toxins. Bioactive foods are required for boosting immunity. In the present study, eight rice (Oryza sativa L.) varieties were selected from the repository of 23,250 Indian germplasm accessions. The immunomodulatory effects of these rice varieties were assessed in vitro and in vivo. Layacha-rice-methanolic-extract (Larimex) significantly enhanced innate (>20%) and adaptive (>10%) immune responses evinced from higher bacterial phagocytosis by macrophages, increased mitogen-induced T-cell proliferation (11%) and scavenged reactive oxygen species (ROS) (70–80%). Larimex activated transcription factor Nrf2 and its downstream genes NQO1, HO1 and Txnrd1 in immune cells. Larimex significantly improved immune responses (>20%) only in cells from wild type but not Nrf2 knock-out mice indicating its causal role in boosting immunity. Untargeted metabolomics of Layacha rice showed preponderance of metabolic pathways and bioactive compounds, which activate Nrf2 in mammalian cells. Layacha rice can be a suitable food for boosting immunity.

Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus)

Antimicrobial peptides (AMPs) are members of humoral immunity and particpate in resisting microbial invasion. In this study, an AMP gene hepcidin was obtained from the oriental loach Misgurnus anguillicaudatus and named Ma-Hep. This Ma-Hep encodes a peptide of 90 amino acids, with a predicted active peptide segment (Ma-sHep) of 25 amino acids at C terminus. Stimulation by a bacterial pathogen Aeromonas hydrophila resulted in significant up-regulation of Ma-Hep transcripts in loach midgut, head kidney, and gill. Ma-Hep and Ma-sHep proteins were expressed in Pichia pastoris and their antibacterial activity was examined. Results showed that Ma-sHep possessed stronger antibacterial activity against various Gram-positive and Gram-negative bacteria, compared to Ma-Hep. Scanning electron microscopy showed that Ma-sHep might kill bacteria by destroying bacterial cell membranes. Moreover, we found that Ma-sHep had an inhibitory effect on blood cell apoptosis induced by A. hydrophila and facilitated the bacterial phagocytosis and clearance in loach. Histopathological analysis indicated Ma-sHep could protect liver and gut of loach from bacterial infection. Ma-sHep has high thermal stability and PH stability, which is conducive to further feed addition. Feed supplemented with Ma-sHep expressing yeast improved the intestinal flora of loach by increasing the dominant bacteria and decreasing the harmful bacteria. Feed supplemented with Ma-sHep expressing yeast also regulated the expression of inflammatory related factors in various tissues of loach and reduced the mortality of loach upon bacterial infection. These findings show that the antibacterial peptide Ma-sHep is involved in the antibacterial defense of loach and can be used as a candidate for new antimicrobial agents in aquaculture.

Differential effects of dexmedetomidine on Gram-positive and Gram-negative bacterial killing and phagocytosis

Dexmedetomidine is a commonly used sedative in perioperative and intensive care settings with purported immunomodulatory properties. Since its effects on immune functions against infections have not been extensively studied, we tested the effects of dexmedetomidine on Gram-positive [Staphylococcus aureus and Enterococcus faecalis] and Gram-negative bacteria [Escherichia coli], and on effector functions of human monocytes THP-1 cells against them. We evaluated phagocytosis, reactive oxygen species (ROS) formation, and CD11b activation, and performed RNA sequencing analyses. Our study revealed that dexmedetomidine improved Gram-positive but mitigated Gram-negative bacterial phagocytosis and killing in THP-1 cells. The attenuation of Toll-like receptor 4 (TLR4) signaling by dexmedetomidine was previously reported. Thus, we tested TLR4 inhibitor TAK242. Similar to dexmedetomidine, TAK242 reduced E. coli phagocytosis but enhanced CD11b activation. The reduced TLR4 response potentially increases CD11b activation and ROS generation and subsequently enhances Gram-positive bacterial killing. Conversely, dexmedetomidine may inhibit the TLR4-signaling pathway and mitigate the alternative phagocytosis pathway induced by TLR4 activation through LPS-mediated Gram-negative bacteria, resulting in worsened bacterial loads. We also examined another α2 adrenergic agonist, xylazine. Because xylazine did not affect bacterial clearance, we proposed that dexmedetomidine may have an off-target effect on bacterial killing process, potentially involving crosstalk between CD11b and TLR4. Despite its potential to attenuate inflammation, we provide a novel insight into potential risks of dexmedetomidine use during Gram-negative infections, highlighting the differential effect of dexmedetomidine on Gram-positive and Gram-negative bacteria.

44 Dual Proteomic Exploration of the Mechanisms Underlying Brake Dust-Induced Impairment of Phagocytic Capacity in Macrophages

Abstract Exposure to brake abrasion dust (BAD) reduces the phagocytic capacity of macrophages, possibly contributing to increased risk of airway infections in polluted regions. The mechanisms that underlie this toxicity remain to be characterised and, considering that pre-exposure of respiratory bacteria to alternative traffic-related particulates enhances their dissemination into the lungs of mice, may include changes in the virulence of pathogens themselves. Dual proteomic screening (TMT-labelled UPLC-MS) was performed for RAW 264.7 monocyte-derived macrophages and S.aureus (USA300) following 24h exposures to BAD (0-16 ug/ml). In S.aureus, expression of agr-regulated, anti-leukocytic virulence factors (lipases, staphopains, hemolysins) increased in a dose-dependent manner as well as proteins required for epithelial invasion (serine proteases) and antibiotic resistance. RAW 264.7 exhibited a predominantly adaptive response following exposures, enhancing expression of proteins required for metal homeostasis, NFkB-mediated inflammation and endosomal maturation. However, disrupted expression of proteins required for electron transport chain activity and Fe-S cluster synthesis accompanied significant dose-dependent reductions in ATP production (60-82%, p< 0.001) while transition electron microscopy (TEM) detected disrupted mitochondrial membrane morphology. TEM also identified reductions in pseudopodia length in exposed macrophages (54% shorter than control, p< 0.05), highlighting a mechanism by which BAD exposure may decrease the incidence of host-pathogen interactions. These data highlight dual mechanisms by which BAD emissions could impair bacterial phagocytosis and enhance susceptibility to airway infections. Through avoidance and suppression of host anti-microbial mechanisms by the bacteria and, as actin polymerisation depends on ATP availability, loss of mitochondrial function and downstream pseudopodia projection in macrophages.

DANGER Signals Activate G -Protein Receptor Kinases Suppressing Neutrophil Function and Predisposing to Infection After Tissue Trauma.

Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition. Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation. We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models. Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury. Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Epithelial TLR9 upregulation by respiratory viruses impairs pulmonary antibacterial immunity

Abstract While much is known about signaling downstream of the dsDNA sensor TLR9, regulation of its expression is underexplored and has significant consequences for innate immune responses to pathogens. Mice lacking TLR9 surprisingly fare better upon coinfection with influenza and Staphylococcus aureus, and other groups have shown protection from S. aureus alone as well. These TLR9KO mice display lower inflammatory cytokine production, reduced cellular infiltration into airspaces, and increased survival. While macrophages taken from influenza-infected TLR9KO mice display increased bacterial phagocytosis and killing, inhibition of TLR9 on wild-type murine macrophages ex vivo surprisingly decreased their ability to phagocytize and kill S. aureus. Bone marrow chimera experiments revealed that lack of TLR9 in non-hematopoietic cells was responsible for the protection from bacterial super-infection seen in whole-body TLR9KO mice. To assess which structural cell types were important for this protection, we bred epithelial- and endothelial-specific TLR9KO mice, which revealed a role for epithelial cells but not endothelium. Interestingly, we also found that influenza infection caused upregulation of TLR9 in epithelial cells. Moreover, infection with two other respiratory viruses also caused epithelial cells to increase TLR9 expression, as did treatment with the antiviral mediator interferon beta (IFNB). IFNB has long been known to regulate susceptibility to bacterial coinfection during influenza, and these results suggest that it does so in part by upregulating TLR9. Together, these data show a role for epithelial TLR9 upregulation, likely driven by IFNB, in the pathogenesis of respiratory viral/bacterial coinfection. Funding for this research was made possible in part by the Michigan Postdoctoral Pioneer Program at the University of Michigan Medical School, and by NIH grants NHLBI T32HL007517 (Helen Rich) and NHLBI R35HL144481 (Beth Moore).

Blocking purinergic signaling alters platelet and macrophage crosstalk and phagocytic ability in the peritoneal cavity during intra-abdominal sepsis

Intra-abdominal sepsis is a life-threatening complex syndrome caused by dysregulation of the host response to infection and is one of the major complications of intra-abdominal surgery. To date, only supportive therapies such as ventilation support, antibiotics and organ replacement are available. However, an increase in antibiotic resistance and the invasiveness of organ transplant has necessitated therapies targeting pathophysiological mechanisms. Platelets, which express the purinergic receptors P2Y1 and P2Y12, mediate macrophage polarization and secretion of pro-inflammatory markers contributing to tissue injury during sepsis. Previously, our group has shown that blocking purinergic signaling through P2Y1 and P2Y12 antagonism significantly lowered inflammatory cytokine levels and improved survival in a murine model of sepsis in a sex specific manner. We hypothesize that the inhibition of P2Y1 and P2Y12 signaling pathways improves the sex-specific outcomes of intra-abdominal sepsis by modulating the crosstalk between platelets and macrophages. Male and female mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis followed by intraperitoneal administration of ticagrelor (P2Y12 antagonist) or MRS2279 (P2Y1 antagonist). The peritoneal cavity fluid (PCF) was collected 4 and 24 hours post-surgery and analyzed. At 4 hours post-surgery, PCF macrophages exhibited increased bacterial phagocytosis from ticagrelor and MRS2279 treated groups in female mice only compared to CLP group. At 24 hours post-CLP surgery, platelet counts did not change but soluble p-selectin and TBX-2 levels were significantly decreased in male groups treated with either ticagrelor or MRS2279, compared to controls. In contrast, soluble p-selectin and TBX-2 levels did not change in female samples.Thus, we conclude that pharmacologically blocking P2Y1 and P2Y12 receptors can enhance the ex-vivo phagocytic activity of macrophages in a sex-related manner, and overall modulate platelet secretion and influence macrophages’ recruitment and activation in the PC during sepsis. National Institute of Health AI156627-01 to EL This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differences in bacteria nanomotion profiles and neutrophil nanomotion during phagocytosis.

The main goal of this work is to highlight the connection between nanomotion and the metabolic activity of living cells. We therefore monitored the nanomotion of four different clinical strains of bacteria (prokaryotes) and the bacterial phagocytosis by neutrophil granulocytes (eukaryotes). All clinical strains of bacteria, regardless of their biochemical profile, showed pronounced fluctuations. Importantly, the nature of their nanomotions was different for the different strains. Flagellated bacteria (Escherichia coli, Proteus mirabilis) showed more pronounced movements than the non-flagellated forms (Staphylococcus aureus, Klebsiella pneumoniae). The unprimed neutrophil did not cause any difference in cantilever oscillations with control. However, in the process of phagocytosis of S. aureus (metabolically active state), a significant activation of neutrophil granulocytes was observed and cell nanomotions were maintained at a high level for up to 30 min of observation. These preliminary results indicate that nanomotion seems to be specific to different bacterial species and could be used to monitor, in a label free manner, basic cellular processes.