Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Radiotherapy orchestrates natural killer cell dependent antitumor immune responses through CXCL8.

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.

Cytokine profiles in the detection of severe lung involvement in hospitalized patients with COVID-19: The IL-8/IL-32 axis.

Coronavirus disease 2019 (COVID-19) is an infectious respiratory disorder caused by a new coronavirus called SARS-CoV-2. The pathophysiology of severe COVID-19 is associated with a "cytokine storm". IL-32 is a key modulator in the pathogenesis of various clinical conditions and is mostly induced by IL-8. IL-32 modulates important inflammatory pathways (including TNF-α, IL-6 and IL-1b), contributing to the pathogenesis of inflammatory diseases. Il-32 was never evaluated before in COVID-19 patients stratifying as mild-moderate and severe patients. A total of 64 COVID-19 patients, 27 healthy controls were consecutively enrolled in the study. Serum concentrations of biomarkers including IL-1ß, IL-10, IFN-γ, TNF-α and IL-6 were quantified by bead-based multiplex analysis and Serum concentration of IL-8 and IL-32 were determined by enzyme-linked immunosorbent assay (ELISA) kits. Interestingly, among the blood parameters, neutrophil and lymphocyte counts were significantly lower in severe COVID-19 patients than in the other, on the contrary, CRP was significantly higher in severe patients than in other groups. The cytokines that best distinguished controls from COVID-19 patients were IL-8 and IL-32, while IL-6 resulted the better variables for discriminate severe group. The best model performance for severe group was obtained by the combination of IL-32, IL-6, IFN-γ, and CRP serum concentration showing an AUC = 0.83. A cut off of 15 pg/ml of IL-6 greatly discriminate survivor from death patients. New insights related to the cytokine storm in COVID-19 patients, highlighting different severity of disease infection.

Sulfation Pattern Dependent Iron(III) Mediated Interleukin-8 Glycan Binding.

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.

The Betacoronavirus PHEV Replicates and Disrupts the Respiratory Epithelia and Upregulates Key Pattern Recognition Receptor Genes and Downstream Mediators, Including IL-8 and IFN-λ.

The upper respiratory tract is the primary site of infection by porcine hemagglutinating encephalomyelitis virus (PHEV). In this study, primary porcine respiratory epithelial cells (PRECs) were cultured in an air-liquid interface (ALI) to differentiate into a pseudostratified columnar epithelium, proliferative basal cells, M cells, ciliated cells, and mucus-secreting goblet cells. ALI-PRECs recreates a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. PHEV replicated actively in this environment, inducing cytopathic changes and progressive disruption of the mucociliary apparatus. The innate immunity against PHEV was comparatively evaluated in ALI-PREC cultures and tracheal tissue sections derived from the same cesarean-derived, colostrum-deprived (CDCD) neonatal donor pigs. Increased expression levels of TLR3 and/or TLR7, RIG1, and MyD88 genes were detected in response to infection, resulting in the transcriptional upregulation of IFN-λ1 in both ALI-PREC cultures and tracheal epithelia. IFN-λ1 triggered the upregulation of the transcription factor STAT1, which in turn induced the expression of the antiviral IFN-stimulated genes OAS1 and Mx1. No significant modulation of the major proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α) was detected in response to PHEV infection. However, a significant upregulation of different chemokines was observed in ALI-PREC cultures (CCL2, CCL5, CXCL8, and CXCL10) and tracheal epithelium (CXCL8 and CXCL10). This study shed light on the molecular mechanisms driving the innate immune response to PHEV at the airway epithelium, underscoring the important role of respiratory epithelial cells in the maintenance of respiratory homeostasis and on the initiation, resolution, and outcome of the infectious process. IMPORTANCE The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro, on air-liquid interface porcine respiratory cell cultures. The results from the study underscore the important role of respiratory epithelial cells in maintaining respiratory homeostasis and on the initiation, resolution, and outcome of the PHEV infectious process.

Elevated Monocytic Interleukin-8 Expression under Intermittent Hypoxia Condition and in Obstructive Sleep Apnea Patients.

Obstructive sleep apnea (OSA) is a disease with great cardiovascular risk. Interleukin-8 (IL-8), an important chemokine for monocyte chemotactic migration, was studied under intermittent hypoxia condition and in OSA patients. Monocytic THP-1 cells were used to investigate the effect of intermittent hypoxia on the regulation of IL-8 by an intermittent hypoxic culture system. The secreted protein and mRNA levels were studied by means of enzyme-linked immunosorbent assay and RT/real-time PCR. The chemotactic migration of monocytes toward a conditioned medium containing IL-8 was performed by means of the transwell filter migration assay. Peripheral venous blood was collected from 31 adult OSA patients and RNA was extracted from the monocytes for the analysis of IL-8 expression. The result revealed that intermittent hypoxia enhanced the monocytic THP-1 cells to actively express IL-8 at both the secreted protein and mRNA levels, which subsequently increased the migration ability of monocytes toward IL-8. The ERK, PI3K and PKC pathways were demonstrated to contribute to the activation of IL-8 expression by intermittent hypoxia. In addition, increased monocytic IL-8 expression was found in OSA patients, with disease severity dependence and diurnal changes. This study concluded the monocytic IL-8 gene expression can be activated by intermittent hypoxia and increased in OSA patients.

PD-1 blockade counteracts post-COVID-19 immune abnormalities and stimulates the anti-SARS-CoV-2 immune response.

A substantial proportion of patients who have recovered from coronavirus disease-2019 (COVID-19) experience COVID-19-related symptoms even months after hospital discharge. We extensively immunologically characterized patients who recovered from COVID-19. In these patients, T cells were exhausted, with increased PD-1+ T cells, as compared with healthy controls. Plasma levels of IL-1ß, IL-1RA, and IL-8, among others, were also increased in patients who recovered from COVID-19. This altered immunophenotype was mirrored by a reduced ex vivo T cell response to both nonspecific and specific stimulation, revealing a dysfunctional status of T cells, including a poor response to SARS-CoV-2 antigens. Altered levels of plasma soluble PD-L1, as well as of PD1 promoter methylation and PD1-targeting miR-15-5p, in CD8+ T cells were also observed, suggesting abnormal function of the PD-1/PD-L1 immune checkpoint axis. Notably, ex vivo blockade of PD-1 nearly normalized the aforementioned immunophenotype and restored T cell function, reverting the observed post-COVID-19 immune abnormalities; indeed, we also noted an increased T cell-mediated response to SARS-CoV-2 peptides. Finally, in a neutralization assay, PD-1 blockade did not alter the ability of T cells to neutralize SARS-CoV-2 spike pseudotyped lentivirus infection. Immune checkpoint blockade ameliorates post-COVID-19 immune abnormalities and stimulates an anti-SARS-CoV-2 immune response.

Bacterial membrane vesicles and phages in blood after consumption of lacticaseibacillus rhamnosus JB-1.

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.

LOX-1-Expressing Immature Neutrophils Identify Critically-Ill COVID-19 Patients at Risk of Thrombotic Complications.

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.

In vitro induction of interleukin-8 by SARS-CoV-2 Spike protein is inhibited in bronchial epithelial IB3-1 cells by a miR-93-5p agomiR.

One of the major clinical features of COVID-19 is a hyperinflammatory state, which is characterized by high expression of cytokines (such as IL-6 and TNF-α), chemokines (such as IL-8) and growth factors and is associated with severe forms of COVID-19. For this reason, the control of the "cytokine storm" represents a key issue in the management of COVID-19 patients. In this study we report evidence that the release of key proteins of the COVID-19 "cytokine storm" can be inhibited by mimicking the biological activity of microRNAs. The major focus of this report is on IL-8, whose expression can be modified by the employment of a molecule mimicking miR-93-5p, which is able to target the IL-8 RNA transcript and modulate its activity. The results obtained demonstrate that the production of IL-8 protein is enhanced in bronchial epithelial IB3-1 cells by treatment with the SARS-CoV-2 Spike protein and that IL-8 synthesis and extracellular release can be strongly reduced using an agomiR molecule mimicking miR-93-5p.

DNAM-1/CD226 is functionally expressed on acute myeloid leukemia (AML) cells and is associated with favorable prognosis.

DNAM-1 is reportedly expressed on cytotoxic T and NK cells and, upon interaction with its ligands CD112 and CD155, plays an important role in tumor immunosurveillance. It has also been reported to be functionally expressed by myeloid cells, but expression and function on malignant cells of the myeloid lineage have not been studied so far. Here we analyzed expression of DNAM-1 in leukemic cells of acute myeloid leukemia (AML) patients. We found substantial levels of DNAM-1 to be expressed on leukemic blasts in 48 of 62 (> 75%) patients. Interaction of DNAM-1 with its ligands CD112 and CD155 induced release of the immunomodulatory cytokines IL-6, IL-8 IL-10 and TNF-α by AML cells and DNAM-1 expression correlated with a more differentiated phenotype. Multivariate analysis did not show any association of DNAM-1 positivity with established risk factors, but expression was significantly associated with clinical disease course: patients with high DNAM-1 surface levels had significantly longer progression-free and overall survival compared to DNAM-1low patients, independently whether patients had undergone allogenic stem cell transplantation or not. Together, our findings unravel a functional role of DNAM-1 in AML pathophysiology and identify DNAM-1 as a potential novel prognostic maker in AML.

Phosphodiesterase 4 mediates interleukin-8-induced heterologous desensitization of the ß2 -adrenergic receptor.

Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.

Generation and characterization of a humanized anti-IL-17A rabbit monoclonal antibody.

Interleukin-17A (IL-17A) produced by Th17 cells, contributes to the pathogenesis of various autoimmune diseases by stimulating the release of cytokines and chemokines and its regulation. Anti-IL-17A antibody which blocks the function of IL-17A has been proved to be an effective treatment of autoimmune disease. The aim of our study was to generate a potential humanized anti-IL-17A therapeutic monoclonal antibody (mAb) through a comprehensive panel of in vitro and in vivo biological activity studies, as well as physicochemical characterization. HZD37-5, a humanized monoclonal antibody specifically recognizing N78 loci of IL-17A, binds to human and rhesus monkeys, blocks IL-17 induced signal transduction and the release of IL-6, IL-8, CXCL-1 and G-GSF. In an in vivo efficacy mouse model, HZD37-5 significantly inhibited human IL-17A induced-keratinocyte chemoattractant (KC) secretion in a dose-dependent manner. The pharmacokinetics (PK) study result of HZD37-5 in rhesus monkeys indicated that HZD37-5 had favorable PK characteristics with limited distribution (78.0-78.8 ml/kg), slow elimination (5.00-6.45 ml/day/kg), long half-life (9.1-10.7 days) and high bioavailability (103%) following a single IV or SC dose at 1.5 mg/kg. These findings provided a comprehensive preclinical characterization of HZD37-5 and supported that it may be developed as a potential therapeutic for the treatment of autoimmune diseases, including psoriasis, psoriatic arthritis, axial spondyloarthritis, etc.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Identification of a Family of Vibrio Type III Secretion System Effectors That Contain a Conserved Serine/Threonine Kinase Domain.

Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse Vibrio species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. IMPORTANCE Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse Vibrio species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and Shigella NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of Vibrio effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.

Immunoglobulin G Immune Complexes May Contribute to Neutrophil Activation in the Course of Severe Coronavirus Disease 2019.

Severe coronavirus disease 2019 (COVID-19) is associated with an overactive inflammatory response mediated by macrophages. Here, we analyzed the phenotype and function of neutrophils in patients with COVID-19. We found that neutrophils from patients with severe COVID-19 express high levels of CD11b and CD66b, spontaneously produce CXCL8 and CCL2, and show a strong association with platelets. Production of CXCL8 correlated with plasma concentrations of lactate dehydrogenase and D-dimer. Whole blood assays revealed that neutrophils from patients with severe COVID-19 show a clear association with immunoglobulin G (IgG) immune complexes. Moreover, we found that sera from patients with severe disease contain high levels of immune complexes and activate neutrophils through a mechanism partially dependent on FcγRII (CD32). Interestingly, when integrated in immune complexes, anti-severe acute respiratory syndrome coronavirus 2 IgG antibodies from patients with severe COVID-19 displayed a higher proinflammatory profile compared with antibodies from patients with mild disease. Our study suggests that IgG immune complexes might promote the acquisition of an inflammatory signature by neutrophils, worsening the course of COVID-19.

Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

Inflammation protein quantification by multiple reaction monitoring mass spectrometry in lipopolysaccharide-stimulated THP-1 cells.

RATIONALE: Inflammation is a cascade of events mediated by a cytokine network triggering the cellular response. In order to monitor the modulation of the crucial inflammatory proteins, e.g., Tumour Necrosis Factor-α (TNF-α), Interferon-γ (INF-γ), Interleukin-8 (IL-8) and Interleukin-10 (IL-10), upon stimulation with endotoxins, differentiated and undifferentiated THP-1 cells were treated with lipopolysaccharides (LPSs) from E. coli, key cell wall components of Gram-negative bacteria. METHODS: The multiple reaction monitoring mass spectrometry (MRM-MS) method was optimized by using the standard proteins to be quantified, in order to construct external calibration curves and define the analytical parameters. The developed method was used to quantify the above-mentioned inflammatory proteins in THP-1 differentiated cells upon stimulation with LPSs with high accuracy, sensitivity, and robustness. RESULTS: The analysis of such proteins in MRM mode allowed the kinetics of stimulation along the time up to 24 h to be followed and the MS results were found to be comparable with those obtained by Western-blotting. A significant increase in TNF-α release triggered a cascade mechanism leading to the production of INF-γ and IL-8. IL-10, instead, was found to be constant throughout the process. CONCLUSIONS: The developed MRM-MS method allowed the quantification of TNF-α, INF-γ, IL-8 and IL-10 along a time-course from 2 to 24 h. Hence, a trace of the kinetics of the inflammatory response in THP-1 cells upon stimulation with E. coli LPSs was obtained. Finally, the extensibility of the developed MRM method to serum samples and other matrices demonstrated the versatility of the approach and the possibility to quantify multiple target proteins in different biological samples by using a few microliters in a single analysis.

Impacts of anti-inflammatory phosphodiesterase inhibitors on a murine model of chronic pulmonary inflammation.

Chronic obstructive pulmonary disease (COPD) often tends to respond poorly to glucocorticoid (GC) therapy. Reduced Histone deacetylase-2 (HDAC-2) activity is an important mechanism behind this GC insensitivity. In this study, we investigated the effects of three phosphodiesterase inhibitors (PDEIs), with an anti-inflammatory propensity, on cigarette smoke (CS)-induced pulmonary inflammation and HDAC-2 activity. Male C57BL/6 mice were exposed to cigarette smoke (CS) over the course of 30 weeks. Administration of the PDEIs commenced from the 29th week and followed a schedule of once daily treatments, 5 days a week, for 2 weeks. Roflumilast (ROF) was administered intragastrically (5 mg·kg-1 ), while pentoxifylline (PTX) (10 mg·kg-1 ) and theophylline (THEO) (10 mg·kg-1 ) were administered intraperitoneally, either alone or in combination with a GC (triamcinolone acetonide or TRI, 5 mg·kg-1 , i.m., single injection). Lung morphometry, as well as the activity of HDAC-2, pro-inflammatory cytokines and reactive oxygen species (ROS) were assessed at the end of the 30-week course. CS exposure was associated with a reduction in HDAC-2 activity and the up-regulation of ROS expression. PTX, ROF, and THEO administration led to the partial restoration of HDAC-2 activity, which was favorably associated with the reduction of ROS expression. However, combining TRI to any of these PDEIs did not synergistically augment HDAC-2 activity. Inactivation of HDAC-2 due to long-term CS exposure is closely related to exaggerated oxidative stress, and this reduced HDAC-2 activity could partially be restored through the use of PDEIs. This finding provides a potential novel approach for further clinical research.