Extensive Inflammatory Response Research Articles

Gram-negative anaerobes elicit a robust keratinocytes immune response with potential insights into HS pathogenesis.

Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes invivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.

Individually or as a Team-The Immunological Milieu in the Lung Caused by Migrating Single-Sex or Mixed-Sex Larvae of Schistosoma mansoni.

While the lung is considered an efficient site for stopping the larvae of the acute Schistosoma spp. infection phase from migrating through extensive inflammatory responses in the surrounding tissues, little is known about these processes. To date, the highest resistance to infection has been achieved in experimental studies with radiation-attenuated cercariae immunization, which elicits a strong Th1/Th2 response in the lung and results in up to 80% protection. Based on our own studies demonstrating a systemic, unpolarized Th1/Th2 response resulting from infection with male or female Schistosoma mansoni, we hypothesize that this atypical immune response is already detectable during the pulmonary passage of parasite larvae. Therefore, we examined the immune milieu in the lungs of mice caused by migrating schistosome larvae, either male or female (single-sex groups) or male + female (bisexual control), 4 and 16 days after infection in bronchoalveolar lavage and lung tissue by flow cytometry, qPCR, and multiplex analyzes. Our results show only minor differences in the inflammatory profile between the single-sex groups but significant differences compared with the bisexual control group. Both single-sex infected groups have increased expression of inflammatory markers in lung tissue, higher numbers of cytotoxic T cells (day 4 post-infection) and more T helper cells (day 16 post-infection), compared with the bisexual control group. A single-sex infection, regardless of whether it is an infection with male or female cercariae, causes an immune milieu in the lung that is clearly different from an infection with both sexes. In terms of identifying therapeutic targets to achieve resistance to re-infection, it is of great scientific interest to identify the differences in the inflammatory potential of male or female and male + female parasites.

Abstract B038: Targeting a Treg-specific deubiquitinase module for antitumor immune therapy

Abstract Checkpoint immunotherapies have transformed the treatment landscape for multiple human malignancies, but still with very limited success in treatment of many human cancers. A major hurdle is mediated by regulatory T (Treg) cells, which suppress the function of antitumor effector T cells. However, current approaches in targeting Treg to activate antitumor immune responses have shown only transient efficacy and are highly unspecific. Importantly, our recent studies, through unbiased CRISPR screening, identified the death from cancer cell signature gene USP22 required for optimal FoxP3 expression. USP22 genetic inhibition resulted in an approximately 15-25% reduction of FoxP3 expression in Tregs and partially diminishes Treg suppressive functions, which consequently results in elevated antitumor immunity against a broad spectrum of cancer types in mice. Therefore, a 20% reduction in FoxP3 protein expression, in this case, by USP22 inhibition, creates an ideal therapeutic window to boost antitumor immunity without triggering extensive autoimmune inflammatory responses (Nature, 2020). More recently, we further demonstrated that tumor microenvironment factors, including TGF-β, hypoxia, and tumor metabolites, induce Treg fitness/adaptation through selectively upregulating a deubiquitinase module, including USP21 and USP22 (Science Advances, 2022). Our ongoing study has identified USP22-specific small molecule inhibitors that largely diminish Treg fitness and dramatically inhibit tumor growth at a clinically relevant setting. In addition, our recent unpublished single-cell RNA-seq characterization revealed a novel function of the Treg-specific deubiquitination module in Treg terminal differentiation into a unique immune suppressive subpopulation, specifically in the tumor microenvironment. Collectively, our study defines a novel Treg deubiquitination module in tumor immunosurveillance and provides a rationale for the first Treg-specific targeting in antitumor immune therapy. Citation Format: Deyu Fang. Targeting a Treg-specific deubiquitinase module for antitumor immune therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B038.

A finite element model of contusion spinal cord injury in rodents

Traumatic spinal cord injuries result from high impact forces acting on the spine and are proceeded by an extensive secondary inflammatory response resulting in motor, sensory, and autonomic dysfunction. Experimental in vivo traumatic spinal cord injuries in rodents using a contusion model have been extremely useful in elucidating the underlying pathophysiology of these injuries. However, the relationship between the pathophysiology and the biomechanical factors is still not well understood. Therefore, the aim of this research is to provide a comprehensive analysis of the biomechanics of traumatic spinal cord injury in a rat contusion model. This is achieved through the development and validation of a finite element model of the thoracic rat spinal cord and subsequently simulating controlled cortical impact-induced traumatic spinal cord injury. The effects of impactor velocity, depth, and geometry on the resulting stresses and strains within the spinal cord are investigated. Our results show that increasing impactor depth results in larger stresses and strains within the spinal cord tissue as expected. Further, for the first time ever our results show that impactor geometry (spherical versus cylindrical) plays an important role in the distribution and magnitude of stresses and strains within the cord. Therefore, finite element modelling can be a powerful tool used to predict stresses and strains that occur in spinal cord tissue during trauma.

In Chagas disease, transforming growth factor beta neutralization reduces Trypanosoma cruzi infection and improves cardiac performance.

Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi infection. During CCC, the parasite remains inside the cardiac cells, leading to tissue damage, involving extensive inflammatory response and irregular fibrosis. Among the fibrogenic factors is transforming growth factor-β (TGF-β), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-β is involved in CCC onset and progression, with increased serum levels and activation of its signaling pathways in the cardiac tissue, which crucially contributes to fibrosis. Inhibition of the TGF-β signaling pathway attenuates T. cruzi infection and prevents cardiac damage in an experimental model of acute Chagas disease. The aim of this study was to investigate the effect of TGF-β neutralization on T. cruzi infection in both in vitro and in vivo pre-clinical models, using the 1D11 monoclonal antibody. To this end, primary cultures of cardiac cells were infected with T. cruzi trypomastigote forms and treated with 1D11. For in vivo studies, 1D11 was administered in different schemes for acute and chronic phase models (Swiss mice infected with 104 parasites from the Y strain and C57BL/6 mice infected with 102 parasites from the Colombian strain, respectively). Here we show that the addition of 1D11 to cardiac cells greatly reduces cardiomyocyte invasion by T. cruzi and the number of parasites per infected cell. In both acute and chronic experimental models, T. cruzi infection altered the electrical conduction, decreasing the heart rate, increasing the PR interval and the P wave duration. The treatment with 1D11 reduced cardiac fibrosis and reversed electrical abnormalities improving cardiac performance. Taken together, these data further support the major role of the TGF-β signaling pathways in T. cruzi-infection and their biological consequences on parasite/host interactions. The therapeutic effects of the 1D11 antibody are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-β neutralization.

Small extracellular vesicles derived from hypoxic preconditioned dental pulp stem cells ameliorate inflammatory osteolysis by modulating macrophage polarization and osteoclastogenesis.

Extensive macrophage inflammatory responses and osteoclast formation are predominant during inflammatory or infective osteolysis. Mesenchymal stem cell (MSC)-derived small extracellular vesicles (MSC-sEV) have been shown to exert therapeutic effects on bone defects. However, cultured MSCs are typically exposed to normoxia (21% O2) in vitro, which differs largely from the oxygen concentration in vivo under hypoxic conditions. It is largely unknown whether sEV derived from dental pulp stem cells (DPSCs) cultured under hypoxic conditions (Hypo-sEV) exert better therapeutic effects on lipopolysaccharide (LPS)-induced inflammatory osteolysis than those cultured under normoxic conditions (Nor-sEV) by simultaneously inhibiting the macrophage inflammatory response and osteoclastogenesis. In this study, we show that hypoxia significantly induces the release of sEV from DPSCs. Moreover, Hypo-sEV exhibit significantly improved efficacy in promoting M2 macrophage polarization and suppressing osteoclast formation to alleviate LPS-induced inflammatory calvarial bone loss compared with Nor-sEV. Mechanistically, hypoxia preconditioning markedly alters the miRNA profiles of DPSC-sEV. MiR-210-3p is enriched in Hypo-sEV, and can simultaneously induce M2 macrophage generation and inhibit osteoclastogenesis by targeting NF-κB1 p105, which attenuates osteolysis. Our study suggests a promising potential for hypoxia-induced DPSC-sEV to treat inflammatory or infective osteolysis and identifies a novel role of miR-210-3p in concurrently hindering osteoclastogenesis and macrophage inflammatory response by inhibiting NF-kB1 expression.

MicroRNA-451 Attenuates the Inflammatory Response of Activated Microglia by Downregulating Nucleotide Binding Oligomerization Domain-Like Receptor Protein 3

Spinal cord injury is the most common problem encountered during spinal surgery. After the initial trauma, the disruption of the blood-brain barrier and subsequent microglia activation result in extensive inflammatory responses. Inflammasomes are large protein complexes that are essential during inflammation. One of the most studied inflammasome components, nucleotide binding oligomerization domain-like receptor protein 3 (NLRP; nucleotide binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3), is widely expressed in the central nervous system. Previous research has shown that microRNA-451 (miR-451) might play a role in regulating inflammatory conditions. Using bioinformatics analysis, we found that NLRP3 is a direct target of miR-451. This in silico prediction was confirmed using dual-luciferase reporter gene assays. To further demonstrate that miR-451 influenced microglial NLRP3 production, we activated microglial cells with lipopolysaccharides. Activating microglial cells with lipopolysaccharides resulted in the production of NLRP3 inflammasomes and the secretion of the proinflammatory cytokines interleukin-1β and interleukin-18. We were able to demonstrate that overexpression of miR-451 suppressed this NLRP3-induced proinflammatory cascade of events. Our findings have highlighted the potential anti-inflammatory role of miR-451 in reducing the secondary neuronal damage after spinal cord injury.

Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells.

ABSTRACTThe pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection.IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.

The expression of STEAP4 in peripheral blood predicts the outcome of septic patients.

BackgroundSepsis is a systemic disease characterized by extensive inflammatory responses and impaired organ function, which are characteristics that make it easily missed and complex to treat. A large number of laboratory and clinical studies on the diagnosis and treatment of sepsis have been continuously carried out, confirming the importance of mitochondrial function during the development of sepsis. STEAP4 is an important metalloreductase in mitochondria, which is involved in the biogenesis and respiratory chain of mitochondria. The role of STEAP4 in inflammation remains controversial. Research in this field may contribute to the development of new diagnostic and treatment options for sepsis.MethodsThe expression of STEAP4 was measured in the peripheral blood of patients with severe sepsis and compared with healthy controls. Cell and mouse inflammatory models were established to detect the expression of STEAP4 and other inflammatory cytokines.Results(I) The expression of STEAP4 in the peripheral blood of patients with severe sepsis is higher than that of healthy volunteers (P<0.01), which is related to the SOFA score and transaminase. (II) STEAP4 has a certain predictive effect on the outcome of patients [area under curve (AUC) =0.696, P<0.05, 95% CI: 0.528 to 0.833]. (III) Inflammation led to increased expression of STEAP4 gene in RAW264.7 cells and mouse liver tissue.ConclusionsThe expression of STEAP4 is elevated in the early stage of sepsis and the degree of its elevation can be used to predict the clinical outcome of sepsis patients.

YAP-Dependent Induction of CD47-Enriched Extracellular Vesicles Inhibits Dendritic Cell Activation and Ameliorates Hepatic Ischemia-Reperfusion Injury.

Hepatic ischemia-reperfusion injury (IRI) is the most common cause of liver damage leading to surgical failures in hepatectomy and liver transplantation. Extensive inflammatory reactions and oxidative responses are reported to be the major processes exacerbating IRI. The involvement of Yes-associated protein (YAP) in either process has been suggested, but the role and mechanism of YAP in IRI remain unclear. In this study, we constructed hepatocyte-specific YAP knockout (YAP-HKO) mice and induced a hepatic IRI model. Surprisingly, the amount of serum EVs decreased in YAP-HKO compared to WT mice during hepatic IRI. Then, we found that the activation of YAP increased EV secretion through F-actin by increasing membrane formation, while inhibiting the fusion of multivesicular body (MVB) and lysosomes in hepatocytes. Further, to explore the essential elements of YAP-induced EVs, we applied mass spectrometry and noticed CD47 was among the top targets highly expressed on hepatocyte-derived EVs. Thus, we enriched CD47+ EVs by microbeads and applied the isolated CD47+ EVs on IRI mice. We found ameliorated IRI symptoms after CD47+ EV treatment in these mice, and CD47+ EVs bound to CD172α on the surface of dendritic cells (DCs), which inhibited DC activation and the cascade of inflammatory responses. Our data showed that CD47-enriched EVs were released in a YAP-dependent manner by hepatocytes, which could inhibit DC activation and contribute to the amelioration of hepatic IRI. CD47+ EVs could be a potential strategy for treating hepatic IRI.

Final results from a 90-day quantitative inhalation toxicology study evaluating the dose-response and fate in the lung and pleura of chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite or amosite asbestos: Histopathological examination, confocal microscopy and collagen quantification of the lung and pleural cavity

The final results from this multi-dose, 90-day inhalation toxicology study in the rat with life-time post-exposure observation have shown a significant fundamental difference in pathological response and tumorgenicity between brake dust generated from brake pads manufactured with chrysotile or from chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos.The groups exposed to brake dust showed no significant pathological or tumorigenic response in the respiratory track compared to the air control group at exposure concentrations and deposited doses well above those at which humans have been exposed. Slight alveolar/interstitial macrophage accumulation of particles was noted. Wagner grades were 1–2 (1 = control group), similar to the TiO2 particle control group.Chrysotile was not biopersistent, exhibiting in the lung a deterioration of its matrix which results in breakage into particles and short fibers which can be cleared by alveolar macrophages and which can continue to dissolve. Particle-laden macrophage accumulation was observed, leading to a very-slight interstitial inflammatory response (Wagner grade 1–3). There was no peribronchiolar inflammation, occasional very-slight interstitial fibrosis (Wagner grade 4), and no exposure-related tumorigenic response.The pathological response of crocidolite and amosite compared to the brake dust and chrysotile was clearly differentiated by the histopathology and the confocal analysis. Crocidolite and amosite induced persistent inflammation, microgranulomas, persistent fibrosis (Wagner grades 4), and a dose-related lung tumor response. Confocal microscopy quantified extensive inflammatory response and collagen development in the lung, visceral and parietal pleura as well as pleural adhesions.These results provide a clear foundation for differentiating the innocuous effects of brake dust exposure from the adverse effects following amphibole asbestos exposure.

Cranial Mandibular Fibrosis Syndrome in Adult Farmed Rainbow Trout Oncorhynchus mykiss.

An unusual condition affecting market size rainbow trout was investigated. This condition was prevalent for several years at low levels but affected a large proportion of stock during 2018 and 2019. Chronic fibrosis affecting cranial tissues and the jaw was observed in samples collected in 2018. A larger sampling was then conducted in 2019 to investigate the presence of an infectious agent(s). An extensive inflammatory response in the mandibular region was the main finding, however infectious agents in the lesions were not identified through classical virology and bacteriology analysis. Tetracapsuloides bryosalmonae infection, calcinosis, and a Gram-positive bacterial infection of a single fish cardiac tissue was observed, however, a correlation of these pathologies and the cranial mandibular fibrosis (CMF) syndrome was not established. The gene expression of a panel of 16 immune-related genes was studied. Among these, tgf-b, sIgM, il11, hspa, and the antimicrobial peptides lys and cath1 were up-regulated in jaw sections of CMF-affected fish, showing a strong positive correlation with the severity of the lesions. Idiopathic chronic fibrosis with the activation of the Tfg-B pathway and local hyper-immunoglobulaemia was therefore diagnosed. Initiating factors and causative agent(s) (biotic or abiotic) of CMF remain, at present, unclear.

Hemoperfusion with HA380 in acute type A aortic dissection patients undergoing aortic arch operation (HPAO): a randomized, controlled, double-blind clinical trial

BackgroundCardiopulmonary bypass (CPB) is an important cause of significant systemic inflammatory response syndrome (SIRS) in the surgical treatment of acute type A aortic dissection (ATAAD). In patients with arch vessel involvement, extensive surgical repairs often necessitate prolonged use of CPB and results in extensive inflammatory responses. Cytokines and chemokines released during CPB contribute to the progression of SIRS, increase perioperative complications, and negatively impact surgical outcomes. A cytokine adsorber (HA380) is expected to reduce the level of cytokines during CPB, which may decrease both intraoperative and postoperative inflammation. The purpose of this study is to investigate if HA380 is able to reduce the levels of inflammatory cytokines and decrease perioperative complications in ATAAD patients undergoing CPB and deep hypothermic circulatory arrest (DHCA).MethodsThis study is a single-center, randomized, controlled, double-blind clinical trial. The study aims to recruit 88 patients with ATAAD and aortic arch involvement who will undergo CPB and DHCA to repair the dissected aorta. Patients will be randomized equally into the CPB/DHCA only group (control group) and the CPB/DHCA + HA380 hemoperfusion group (intervention group), with 44 patients each. Patients in the control group will undergo CPB and DHCA only, while patients in the intervention group will undergo continuous hemoperfusion with HA380, in addition to CPB and DHCA. The primary outcome is a composite of major perioperative complications. The secondary outcomes include related inflammatory markers, coagulation parameters, and minor perioperative complications. To comprehensively evaluate the effect of hemoperfusion on the perioperative outcomes, we will also determine if there are differences in perioperative all-cause mortality, length of ICU stay, and total hospitalization costs.DiscussionIn the current trial, hemoperfusion will be applied in patients undergoing CPB and DHCA for repair of the aorta involving the aortic arch. This trial aims to test the safety and efficacy of our hemoperfusion device (HA380) in such settings. Upon completion of the trial, we will determine if HA380 is effective in reducing perioperative proinflammatory cytokine levels. Further, we will also verify if reduction in the proinflammatory cytokine levels, if present, translates to improvement in patient outcomes.Trial registrationClinicalTrials.gov NCT04007484. Registered on 1 July 2019 (retrospectively registered).

Abstract 14296: Differential Left and Right Ventricle Response to Cold Storage Followed by Reperfusion in Heart Transplantation

Introduction: In heart transplantation, the supply of donor hearts is contributed by the short tolerable preservation period with risk for primary graft dysfunction. Understanding the differential biological responses to preservation between the left ventricle (LV) and right ventricle (RV) may provide critical insights. Methods: Murine donor hearts were infused with cold HTK solution and stored on ice for 0h, 4h and 8h followed by 90minutes perfusion with Krebs Buffer. The LV and RV were collected at the end of reperfusion for gene expression and protein analysis by RNA-sequencing and western blot. Results: Differential gene expression analysis showed that RV has 1092 upregulated genes and 291 downregulated genes compared to LV after cold storage followed by perfusion. GO analysis show that gene sets related to immune response (padj=7e-27), cell responses to interferon-beta (padj=8.90e-19) were enriched in RV upregulated genes (figure). We also confirmed the upregulation of immune response genes such IL1-β, Myd88 by qPCR and western blot. Moreover, we found that active β-catenin, a potential upstream regulator of IL1- β, also upregulated in RV. Wnt/β-catenin downstream target genes were also enriched in human donor RVs during prolonged preservation. Importantly, donor hearts from murine Myd88-/- showed reduced differences in the transcriptome of the LV vs RV (figure) and demonstrated improved heart function after prolonged cold storage with reperfusion. Conclusions: The RV demonstrates more extensive inflammatory response to storage compared to the LV. Selective upregulation of Wnt/β-catenin signaling in the RV may be important for enhancing this inflammatory response to preservation. Deficiency of MyD88 mediated innate immune signaling improved cardiac contractility after prolonged preservation and likely have important clinical implications for transplantation.

Comparison of respiratory toxicity of TiO2 and Fe3O4 nanoparticles after intravenous instillation: an experimental study.

Nanomaterials consist of particles smaller than 100 nm - nanoparticles (NPs). Their nano dimensions allow them to penetrate through various membranes and enter into the bloodstream and disseminate into different body organs. Massive expansion of nanotechnologies together with production of new nanoparticles which have not yet been in contact with living organisms may pose a potential health problem. It is therefore necessary to investigate the health impact of NPs after experimental exposure. Comparison of the effect of TiO2 and NPs Fe3O4 in Wistar rats at time intervals 1, 7, 14 and 28 days was performed by studying the cytotoxic effect in the isolated inflammatory cells from bronchoalveolar lavage (BAL). Wistar rats were intravenously (i.v.) given a suspension of NPs TiO2 or Fe3O4 (coated by sodium oleate) via the tail vein. After time intervals of 1, 7, 14 and 28 days, we sacrificed the animals under anaesthesia, performed BAL and isolated the cells. The number of animals in the individual groups was 7-8. We examined the differential count of BAL cells (alveolar macrophages - AM, polymorphonuclear leukocytes - PMN, lymphocytes - Ly); viability and phagocytic activity of AM; the proportion of immature and polynuclear cells and enzymes - cathepsin D - CAT D, lactate dehydrogenase - LDH and acid phosphatase - ACP. We found that TiO2 NPs are relatively inert - without induction of inflammatory and cytotoxic response. Exposure to nanoparticles Fe3O4 induced - under the same experimental conditions - in comparison with the control and TiO2 a more extensive inflammatory and cytotoxic response, albeit only at 1, 7 and 14 days after injection. The results suggest that TiO2 and Fe3O4 nanoparticles used in our study were transferred from the bloodstream to the respiratory tract, but this effect was not observed at 28 days after i.v. injection, probably due to their removal from the respiratory tract.

Viral presence and immunopathology in patients with lethal COVID-19: a prospective autopsy cohort study

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy.MethodsThis prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course.FindingsOur cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41–78). Median disease course (time from onset of symptoms to death) was 22 days (range 5–44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5–44]; ten patients with neutrophilic plugs, 21 days [5–44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets..InterpretationIn patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19.FundingAmsterdam UMC Corona Research Fund.

The cGAS-STING pathway: The role of self-DNA sensing in inflammatory lung disease.

The presence of DNA in the cytosol is usually a sign of microbial infections, which alerts the host innate immune system to mount a defense response. Cyclic GMP-AMP synthase (cGAS) is a critical cytosolic DNA sensor that elicits robust innate immune responses through the production of the second messenger, cyclic GMP-AMP (cGAMP), which binds and activates stimulator of interferon genes (STING). However, cGAS binds to DNA irrespective of DNA sequence, therefore, self-DNA leaked from the nucleus or mitochondria can also serve as a cGAS ligand to activate this pathway and trigger extensive inflammatory responses. Dysregulation of the cGAS-STING pathway is responsible for a broad array of inflammatory and autoimmune diseases. Recently, evidence has shown that self-DNA release and cGAS-STING pathway over-activation can drive lung disease, making this pathway a promising therapeutic target for inflammatory lung disease. Here, we review recent advances on the cGAS-STING pathway governing self-DNA sensing, highlighting its role in pulmonary disease.

Pyridoxine induces monocyte-macrophages death as specific treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy that gradually develops resistance to current chemotherapy treatments. The available chemotherapy drugs show serious non-specific cytotoxicity to healthy normal cells, resulting in relapse and low survival rates. Natural small molecules with less toxicity and high selectivity for AML are urgently needed. In this study, we confirmed that pyridoxine (vitamin B6) selectively induces monocyte macrophages to undergo programmed cell death in two different modes: caspase-3-dependent apoptosis in U937cells or GSDME-mediated pyroptosis in THP-1cells. Further molecular analysis indicated that blocking the caspase pathway could switch the death to MLKL-dependent necroptosis and subsequent extensive inflammatory response. Pyridoxine also delayed the disease progression in a THP-1 leukemia mouse model. In addition, it induced the death of primary AML cells from AML patients by activating caspase-8/3. Overall, our results identify pyridoxine, a low-toxicity natural small molecule, as a potential therapeutic drug for AML treatment.

Mass spectrometric analysis of the in vitro secretome from equine bone marrow-derived mesenchymal stromal cells to assess the effect of chondrogenic differentiation on response to interleukin-1\u03b2 treatment

BackgroundSimilar to humans, the horse is a long-lived, athletic species. The use of mesenchymal stromal cells (MSCs) is a relatively new frontier, but has been used with promising results in treating joint diseases, e.g., osteoarthritis. It is believed that MSCs exert their main therapeutic effects through secreted trophic biomolecules. Therefore, it has been increasingly important to characterize the MSC secretome. It has been shown that the effect of the MSCs is strongly influenced by the environment in the host compartment, and it is a crucial issue when considering MSC therapy. The aim of this study was to investigate differences in the in vitro secreted protein profile between naïve and chondrogenic differentiating bone marrow-derived (BM)-MSCs when exposed to an inflammatory environment.MethodsEquine BM-MSCs were divided into a naïve group and a chondrogenic group. Cells were treated with normal expansion media or chondrogenic media. Cells were treated with IL-1β for a period of 5 days (stimulation), followed by 5 days without IL-1β (recovery). Media were collected after 48 h and 10 days. The secretomes were digested and analyzed by nanoLC-MS/MS to unravel the orchestration of proteins.ResultsThe inflammatory proteins IL6, CXCL1, CXCL6, CCL7, SEMA7A, SAA, and haptoglobin were identified in the secretome after 48 h from all cells stimulated with IL-1β. CXCL8, OSM, TGF-β1, the angiogenic proteins VCAM1, ICAM1, VEGFA, and VEGFC, the proteases MMP1 and MMP3, and the protease inhibitor TIMP3 were among the proteins only identified in the secretome after 48 h from cells cultured in normal expansion media. After 10-day incubation, the proteins CXCL1, CXCL6, and CCL7 were still identified in the secretome from BM-MSCs stimulated with IL-1β, but the essential inducer of inflammation, IL6, was only identified in the secretome from cells cultured in normal expansion media.ConclusionThe findings in this study indicate that naïve BM-MSCs have a more extensive inflammatory response at 48 h to stimulation with IL-1β compared to BM-MSCs undergoing chondrogenic differentiation. This extensive inflammatory response decreased after 5 days without IL-1β (day 10), but a difference in composition of the secretome between naïve and chondrogenic BM-MSCs was still evident.

Vitamin D Treatment Attenuates Heart Apoptosis After Coronary Artery Bypass Surgery: A Double-Blind, Randomized, Placebo-Controlled Clinical Trial.

Vitamin D plays an important role in immune system and in the regulation of inflammatory cytokines. Coronary artery bypass graft (CABG) with cardiopulmonary bypass (CPB) is associated with an extensive inflammatory response. The aim of this study is to examine the effect of vitamin D treatment on the apoptosis and inflammatory changes developed after CABG. This trial was conducted on 70 patients undergoing CABG with CPB. Patients were randomly administered either in placebo or in the group of orally consuming 150 000 IU vitamin D daily for 3 consecutive days before surgery. The right atrium sample was taken to assess caspases 2, 3, and 7 activity using immunohistochemistry method. The serum level of interleukin-10 (IL-10) and insulin-like growth factor 1 (IGF-1) were compared at intervals. The average number of positive cells for caspases 2 and 3 were less in vitamin D group (P = .006 and P < .001, respectively). There was an increase in serum levels of IL-10 after 3 days from vitamin D treatment before surgery (vitamin D group = 4.4 ± 4.9 ng/mL and control group = 1 ± 0.5 ng/mL, P = .001). After operation, IL-10 increased in both groups, higher level in vitamin D group (P < .001). The comparison of serum IGF-1 showed significant difference after 3 days (P = .006) and remained higher in vitamin D group after CPB (P < .001). These findings suggest the apoptosis rate after CPB can be reduced by vitamin D. Vitamin D treatment may improve the inflammatory status before and after surgery. Further studies are needed to confirm the antiapoptotic property of vitamin D and clinical implication.