P6300Endothelial cell activation by pro-inflammatory cytokines exerts novel paracrine effects on co-cultured cardiomyocytes

Abstract Background Anti-inflammatory therapies have failed to meet expectations in recent clinical trials for heart failure, despite the number of studies demonstrating pro-fibrotic, arrhythmogenic and hypertrophic effects of inflammation. To enlighten this, we sought to examine the contribution of non-myocytes in the cardiac inflammatory response. Endothelial cells (EC) can regulate cardiomyocyte (CM) function with multiple soluble factors. While many studies have shown the effects of pro-inflammatory cytokines on EC or CM separately, it is still unclear how the activation of EC can affect CM function. Purpose We studied the effect of pro-inflammatory pre-conditioning of EC on CM in indirect co-culture systems and in an ex vivo model of cardiac tissue. We hypothesised that pro-inflammatory activation of EC would alter the contractility of co-cultured CM. Methods Human cardiac microvascular EC were first pre-conditioned for 24h with Cytomix (1 ng/ml TNF-α, 1 ng/ml IL-1β, 25 ng/ml IL-6 Rα/IL-6 chimera) and co-cultured in a transwell system (pore size: 0.4μm) with adult rat ventricular CM. Co-culture supernatants were screened using a Cytokine Profiler Array. In vitro analysis of calcium handling in CM utilised the optical mapping technique and Fluo-4. Contractility of cardiac tissue was measured ex vivo using myocardial slices of 300 μm, prepared from left ventricles of adult rat and cultured for 24h with field stimulation and a fixed stretch producing a sarcomeric length of 2.2 μm. Results Treatment of EC by Cytomix prior to co-cultures induced a release of CC and CXC chemokines, G-/GM-CSF, ST2 and the adhesion molecule ICAM-1. Using published RNAseq datasets we noticed that adult CM do not constitutively express the receptors for the chemokines identified with the Profiler Array. However, duration of calcium transients in CM was significantly reduced from 454 to 322 ms in co-cultures with EC pre-conditioned by Cytomix, compared to untreated EC. Amplitude and time to peak were unchanged. In contrast, myocardial slices treated with Cytomix demonstrated a significant increase in contractility compared to control (from 3.3 to 5.3 mN/mm2) but no significant change in the duration of contraction (from 467 to 434 ms) or the rate of relaxation. Conclusions Pre-conditioned EC exert paracrine effects on the calcium handling of isolated CM, suggesting pro-inflammatory activation of proximal EC can affect CM function. In myocardial slices, pro-inflammatory stimulation provoked an inotropic response. The divergence of effect with findings in isolated cells may reflect differences in experimental design and multicellularity. Future work will aim to characterise the soluble mediators involved, and selectively target EC in slices to contextualise the effects described in co-cultures. Ultimately, this may inform the development of novel anti-inflammatory strategies for clinical use. Acknowledgement/Funding British Heart Foundation

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Computational Identification of Potential Anti-Inflammatory Natural Compounds Targeting the p38 Mitogen-Activated Protein Kinase (MAPK): Implications for COVID-19-Induced Cytokine Storm

Biomolecules ◽

10.3390/biom11050653 ◽

2021 ◽

Vol 11 (5) ◽

pp. 653

Author(s):

Seth O. Asiedu ◽

Samuel K. Kwofie ◽

Emmanuel Broni ◽

Michael D. Wilson

Keyword(s):

Molecular Docking ◽

P38 Mapk ◽

Inflammatory Cytokines ◽

Binding Energies ◽

Mitogen Activated Protein Kinase ◽

Inflammatory Activity ◽

Computational Techniques ◽

Cytokine Storm ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

Severely ill coronavirus disease 2019 (COVID-19) patients show elevated concentrations of pro-inflammatory cytokines, a situation commonly known as a cytokine storm. The p38 MAPK receptor is considered a plausible therapeutic target because of its involvement in the platelet activation processes leading to inflammation. This study aimed to identify potential natural product-derived inhibitory molecules against the p38α MAPK receptor to mitigate the eliciting of pro-inflammatory cytokines using computational techniques. The 3D X-ray structure of the receptor with PDB ID 3ZS5 was energy minimized using GROMACS and used for molecular docking via AutoDock Vina. The molecular docking was validated with an acceptable area under the curve (AUC) of 0.704, which was computed from the receiver operating characteristic (ROC) curve. A compendium of 38,271 natural products originating from Africa and China together with eleven known p38 MAPK inhibitors were screened against the receptor. Four potential lead compounds ZINC1691180, ZINC5519433, ZINC4520996 and ZINC5733756 were identified. The compounds formed strong intermolecular bonds with critical residues Val38, Ala51, Lys53, Thr106, Leu108, Met109 and Phe169. Additionally, they exhibited appreciably low binding energies which were corroborated via molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) calculations. The compounds were also predicted to have plausible pharmacological profiles with insignificant toxicity. The molecules were also predicted to be anti-inflammatory, kinase inhibitors, antiviral, platelet aggregation inhibitors, and immunosuppressive, with probable activity (Pa) greater than probable inactivity (Pi). ZINC5733756 is structurally similar to estradiol with a Tanimoto coefficient value of 0.73, which exhibits anti-inflammatory activity by targeting the activation of Nrf2. Similarly, ZINC1691180 has been reported to elicit anti-inflammatory activity in vitro. The compounds may serve as scaffolds for the design of potential biotherapeutic molecules against the cytokine storm associated with COVID-19.

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Cerebrospinal fluid biomarkers of neuroinflammation in children with hydrocephalus and shunt malfunction

Fluids and Barriers of the CNS ◽

10.1186/s12987-021-00237-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Carolyn A. Harris ◽

Diego M. Morales ◽

Rooshan Arshad ◽

James P. McAllister ◽

David D. Limbrick

Keyword(s):

Cerebrospinal Fluid ◽

Inflammatory Cytokines ◽

Protein Concentration ◽

Shunt Revision ◽

Csf Shunt ◽

Shunt Failure ◽

Revision Operation ◽

Protein Levels ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

Abstract Background Approximately 30% of cerebrospinal fluid (CSF) shunt systems for hydrocephalus fail within the first year and 98% of all patients will have shunt failure in their lifetime. Obstruction remains the most common reason for shunt failure. Previous evidence suggests elevated pro-inflammatory cytokines in CSF are associated with worsening clinical outcomes in neuroinflammatory diseases. The aim of this study was to determine whether cytokines and matrix metalloproteinases (MMPs) contribute towards shunt failure in hydrocephalus. Methods Using multiplex ELISA, this study examined shunt failure through the CSF protein concentration profiles of select pro-inflammatory and anti-inflammatory cytokines, as well as select MMPs. Interdependencies such as the past number of previous revisions, length of time implanted, patient age, and obstruction or non-obstruction revision were examined. The pro-inflammatory cytokines were IL-1β, IL-2, IL-5, IL-6, IL-8, IL-12, IL-17, TNF-α, GM-CSF, IFN-γ. The anti-inflammatory cytokines were IL-4 and IL-10, and the MMPs were MMP-2, MMP-3, MMP-7, MMP-9. Protein concentration is reported as pg/mL for each analyte. Results Patient CSF was obtained at the time of shunt revision operation; all pediatric (< 18), totaling n = 38. IL-10, IL-6, IL-8 and MMP-7 demonstrated significantly increased concentrations in patient CSF for the non-obstructed subgroup. Etiological examination revealed IL-6 was increased in both obstructed and non-obstructed cases for PHH and congenital hydrocephalic patients, while IL-8 was higher only in PHH patients. In terms of number of past revisions, IL-10, IL-6, IL-8, MMP-7 and MMP-9 progressively increased from zero to two past revisions and then remained low for subsequent revisions. This presentation was notably absent in the obstruction subgroup. Shunts implanted for three months or less showed significantly increased concentrations of IL-6, IL-8, and MMP-7 in the obstruction subgroup. Lastly, only patients aged six months or less presented with significantly increased concentration of IL-8 and MMP-7. Conclusion Non-obstructive cases are reported here to accompany significantly higher CSF cytokine and MMP protein levels compared to obstructive cases for IL-10, IL-6, IL-8, MMP-7 and MMP-9. A closer examination of the definition of obstruction and the role neuroinflammation plays in creating shunt obstruction in hydrocephalic patients is suggested.

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Role of Inflammatory Cytokines in COVID-19 Patients: A Review on Molecular Mechanisms, Immune Functions, Immunopathology and Immunomodulatory Drugs to Counter Cytokine Storm

Vaccines ◽

10.3390/vaccines9050436 ◽

2021 ◽

Vol 9 (5) ◽

pp. 436

Author(s):

Ali A. Rabaan ◽

Shamsah H. Al-Ahmed ◽

Javed Muhammad ◽

Amjad Khan ◽

Anupam A Sule ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Inflammatory Markers ◽

Therapeutic Drug ◽

Molecular Pathways ◽

Cytokine Storm ◽

Immunomodulatory Drugs ◽

Alveolar Cells ◽

Systemic Complications ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a severe pandemic of the current century. The vicious tentacles of the disease have been disseminated worldwide with unknown complications and repercussions. Advanced COVID-19 syndrome is characterized by the uncontrolled and elevated release of pro-inflammatory cytokines and suppressed immunity, leading to the cytokine storm. The uncontrolled and dysregulated secretion of inflammatory and pro-inflammatory cytokines is positively associated with the severity of the viral infection and mortality rate. The secretion of various pro-inflammatory cytokines such as TNF-α, IL-1, and IL-6 leads to a hyperinflammatory response by recruiting macrophages, T and B cells in the lung alveolar cells. Moreover, it has been hypothesized that immune cells such as macrophages recruit inflammatory monocytes in the alveolar cells and allow the production of large amounts of cytokines in the alveoli, leading to a hyperinflammatory response in severely ill patients with COVID-19. This cascade of events may lead to multiple organ failure, acute respiratory distress, or pneumonia. Although the disease has a higher survival rate than other chronic diseases, the incidence of complications in the geriatric population are considerably high, with more systemic complications. This review sheds light on the pivotal roles played by various inflammatory markers in COVID-19-related complications. Different molecular pathways, such as the activation of JAK and JAK/STAT signaling are crucial in the progression of cytokine storm; hence, various mechanisms, immunological pathways, and functions of cytokines and other inflammatory markers have been discussed. A thorough understanding of cytokines’ molecular pathways and their activation procedures will add more insight into understanding immunopathology and designing appropriate drugs, therapies, and control measures to counter COVID-19. Recently, anti-inflammatory drugs and several antiviral drugs have been reported as effective therapeutic drug candidates to control hypercytokinemia or cytokine storm. Hence, the present review also discussed prospective anti-inflammatory and relevant immunomodulatory drugs currently in various trial phases and their possible implications.

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A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

Scientific Reports ◽

10.1038/s41598-021-89882-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hiroto Nakajima ◽

Atsushi Miyashita ◽

Hiroshi Hamamoto ◽

Kazuhisa Sekimizu

Keyword(s):

Inflammatory Cytokines ◽

Immune Cells ◽

Ex Vivo ◽

Large Bubble ◽

Suitable Model ◽

Bacterial Cells ◽

Functional Impairments ◽

Gene Expressions ◽

Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

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Human umbilical cord mesenchymal stem cell-derived extracellular vesicles attenuate experimental autoimmune encephalomyelitis via regulating pro and anti-inflammatory cytokines

Scientific Reports ◽

10.1038/s41598-021-91291-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Samira Ahmadvand Koohsari ◽

Abdorrahim Absalan ◽

Davood Azadi

Keyword(s):

Spinal Cord ◽

Body Weight ◽

Extracellular Vesicles ◽

Inflammatory Cytokines ◽

Clinical Score ◽

Human Umbilical Cord ◽

Therapeutic Effects ◽

Leukocyte Infiltration ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

AbstractThe therapeutic effects of mesenchymal stem cells-extracellular vesicles have been proved in many inflammatory animal models. In the current study, we aimed to investigate the effect of extracellular vesicles (EVs) derived from human umbilical cord-MSC (hUCSC-EV) on the clinical score and inflammatory/anti-inflammatory cytokines on the EAE mouse model. After induction of EAE in C57Bl/6 mice, they were treated intravenously with hUCSC-EV or vehicle. The clinical score and body weight of all mice was registered every day. On day 30, mice were sacrificed and splenocytes were isolated for cytokine assay by ELISA. Cytokine expression of pro-/anti-inflammatory cytokine by real-time PCR, leukocyte infiltration by hematoxylin and eosin (H&E) staining, and the percent of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) positive cells by immunohistochemistry were assessed in the spinal cord. Our results showed that hUCSC-EV-treated mice have lower maximum mean clinical score (MMCS), pro-inflammatory cytokines, and inflammatory score in comparison to the control mice. We also showed that hUCSC-EV administration significantly improved body weight and increased the anti-inflammatory cytokines and the frequency of Treg cells in the spleen. There was no significant difference in the percent of GFAP and MBP positive cells in the spinal cord of experimental groups. Finally, we suggest that intravenous administration of hUCSC-EV alleviate induce-EAE by reducing the pro-inflammatory cytokines, such as IL-17a, TNF-α, and IFN-γ, and increasing the anti-inflammatory cytokines, IL-4 and IL-10, and also decrease the leukocyte infiltration in a model of MS. It seems that EVs from hUC-MSCs have the same therapeutic effects similar to EVs from other sources of MSCs, such as adipose or bone marrow MSCs.

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Focal intra-colon cooling reduces organ injury and systemic inflammation after REBOA management of lethal hemorrhage in rats

Scientific Reports ◽

10.1038/s41598-021-93064-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Awadhesh K. Arya ◽

Kurt Hu ◽

Lalita Subedi ◽

Tieluo Li ◽

Bingren Hu

Keyword(s):

Inflammatory Response ◽

Inflammatory Cytokines ◽

Troponin I ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Balloon Catheter ◽

Organ Injury ◽

Upper Body ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

AbstractResuscitative endovascular balloon occlusion of the aorta (REBOA) is a lifesaving maneuver for the management of lethal torso hemorrhage. However, its prolonged use leads to distal organ ischemia–reperfusion injury (IRI) and systemic inflammatory response syndrome (SIRS). The objective of this study is to investigate the blood-based biomarkers of IRI and SIRS and the efficacy of direct intestinal cooling in the prevention of IRI and SIRS. A rat lethal hemorrhage model was produced by bleeding 50% of the total blood volume. A balloon catheter was inserted into the aorta for the implementation of REBOA. A novel TransRectal Intra-Colon (TRIC) device was placed in the descending colon and activated from 10 min after the bleeding to maintain the intra-colon temperature at 37 °C (TRIC37°C group) or 12 °C (TRIC12°C group) for 270 min. The upper body temperature was maintained at as close to 37 °C as possible in both groups. Blood samples were collected before hemorrhage and after REBOA. The organ injury biomarkers and inflammatory cytokines were evaluated by ELISA method. Blood based organ injury biomarkers (endotoxin, creatinine, AST, FABP1/L-FABP, cardiac troponin I, and FABP2/I-FABP) were all drastically increased in TRIC37°C group after REBOA. TRIC12°C significantly downregulated these increased organ injury biomarkers. Plasma levels of pro-inflammatory cytokines TNF-α, IL-1b, and IL-17F were also drastically increased in TRIC37°C group after REBOA. TRIC12°C significantly downregulated the pro-inflammatory cytokines. In contrast, TRIC12°C significantly upregulated the levels of anti-inflammatory cytokines IL-4 and IL-10 after REBOA. Amazingly, the mortality rate was 100% in TRIC37°C group whereas 0% in TRIC12°C group after REBOA. Directly cooling the intestine offered exceptional protection of the abdominal organs from IRI and SIRS, switched from a harmful pro-inflammatory to a reparative anti-inflammatory response, and mitigated mortality in the rat model of REBOA management of lethal hemorrhage.

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Increased Ex-VIVO Production of Pro-Inflammatory Cytokines by Esophageal Mucosa in Patients with Sensorimotor Esophageal Dysphagia

Gastroenterology ◽

10.1016/s0016-5085(17)31368-9 ◽

2017 ◽

Vol 152 (5) ◽

pp. S328

Author(s):

Francesco Covotta ◽

Andrea Cossu ◽

Danilo Badiali ◽

Ivano Biviano ◽

Adriana Marcheggiano ◽

...

Keyword(s):

Inflammatory Cytokines ◽

Ex Vivo ◽

Esophageal Mucosa ◽

Esophageal Dysphagia ◽

Pro Inflammatory Cytokines

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Benznidazole Anti-Inflammatory Effects in Murine Cardiomyocytes and Macrophages Are Mediated by Class I PI3Kδ

Frontiers in Immunology ◽

10.3389/fimmu.2021.782891 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ágata C. Cevey ◽

Paula D. Mascolo ◽

Federico N. Penas ◽

Azul V. Pieralisi ◽

Aldana S. Sequeyra ◽

...

Keyword(s):

Cell Line ◽

Chagas Disease ◽

Inflammatory Cytokines ◽

Immune Cells ◽

Macrophage Polarization ◽

Catalytic Subunit ◽

M2 Macrophage ◽

Socs3 Expression ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

Benznidazole (Bzl), the drug of choice in many countries for the treatment of Chagas disease, leads to parasite clearance in the early stages of infection and contributes to immunomodulation. In addition to its parasiticidal effect, Bzl inhibits the NF-κB pathway. In this regard, we have previously described that this occurs through IL-10/STAT3/SOCS3 pathway. PI3K pathway is involved in the regulation of the immune system by inhibiting NF-κB pathway through STAT3. In this work, the participation of PI3K in the immunomodulatory effects of Bzl in cardiac and immune cells, the main targets of Chagas disease, was further studied. For that, we use a murine primary cardiomyocyte culture and a monocyte/macrophage cell line (RAW 264.7), stimulated with LPS in presence of LY294002, an inhibitor of PI3K. Under these conditions, Bzl could neither increase SOCS3 expression nor inhibit the NOS2 mRNA expression and the release of NOx, both in cardiomyocytes and macrophages. Macrophages are crucial in the development of Chronic Chagas Cardiomyopathy. Thus, to deepen our understanding of how Bzl acts, the expression profile of M1-M2 macrophage markers was evaluated. Bzl inhibited the release of NOx (M1 marker) and increased the expression of Arginase I (M2 marker) and a negative correlation was found between them. Besides, LPS increased the expression of pro-inflammatory cytokines. Bzl treatment not only inhibited this effect but also increased the expression of typical M2-macrophage markers like Mannose Receptor, TGF-β, and VEGF-A. Moreover, Bzl increased the expression of PPAR-γ and PPAR-α, known as key regulators of macrophage polarization. PI3K directly regulates M1-to-M2 macrophage polarization. Since p110δ, catalytic subunit of PI3Kδ, is highly expressed in immune cells, experiments were carried out in presence of CAL-101, a specific inhibitor of this subunit. Under this condition, Bzl could neither increase SOCS3 expression nor inhibit NF-κB pathway. Moreover, Bzl not only failed to inhibit the expression of pro-inflammatory cytokines (M1 markers) but also could not increase M2 markers. Taken together these results demonstrate, for the first time, that the anti-inflammatory effect of Bzl depends on PI3K activity in a cell line of murine macrophages and in primary culture of neonatal cardiomyocytes. Furthermore, Bzl-mediated increase expression of M2-macrophage markers involves the participation of the p110δ catalytic subunit of PI3Kδ.

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Host F-box protein 22 enhances the uptake of Brucella by macrophages and drives a sustained release of pro-inflammatory cytokines through degradation of the anti-inflammatory effector proteins of Brucella.

10.1101/2021.03.15.435452 ◽

2021 ◽

Author(s):

Girish Radhakrishnan ◽

Varadendra Mazumdar ◽

Kiranmai Joshi ◽

Binita Roy Nandi ◽

Swapna Namani ◽

...

Keyword(s):

Immune Responses ◽

Inflammatory Cytokines ◽

Effector Proteins ◽

Host Protein ◽

Limited Information ◽

Phagocytic Cells ◽

Scf Complex ◽

Enhanced Production ◽

Anti Inflammatory ◽

Pro Inflammatory Cytokines

Brucella species are intracellular bacterial pathogens, causing the world-wide zoonotic disease, brucellosis. Brucella invade professional and non-professional phagocytic cells, followed by resisting intracellular killing and establishing a replication permissive niche. Brucella also modulate the innate and adaptive immune responses of the host for their chronic persistence. The complex intracellular cycle of Brucella majorly depends on multiple host factors but limited information is available on host and bacterial proteins that play essential role in the invasion, intracellular replication and modulation of host immune responses. By employing an siRNA screening, we identified a role for the host protein, FBXO22 in Brucella -macrophage interaction. FBXO22 is the key element in the SCF E3 ubiquitination complex where it determines the substrate specificity for ubiquitination and degradation of various host proteins. Downregulation of FBXO22 by siRNA or CRISPR-Cas9 system, resulted diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella -infected macrophages that resulted induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella , including the anti-inflammatory proteins, TcpB and OMP25 for degradation through the SCF complex. We did not observe any role for another F-box containing protein of SCF complex, β-TrCP in Brucella -macrophage interaction. Our findings unravel novel functions of FBXO22 in host-pathogen interaction and its contribution to pathogenesis of infectious diseases.

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