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Cytokine Dec 2022Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced...
BACKGROUND
Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced by exacerbated immune response in corneal stroma. Myofibroblasts play a key role in this process by producing a disorganized extracellular matrix and inflammatory mediators. However, the immune response profile of myofibroblasts during viral infections is still under study. The aim of this work was to analyze the cytokine profile of human limbal myfibroblasts (HLMs) stimulated with the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) and to identify their signaling pathways.
METHODS
HLMs were isolated from cadaveric sclera-corneal rims and stimulated with poly I:C (10 μg/ml) for 12 h. The secretion of 36 cytokines was measured using the Human Cytokine Array Panel A. The secretion of IFN-β was quantified by ELISA. The expression of pattern recognition receptors (PRRs) such as TLR3, RIG-1 and MDA5 were analyzed by western blot assays. Furthermore, translocation of the nuclear factors NF-κB, IRF3, and IRF7 was assessed by fluorescence staining. In addition, the differentially expressed cytokines were analyzed using the Core Analysis Tool of the Ingenuity Pathway Analysis IPA software.
RESULTS
HLMs stimulated with poly I:C increased (fold change > 2) the secretion of G-CSF, sTREM-1, CXCL1, CCL1, CXCL8, CXCL10, CXCL11, CCL2, CCL5, IL-13, IL-6, IL-1ra, and IFN-β compared with HLMs under basal conditions. Poly I:C stimulation also induced the expression of RIG-1 (p < 0.001), but the expression of TLR3 and MDA5 was unmodified. Finally, HLMs increased nuclear translocation of NF-κB, IRF3, and IRF7 after poly I:C stimulation. Bioinformatic analysis identified canonical signaling pathways associated with cell adhesion and diapedesis, chemokine signaling, and activation of IRFs by cytosolic pattern recognition receptors.
CONCLUSIONS
These results demonstrate that HLMs secrete cytokines involved in immune cell activation and chemotaxis. The data suggest a key role for HLMs during viral infections in cornea and extend our knowledge about the signaling pathways they trigger.
Topics: Antiviral Agents; Cornea; Cytokines; Granulocyte Colony-Stimulating Factor; Humans; Interferon-beta; Interleukin 1 Receptor Antagonist Protein; Interleukin-13; Interleukin-6; Myofibroblasts; NF-kappa B; Poly I-C; RNA, Double-Stranded; Receptors, Pattern Recognition; Toll-Like Receptor 3; Virus Diseases
PubMed: 36183616
DOI: 10.1016/j.cyto.2022.156047 -
International Journal of Molecular... Sep 2022Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier...
Subepithelial human esophageal myofibroblasts (HEMFs) in gastroesophageal reflux disease (GERD) are exposed to luminal contents via impaired squamous epithelium barrier integrity. The supernatant of HEMFs treated with acidic bile salts reflective of in vivo reflux increases squamous epithelial thickness. We aimed to identify the involved mechanisms using an unbiased approach. Acidic-bile-salt-treated primary HEMF cultures (n = 4) were submitted for RNA-Seq and analyzed with Partek Flow followed by Ingenuity Pathway Analysis (IPA). A total of 1165 molecules (579 downregulated, 586 upregulated) were differentially expressed, with most top regulated molecules either extracellular or in the plasma membrane. Increases in HEMF CXCL-8, IL-6, AREG, and EREG mRNA, and protein secretion were confirmed. Top identified canonical pathways were agranulocyte and granulocyte adhesion and diapedesis, PI3K/AKT signaling, CCR5 signaling in macrophages, and the STAT3 pathway. Top diseases and biological functions were cellular growth and development, hematopoiesis, immune cell trafficking, and cell-mediated response. The targets of the top upstream regulator ErbB2 included CXCL-8, IL-6, and AREG and the inhibition of CXCL-8 in the HEMF supernatant decreased squamous epithelial proliferation. Our work shows an inflammatory/immune cell and proliferative pathways activation in HEMFs in the GERD environment and identifies CXCL-8 as a HEMF-derived chemokine with paracrine proliferative effects on squamous epithelium.
Topics: Bile Acids and Salts; Carcinoma, Squamous Cell; Gastroesophageal Reflux; Humans; Interleukin-6; Myofibroblasts; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Messenger
PubMed: 36142285
DOI: 10.3390/ijms231810371 -
American Journal of Respiratory Cell... Jan 2023The mechanisms by which excessive systemic activation of adaptive T lymphocytes, as in cytokine release syndrome (CRS), leads to innate immune cell-mediated acute lung...
The mechanisms by which excessive systemic activation of adaptive T lymphocytes, as in cytokine release syndrome (CRS), leads to innate immune cell-mediated acute lung injury (ALI) or acute respiratory distress syndrome, often in the absence of any infection, remains unknown. Here, we investigated the roles of IFN-γ and IL-17A, key T-cell cytokines significantly elevated in patients with CRS, in the immunopathogenesis of CRS-induced extrapulmonary ALI. CRS was induced in wild-type (WT), IL-17A- and IFN-γ knockout (KO) human leukocyte antigen-DR3 transgenic mice with 10 μg of the superantigen, staphylococcal enterotoxin B, given intraperitoneally. Several ALI parameters, including gene expression profiling in the lungs, were studied 4, 24, or 48 hours later. Systemic T-cell activation with staphylococcal enterotoxin B resulted in robust upregulation of several chemokines, S100A8/A9, matrix metalloproteases, and other molecules implicated in tissue damage, granulocyte as well as agranulocyte adhesion, and diapedesis in the lungs as early as 4 hours, which was accompanied by subsequent neutrophil/eosinophil lung infiltration and severe ALI in IFN-γ KO mice. These pathways were significantly underexpressed in IL-17A KO mice, which manifested mildest ALI and intermediate in WT mice. Neutralization of IFN-γ worsened ALI in WT and IL-17A KO mice, whereas neutralizing IL-17A did not mitigate lung injury in IFN-γ KO mice, suggesting a dominant protective role for IFN-γ in ALI and that IL-17A is dispensable. Ruxolitinib, a Janus kinase inhibitor, increased ALI severity in WT mice. Thus, our study identified novel mechanisms of ALI in CRS and its differential modulation by IFN-γ and IL-17A.
Topics: Humans; Mice; Animals; Interleukin-17; Cytokine Release Syndrome; Interferon-gamma; Cytokines; Lung; Acute Lung Injury; Mice, Knockout; Mice, Inbred C57BL
PubMed: 36125351
DOI: 10.1165/rcmb.2022-0117OC -
Journal of Neuroinflammation Sep 2022Cerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH.
BACKGROUND
Cerebral vasospasm (CV) can contribute to significant morbidity in subarachnoid hemorrhage (SAH) patients. A key unknown is how CV induction is triggered following SAH.
METHODS
Human aneurysmal blood and cerebral spinal fluid were collected for evaluation. To confirm mechanism, c57/bl6 wild type and c57/bl6 IL-6 female knockout (KO) mice were utilized with groups: saline injected, SAH, SAH + IL-6 blockade, SAH IL-6 KO, SAH IL-6 KO + IL-6 administration, SAH + p-STAT3 inhibition. Dual-labeled microglia/myeloid mice were used to show myeloid diapedesis. For SAH, 50 μm blood was collected from tail puncture and administered into basal cisterns. IL-6 blockade was given at various time points. Various markers of neuroinflammation were measured with western blot and immunohistochemistry. Cerebral blood flow was also measured. Vasospasm was measured via cardiac injection of India ink/gelatin. Turning test and Garcia's modified SAH score were utilized. P < 0.05 was considered significant.
RESULTS
IL-6 expression peaked 3 days following SAH (p < 0.05). Human IL-6 was increased in aneurysmal blood (p < 0.05) and in cerebral spinal fluid (p < 0.01). Receptor upregulation was periventricular and perivascular. Microglia activation following SAH resulted in increased caveolin 3 and myeloid diapedesis. A significant increase in BBB markers endothelin 1 and occludin was noted following SAH, but reduced with IL-6 blockade (p < 0.01). CV occurred 5 days post-SAH, but was absent in IL-6 KO mice and mitigated with IL-6 blockade (p < 0.05). IL-6 blockade, and IL-6 KO mitigated effects of SAH on cerebral blood flow (p < 0.05). SAH mice had impaired performance on turn test and poor modified Garcia scores compared to saline and IL-6 blockade. A distinct microglia phenotype was noted day 5 in the SAH group (overlap coefficients r = 0.96 and r = 0.94) for Arg1 and iNOS, which was altered by IL-6 blockade. Day 7, a significant increase in toll-like receptor 4 and Stat3 was noted. This was mitigated by IL-6 blockade and IL-6 KO, which also reduced Caspase 3 (p < 0.05). To confirm the mechanism, we developed a p-STAT3 inhibitor that targets the IL-6 pathway and this reduced NFΚB, TLR4, and nitrotyrosine (p < 0.001). Ventricular dilation and increased Tunel positivity was noted day 9, but resolved by IL-6 blockade (p < 0.05).
CONCLUSION
Correlation between IL-6 and CV has been well documented. We show that a mechanistic connection exists via the p-STAT3 pathway, and IL-6 blockade provides benefit in reducing CV and its consequences mediated by myeloid cell origin diapedesis.
Topics: Animals; Caspase 3; Caveolin 3; Endothelin-1; Female; Gelatin; Humans; Interleukin-6; Mice; Mice, Knockout; Subarachnoid Hemorrhage; Toll-Like Receptor 4; Vasospasm, Intracranial
PubMed: 36114540
DOI: 10.1186/s12974-022-02592-x -
Frontiers in Immunology 2022The obligate intracellular parasite makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has...
The obligate intracellular parasite makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has remained unresolved. Here, we addressed the effects of infection on the trans-endothelial migration (TEM) of dendritic cells (DCs) across polarised brain endothelial monolayers. We report that upregulated expression of leukocyte ICAM-1 is a feature of the enhanced TEM of parasitised DCs. The secreted parasite effector GRA15 induced an elevated expression of ICAM-1 in infected DCs that was associated with enhanced cell adhesion and TEM. Consequently, gene silencing of in primary DCs or deletion of parasite GRA15 reduced TEM. Further, the parasite effector TgWIP, which impacts the regulation of host actin dynamics, facilitated TEM across polarised endothelium. The data highlight that the concerted action of the secreted effectors GRA15 and TgWIP modulate the leukocyte-endothelial interactions of TEM in a parasite genotype-related fashion to promote dissemination. In addition to the canonical roles of endothelial ICAM-1, this study identifies a previously unappreciated role for leukocyte ICAM-1 in infection-related TEM.
Topics: Dendritic Cells; Endothelium; Intercellular Adhesion Molecule-1; Toxoplasma; Transendothelial and Transepithelial Migration
PubMed: 35990682
DOI: 10.3389/fimmu.2022.950914 -
The Journal of Investigative Dermatology Oct 2022
Topics: Blood Platelets; Hemorrhage; Humans; Neutrophils; Transendothelial and Transepithelial Migration
PubMed: 35987718
DOI: 10.1016/j.jid.2022.06.009 -
Clinics and Practice Jul 2022It is an acknowledged fact that SARS-CoV-2 exhibits tropism for the human placenta. A possible mechanism of SARS-CoV-2 entry into host cells is via...
It is an acknowledged fact that SARS-CoV-2 exhibits tropism for the human placenta. A possible mechanism of SARS-CoV-2 entry into host cells is via angiotensin-converting enzyme 2 (ACE2) receptors, which are expressed in trophoblasts, endothelial cells, and macrophages. The present study describes a case of spontaneous miscarriage in the 20th gestational week after maternal SARS-CoV-2 infection. The placenta and various fetal organs were examined for structural alterations and expression of the viral nucleocapsid protein and several immune cell markers via immunohistochemistry (IHC). Histopathological examination of the placenta revealed acute chorioamnionitis, acute subamnionic placentitis, multiple intervillous thrombi, increased fibrinoid deposition, and necrotic changes of the chorionic villi. Immunohistochemistry confirmed the presence of SARS-CoV-2 nucleocapsid protein regions predominantly in the syncytiotrophoblast. Staining of the placental tissue for different markers helped elucidate the distribution of immune cells. Pathomorphological examination of the fetal organs demonstrated changes in microcirculation with the presence of sludge phenomenon and diapedesis haemorrhages, mostly in the lungs, brain, and myocardium. IHC staining of fetal organs revealed expression of SARS-CoV-2 nucleocapsid protein, which was detected to the highest extent in the brain, lungs, and liver. The findings of the present report support the hypothesis of possible vertical transmission of SARS-CoV-2 from mother to fetus.
PubMed: 35892447
DOI: 10.3390/clinpract12040061 -
Blood Jul 2022
Topics: Humans; Inflammation; Ion Channels; Leukocytes; Transendothelial and Transepithelial Migration
PubMed: 35862093
DOI: 10.1182/blood.2022016596 -
International Journal of Molecular... Jun 2022In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing...
In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing exacerbated cell responses and tissue damage, novel therapeutic agents are sought. 4aa and 4ba, two pyridazinone-scaffold-based phosphodiesterase-IV inhibitors are compared in vitro to zardaverine for their ability to: (1) modulate production of pro-inflammatory mediators, reactive oxygen species (ROS), and phagocytosis; (2) modulate degranulation by PMNs after transepithelial lung migration. Compound 4ba and zardaverine were tested in vivo for their ability to limit tissue recruitment of PMNs in a murine air pouch model. In vitro treatment of lipopolysaccharide-stimulated PMNs with compounds 4aa and 4ba inhibited the release of interleukin-8, tumor necrosis factor-α, and matrix metalloproteinase-9. PMNs phagocytic ability, but not ROS production, was reduced following treatment. Using a lung inflammation model, we proved that PMNs transmigration led to reduced expression of the CD16 phagocytic receptor, which was significantly blunted after treatment with compound 4ba or zardaverine. Using the murine air pouch model, LPS-induced PMNs recruitment was significantly decreased upon addition of compound 4ba or zardaverine. Our data suggest that new pyridazinone derivatives have therapeutic potential in inflammatory diseases by limiting tissue recruitment and activation of PMNs.
Topics: Animals; Lipopolysaccharides; Mice; Neutrophils; Phagocytosis; Reactive Oxygen Species; Transendothelial and Transepithelial Migration
PubMed: 35806233
DOI: 10.3390/ijms23137226 -
Scientific Reports Jul 2022The protozoan parasite Toxoplasma gondii (T. gondii) causes one of the most common human zoonotic diseases and infects approximately one-third of the global population....
The protozoan parasite Toxoplasma gondii (T. gondii) causes one of the most common human zoonotic diseases and infects approximately one-third of the global population. T. gondii infects nearly every cell type and causes severe symptoms in susceptible populations. In previous laboratory animal studies, T. gondii movement and transmission were not analyzed in real time. In a three-dimensional (3D) microfluidic assay, we successfully supported the complex lytic cycle of T. gondii in situ by generating a stable microvasculature. The physiology of the T. gondii-infected microvasculature was monitored in order to investigate the growth, paracellular and transcellular migration, and transmission of T. gondii, as well as the efficacy of T. gondii drugs.
Topics: Animals; Microfluidics; Toxoplasma; Toxoplasmosis; Transendothelial and Transepithelial Migration; Zoonoses
PubMed: 35794197
DOI: 10.1038/s41598-022-15305-4