The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
BACKGROUND - Vascular dysfunction is commonly seen during severe viral infections. Endothelial nitric oxide synthase (eNOS), has been postulated to play an important role in regulating vascular homeostasis as well as propagation of the inflammatory reaction. We hypothesized that the loss of eNOS would negatively impact toll-like receptor 3 (TLR3) signaling and worsen vascular function to viral challenge.
METHODS - Human microvascular endothelial cells (HMVECs) were exposed to either control or eNOS siRNA and then treated with Poly I:C, a TLR3 agonist and mimicker of dsRNA viruses. Cells were assessed for protein-protein associations, cytokine and chemokine analysis as well as transendothelial electrical resistance (TEER) as a surrogate of permeability.
RESULTS - HMVECs that had reduced eNOS expression had a significantly elevated increase in IL-6, IL-8 and IP-10 production after Poly I:C. In addition, the knockdown of eNOS enhanced the change in TEER after Poly I:C stimulation. Western blot analysis showed enhanced phosphorylation of p38 in sieNOS treated cells with Poly I:C compared to siControl cells. Proximity ligation assays further demonstrated direct eNOS-p38 protein-protein interactions. The addition of the p38 inhibitor, SB203580, in eNOS knockdown cells reduced both cytokine production after Poly I:C, and as well as mitigated the reduction in TEER, suggesting a direct link between eNOS and p38 in TLR3 signaling.
CONCLUSIONS - These results suggest that reduction of eNOS increases TLR3-mediated inflammation in human endothelial cells in a p38-dependent manner. This finding has important implications for understanding the pathogenesis of severe viral infections and the associated vascular dysfunction.
Malignant pleural effusion (MPE) is a frequent metastatic manifestation of human cancers. While we previously identified KRAS mutations as molecular culprits of MPE formation, the underlying mechanism remained unknown. Here, we determine that non-canonical IKKα-RelB pathway activation of KRAS-mutant tumor cells mediates MPE development and this is fueled by host-provided interleukin IL-1β. Indeed, IKKα is required for the MPE-competence of KRAS-mutant tumor cells by activating non-canonical NF-κB signaling. IL-1β fuels addiction of mutant KRAS to IKKα resulting in increased CXCL1 secretion that fosters MPE-associated inflammation. Importantly, IL-1β-mediated NF-κB induction in KRAS-mutant tumor cells, as well as their resulting MPE-competence, can only be blocked by co-inhibition of both KRAS and IKKα, a strategy that overcomes drug resistance to individual treatments. Hence we show that mutant KRAS facilitates IKKα-mediated responsiveness of tumor cells to host IL-1β, thereby establishing a host-to-tumor signaling circuit that culminates in inflammatory MPE development and drug resistance.
BACKGROUND - Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood.
OBJECTIVE AND METHODS - We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18-49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination.
RESULTS - Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination.
CONCLUSIONS - Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed.
TRIAL REGISTRATION - ClinicalTrials.gov NCT01573312.
Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.
© Society for Leukocyte Biology.
The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.
Suppressor of cytokine signaling (SOCS)-3 has been suggested to regulate CXCR4 signaling in a variety of human cell lines. In mice, conditional SOCS3 inactivation in hematopoietic cells including B-lineage lymphocytes has been reported to exacerbate CXCR4-signaling and focal adhesion kinase phosphorylation, which resulted in altered immature B cell distribution in bone marrow (BM) due to sustained α4β1 integrin-mediated adhesion to the extracellular matrix. However, a recent study examining conditional SOCS3 deletion specifically in B-lineage cells failed to detect significant roles in B-lineage cell retention in BM. In this study we carefully examined the role played by SOCS3 in CXCR4 signaling in developing B cell subsets. We show that in mice conditionally deficient in SOCS3 exclusively in B cells (Socs3fl/fl Mb1cre/+) there was no detectable difference in B cell development in BM and in periphery. We show that SOCS3 deficient and sufficient immature B cell subsets are similarly distributed between BM parenchyma and sinusoids, and are equally competent at exiting BM into peripheral blood. Furthermore, we found no significant differences in CXCR4 desensitization upon ligand exposure in developing B lymphocyte subsets. Consequently, SOCS3-deficient and sufficient B-lineage cell migration towards CXCL12 in vitro was undistinguishable, and B-lineage cell amoeboid motility within BM parenchyma was also unaffected by SOCS3-deficiency. Thus we conclude that SOCS3 has no detectable influence on biological processes known to be controlled by CXCR4 signaling.
Nuclear LASP-1 (LIM and SH3 protein-1) has a direct correlation with overall survival of breast cancer patients. In this study, immunohistochemical analysis of a human breast TMA showed that LASP-1 is absent in normal human breast epithelium but the expression increases with malignancy and is highly nuclear in aggressive breast cancer. We investigated whether the chemokines and growth factors present in the tumor microenvironment could trigger nuclear translocation of LASP-1.Treatment of human breast cancer cells with CXCL12, EGF and HRG, and HMEC-CXCR2 cells with CXCL8 facilitated nuclear shuttling of LASP-1. Data from the biochemical analysis of the nuclear and cytosolic fractions further confirmed the nuclear translocation of LASP-1 upon chemokine and growth factor treatment. CXCL12-dependent nuclear import of LASP-1 could be blocked by CXCR4 antagonist, AMD-3100. Knock down of LASP-1 resulted in alterations in gene expression leading to an increased level of cell-junction and extracellular matrix proteins and an altered cytokine secretory profile. Three-dimensional cultures of human breast cancer cells on Matrigel revealed an altered colony growth, morphology and arborization pattern in LASP-1 knockdown cells. Functional analysis of the LASP-1 knockdown cells revealed increased adhesion to collagen IV and decreased invasion through the Matrigel. Proteomic analysis of immunoprecipitates of LASP-1 and subsequent validation approaches revealed that LASP-1 associated with the epigenetic machinery especially UHRF1, DNMT1, G9a and the transcription factor Snail1. Interestingly, LASP-1 associated with UHRF1, G9a, Snail1 and di- and tri-methylated histoneH3 in a CXCL12-dependent manner based on immunoprecipitation and proximity ligation assays. LASP-1 also directly bound to Snail1 which may stabilize Snail1. Thus, nuclear LASP-1 appears to functionally serve as a hub for the epigenetic machinery.
Monophosphoryl lipid A (MPLA) is a TLR4 agonist that is used as an immunomodulator in human vaccines; additionally, it has been shown to be protective in models of sepsis. As endothelial cells regulate inflammation, we hypothesized that MPLA would decrease activation of human umbilical vein endothelial cells (HUVECs) to LPS. We studied HUVECs challenged with LPS (100 ng/ml), MPLA (0.001-100 µg/ml) or a combination. Secretion of IL-6, RANTES (CCL5) and IP-10 (CXCL10) were assessed by ELISA. Activation of MAPK phosphorylation and cytokine transcription were assessed by Western blot analysis and PCR, respectively. MPLA alone was a weak stimulator of myeloid differentiation primary response protein 88-dependent IL-6 and did not induce TIR-domain-containing adapter-inducing IFN-β (TRIF)-dependent chemokine responses. MPLA significantly reduced LPS-mediated IL-6 production. This inhibitory effect was also conferred for the TRIF-dependent chemokines RANTES and IP-10. Inhibition of LPS-mediated activation by MPLA was associated with reduced p38 phosphorylation and mRNAs encoding inflammatory cytokines. MPLA inhibition of LPS signaling appeared to be at the level of the TLR4 receptor, acting as a receptor antagonist with weak agonistic properties. This study provides evidence of a novel mechanism for the inhibitory effect of MPLA on LPS-induced endothelial activation.
© The Author(s) 2014.
Dormant or slow-cycling disseminated tumor cells (DTCs) in bone marrow (BM) are resistant to conventional therapy in various cancers including head and neck squamous cell carcinoma (HNSCC), although the molecular mechanisms remain largely unknown. This study aimed to identify the intrinsic molecular mechanisms underlying drug resistance in BM-DTCs. We used in vivo selection of the human HNSCC cell line HEp3, which mimics non-proliferative BM-DTCs in mice, to establish BM-DTC-derived (BM-HEp3) and lung metastases-derived (Lu-HEp3) sublines. Both sublines had higher migration activity and shortened survival in a murine xenograft model compared with parental (P-HEp3) cells. Slow-cycling BM-HEp3 cells had intrinsically enhanced cisplatin resistance compared with Lu-HEp3 cells, which also manifested this resistance but proliferated rapidly. The drug resistance and slow-cycling state of BM-HEp3 cells depended on enhanced positive feedback of the signaling axis of stromal cell-derived factor-1 (SDF-1)-C-X-C chemokine receptor-4 (CXCR4) via their overexpression. Interestingly, BM-DTCs highly expressed transforming growth factor-beta 2 (TGF-β2) to maintain SDF-1-CXCR4 overexpression. Inhibition of SDF-1-CXCR4 signaling by down-regulating TGF-β2 fully reversed the drug resistance of BM-HEp3 cells via reactivation of cell proliferation. These data suggest that the intrinsic TGF-β2-triggered SDF-1-CXCR4 signaling axis is crucial for drug resistance dependent on a slow-cycling state in BM-DTCs.
Sepsis is strongly associated with patency of the ductus arteriosus (PDA) in critically ill newborns. Inflammation and the aminoglycoside antibiotics used to treat neonatal sepsis cause smooth muscle relaxation, but their contribution to PDA is unknown. We examined whether: 1) lipopolysaccharide (LPS) or inflammatory cytokines cause relaxation of the ex vivo mouse DA; 2) the aminoglycosides gentamicin, tobramycin, or amikacin causes DA relaxation; and 3) newborn infants treated with aminoglycosides have an increased risk of symptomatic PDA (sPDA). Changes in fetal mouse DA tone were measured by pressure myography in response to LPS, TNF-α, IFN-γ, macrophage-inflammatory protein 2, IL-15, IL-13, CXC chemokine ligand 12, or three aminoglycosides. A clinical database of inborn patients of all gestations was analyzed for association between sPDA and aminoglycoside treatment. Contrary to expectation, neither LPS nor any of the inflammatory mediators caused DA relaxation. However, each of the aminoglycosides caused concentration-dependent vasodilation in term and preterm mouse DAs. Pretreatment with indomethacin and N-(G)-nitro-L-arginine methyl ester did not prevent gentamicin-induced DA relaxation. Gentamicin-exposed DAs developed less oxygen-induced constriction than unexposed DAs. Among 488,349 infants who met the study criteria, 40,472 (8.3%) had sPDA. Confounder-adjusted odds of sPDA were higher in gentamicin-exposed infants, <25 wk and >32 wk. Together, these findings suggest that factors other than inflammation contribute to PDA. Aminoglycoside-induced vasorelaxation and inhibition of oxygen-induced DA constriction support the paradox that antibiotic treatment of sepsis may contribute to DA relaxation. This association was also found in newborn infants, suggesting that antibiotic selection may be an important consideration in efforts to reduce sepsis-associated PDA.
Copyright © 2014 the American Physiological Society.