Ameliorate impacts of scopoletin against vancomycin-induced intoxication in rat model through modulation of Keap1-Nrf2/HO-1 and IκBα-P65 NF-κB/P38 MAPK signaling pathways: Molecular study, molecular docking evidence and network pharmacology analysis.

Nephrotoxicity is an indication for the damage of kidney-specific detoxification and excretion mechanisms by exogenous or endogenous toxicants. Exposure to vancomycin predominantly results in renal damage and losing the control of body homeostasis. Vancomycin-treated rats (200 mg/kg/once daily, for seven consecutive days, i.p.) revealed significant increase in serum pivotal kidney function, oxidative stress, and inflammatory biomarkers. Histologically, vancomycin showed diffuse acute tubular necrosis, denudation of epithelium and infiltration of inflammatory cells in the lining tubular epithelium in cortical portion. In the existing study, the conservative consequences of scopoletin against vancomycin nephrotoxicity was investigated centering on its capacity to alleviate oxidative strain and inflammation through streamlining nuclear factor (erythroid-derived-2) like 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling and prohibiting the nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (p38 MAPK) pathway. With respect to vancomycin group, scopoletin pretreatment (50 mg/kg/once daily, i.p.) efficiently reduced kidney function, oxidative stress biomarkers and inflammatory mediators. Moreover, histological and immunohistochemical examination of scopoletin-treated group showed remarkable improvement in histological structure and reduced vancomycin-induced renal expression of iNOS, NF-κB and p38 MAPK. In addition, scopoletin downregulated (Kelch Like ECH Associated Protein1) Keap1, P38MAPK and NF-κB expression levels while upregulated renal expression levels of regulatory protein (IκBα), Nrf2 and HO-1. Furthermore, molecular docking and network approach were constructed to study the prospect interaction between scopoletin and the targeted proteins that streamline oxidative stress and inflammatory pathways. The present investigations elucidated that scopoletin co-treatment with vancomycin may be a rational curative protocol for mitigation of vancomycin-induced renal intoxication.

Anti-inflammatory effects of oleic acid and the anthocyanin keracyanin alone and in combination: effects on monocyte and macrophage responses and the NF-κB pathway.

Monocyte recruitment and activation of macrophages are essential for homeostasis but are also related to the development and progression of cardiometabolic diseases. The management of inflammation with dietary components has been widely investigated. Two components that may influence inflammation are unsaturated fatty acids such as oleic acid (OA; 18:1cis-9) and antioxidant compounds like anthocyanins. Molecular and metabolic effects of such bioactive compounds are usually investigated in isolation, whereas they may be present in combination in foods or the diet. Considering this, we aimed to analyze the effects of OA and the anthocyanin keracyanin (AC) alone and in combination on toll-like receptor-mediated inflammatory responses in monocytes and macrophages. For this, THP-1-derived macrophages and monocytes were exposed to 3 treatments: OA, AC, or the combination (OAAC) and then stimulated with lipopolysaccharide. Inflammation-related gene expression and protein concentrations of IL-1ß, TNF-α, IL-6, MCP-1, and IL-10 were assessed. Also, NFκBp65, IκBα, and PPAR-γ protein expression were determined. OA, AC, and OAAC decreased pNFκBp65, PPARγ, IκBα, TNF-α, IL-1ß, IL-6, and MCP-1 and increased IL-10. MCP-1 protein expression was lower with OAAC than with either OA and AC alone. Compared to control, OAAC decreased mRNA for TLR4, IκKα, IκBα, NFκB1, MCP-1, TNF-α, IL-6, and IL-1ß more than OA or AC did alone. Also, IL-10 mRNA was increased by OAAC compared with control, OA, and AC. In summary, OA and AC have anti-inflammatory effects individually but their combination (OAAC) exerts a greater effect.

A system-view of Bordetella pertussis booster vaccine responses in adults primed with whole-cell versus acellular vaccine in infancy.

The increased incidence of whooping cough worldwide suggests that current vaccination against Bordetella pertussis infection has limitations in quality and duration of protection. The resurgence of infection has been linked to the introduction of acellular vaccines (aP), which have an improved safety profile compared with the previously used whole-cell (wP) vaccines. To determine immunological differences between aP and wP priming in infancy, we performed a systems approach of the immune response to booster vaccination. Transcriptomic, proteomic, cytometric, and serologic profiling revealed multiple shared immune responses with different kinetics across cohorts, including an increase of blood monocyte frequencies and strong antigen-specific IgG responses. Additionally, we found a prominent subset of aP-primed individuals (30%) with a strong differential signature, including higher levels of expression for CCL3, NFKBIA, and ICAM1. Contrary to the wP individuals, this subset displayed increased PT-specific IgE responses after boost and higher antigen-specific IgG4 and IgG3 antibodies against FHA and FIM2/3 at baseline and after boost. Overall, the results show that, while broad immune response patterns to Tdap boost overlap between aP- and wP-primed individuals, a subset of aP-primed individuals present a divergent response. These findings provide candidate targets to study the causes and correlates of waning immunity after aP vaccination.

Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Dominant-negative NFKBIA mutation promotes IL-1ß production causing hepatic disease with severe immunodeficiency.

Although IKK-ß has previously been shown as a negative regulator of IL-1ß secretion in mice, this role has not been proven in humans. Genetic studies of NF-κB signaling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppressing IL-1ß expression. Here, we report an infant with a clinical pathology comprising neutrophil-mediated autoinflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation of NFKBIA, resulting in a L34P IκBα variant that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1ß secretion was elevated in the patient's stimulated leukocytes, in her induced pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hypersecretion of IL-1ß correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1ß release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, while decreasing neutrophil activation and associated IL-1ß secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signaling in the prevention of neutrophil-dependent autoinflammatory diseases. These findings also highlight the therapeutic potential of IL-1 inhibitors in treating complications arising from systemic NF-κB inhibition.

Maternal administration of probiotics promotes brain development and protects offspring's brain from postnatal inflammatory insults in C57/BL6J mice.

Neonatal morbidities are associated with long term neurological deficits in life and have also been associated with dysbiosis. We tested whether optimizing the neonate's microbiome through maternal probiotic supplementation can improve offspring's neurodevelopmental outcomes. Maternal LB supplementation, carried out by giving Lactobacillus acidophilus and Bifidobacterium infantis (LB) to pregnant C57/BL6J mice daily from E16 to weaning, significantly suppressed postnatal peripheral proinflammatory insult-induced systemic inflammation and normalized compromised blood-brain barrier permeability and tight junction protein expression in the offspring at pre-weaned age. Maternal LB exposure also regulated markers associated with leukocyte transendothelial migration, extracellular matrix injury and neuroinflammation. The suppressed neuroinflammation by maternal LB supplementation was associated with reduced astrocyte/microglia activation and downregulation of the transcriptional regulators CEBPD and IκBα. Furthermore, maternal LB supplementation promoted neuronal and oligodendrocyte progenitor cell development. Our study demonstrates the efficacy of maternal LB supplementation in modulating systemic and central nervous system inflammation as well as promoting neural/oligodendrocyte progenitor development in the offspring. This evidence suggests that maternal probiotic supplementation may be a safe and effective strategy to improve neurological outcomes in the offspring.

Elevated EPSTI1 promote B cell hyperactivation through NF-κB signalling in patients with primary Sjögren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterised by aberrant B cell hyperactivation, whose mechanism is partially understood. METHODS: We performed whole transcriptome sequencing of B cells from three pSS patients and three matched healthy controls (HC). Differentially expression genes (DEGs) were confirmed with B cells from 40 pSS patients and 40 HC by quantitative PCR and western blot. We measured the proliferation potential and immunoglobulins production of siRNA-transfected or plasmid-transfected B cells stimulated with cytosine-phosphate-guanine (CpG) or anti-IgM. We also explored Toll-like receptor 9 (TLR9) signalling to reveal the potential mechanism of B cell hyperactivation in pSS. RESULTS: We identified 77 upregulated and 32 downregulated DEGs in pSS B cells. We confirmed that epithelial stromal interaction (EPST1) expression in pSS B cells was significantly higher than that from HCs. EPSTI1-silencing B cells stimulated with CpG were less proliferated and produced lower level of IgG and IgM comparing with control B cells. EPSTI1-silencing B cells expressed lower level of p-p65 and higher level of IκBα, and B cells with overexpressed EPSTI1 showed higher level of p-p65 and lower level of IκBα. Finally, IκBα degradation inhibitor Dehydrocostus Lactone treatment attenuated p65 phosphorylation promoted by EPSTI1. CONCLUSION: Elevated EPSTI1 expression in pSS B cells promoted TLR9 signalling activation and contributed to the abnormal B cell activation, which was promoted by facilitating p65 phosphorylation and activation of NF-κB signalling via promoting IκBα degradation. EPSTI1 might be implicated in pSS pathogenesis and was a potential therapeutic target of pSS.

IκBα phosphorylation and associated NF-κB activation are essential events in lymphocyte activation, proliferation, and anti-bacterial adaptive immune response of Nile tilapia.

Inhibitory protein IκBα plays a crucial role in the inflammatory process and immune response by regulating the activity of transcription factor NF-κB. In teleost, great progress has been achieved regarding NF-κB signaling for innate immunity, but whether this pathway modulates adaptive immunity, and how, remains largely unclear. In this study, after characterizing the sequence, structure, and phylogeny of Nile tilapia Oreochromis niloticus IκBα (defined as On-IκBα), we investigated the association between IκBα-regulated NF-κB activation and the lymphocyte-mediated adaptive immune response in Nile tilapia. We found that On-IκBα was evolutionarily conserved, and its mRNA was expressed widely in various tissues, with most abundance in the trunk kidney. mRNA expression of On-IκBα was significantly upregulated in spleen at both innate and adaptive immune stages after Aeromonas hydrophila infection. Moreover, phosphorylation of On-IκBα and the downstream On-NF-κB p65 was obviously elevated in spleen leukocytes at 3, 5, or 8 days after A. hydrophila infection, indicating the activation of NF-κB signaling. Correlating with the augmented protein phosphorylation, leukocyte proliferation was enhanced during the same immune stage, suggesting the potential association of IκBα and IκBα-regulated NF-κB signaling in the primary adaptive immune response. Although lymphocyte activation by the T cell-specific mitogen PHA did not alter On-IκBα mRNA expression significantly, lymphocyte activation by the agonist PMA obviously elevated On-IκBα and OnNF-κB p65 phosphorylation in spleen leukocytes. Together, the results suggest that IκBα phosphorylation and its regulated NF-κB activation are essential events associated with lymphocyte activation, proliferation, and anti-bacterial adaptive immune response in Nile tilapia. Our study aids to understand the regulatory mechanism of adaptive immunity in teleost.

The immune-adjuvant activity and the mechanism of resveratrol on pseudorabies virus vaccine in a mouse model.

Resveratrol had shown various properties before, like immunomodulatory, anti-inflammatory and antiviral activities. Based on these properties, the present study was designed to evaluate the effects and mechanism of resveratrol as an immune-adjuvant for pseudorabies virus (PRV) vaccine. We found that oral administration of resveratrol to mice significantly increased the number of T lymphocytes in the spleen, and elevated the concentrations of antibodies and cytokines in the serum. Resveratrol (30 mg/kg) could enhance phagocytic capacity of peritoneal macrophage (PM) by boosting the percentage of phagocytosis, phagocytic index and the level of lysozyme. Resveratrol also enhanced antigen presentation function of PM by upregulating the expressions of CD86 and MHC-II. Further study revealed that resveratrol could increase the protein levels of TLR4, Ikk, IκBα, NF-κB and JNK when compared with non-adjuvant group. These results provide further insight into the mechanism of action in adjuvant activity of resveratrol, and also offer preclinical evidence for development as a PRV vaccine adjuvant.

Triptolide attenuates lipopolysaccharide-induced inflammatory responses in human endothelial cells: involvement of NF-κB pathway.

BACKGROUND: Endothelial cell inflammation is a central event in the pathogenesis of numerous cardiovascular diseases, including sepsis and atherosclerosis. Triptolide, a principal bioactive ingredient of Traditional Chinese Medicine Tripterygium wilfordii Hook.F., displays anti-inflammatory actions in vivo. However, the mechanisms underlying these beneficial effects remain undetermined. The present study investigated the effects and possible mechanisms of triptolide on lipopolysaccharide (LPS)-induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). METHODS: The effects of triptolide on the LPS-induced production and expression of inflammatory molecules, monocyte adhesion and activation of nuclear factor (NF)-κB pathway were examined in cultured HUVECs. RESULTS: In cultured HUVECs, pre-treatment with triptolide dose-dependently attenuated LPS-induced cytokine and chemokine production, adhesion molecule expression and monocyte adhesion. Mechanistically, triptolide was found to dose-dependently inhibit the LPS-induced increases in the DNA binding activity of NF-κB p65 associated with attenuating IκBα phosphorylation and its degradation. Additionally, the present study revealed that triptolide inhibited LPS-triggered NF-κB transcriptional activation in a dose-dependent manner. CONCLUSIONS: The results of the present study indicated that triptolide suppresses the inflammatory response of endothelial cells possibly via inhibition of NF-κB activation.

Directed self-assembly of herbal small molecules into sustained release hydrogels for treating neural inflammation.

Self-assembling natural drug hydrogels formed without structural modification and able to act as carriers are of interest for biomedical applications. A lack of knowledge about natural drug gels limits there current application. Here, we report on rhein, a herbal natural product, which is directly self-assembled into hydrogels through noncovalent interactions. This hydrogel shows excellent stability, sustained release and reversible stimuli-responses. The hydrogel consists of a three-dimensional nanofiber network that prevents premature degradation. Moreover, it easily enters cells and binds to toll-like receptor 4. This enables rhein hydrogels to significantly dephosphorylate IκBα, inhibiting the nuclear translocation of p65 at the NFκB signalling pathway in lipopolysaccharide-induced BV2 microglia. Subsequently, rhein hydrogels alleviate neuroinflammation with a long-lasting effect and little cytotoxicity compared to the equivalent free-drug in vitro. This study highlights a direct self-assembly hydrogel from natural small molecule as a promising neuroinflammatory therapy.

Activation of NF-κB in B cell receptor signaling through Bruton's tyrosine kinase-dependent phosphorylation of IκB-α.

The antigen-mediated triggering of B cell receptor (BCR) activates the transcription factor NF-κB that regulates the expression of genes involved in B cell differentiation, proliferation, and survival. The tyrosine kinase Btk is essentially required for the activation of NF-κB in BCR signaling through the canonical pathway of IKK-dependent phosphorylation and proteasomal degradation of IκB-α, the main repressor of NF-κB. Here, we provide the evidence of an additional mechanism of NF-κB activation in BCR signaling that is Btk-dependent and IKK-independent. In DeFew B lymphoma cells, the anti-IgM stimulation of BCR activated Btk and NF-κB p50/p65 within 0.5 min in absence of IKK activation and IκB-α degradation. IKK silencing did not affect the rapid activation of NF-κB. Within this short time, Btk associated and phosphorylated IκB-α at Y289 and Y305, and, concomitantly, p65 translocated from cytosol to nucleus. The mutant IκB-α Y289/305A inhibited the NF-κB activation after BCR triggering, suggesting that the phosphorylation of IκB-α at tyrosines 289 and 305 was required for NF-κB activation. In primary chronic lymphocytic leukemia cells, Btk was constitutively active and associated with IκB-α, which correlated with Y305-phosphorylation of IκB-α and increased NF-κB activity compared with healthy B cells. Altogether, these results describe a novel mechanism of NF-κB activation in BCR signaling that could be relevant for Btk-targeted therapy in B-lymphoproliferative disorders. KEY MESSAGES: Anti-IgM stimulation of BCR activates NF-κB p50/p65 within 30 s by a Btk-dependent and IKK-independent mechanism. Btk associates and phosphorylates IκB-α at Y289 and Y305, promoting NF-κB activation. In primary CLLs, the binding of Btk to IκB-α correlates with tyrosine phosphorylation of IκB-α and increased NF-κB activity.

miR-34a: a new player in the regulation of T cell function by modulation of NF-κB signaling.

NF-κB functions as modulator of T cell receptor-mediated signaling and transcriptional regulator of miR-34a. Our in silico analysis revealed that miR-34a impacts the NF-κB signalosome with miR-34a binding sites in 14 key members of the NF-κB signaling pathway. Functional analysis identified five target genes of miR-34a including PLCG1, CD3E, PIK3CB, TAB2, and NFΚBIA. Overexpression of miR-34a in CD4+ and CD8+ T cells led to a significant decrease of NFΚBIA as the most downstream cytoplasmic NF-κB member, a reduced cell surface abundance of TCRA and CD3E, and to a reduction of T cell killing capacity. Inhibition of miR-34a caused an increase of NFΚBIA, TCRA, and CD3E. Notably, activation of CD4+ and CD8+ T cells entrails a gradual increase of miR-34a. Our results lend further support to a model with miR-34a as a central NF-κB regulator in T cells.

Beta Adrenergic Receptors Stimulation Attenuates Phosphorylation of NF-κB and IκBα in Hyperglycemic Endothelial Cells.

BACKGROUND/AIMS: NF-κB induces transcription of a number of genes, associated with inflammation and apoptosis. In this study, we have investigated the effect of ß-adrenergic receptor stimulation on NF-κB and IκBα in HUVECs. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in high and low glucose concentrations. All HUVECs were treated with different concentrations of isoproterenol and propranolol for different time periods. The analytical procedures consisted of Western Blot, ELISA, DCFH-DA and TUNEL assays. RESULTS: Isoproterenol (agonist of a beta-adrenergic receptor) significantly reduced phosphorylation at Ser-536 of NF-κB; and Ser-32 and Ser-36 of IκBα in hyperglycemic HUVECs. Isoproterenol also significantly reduced apoptosis and ROS generation. No effect of IκBα was observed on Tyr-42 phosphorylation. The effect of isoproterenol was reversed by the antagonist propranolol. We also checked if NF-κB inhibitor MG132 causes any change at the level of apoptosis. However, we observed an almost similar effect. CONCLUSION: Given data demonstrates that beta-adrenergic receptors stimulation has a protective effect on HUVECs that might be occuring via NF-κß and IκBα pathway.

Persistent IKKα phosphorylation induced apoptosis in UVB and Poly I:C co-treated HaCaT cells plausibly through pro-apoptotic p73 and abrogation of IκBα.

Toll-like receptor 3 (TLR3), a member of pattern recognition receptors, is reported to initiate skin inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. Recently, we have discovered the NF-κB pathway activated by TLR3 is involved in apoptosis of UVB-Poly I:C-treated HaCaT cells. The real culprit for apoptosis has not been precisely identified since the system of NF-κB pathway is complex. In this study, we silenced main transcriptional factors in NF-κB family, RelA, RelB and c-Rel, but to our surprise the results show that none of them participate in apoptosis induction in UVB-Poly I:C-treated HaCaT cells. Therefore, we moved to investigate the apoptosis-associated molecules in the upstream of NF-κB pathway. We firstly checked the expression of IκBα, an NF-κB inhibitor. UVB (4.8 mJ/cm2) and Poly I:C (0.3 µg/mL) co-treatment decreased IκBα expression level in a time-dependent manner. Silencing IκBα with siRNA further enhanced UVB-Poly I:C-induced cell death. We then investigated IκB kinase (IKK) complex that contributes to the degradation of IκBα. IKK is composed of IKKα, IKKß and NEMO. Treatment with IKK-16, an IKKα/ß inhibitor, significantly diminished UVB-Poly I:C-induced IκBα degradation and thus apoptosis. Silencing either IKKα or NEMO but not IKKß with corresponding siRNA inhibited apoptosis. Tumor repressor p73, a homologue of p53, is reported to mediate IKKα-induced apoptosis in DNA damage response. Silencing p73 reduced cell apoptosis in UVB-Poly I:C-treated HaCaT cells. In summary, UVB and Poly I:C co-treatment activates IKKα and NEMO, which diminishes anti-apoptotic IκBα, resulting in enhancement of apoptosis through p73. The findings partially clarify the possible molecular mechanism of pro-apoptotic NF-κB pathway activated by TLR3 in the fate of UVB-irradiated epidermis.

Sinomenine relieves oxygen and glucose deprivation-induced microglial activation via inhibition of the SP1/miRNA-183-5p/IκB-α signaling pathway.

Studies have shown that the inflammatory activation of miroglia (MG) and nuclear factor kappa B ( NF-κB ) play a dominant role in inflammatory response. Previous studies have shown that sinomenine, an anti-inflammatory agent extracted from Sinomenium acutum, can directly protect neurons against cerebral ischemia injury. However, there are no reports on its effect on ischemia/reperfusion-induced inflammatory activation of MG. In the present study, an in vitro ischemia/reperfusion model was developed with mouse BV-2 microglia cells, a model of oxygen-glucose deprivation/reperfusion (OGD/R), and the inhibitory effect of sinomenine pretreatment on inflammatory activation was confirmed through measurement of inflammatory indicators. Mechanistically, sinomenine suppressed OGD/R-induced inflammatory activation through the SP1/miRNA-183-5p/IκB-α pathway. In conclusion, this study shows that sinomenine effectively inhibits OGD/R-induced inflammatory activation in MG by suppressing the activation of transcription specificity protein 1 (SP 1). This finding is of significance for the clinical use of sinomenine in treating cerebral ischemia/reperfusion injury.

Tie2 signalling through Erk1/2 regulates TLR4 driven inflammation.

The inflammatory response is essential for eradication of lipopolysaccharide (LPS) presenting microbial invaders but requires exquisite regulation to prevent detrimental vascular inflammation. Endothelial cells play active roles in both the initiation of inflammation, through the detection of LPS by Toll-like Receptor 4 (TLR4), and the resolution of inflammation, through the actions of the receptor tyrosine kinase, Tie2. The process by which Tie2 attenuates LPS-TLR4 driven inflammation is poorly understood. To investigate the effects of Tie2 on TLR4 signalling, Nf-κB activation was monitored in cells expressing Tie2 mutants harboring tyrosine (Y) to phenylalanine (F) substitutions in the cytoplasmic domain. Tie2 attenuated LPS induced Nf-κB activation in a manner requiring Tie2 kinase activation, the carboxy-terminal tyrosine residue Y1100 and downstream Erk1/2 signalling. Tyrosine 1100 was also required for the Tie2 dependent decrease in expression of the TLR4 signalling proteins, TRAF6 and IRAK1 and stabilization of the Nf-κB inhibitor, IκBα. In contrast, upregulation of known TLR4 antagonist miRNA-146b-5p required all three tyrosine phosphorylation sites in Tie2. Finally, we confirmed in an in vivo model that activation of Tie2 signalling reduces LPS mediated inflammation. Our results show that Y1100 initiated Erk1/2 signalling is essential for the anti-inflammatory effect of Tie2 on TLR4 mediated inflammation.

Structural characterization and immunomodulatory activity of a novel polysaccharide from Ficus carica.

A novel polysaccharide (FCPW80-2) with a molecular weight of 1.21 × 105 Da was first isolated from Ficus carica through hot water extraction and several chromatographic methods. The structure of FCPW80-2 was determined by chemical and instrumental analysis. The results showed that the backbone of FCPW80-2 consists of (1→5)-linked α-l-Ara, (1→3,6)-linked ß-d-Man and (1→4,6)-linked ß-d-Gal. The branches of FCPW80-2 consist of (1→4)-linked α-d-Glc and (1→3)-linked ß-l-Rha terminated with (1→)-linked ß-d-Glc. In vitro immunomodulatory activity assays revealed that FCPW80-2 could markedly promote the secretion of cytotoxic molecules (NO) and cytokines (TNF-α and IL-6) as well as the phagocytosis of RAW264.7 macrophages. Moreover, TLR2 was found to be a pattern recognition receptor (PRR) of FCPW80-2, and its related mitogen-activated protein kinases (MAPKs), including p-ERK, p-JNK and p-p38, were rapidly upregulated by FCPW80-2 in RAW264.7 macrophages. Furthermore, FCPW80-2 could not only upregulate the expression of p-p65 and p-IκB-α, but also cause the translocation of nuclear factor-kappa B (NF-κB) p65 from cytosol to nuclei in RAW264.7 macrophages. The results demonstrated that MAPK and NF-κB signalling pathways participated in FCPW80-2-induced macrophage activation and FCPW80-2 could be developed as a potential immunomodulating functional food.

PGE2 downregulates LPS-induced inflammatory responses via the TLR4-NF-κB signaling pathway in bovine endometrial epithelial cells.

Postpartum bacterial infections of the uterus cause endometritis in dairy cows. Inflammatory responses to bacterial infections in the bovine uterus were generated through pattern recognition receptors (PRRs) that bind to pathogen-associated molecules such as lipopolysaccharide (LPS) from Escherichia coli. Among these PRRs, Toll-like receptor 4 (TLR4) is primarily responsible for LPS recognition, which triggers inflammatory responses via mitogen-activated protein kinases (MAPKs) and NF-κB signaling activation, resulting in the expression of inflammatory mediators in mammals such as IL-8 and IL-6. Previous studies indicate that PGE2 plays an important role in bacterial endometritis, although details on the mechanism underlying how it regulates LPS-induced inflammatory responses in bovine endometrial epithelial cells (bEECs) remain elusive. In the present study, bEECs were pre-treated with exogenous PGE2 and/or PGF2α prior to LPS stimulation. With PGE2 pre-treatment, we observed an augmentation in LPS-stimulated PKA, ERK, and IκBα phosphorylation and cyclooxygenase-2 (COX-2) and anti-inflammatory cytokine IL-6 expression and downregulation of prostaglandin E2 receptor 4 (EP4) and TLR4 in bEECs. These results indicate that LPS-induced inflammatory responses through TLR4 signaling in bEECs could be downregulated by exogenous PGE2 pre-treatment, but not PGF2α.

Transcriptome of Porcine PBMCs over Two Generations Reveals Key Genes and Pathways Associated with Variable Antibody Responses post PRRSV Vaccination.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a virus susceptible to antibody dependent enhancement, causing reproductive failures in sows and preweaning mortality of piglets. Modified-live virus (MLV) vaccines are used to control PRRS in swine herds. However, immunized sows and piglets often generate variable antibody levels. This study aimed to detect significant genes and pathways involved in antibody responsiveness of pregnant sows and their offspring post-PRRSV vaccination. RNA sequencing was conducted on peripheral blood-mononuclear cells (PBMCs), which were isolated from pregnant sows and their piglets with high (HA), median (MA), and low (LA) PRRS antibody levels following vaccination. 401 differentially expressed genes (DEGs) were identified in three comparisons (HA versus MA, HA versus LA, and MA versus LA) of sow PBMCs. Two novel pathways (complement and coagulation cascade pathway; and epithelial cell signaling in H. pylori infection pathway) revealed by DEGs in HA versus LA and MA versus LA were involved in chemotactic and proinflammatory responses. TNF-α, CCL4, and NFKBIA genes displayed the same expression trends in subsequent generation post-PRRS-MLV vaccination. Findings of the study suggest that two pathways and TNF-α, CCL4, and NFKBIA could be considered as key pathways and potential candidate genes for PRRSV vaccine responsiveness, respectively.