Elevated Expression Of IL-6 Research Articles

Abstract 716: Elabela Ameliorates Atherogenesis Through Restoring The M1/M2 Macrophage Balance In Apoe -/- Mice

Atherosclerosis is the leading cause of death in patients with cardiovascular disease worldwide. ELABELA, an early endogenous ligand for the G protein-coupled receptor APJ (apelin peptide jejunum, apelin receptor), maybe a novel therapeutic target for multiple cardiovascular diseases. The major goal of the present study was to test the potential therapeutic action of ELABELA in atherogenesis and the underlying mechanisms. ELABELA-21 (ELA-21) was used to treat low-density lipoprotein (ox-LDL)-incubated macrophages to detect macrophage foaminess. ApoE -/- mice were fed a high-fat diet (HFD) for 16 weeks and received subcutaneous injections of ELA-21 peptides (1 mg/kg/day) in the last 4 weeks to detect atherosclerotic plaque area, macrophage infiltration, and the balance between M1 and M2 macrophages. ELA-21 significantly ameliorated atherosclerosis in HFD-fed ApoE -/- mice, as characterized by significantly decreased atherosclerotic plaque area (40%, assessed by Oil-red-O staining), inhibited macrophage infiltration (45%, assessed by CD68 staining), and increased collagen content (65%, assessed by α-SMA staining). By RT-qPCR, ELA-21 treatment inhibited the mRNA expression of Icam-1 (49%), iNOS (53%), IL-1β (59%), IL-6 (55%), and Mc p-1 (80%) in the aorta of HFD-fed ApoE -/- mice. In vitro , ELA-21 treatment inhibited macrophage-derived foam cell formation (75.5%) induced by ox-LDL assessed by Oil-red-O staining. Additionally, in macrophages from the abdominal cavity of HFD-fed ApoE -/- mice, ELA-21 treatment abolished the lipid accumulation (50%) assessed by Oil-red-O staining, and modulated the balance between M1 and M2 macrophages toward a more anti-inflammatory status in HFD-fed ApoE -/- mice, as characterized by the significantly decreased mRNA levels of IL-1β (94%), Tnf-α (55%), iNOS (99%), and IL-12β (99%) (the markers of M1 macrophages), and elevated mRNA expression of Fizz1 (17777%), IL-10 (529%), and Cd206 (24%)(the markers of M2 macrophages). Collectively, our data highlighted an anti-atherosclerotic effect of ELA-21 by restoring the M1/M2 macrophage balance.

Kininase I inhibition reduces B1R mediated signaling and attenuates angiotensin II-induced hypertension

Objectives: Kininase I, or carboxypeptidase M and N (CPM/CPN), metabolizes bradykinin and kallidin into endogenous kinin B1 receptor (B1R) agonists. B1R expression in the brain is linked to neuroinflammation and autonomic dysfunction, and the development of hypertension. The metalloprotease aminopeptidase N (CD13) can interact with B1R to enhance proinflammatory cytokine production. However, the exact role of kininase I in the development of hypertension has not been yet investigated. In the present study, we tested the hypothesis that inhibition of kininase I will prevent the activation of the B1R, and reduce CD13 signaling, thus ultimately attenuating the progression of hypertension. Methods and Results: To inhibit the kininase I activity, we have utilized carboxypeptidase N gene deleted knockout (CPNKO) mice. Twelve-week-old male and female C57BL/6NJ wild-type (WT) and CPNKO mice, were administered angiotensin II (Ang II, 600 ng/kg/min; 2 weeks) or saline (vehicle) via osmotic minipumps. The mean arterial blood pressure (MAP) was measured using radio telemetry probes for 2 weeks. CPNKO mice did not show any significant differences in MAP at baseline. Ang II-infusion significantly increased the MAP in WT mice compared to saline infused control mice (147 ±5 vs 102 ±2 mmHg, n=5, p<0.05), which was attenuated in CPNKO mice (125 ±4 mmHg, n=5, p<0.01). Interestingly, there were no significant differences in MAP following Ang II-infusion in male and female CPNKO mice. Ang II treatment resulted in sympathoexcitation in WT mice, as indicated by increased urinary norepinephrine levels (446 ±38 ng/ml, p<0.05 vs. control), but this increase was blunted in CPNKO mice infused with Ang II (246 ±14 ng/ml, p<0.05 vs. WT+Ang II). WT mice treated with Ang II had increased inflammation, as shown by elevated gene expression (measured by qRT-PCR) of TNF, IL-1β and IL-6 (5-, 3.6-, and 4-fold, respectively, n=6, p<0.01 vs. WT) in the hypothalamic paraventricular nucleus (PVN). The expression of these inflammatory genes was attenuated in CPNKO mice with Ang II infusion. Immunofluorescence staining showed that Ang II-infusion also significantly increased the expression of B1R, CPM, and CD13 in the PVN, a central regulatory center for cardiovascular and autonomic control. CPM has been shown to interact directly with B1R. To further determine interactions between B1R and CPM, we used proximity ligation assay (PLA) to elucidate close (<40 nM) protein-protein interactions, as indicated by fluorescent PLA signals. Our data suggest that direct B1R-CPM interactions occurring within the PVN as the PLA signals are significantly increased by Ang II treatment in WT mice (p<0.05, n=3), and blunted in CPNKO mice. Conclusions: Kininase I inhibition by CPN gene deletion attenuates Ang II-induced hypertension by reducing B1R and CD13 acti vation, sympathoexcitation, and inflammation. Our data suggest that targeting kininase I may be a novel therapeutic target for hypertension. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autophagy induction protects the perivascular adipose tissue during hyperaldosteronism-induced hypertension

Hyperaldosteronism (HA), characterized by an autonomous production of aldosterone, promotes increased blood pressure and cardiovascular damage. The perivascular adipose tissue (PVAT) participates of the control of vascular function and is inflamed in HA. Autophagy is a recycling process of unnecessary or dysfunctional components and it is important for inflammatory tolerance, but whether the autophagic flux is dysregulated in the PVAT during HA, which would contribute to the inflammation and dysfunction of this tissue, is still to be elucidated. Herein, we hypothesize that treatment with autophagy inducer will rescue the autophagic unbalance, subsequently decreasing inflammation, and improving PVAT function during HA. 10-12-weeks-old male C57BL/6J mice were infused with aldosterone for 14 days (600ug/Kg/day via osmotic mini-pump). Vascular function was studied in PVAT-intact thoracic aortae and blood pressure was analyzed by the tail-cuff. Brown adipocyte cells (WT1) were used to evaluate the molecular mechanisms by which HA induces PVAT dysfunction. HA promoted an increase in systolic blood pressure [mmHg – before HA: 123.8±0.9 vs. after HA: 149.5±1.1]. Furthermore, PVAT decreased the contractile response [Emax (% KCl)] in control arteries [Control PVAT (-): 137.2±1.9 vs. Control PVAT (+): 107.4±2.7], whereas HA promoted loss of PVAT anti-contractile effect [HA PVAT (-): 159.9±2.9 vs. HA PVAT (+): 164.1±3.4]. We found that PVAT from HA mice presents an unbalance in LC3II/LC3I ratio, an important autophagy marker, inflammation, characterized by elevated TNF-α, IL-6, IL-1β, IL-17, and IFN-γ gene expression, and elevated reactive oxygen species (ROS). To induce autophagy, we treated mice with TB-peptide (16mg/Kg/day via ip for 7 days). TB-peptide partially improved the PVAT function of mice with HA [HA PVAT (+): 122.5±4.1]. On mechanistic levels, aldosterone (100nM, 12-96 h) induced an oscillatory autophagy flux in WT1 cells, characterized by an initial increase in LC3II/LC3I ratio at 12 and 24 h, followed by a decrease at 48 and 96 h. These effects were not observed in the presence of the mineralocorticoid receptor antagonist (Eplerenone, 1μM). Aldosterone also led to the expression of pro-inflammatory genes (TNF-α, IL-6, IL-1β, IL-17, and IFN-γ) and elevated ROS, such effects were prevented by inducing autophagy with TB-peptide. In summary, these findings reveal a new mechanism in vascular injury associated with HA, aldosterone impairs autophagic flux in PVAT, inducing inflammation, oxidative stress, and dysfunction. Our study places PVAT and autophagic process as attractive targets to reduce cardiovascular risk in HA. FAPESP (2022/06639-2), NHLBI-R00 (R00HL14013903), AHA-CDA (CDA857268), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling.

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

Expression and significance of IL-6 and IL-8 in canine mammary gland tumors

Mammary gland tumors are the most common malignant diseases which seriously threaten the health of women and female dogs. There is a lack of an effective tumor marker which can effectively assist in the early diagnosis, and prognosis of mammary gland tumors in veterinary clinical medicine. IL-6, and IL-8 as immunosuppressive factors may stimulate tumor cells growth, contribute to loco-regional relapse and metastasis that might be utilized as a marker for immunity status and monitoring of the course of tumor. The present study aimed to investigate the expression of serum/tissue IL-6, IL-8 and IL-10 in canine mammary gland tumors using Enzyme linked immunosorbent assay(ELISA), Western blot and Immunohistochemistry assay(IHC) to determine whether it is associated with tumor progression. The results showed that levels of IL-6, IL-8 and IL-10 in serum were higher in malignant tumor group than that in benign tumor and control group; the expression levels of IL-6 and IL-8 were significantly elevated in grade III than in grade I and II and was related to metastasis. Likewise, IL-6 and IL-8 were also highly expressed in malignant tumor tissues. Elevated expression of IL-6 was associated with histopathological grade and metastases in malignant tumors. Moreover, high expression of IL-6 occurred in the Basal-like subtypes whereas high expression of IL-8 occurred in the Luminal B subtypes. The results of this study indicated that changes of IL-6 and IL-8 in the tumor microenvironments were closely related to the diseases status and may be used as a potential diagnostic or biomarker in canine mammary gland tumors.

The two-component system histidine kinase EnvZ contributes to Avian pathogenic Escherichia coli pathogenicity by regulating biofilm formation and stress responses

EnvZ, the histidine kinase (HK) of OmpR/EnvZ, transduces osmotic signals in Escherichia coli K12 and affects the pathogenicity of Shigella flexneri and Vibrio cholera. Avian pathogenic E. coli (APEC) is an extra-intestinal pathogenic E. coli (ExPEC), causing acute and sudden death in poultry and leading to severe economic losses to the global poultry industry. How the functions of EnvZ correlate with APEC pathogenicity was still unknown. In this study, we successfully constructed the envZ mutant strain AE17ΔenvZ and the inactivation of envZ significantly reduced biofilms and altered red, dry, and rough (rdar) morphology. In addition, AE17ΔenvZ was significantly less resistant to acid, alkali, osmotic, and oxidative stress conditions. Deletion of envZ significantly enhanced sensitivity to specific pathogen-free (SPF) chicken serum and increased adhesion to chicken embryonic fibroblast DF-1 cells and elevated inflammatory cytokine IL-1β, IL6, and IL8 expression levels. Also, when compared with the WT strain, AE17ΔenvZ attenuated APEC pathogenicity in chickens. To explore the molecular mechanisms underpinning envZ in APEC17, we compared the WT and envZ-deletion strains using transcriptome analyses. RNA-Seq results identified 711 differentially expressed genes (DEGs) in the envZ mutant strain and DEGs were mainly enriched in outer membrane proteins, stress response systems, and TCSs. Quantitative real-time reverse transcription PCR (RT-qPCR) showed that EnvZ influenced the expression of biofilms and stress responses genes, including ompC, ompT, mlrA, basR, hdeA, hdeB, adiY, and uspB. We provided compelling evidence showing EnvZ contributed to APEC pathogenicity by regulating biofilms and stress response expression.

Sevoflurane activates the IL-6/HO-1 pathway to promote macrophage M2 polarization and prostate cancer lung metastasis.

Sevoflurane (Sev) is commonly used for cancer surgeries, but its effect on cancer cell biology remains unclear. This study aims to delineate the mechanistic actions underlying the oncogenic and metastatic potential of Sev on macrophage M2 polarization and lung metastasis in prostate cancer (PCa). We performed bioinformatics analysis to predict Sev- and PCa-related targets HO-1 and IL-6, and validated their expression in response to Sev exposure. The culture supernatant of macrophages differentiated from mouse bone marrow was co-cultured with mouse PCa cell line RM-1. HO-1 and IL-6 was found to be upregulated in macrophages exposed to Sev. Ectopic expression or knockdown of HO-1 and IL-6 was introduced to macrophages to probe their effect on macrophage polarization, migratory and invasive capacities of PCa cells and lung metastasis in tumor-bearing mice. It was demonstrated that Sev augmented macrophage M2 polarization and migratory and invasive potential of PCa cells as well as lung metastasis, which was associated with HO-1 upregulation. Mechanistic investigation indicated that Sev elevated IL-6 expression to enhance HO-1 expression. In vivo experiments verified that Sev facilitated macrophage M2 polarization and lung metastasis of RM-1 cells via IL-6/HO-1 in mice. Taken together, our work reveals a novel IL-6/HO-1-mediated regulatory network underlying Sev function that may be a viable target for preventing lung metastasis of PCa.

Preliminary verification of the anti-hypoxia mechanism of Gentiana straminea maxim based on UPLC-triple TOF MS/MS and network pharmacology

BackgroundAnoxia is characterized by changes in the morphology, metabolism, and function of tissues and organs due to insufficient oxygen supply or oxygen dysfunction. Gentiana straminea Maxim (G.s Maxim) is a traditional Tibetan medicine. Our previous work found that G.s Maxim mediates resistance to hypoxia, and we found that the ethyl acetate extract had the best effect. Nevertheless, the primary anti-hypoxia components and mechanisms of action remain unclear.MethodsCompounds from the ethyl acetate extraction of G.s Maxim were identified using UPLC-Triple TOF MS/MS. Then Traditional Chinese Medicine Systematic Pharmacology Database was used to filtrate them. Network pharmacology was used to forecast the mechanisms of these compounds. Male specific pathogen-free Sprague Dawley rats were randomly divided into six groups: (1) Control; (2) Model; (3) 228 mg/kg body weight Rhodiola capsules; (4) 6.66 g/kg body weight the G.s Maxim’s ethyl acetate extraction; (5) 3.33 g/kg body weight the G.s Maxim’s ethyl acetate extraction; (6) 1.67 g/kg body weight the G.s Maxim’s ethyl acetate extraction. After administering intragastric ally for 15 consecutive days, an anoxia model was established using a hypobaric oxygen chamber (7000 m, 24 h). Then Histology, enzyme-linked immunosorbent assays, and western blots were performed to determine these compounds’ anti-hypoxic effects and mechanisms. Finally, we performed a molecular docking test to test these compounds using Auto Dock.ResultsEight drug-like compounds in G.s Maxim were confirmed using UPLC-Triple TOF MS/MS and Lipinski’s rule. The tumor necrosis factor (TNF) signaling pathway, the hypoxia-inducible factor 1 (HIF-1) signaling pathway, and the nuclear factor kappa-B (NF-κB) signaling pathway was signaling pathways that G.s Maxim mediated anti-anoxia effects. The critical targets were TNF, Jun proto-oncogene (JUN), tumor protein p53 (TP53), and threonine kinase 1 (AKT1). Animal experiments showed that the ethyl acetate extraction of G.s Maxim ameliorated the hypoxia-induced damage of hippocampal nerve cells in the CA1 region and reversed elevated serum expression of TNF-α, IL-6, and NF-κ B in hypoxic rats. The compound also reduced the expression of HIF-1α and p65 and increased the Bcl-2/Bax ratio in brain tissue. These findings suggest that G.s Maxim significantly protects against brain tissue damage in hypoxic rats by suppressing hypoxia-induced apoptosis and inflammation. Ccorosolic acid, oleanolic acid, and ursolic acid had a strong affinity with core targets.ConclusionsThe ethyl acetate extraction of G.s Maxim mediates anti-hypoxic effects, possibly related to inhibiting apoptosis and inflammatory responses through the HIF-1/NF-κB pathway. The primary active components might be corosolic, oleanolic, and ursolic acids.

Poly I:C promotes malate to enhance innate immune response against bacterial infection

Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of double-stranded RNA (dsRNA) that activates anti-infective innate immunity. The underlying mechanisms are identified as targeting pattern recognition receptors and Th1-inducing. However, whether poly I:C manipulates metabolism to implement this anti-infective function is unknown. Here, GC-MS based metabolomics was used to characterize metabolic profiles induced by different doses of poly I:C. Analysis on the dose-dependent metabolomes shows that elevation of the TCA cycle and malate with the increasing dose of ploy I:C forms the most characteristic feature of the poly I:C stimulation. Exogenous malate activates the TCA cycle and elevates survival of zebrafish infected with Vibrio alginolyticus, which is related to the elevated expression of il-1b, il-6, il-8, tnf-a, and c3b. These results reveal a previously unknown regulation of poly I:C that boosts the TCA cycle to enhance innate immunity against bacterial infection.

Abstract 882: A novel p73 C-terminal isoform, p73α1, regulates inflammation and tumor suppression via the Notch1 pathway

Abstract p73, a member of the p53 family, is expressed as two N-terminal isoforms: TA, a tumor suppressor and ΔN, an oncoprotein. Alternative RNA splicing at the 3` end gives rise to multiple C-terminal isoforms, but their function is largely unknown. To address this, CRISPR was used to knock out exon 12 (E12) in several cell lines and mice, leading to isoform switching from p73α to p73α1. p73α1 is a novel isoform whose expression we detected in multiple cancer cell lines. We found that knockout of E12 inhibits the cell proliferation and migration abilities of H1299 and MIA PaCa-2 cancer cells and E12-/- MEFs exhibit increased cellular senescence. Similarly, ectopic expression of p73α1 in H1299 cells suppresses cell proliferation and knockdown of p73α1 restores the cell proliferative and migratory capacities of H1299 E12-/- cells. Consistently, we found that E12+/- mice are not prone to spontaneous tumors, unlike Trp73+/- mice, but instead are prone to chronic systemic inflammation and show elevated IL-1β and IL-6 expression in inflamed tissues. We then identified Notch1, a master regulator of the inflammatory response, as a mediator of p73α1-induced inflammation and tumor suppression, and found it to be elevated in inflamed E12+/- mouse tissues. Furthermore, we found that knockdown of Notch1 enhances cell proliferation and migration in H1299 E12-/- cells. Together, these data suggest that p73α1 plays a role in the inflammatory response and tumor suppression via Notch1. Citation Format: Kyra Laubach, Wenshen Yan, Xiangmudong Kong, Mingyi Chen, Jin Zhang, Xinbin Chen. A novel p73 C-terminal isoform, p73α1, regulates inflammation and tumor suppression via the Notch1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 882.

Mechanism of electroacupuncture against intestinal inflammation in mice with Parkinson's di-sease by NF-κB/IL-6 pathway

To observe the effects of electroacupuncture (EA) at "Fengfu"(GV16), "Taichong"(LR3), and "Zusanli"(ST36) on the tyrosine hydroxylase (TH), nuclear factor-kappa B (NF-κB), interleukin (IL)-6, and zonula occludens-1 (ZO-1) in mice with Parkinson's disease (PD), and to explore the underlying mechanism of EA in the treatment of PD. C57BL/6 mice were randomly divided into control, model, and EA groups, with 10 mice in each group. The PD model was induced by rotenone (i.g.) in mice for 28 d. EA was applied to GV16, LR3 and ST36 of mice in the EA group for 30 min, once daily for 14 d. The behavioral changes of mice in each group before and after treatment were observed and scored. The total distance traveled autonomously of mice was detected in the open field test. TH expression in the substantia nigra (SN) was measured by immunohistochemistry, and the changes in colon tissue structure were observed by HE staining. The intestinal mRNA expression of ZO-1, NF-κB, and IL-6 was detected by quantitative real-time PCR, and the colon tissue protein expression of NF-κB was detected by Western blot. The colon tissue concentration of IL-6 was detected by ELISA. Compared with the control group, the pre- and post-treatment behavioral scores of the model group and the pre-treatment behavioral score of the EA group were increased (P<0.01). Compared with the model group, the EA group showed reduced behavioral score after treatment (P<0.01). Compared with the control group, the model group showed reduced goblet cells and crypts and thinner muscle layer in the intestinal specimens of mice. Compared with the model group, the EA group showed intact surface villi, increased goblet cells and crypts, and thickened muscle layer. Compared with the control group, the model group exhibited reduced total distance traveled in the open field test, TH expression in SN, and ZO-1 mRNA expression (P<0.01), and elevated mRNA and protein expression of NF-κB and IL-6, and the ratio of p-NF-κB/NF-κB in the intestinal tract (P<0.01). Compared with the model group, the EA group displayed increased total distance traveled, TH expression in SN, and ZO-1 mRNA expression (P<0.01), and declining mRNA and protein expression of NF-κB and IL-6, and the ratio of p-NF-κB/NF-κB in the colon tissue (P<0.01, P<0.05). EA at GV16, LR3, and ST36 can regulate the expression of NF-κB/IL-6, inhibit the transmission of the colon tissue inflammatory response, repair the intestinal barrier function, and potentiate the TH activity, thereby improving the behavioral performance of PD mice.

Lack of STAT6 enhances murine acute lung injury through NLRP3/p38 MAPK signaling pathway in macrophages

BackgroundSignal transducer and activator of transcription 6 (STAT6) is an intracelluar transcriotion factor and NLRP3 (Nod-like receptor containing a pyrin domain 3) is a component of NLRP3 inflammasome in pyroptotic cells. There was increased activation of STAT6 and expression of NLRP3 in mice with murine acute lung injury (ALI). However, it is unknown their roles in the development of murine ALI. We in this study, investigated the effects of STAT6 signaling on murine ALI and pyroptosis in STAT6 knock-out (KO) mice and macrophages.ResultsSTAT6 was activated in the lung tissues of mice 2 days after intratracheal treatmemt with 5 mg/kg LPS. Lack of STAT6 expression in KO mice induced more severe lung inflammation, associated with elevated neutrophil influx and expression of TNF-alpha, IL-6 and IL-1beta in the inflamed lung tissues. In addition, the expression of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), p-p38 MAPK (p38 mitogen-activated protein kinase) and ratio of LC3-II/I (microtubule-associated protein-1 light chain-3) was increased, accompanied with the increased polarization of Siglec-F(−) subtype macrophages in KO mice with ALI. Further studies in bone marrow-derived macrophages (BMDMs) revealed that lack of STAT6 increased the expression of NLRP3 and p-p38 MAPK, in association with elevated expression of TNF-alpha, IL-1beta and Calreticulin in LPS-treated KO BMDMs.ConclusionsLack of STAT6 exacerbated murine ALI through improving the expression of NLRP3 and activation of p38 MAPK in macrophages. STAT6 has an immune suppressive role in the development of ALI and would be a promising therapeutic target in the treatment of ALI and possibly among patients with acute respiratory distress syndrome (ARDS).

Mitochondrial transcription factor B2 overexpression increases M2 macrophage infiltration via cytosolic mitochondrial DNA-stimulated Interleukin-6 secretion in ovarian cancer

ABSTRACT Mitochondrial transcription factor B2 (TFB2M) is a protein modulating both mitochondrial DNA (mtDNA) transcription and compacting. In this study, we explored the expression profile of TFB2M in ovarian cancer, its association with infiltration of tumor-associated macrophages (TAMs), and its influence on macrophage polarization. Serial sections of ovarian cancer tissue arrays were stained to detect TFB2M and CD163 expression. Epithelial ovarian cancer cell line OVISE and CAOV4 were used to assess the influence of TFB2M on IL-6 expression. THP-1 cells were utilized as an in vitro model for macrophage migration and polarization. Results showed that higher TFB2M expression is associated with poor survival in ovarian cancer patients. IHC staining confirmed a moderately positive correlation between TFB2M expression and the infiltration of CD163-positive cells in 68 primary ovarian cancer cases. TFB2M overexpression was associated with increased mtDNA outside the mitochondria and elevated IL-6 expression in ovarian cancer cells. When cytosolic mtDNA was selectively inhibited by DNase I, TFB2M-induced IL-6 upregulation was canceled. TFB2M overexpression could activate the nuclear factor kappa-B (NF-κB) signaling pathway via promoting nucleus entry of p65 and p-p65, which was abrogated by inhibiting cytosolic mtDNA, TLR9, or NF-κB signaling pathway. Conditioned medium from OIVSE cells with TFB2M overexpression could induce macrophage migration and M2 polarization. However, these inducing effects were abrogated by DNase I, TLR9 inhibitor, and anti-IL-6 R pretreatment. In conclusion, this study showed a novel role of TFB2M in the immunosuppressive tumor microenvironment. It promotes M2 macrophage infiltration via a cytosolic mtDNA/TLR9/NF-κB/IL-6 pathway in ovarian cancer.

Brain Kinin B1 Receptor Antagonism Attenuates Angiotensin II‐Induced Neurogenic Hypertension

Hypertension remains the leading cause of death globally, accounting for 10.4 million deaths per year. Recent evidence has shown that the kallikrein‐kinin system may play a vital role in the neural control of hypertension, through interactions with key regulatory systems such as the renin‐angiotensin system (RAS). We previously showed that kinin B1 receptor (B1R) activation promotes the development of DOCA‐salt‐induced neurogenic hypertension. However, accumulated evidence has shown that elevated levels of angiotensin II (Ang II) in the brain or upregulated neuronal angiotensin II type I receptor (AT1R) expression, results in increased neuroinflammation and the development of neurogenic hypertension. Recently, we presented data supporting B1R blockade prevents Ang II induced inflammation and oxidative stress in primary hypothalamic neurons. Yet, the interactions between the B1R and AT1R have not been fully studied. In this study, we hypothesize that Ang II treatment will increase blood pressure through B1R mediated mechanisms. Ang II infusion (600 ng/kg/min, SC, 2 weeks) increased blood pressure (radiotelemetry) in wild‐type (WT) male mice (145 ±13 mmHg vs. 103 ±6, n=6, p&lt;0.01). This increase in blood pressure was attenuated in B1R knockout (B1RKO) mice with Ang II infusion (118 ±12 mmHg, n=6, p&lt;0.01). Interestingly, our data showed no significant differences in B1RKO male and female mice with Ang II‐infusion. Additionally, B1RKO mice show reduced Ang II‐induced AT1R expression in the brain. Using ultra‐sensitive mass spectrometry detection, we found that Ang II‐induced an increase in B1R specific agonist Lys‐Des‐Arg9‐Bradykinin (LDABK) within the hypothalamus in WT mice (p&lt;0.01, n=5). More specifically, immunofluorescence staining showed that B1R is upregulated within neurons in the paraventricular nucleus (PVN) of the hypothalamus, a central regulatory center for cardiovascular and autonomic control. Ang II‐treated mice had increased inflammation (qRT‐PCR), as shown by elevated gene and protein expression of TNFα, IL‐1β, and IL‐6 (6‐, 5‐ and 8‐fold respectively, n=5, p&lt;0.01 vs. control) in the PVN, which was attenuated in B1RKO mice. To further determine interactions between AT1R and B1R, we used proximity ligation assay (PLA), to elucidate close proximity (&lt;40 nM) receptor‐receptor interactions indicated by fluorescent PLA signal. Our results showed that AT1R‐B1R close proximity interactions are detected within the PVN, and the PLA signals are significantly increased by Ang II treatment in WT mice (p&lt;0.01, n=5). Furthermore, to supplement our findings we isolated cultured primary hypothalamic neurons from WT and B1RKO mice and investigated the interaction between Ang II and B1R activation. Primary WT neurons treated with Ang II (300 nM, 24 hr) showed increased B1R expression, inflammation, and AT1R‐B1R interaction. However, B1RKO neurons or WT neurons pretreated with R715 (a B1R specific peptide antagonist) showed an attenuation of these effects. These results indicate B1R blockade attenuates Ang II‐induced hypertension through novel B1R and AT1R interactions. Collectively, our data provide distinct evidence suggesting B1R blockade could serve as a therapeutic target in the treatment of hypertension.

Exacerbated Vascular Inflammation Following Traumatic Hemorrhage in Obese Rats

IntroductionObesity increases the risk for morbidity and mortality in trauma patients. These complications are associated with prolonged tissue hypoperfusion, hypercoagulability, and edema that reflect profound vascular damage. Traumatic hemorrhage acutely attenuates vascular responsiveness, but the degree of this dysfunction during the obese state is not known. Traumatic hemorrhage also increases circulating inflammatory cytokines; however, the local inflammatory response in vascular cells is unknown. We hypothesized that obesity potentiates trauma‐induced vascular inflammation and dysfunction.MethodsMale Sprague‐Dawley rats (~250g) fed either normal chow (NC; 13.5% kcal fat, n=20), moderate‐fat (MF; 33% kcal fat, n=20), or high‐fat (HF; 60% kcal fat, n=20) diet for 6‐8 weeks were assigned to a traumatic hemorrhage protocol or sham control. Under anesthesia, hemorrhage was induced by the laceration of a mesenteric artery and a Grade V splenic injury. Hypotension (mean arterial pressure = 30–40 mm Hg) was maintained for 30 minutes. Vasoconstrictor and vasodilator properties were assessed ex vivo in isolated mesenteric arteries pre‐ and post‐ hemorrhage. Carotid arteries collected after hemorrhage or sham control were flushed with trizol to collect endothelial cells (ECs). Gene expression was assessed in ECs and the remaining carotid artery (media + adventitia) cells for pro‐inflammatory interleukin (IL)‐1β, and IL‐6, pro‐oxidant NADPH oxidase 2 (NOX2), and α‐adrenergic receptor.ResultsIn NC rats, hemorrhage attenuated norepinephrine‐induced vasoconstriction and endothelium‐dependent dilation. This was accompanied by elevated IL‐1β expression in carotid artery ECs and elevated IL‐1β, IL‐6, NOX2, and α‐adrenergic receptor expression in the carotid artery media + adventitia post‐hemorrhage versus sham control. In MF and HF rats, norepinephrine‐induced vasoconstriction and endothelium‐dependent dilation did not change pre‐ to post‐hemorrhage. In sham animals, endothelial cell IL‐1β and NOX2 expression were higher in HF compared with NC rats. Similar to NC rats, the MF and HF rats had higher IL‐1β expression in carotid artery ECs, and IL‐1β, IL‐6, NOX2, and α‐adrenergic receptor expression in the carotid artery media + adventitia post‐hemorrhage compared with sham. The hemorrhage‐induced elevation of IL‐1β in carotid artery ECs and media + adventitia was greater in HF rats compared with NC and MF rats.ConclusionOur clinically‐relevant model of traumatic hemorrhage attenuates vascular responsiveness and induces vascular inflammation. The attenuated vascular responsiveness following hemorrhage is absent in MF and HF rats, while the elevated vascular inflammation persists. HF rats have an amplified arterial inflammatory response to hemorrhage, both in ECs and in cells of the media + adventitia. The augmented vascular inflammatory response to hemorrhage in HF rats may explain the worse outcomes in obese trauma patients.

Effect of Airborne Particulate Matter on the Immunologic Characteristics of Chronic Rhinosinusitis with Nasal Polyps.

The inflammatory mechanisms of environmental pollutants in chronic rhinosinusitis (CRS) have recently been proposed. However, the mechanisms underlying the inflammatory effects of particulate matter (PM) on nasal polyp (NP) tissues remain unknown. Here we investigated the mechanism underlying the inflammatory effects of PM10 on human nasal polyp-derived fibroblasts (NPDFs). We isolated NPDFs from human NP tissues obtained from patients with CRS with NPs (CRSwNP). The NPDFs were exposed to PM10 in vitro. Immunologic characteristics were assessed using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, Western blot, and flow cytometry. Additionally, we investigated the effect of NPDF-conditioned media (CM) on the expression of CD4+ T cell inflammatory mediators. PM10-treated NPDFs significantly upregulated interleukin (IL)-6, IL-4, and IL-33 expression and CXCL1 protein levels than PM10-treated normal tissues. MAP kinase, AP-1, and NF-kB were the primary cell signaling proteins. Immune cells in NPDF-CM had elevated IL-13, IL-17A, and IL-10 expression, but no significant difference in IFN-γ, TNF-α, and IL-4 expression. Moreover, under a Th2 inducing condition, NPDF-CM-treated CD4+ T cells had increased expression of IL-13, IL-10, and IL-17, which was reversed on ST2 inhibitor addition. Our study suggests that PM10 exposure could significantly increase the Th2 inflammatory pathway in NP tissues, specifically the IL-33/ST2 pathway-mediated immune response.

Hypoxic Processes Induce Complement Activation via Classical Pathway in Porcine Neuroretinas.

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher C3+ and MAC+ cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of C1q+ microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated TNF, IL6, and IL8 mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases.

Novel NFκB Inhibitor SC75741 Mitigates Chondrocyte Degradation and Prevents Activated Fibroblast Transformation by Modulating miR-21/GDF-5/SOX5 Signaling.

Osteoarthritis (OA) is a common articular disease manifested by the destruction of cartilage and compromised chondrogenesis in the aging population, with chronic inflammation of synovium, which drives OA progression. Importantly, the activated synovial fibroblast (AF) within the synovium facilitates OA through modulating key molecules, including regulatory microRNAs (miR’s). To understand OA associated pathways, in vitro co-culture system, and in vivo papain-induced OA model were applied for this study. The expression of key inflammatory markers both in tissue and blood plasma were examined by qRT-PCR, western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assays. Herein, our result demonstrated, AF-activated human chondrocytes (AC) exhibit elevated NFκB, TNF-α, IL-6, and miR-21 expression as compared to healthy chondrocytes (HC). Importantly, AC induced the apoptosis of HC and inhibited the expression of chondrogenesis inducers, SOX5, TGF-β1, and GDF-5. NFκB is a key inflammatory transcription factor elevated in OA. Therefore, SC75741 (an NFκB inhibitor) therapeutic effect was explored. SC75741 inhibits inflammatory profile, protects AC-educated HC from apoptosis, and inhibits miR-21 expression, which results in the induced expression of GDF-5, SOX5, TGF-β1, BMPR2, and COL4A1. Moreover, ectopic miR-21 expression in fibroblast-like activated chondrocytes promoted osteoblast-mediated differentiation of osteoclasts in RW264.7 cells. Interestingly, in vivo study demonstrated SC75741 protective role, in controlling the destruction of the articular joint, through NFκB, TNF-α, IL-6, and miR-21 inhibition, and inducing GDF-5, SOX5, TGF-β1, BMPR2, and COL4A1 expression. Our study demonstrated the role of NFκB/miR-21 axis in OA progression, and SC75741’s therapeutic potential as a small-molecule inhibitor of miR-21/NFκB-driven OA progression.

LncRNA NEAT1 regulates MMP-16 by targeting miR-200a/b to aggravate inflammation in asthma

Asthma is a common respiratory disease which is characterized by persistent airway inflammation. Abnormal expression of long non-coding RNAs (lncRNAs) is observed in asthma. However, whether lncRNA nuclear-enriched abundant transcript 1 (NEAT1) regulates asthmatic inflammation and its mechanism still needs to be further investigated. The expression levels of inflammatory factors (tumour necrosis factor (TNF)-α, interleukin (IL)-4, IL-13, and IL-10) were detected using reverse transcription quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). MTT and flow cytometry assays were employed to determine cell proliferation and apoptosis, respectively. Dual luciferase reporter assay was performed to verify the relationship between miR-200a/b and MMP-16 or NEAT1. NEAT1 silencing markedly reduced TNF-α, IL-4, and IL-13 levels, while elevated IL-10 expression, suppressed cell proliferation, and promoted cell apoptosis. However, NEAT1 overexpression elicited the opposite effects on cell proliferation and inflammation cytokines secretion. What is more, NEAT1 negatively regulated miR-200a/b expression, and MMP16 was a target gene of miR-200a/b. miR-200a/b overexpression suppressed inflammation, cell proliferation, and enhanced cell apoptosis through regulation of MMP16. Moreover, MMP-16 overexpression or miR-200a/b inhibition abolished the regulatory effect of sh-NEAT1 on cell inflammation and apoptosis in BEAS-2B cells. NEAT1 acted as the role of sponge for miR-200a/b to regulate MMP-16 expression, thereby promoting asthma progression, suggesting that NEAT1 might have great potential as therapeutic target for asthma.

Bacillus subtilis LCBS1 supplementation and replacement of fish meal with fermented soybean meal in bullfrog (Lithobates catesbeianus) diets: Effects on growth performance, feed digestibility and gut health

Two consecutive feeding trials were conducted to evaluate the effects of Bacillus subtilis LCBS1 supplementation and substituting fish meal (FM) with fermented soybean meal (FSM) in terms of growth performance, feed digestibility, intestinal morphology, and microbial composition of bullfrogs. In trial 1, bullfrogs received either a soybean meal (SM)-based basal diet (control check, CK group) or probiotic diet (basal diet was supplemented with 1 × 107 CFU/g of B. subtilis LCBS1, BS group). Each diet was fed to three replicates of bullfrogs (29.61 ± 0.07 g) for 58 days. Evaluation of B. subtilis LCBS1 FSM as a FM replacer in bullfrog diet was conducted in trial 2. A diet containing 20% FM formed the basal diet, and 60% and 100% of the FM was replaced with SM or FSM (FM, SM60, FSM60, SM100, and FSM100 diets). Each diet was fed to three replicates of bullfrogs (23.3 ± 0.3 g) for 63 days. As a result, in trial 1, BS treatments significantly increased weight gain (WG) and whole-body protein deposition. Significant improvements in villus height (VH), muscularis thickness (MT), and digestibilities of dry matter (DM), crude protein (CP) and phosphorus (P) were observed in the BS group compared with the control group. Probiotic supplementation resulted in elevated IL-4 and IL-10 expression and decreased TNF-α and IL-17 expression. A remarkable decrease in serum D-lactate concentration and diamine oxidase activity was detected in the BS group. Proteobacteria, Fusobacteria, and Firmicutes were the three most abundant bacterial phyla in each group, while Enterobacter and Bacillus were the most abundant genera in the CK and BS groups, respectively. In trial 2, compared with bullfrogs that received the SM diets, bullfrogs fed either the FM or FSM diets showed greater WG, feed efficiency, hind leg index, whole-body CP content, and digestibilities of DM, CP, calcium, P, and phytate P, and increased VH, villus thickness, and MT. Serum urea nitrogen and ammonia concentrations were significantly higher in the SM group than the FM or FSM group. Overall, dietary B. subtilis LCBS1 supplementation improved growth and digestion in bullfrogs and attenuated the intestinal structural damage and microflora dysbacteriosis caused by SM-based diets. Substituting 60–100% of FM with SM impaired the growth performance, digestion ability, and jejunal morphology of bullfrogs, while replacing equivalent FM with FSM effectively ameliorated these traits.