Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides

Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The K_D value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.     

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis,and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.     

Fenofibrate Regulates Visceral Obesity and Nonalcoholic Steatohepatitis in Obese Female Ovariectomized C57BL/6J Mice

Fibrates, including fenofibrate, are a class of hypolipidemic drugs that activate peroxisome proliferator-activated receptor α (PPARα), which in-turn regulates the expression of lipid and lipoprotein metabolism genes. We investigated whether fenofibrate can reduce visceral obesity and nonalcoholic fatty liver disease via adipose tissue PPARα activation in female ovariectomized (OVX) C57BL/6J mice fed a high-fat diet (HFD), a mouse model of obese postmenopausal women. Fenofibrate reduced body weight gain (−38%, p < 0.05), visceral adipose tissue mass (−46%, p < 0.05), and visceral adipocyte size (−20%, p < 0.05) in HFD-fed obese OVX mice. In addition, plasma levels of alanine aminotransferase and aspartate aminotransferase, as well as free fatty acids, triglycerides, and total cholesterol, were decreased. Fenofibrate also inhibited hepatic lipid accumulation (−69%, p < 0.05) and infiltration of macrophages (−72%, p < 0.05), while concomitantly upregulating the expression of fatty acid β-oxidation genes targeted by PPARα and decreasing macrophage infiltration and mRNA expression of inflammatory factors in visceral adipose tissue. These results suggest that fenofibrate inhibits visceral obesity, as well as hepatic steatosis and inflammation, in part through visceral adipose tissue PPARα activation in obese female OVX mice.     

Resolvin D1 Decreases Severity of Streptozotocin-Induced Type 1 Diabetes Mellitus by Enhancing BDNF Levels,Reducing Oxidative Stress,and Suppressing Inflammation

Type 1 diabetes mellitus is an autoimmune disease characterized by increased production of pro-inflammatory cytokines secreted by infiltrating macrophages and T cells that destroy pancreatic β cells in a free radical-dependent manner that causes decrease or absence of insulin secretion and consequent hyperglycemia. Hence, suppression of pro-inflammatory cytokines and oxidative stress may ameliorate or decrease the severity of diabetes mellitus. To investigate the effect and mechanism(s) of action of RVD1, an anti-inflammatory metabolite derived from docosahexaenoic acid (DHA), on STZ-induced type 1 DM in male Wistar rats, type 1 diabetes was induced by single intraperitoneal (i.p) streptozotocin (STZ-65 mg/kg) injection. RVD1 (60 ng/mL, given intraperitoneally) was administered from day 1 along with STZ for five consecutive days. Plasma glucose, IL-6, TNF-α, BDNF (brain-derived neurotrophic factor that has anti-diabetic actions), LXA4 (lipoxin A4), and RVD1 levels and BDNF concentrations in the pancreas, liver, and brain tissues were measured. Apoptotic (Bcl2/Bax), inflammatory (COX-1/COX-2/Nf-κb/iNOS/PPAR-γ) genes and downstream insulin signaling proteins (Gsk-3β/Foxo1) were measured in the pancreatic tissue along with concentrations of various antioxidants and lipid peroxides. RVD1 decreased severity of STZ-induced type 1 DM by restoring altered plasma levels of TNF-α, IL-6, and BDNF (p < 0.001); expression of pancreatic COX-1/COX-2/PPAR-γ genes and downstream insulin signaling proteins (Gsk-3β/Foxo1) and the concentrations of antioxidants and lipid peroxides to near normal. RVD1 treatment restored expression of Bcl2/Pdx genes, plasma LXA4 (p < 0.001) and RVD1 levels and increased brain, pancreatic, intestine, and liver BDNF levels to near normal. The results of the present study suggest that RVD1 can prevent STZ-induced type 1 diabetes by its anti-apoptotic, anti-inflammatory, and antioxidant actions and by activating the Pdx gene that is needed for pancreatic β cell proliferation.     

Suppression of Hypoxia-Inducible Factor 1α by Low-Molecular-Weight Heparin Mitigates Ventilation-Induced Diaphragm Dysfunction in a Murine Endotoxemia Model

Mechanical ventilation (MV) is required to maintain life for patients with sepsis-related acute lung injury but can cause diaphragmatic myotrauma with muscle damage and weakness, known as ventilator-induced diaphragm dysfunction (VIDD). Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in inducing inflammation and apoptosis. Low-molecular-weight heparin (LMWH) was proven to have anti-inflammatory properties. However, HIF-1α and LMWH affect sepsis-related diaphragm injury has not been investigated. We hypothesized that LMWH would reduce endotoxin-augmented VIDD through HIF-1α. C57BL/6 mice, either wild-type or HIF-1α–deficient, were exposed to MV with or without endotoxemia for 8 h. Enoxaparin (4 mg/kg) was administered subcutaneously 30 min before MV. MV with endotoxemia aggravated VIDD, as demonstrated by increased interleukin-6 and macrophage inflammatory protein-2 levels, oxidative loads, and the expression of HIF-1α, calpain, caspase-3, atrogin-1, muscle ring finger-1, and microtubule-associated protein light chain 3-II. Disorganized myofibrils, disrupted mitochondria, increased numbers of autophagic and apoptotic mediators, substantial apoptosis of diaphragm muscle fibers, and decreased diaphragm function were also observed (p < 0.05). Endotoxin-exacerbated VIDD and myonuclear apoptosis were attenuated by pharmacologic inhibition by LMWH and in HIF-1α–deficient mice (p < 0.05). Our data indicate that enoxaparin reduces endotoxin-augmented MV-induced diaphragmatic injury, partially through HIF-1α pathway inhibition.     

Different Role of Tumor Necrosis Factor-α Polymorphism in Non-Hodgkin Lymphomas among Caucasian and Asian Populations: A Meta-Analysis

Tumor necrosis factor-α (TNF-α) is an immunoregulatory cytokine involved in B- and T-cell function, and also plays an important role in inflammation and cancer. TNF-α-308G>A has been associated with constitutively elevated TNF-α expression. Several studies have reported the association between the TNF-α-308G>A polymorphism and non-Hodgkin lymphomas (NHL) risk, however, results are still inconsistent. To solve these conflicts, we conducted the first meta-analysis to assess the effect of TNF-α-308G>A polymorphism on the risk of NHL and various subtypes (additive model) including 10,619 cases and 12,977 controls in Caucasian and Asian populations. Our meta-analysis indicated that TNF-α-308G>A polymorphism is not associated with NHL risk when pooling all studies together (OR = 1.06, 95% CI: 0.92–1.23, p = 0.413). In stratified analyses, we found TNF-α-308A allele was significantly associated with higher risk of NHL, B-cell lymphomas (BCL), T-cell lymphomas (TCL) and diffuse large B-cell lymphomas (DLBCL) in Caucasians (OR = 1.22, 95% CI: 1.06–1.40, p = 0.007; OR = 1.18, 95% CI: 1.03–1.34, p = 0.014; OR = 1.20, 95% CI: 1.01–1.42, p = 0.040; OR = 1.21, 95% CI: 1.11–1.32, p < 0.001, respectively). Interestingly, it was associated with decreased risk of NHL, BCL and DLBCL in Asians (OR = 0.75, 95% CI: 0.66–0.86, p < 0.001; OR = 0.70, 95% CI: 0.52–0.94, p = 0.018; OR = 0.70, 95% CI: 0.57–0.86, p = 0.001). These findings also suggest TNF-α might play a distinct role in pathogenesis of NHL in different populations.     

Characterization of a Cutibacterium acnes Camp Factor 1-Related Peptide as a New TLR-2 Modulator in In Vitro and Ex Vivo Models of Inflammation

Cutibacterium acnes (C. acnes) has been implicated in inflammatory acne where highly mutated Christie–Atkins–Munch–Petersen factor (CAMP)1 displays strong toll like receptor (TLR)-2 binding activity. Using specific antibodies, we showed that CAMP1 production was independent of C. acnes phylotype and involved in the induction of inflammation. We confirmed that TLR-2 bound both mutated and non-mutated recombinant CAMP1, and peptide array analysis showed that seven peptides (A14, A15, B1, B2, B3, C1 and C3) were involved in TLR-2 binding, located on the same side of the three-dimensional structure of CAMP1. Both mutated and non-mutated recombinant CAMP1 proteins induced the production of C-X-C motif chemokine ligand interleukin (CXCL)8/(IL)-8 in vitro in keratinocytes and that of granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, IL-1β and IL-10 in ex vivo human skin explants. Only A14, B1 and B2 inhibited the production of CXCL8/IL-8 by keratinocytes and that of (GM-CSF), TNF-α, IL-1β and IL-10 in human skin explants stimulated with rCAMP1 and C. acnes. Following pretreatment with B2, RNA sequencing on skin explants identified the 10 genes displaying the strongest differential expression as IL6, TNF, CXCL1, CXCL2, CXCL3, CXCL8, IL-1β, chemokine ligand (CCL)2, CCL4 and colony stimulating factor (CSF)2. We, thus, identified a new CAMP1-derived peptide as a TLR-2 modulator likely to be a good candidate for clinical evaluation.     

Protein Structures among Bio-Ethanol Co-Products and Its Relationships with Ruminal and Intestinal Availability of Protein in Dairy Cattle

The objectives of this study were to reveal molecular structures of protein among different types of the dried distillers grains with solubles (100% wheat DDGS (WDDGS); DDGS blend1 (BDDGS1, corn to wheat ratio 30:70%); DDGS blend2 (BDDGS2, corn to wheat ratio 50:50 percent)) and different batches within DDGS type using diffuse reflectance infrared Fourier transform spectroscopy (DRIFT). Compared with BDDGS1 and BDDGS2, wheat DDGS had higher (p < 0.05) peak area intensities of protein amide I and II and amide I to II intensity ratio. Increasing the corn to wheat ratio form 30:70 to 50:50 in the blend DDGS did not affect amide I and II area intensities and their ratio. Amide I to II peak intensity ratio differed (p < 0.05) among the different batches within WDDGS and BDDGS1. Compared with both blend DDGS types, WDDGS had higher α-helix and β-sheet ratio (p < 0.05), while α-helix to β-sheet ratio was similar among the three DDGS types. The α-helix to β-sheet ratio differed significantly among batches within WDDGS. Principal component analysis (PCA) revealed that protein molecular structures in WDDGS differed from those of BDDGS1 and between different batches within BDDGS1 and BDDGS2. The α-helix to β-sheet ratios of protein in all DDGS types had an influence on availability of protein at the ruminal level as well as at the intestinal level. The α-helix to β-sheet ratio was positively correlated to rumen undegraded protein (r = 0.41, p < 0.05) and unavailable protein (PC; r = 0.59, p < 0.05).     

Anti-Inflammatory Mechanism of Neural Stem Cell Transplantation in Spinal Cord Injury

Neural stem cell (NSC) transplantation has been proposed to promote functional recovery after spinal cord injury. However, a detailed understanding of the mechanisms of how NSCs exert their therapeutic plasticity is lacking. We transplanted mouse NSCs into the injured spinal cord seven days after SCI, and the Basso Mouse Scale (BMS) score was performed to assess locomotor function. The anti-inflammatory effects of NSC transplantation was analyzed by immunofluorescence staining of neutrophil and macrophages and the detection of mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-12 (IL-12). Furthermore, bone marrow-derived macrophages (BMDMs) were co-cultured with NSCs and followed by analyzing the mRNA levels of inducible nitric oxide synthase (iNOS), TNF-α, IL-1β, IL-6 and IL-10 with quantitative real-time PCR. The production of TNF-α and IL-1β by BMDMs was examined using the enzyme-linked immunosorbent assay (ELISA). Transplanted NSCs had significantly increased BMS scores (p < 0.05). Histological results showed that the grafted NSCs migrated from the injection site toward the injured area. NSCs transplantation significantly reduced the number of neutrophils and iNOS+/Mac-2+ cells at the epicenter of the injured area (p < 0.05). Meanwhile, mRNA levels of TNF-α, IL-1β, IL-6 and IL-12 in the NSCs transplantation group were significantly decreased compared to the control group. Furthermore, NSCs inhibited the iNOS expression of BMDMs and the release of inflammatory factors by macrophages in vitro (p < 0.05). These results suggest that NSC transplantation could modulate SCI-induced inflammatory responses and enhance neurological function after SCI via reducing M1 macrophage activation and infiltrating neutrophils. Thus, this study provides a new insight into the mechanisms responsible for the anti-inflammatory effect of NSC transplantation after SCI.     

Epithelial Regeneration Ability of Crohn’s Disease Assessed Using Patient-Derived Intestinal Organoids

Little is known about the ability for epithelial regeneration and wound healing in patients with inflammatory bowel diseases. We evaluated the epithelial proliferation and wound healing ability of patients with Crohn’s disease (CD) using patient-derived intestinal organoids. Human intestinal organoids were constructed in a three-dimensional intestinal crypt culture of enteroscopic biopsy samples from controls and CD patients. The organoid-forming efficiency of ileal crypts derived from CD patients was reduced compared with those from control subjects (p < 0.001). Long-term cultured organoids (≥6 passages) derived from controls and CD patients showed an indistinguishable microscopic appearance and culturing behavior. Under TNFα-enriched conditions (30 ng/mL), the organoid reconstitution rate and cell viability of CD patient-derived organoids were significantly lower than those of the control organoids (p < 0.05 for each). The number of EdU+ cells was significantly lower in TNFα-treated organoids derived from CD patients than in TNFα-treated control organoids (p < 0.05). In a wound healing assay, the unhealed area in TNFα-treated CD patient-derived organoids was significantly larger than that of TNFα-treated control organoids (p < 0.001). The wound healing ability of CD patient-derived organoids is reduced in TNFα-enriched conditions, due to reduced cell proliferation. Epithelial regeneration ability may be impaired in patients with CD.     

Specific Cerebrospinal Fluid SerpinA1 Isoform Pattern in Alzheimer’s Disease

SerpinA1 (α1-antitrypsin) is a soluble glycoprotein, the cerebrospinal fluid (CSF) isoforms of which showed disease-specific changes in neurodegenerative disorders that are still unexplored in Alz-heimer’s disease (AD). By means of capillary isoelectric focusing immunoassay, we investigated six serpinA1 isoforms in CSF samples of controls (n = 29), AD-MCI (n = 29), AD-dem (n = 26) and Lewy body disease (LBD, n = 59) patients and correlated the findings with CSF AD core biomarkers (Aβ42/40 ratio, p-tau, t-tau). Four CSF serpinA1 isoforms were differently expressed in AD patients compared to controls and LBD patients, especially isoforms 2 and 4. AD-specific changes were found since the MCI stage and significantly correlated with decreased Aβ42/40 (p < 0.05) and in-creased p-tau and t-tau levels in CSF (p < 0.001). Analysis of serpinA1 isoform provided good di-agnostic accuracy in discriminating AD patients versus controls (AUC = 0.80) and versus LBD patients (AUC = 0.92), with best results in patients in the dementia stage (AUC = 0.97). SerpinA1 isoform expression is altered in AD patients, suggesting a common, albeit disease-specific, in-volvement of serpinA1 in most neurodegenerative disorders.     

Role of Pannexin 1 ATP-Permeable Channels in the Regulation of Signaling Pathways during Skeletal Muscle Unloading

Skeletal muscle unloading results in atrophy. We hypothesized that pannexin 1 ATP-permeable channel (PANX1) is involved in the response of muscle to unloading. We tested this hypothesis by blocking PANX1, which regulates efflux of ATP from the cytoplasm. Rats were divided into six groups (eight rats each): non-treated control for 1 and 3 days of the experiments (1C and 3C, respectively), 1 and 3 days of hindlimb suspension (HS) with placebo (1H and 3H, respectively), and 1 and 3 days of HS with PANX1 inhibitor probenecid (PRB; 1HP and 3HP, respectively). When compared with 3C group there was a significant increase in ATP in soleus muscle of 3H and 3HP groups (32 and 51%, respectively, p < 0.05). When compared with 3H group, 3HP group had: (1) lower mRNA expression of E3 ligases MuRF1 and MAFbx (by 50 and 38% respectively, p < 0.05) and MYOG (by 34%, p < 0.05); (2) higher phosphorylation of p70S6k and p90RSK (by 51 and 35% respectively, p < 0.05); (3) lower levels of phosphorylated eEF2 (by 157%, p < 0.05); (4) higher level of phosphorylated GSK3β (by 189%, p < 0.05). In conclusion, PANX1 ATP-permeable channels are involved in the regulation of muscle atrophic processes by modulating expression of E3 ligases, and protein translation and elongation processes during unloading.     

Increased LRG1 Levels in Overweight and Obese Adolescents and Its Association with Obesity Markers,Including Leptin,Chemerin, and High Sensitivity C-Reactive Protein

Leucine-rich α-2 glycoprotein1 (LRG1) is a member of the leucine-rich repeat (LRR) family that is implicated in multiple diseases, including cancer, aging, and heart failure, as well as diabetes and obesity. LRG1 plays a key role in diet-induced hepatosteatosis and insulin resistance by mediating the crosstalk between adipocytes and hepatocytes. LRG1 also promotes hepatosteatosis by upregulating de novo lipogenesis in the liver and suppressing fatty acid β-oxidation. In this study, we investigated the association of LRG1 with obesity markers, including leptin and other adipokines in adolescents (11–14 years; n = 425). BMI-for-age classification based on WHO growth charts was used to define obesity. Plasma LRG1 was measured by ELISA, while other markers were measured by multiplexing assay. Median (IQR) of LRG1 levels was higher in obese (30 (25, 38) µg/mL) and overweight (30 (24, 39) µg/mL) adolescents, compared to normal-weight participants (27 (22, 35) µg/mL). The highest tertile of LRG1 had an OR [95% CI] of 2.55 [1.44, 4.53] for obesity. LRG1 was positively correlated to plasma levels of high sensitivity c-reactive protein (HsCRP) (ρ = 0.2), leptin (ρ = 0.2), and chemerin (ρ = 0.24) with p < 0.001. Additionally, it was positively associated with plasma level of IL6 (ρ = 0.17) and IL10 (ρ = 0.14) but not TNF-α. In conclusion, LRG1 levels are increased in obese adolescents and are associated with increased levels of adipogenic markers. These results suggest the usefulness of LRG1 as an early biomarker for obesity and its related pathologies in adolescents.     

Wnt3a Promotes the Vasculogenic Mimicry Formation of Colon Cancer via Wnt/β-Catenin Signaling

Our previous study provided evidence that non-canonical Wnt signaling is involved in regulating vasculogenic mimicry (VM) formation. However, the functions of canonical Wnt signaling in VM formation have not yet been explored. In this study, we found the presence of VM was related to colon cancer histological differentiation (p < 0.001), the clinical stage (p < 0.001), and presence of metastasis and recurrence (p < 0.001). VM-positive colon cancer samples showed increased Wnt3a expression (p < 0.001) and β-catenin nuclear expression (p < 0.001) compared with the VM-negative samples. In vitro, over-regulated Wnt3a expression in HT29 colon cancer cells promoted the capacity to form tube-like structures in the three-dimensional (3-D) culture together with increased expression of endothelial phenotype-associated proteins such as VEGFR2 and VE-cadherin. The mouse xenograft model showed that Wnt3a-overexpressing cells grew into larger tumor masses and formed more VM than the control cells. In addition, the Wnt/β-catenin signaling antagonist Dickkopf-1(Dkk1) can reverse the capacity to form tube-like structures and can decrease the expressions of VEGFR2 and VE-cadherin in Wnt3a-overexpressing cells. Taken together, our results suggest that Wnt/β-catenin signaling is involved in VM formation in colon cancer and might contribute to the development of more accurate treatment modalities aimed at VM.     

β-Conglycinin Reduces the Tight Junction Occludin and ZO-1 Expression in IPEC-J2

Soybean allergy presents a health threat to humans and animals. The mechanism by which food/feed allergen β-conglycinin injures the intestinal barrier has not been well understood. In this study, the changes of epithelial permeability, integrity, metabolic activity, the tight junction (TJ) distribution and expression induced by β-conglycinin were evaluated using IPEC-J2 model. The results showed a significant decrease of trans-epithelial electrical resistance (TEER) (p < 0.001) and metabolic activity (p < 0.001) and a remarkable increase of alkaline phosphatase (AP) activity (p < 0.001) in a dose-dependent manner. The expression levels of tight junction occludin and ZO-1 were decreased (p < 0.05). The reduced fluorescence of targets and change of cellular morphology were recorded. The tight junction occludin and ZO-1 mRNA expression linearly declined with increasing β-conglycinin (p < 0.001).