Inhibition Of Toll-like Receptor Research Articles

FRI0016 R835, A NOVEL IRAK1/4 DUAL INHIBITOR IN CLINICAL DEVELOPMENT, BLOCKS TOLL-LIKE RECEPTOR 4 (TLR4) SIGNALING IN HUMAN AND MOUSE

Background:Toll-Like Receptors (TLR) and Interleukin-1 Receptors (IL-1R) play a critical role in the innate immune response as microbial sensors, providing a bridge between the innate and adaptive immunity (1). Interleukin receptor associated kinases (IRAK) 1 and 4 are serine/threonine kinases that are essential for signaling downstream of most TLRs and IL-1Rs and the resulting production of pro-inflammatory cytokines (2). Suppression of TLR and IL-1R signaling through inhibition of IRAK1/4 kinases is a promising therapeutic approach for the treatment of inflammatory and autoimmune diseases.Objectives:The aim of the study was to characterize the effects of R835, a novel small molecule inhibitor of IRAK1/4, on TLR4 signaling.Methods:R835 was identified in cell-based assays measuring cytokine production induced by LPS (TLR4 ligand). Inhibition of IRAK1 and IRAK4 kinases by R835 was confirmed in biochemical assays and cell lysates. The ability of R835 to inhibit TLR4 signaling was further evaluated in human and mouse whole blood assays. R835 was tested in a mouse model of LPS-induced cytokine release. Mice were pre-treated orally with vehicle or R835 prior to challenge with LPS and serum cytokine levels were monitored over a 24-hour period.Results:We have identified R835, a selective small molecule inhibitor of IRAK1 and IRAK4. R835 blocked LPS/TLR4 signaling and the resulting production of proinflammatory cytokines in both human and mouse cells and whole blood. R835 suppressed serum cytokine elevation in mice challenged with LPS.Conclusion:Our study demonstrates that R835, through inhibition of IRAK1/4 kinase activity, blocks LPS-induced cytokine production in vitro and in vivo. In a recent phase 1 study, R835 substantially reduced the increase of serum cytokines after an intravenous LPS challenge in healthy volunteers. Importantly, this shows that the pharmacological inhibition of IRAK1/4 pathway by R835 in humans mirrors the results obtained in mice. To our knowledge, R835 is the first dual IRAK1/4 inhibitor to enter clinical development and demonstrate inhibition of TLR4-induced cytokines in both mice and humans. R835 is a promising clinical candidate that will allow the exploration of IRAK1/4 inhibition in the treatment of cytokine-driven rheumatic and autoimmune diseases.

Tetramethylpyrazine attenuates endotoxin-induced retinal inflammation by inhibiting microglial activation via the TLR4/NF-κB signalling pathway

Ocular inflammation is a common pathological condition of a series of retinal degenerative diseases. Tetramethylpyrazine (TMP), a Chinese herbal extraction, is widely used in the treatment of several ocular diseases in Eastern countries. However, the exact mechanisms correlating the vision protective effects of TMP have not been elucidated. Thus, this study aimed to investigate TMP’s molecular targets in anti-inflammatory activity in endotoxin lipopolysaccharide (LPS)-induced retinal inflammation both in vitro and in vivo. The primary cultured retinal microglial cells were pretreated with TMP and then activated by LPS. We found pretreatment with TMP significantly inhibited LPS-induced upregulation of CD68, a marker of mononuclear microglia activation. The morphological changes induced by LPS were also inhibited by the TMP pretreatment. Moreover, Toll like receptor 4 (TLR4), phosphorylation of inhibitor of NF-κB alpha (p-IκB-α) and the translocation of nuclear factor kappa B p65 (NF-κB p65) were significantly downregulated in retinal microglial cells with TMP pretreatment, which indicated that TMP might suppress LPS-induced retinal microglial activation through TLR4/NF-κB signalling pathway. And these results were confirmed in vivo. Pretreatment with TMP inhibited microglial activation, migration and regeneration, especially in ganglion cell layer (GCL). In addition to the inhibition of TLR4, TMP significantly inhibited the translocation of NF-κB p-65 to the nucleus in vivo. The downstream genes of NF-κB, such as the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), were significantly downregulated by TMP pretreatment in the retina. Accordingly, the increased expression of cleaved caspase-3 and the decreased ratio of B-cell lymphoma-2 (Bcl-2) to Bcl-2 associated X Protein (Bax) were significantly attenuated by TMP. TUNEL assay also demonstrated that TMP exerted neuroprotective effects in the retina. Therefore, this study elucidated a novel mechanism that TMP inhibits retinal inflammation by inhibiting microglial activation via a TLR4/NF-κB signalling pathway.

Inhibition of TLR4 Induces M2 Microglial Polarization and Provides Neuroprotection via the NLRP3 Inflammasome in Alzheimer's Disease.

Accumulating evidence has indicated that activation of microglia and neuroinflammation reaction play a prominent role in Alzheimer’s disease (AD). Inhibition of toll-like receptor 4 (TLR4) has been shown to be associated with immune responses and brain damage, but its effects on AD remain unclear. This study mainly aimed to investigate the protective effect of TAK-242 (TLR4-specific inhibitor) on microglial polarization and neuroprotection in an AD mouse model and the underlying mechanisms. We found that APP/PS1 transgenic AD mice exhibited a dramatic increase in TLR4 levels concomitant with a significantly higher expression of inflammatory microglia compared to C57BL/6 wild-type mice. Furthermore, inhibition of TLR4 by TAK-242 administration significantly improved neurological function, decreased the level of Bax, and caused a significant reduction in the levels of M1-markers (iNOS and TNFα), while the expressions of M2-phenotype markers (Trem-2 and Arg-1) were increased both in vivo and in vitro. Furthermore, TAK-242 treatment enhanced BV2 microglial phagocytosis. Moreover, Aβ25–35 caused the upregulation of inflammatory cytokine production, MyD88, NF-kappaB-p65, and NLRP3, which could be ameliorated by NLRP3-siRNA or TAK-242. These findings indicated that TLR4 inhibition provided neuroprotection and promoted a microglial switch from the inflammatory M1 phenotype to the protective M2 phenotype in AD. The mechanism involved may be related to modulation of the MyD88/NF-kappaB/NLRP3 signaling pathway.

Dexmedetomidine‑mediated regulation of miR‑17‑3p in H9C2 cells after hypoxia/reoxygenation injury

Patients with heart disease often suffer from ischemia, which can be treated by reperfusion. However, this treatment can lead to the development of ischemia/reperfusion (I/R) injury, an inflammatory condition that can cause further heart damage. Dexmedetomidine (Dex), an α2-adrenoceptor agonist, and the microRNA (miR)-17-3p, have both been suggested to alleviate I/R injury and cardiac tissue inflammation. The aim of the present study was to investigate whether Dex and miR-17-3p could act together to prevent I/R injury. H9C2 cells, a myoblast cell line used as a model of rat cardiomyocytes, were cultured in a hypoxic environment for 3 h, and then reoxygenated for 3 h. This hypoxia/reoxygenation (H/R) was used to model I/R. Cell Counting kit-8 was used to determine cell viability and an annexin V-FITC/propidium iodide apoptosis kit used to analyze cell apoptosis. A dual luciferase reporter assay was used to determine the interaction between miR-17-3p and toll-like receptor 4 (TLR4). Western blotting and reverse transcription-quantitative PCR were used to determine protein levels and mRNA expression of TLR4 and galectin-3. A concentration of 0.1-10 µmol/l Dex attenuated H/R injury, which was accompanied by increased miR-17-3p levels. Additionally, the inhibition of miR-17-3p exacerbated H/R injury and reduced the effect of Dex on H/R injury. H/R led to an increased galectin-3 level compared with that in control cells, and Dex or miR-17-3p inhibitor did not markedly affect the level of galectin-3, indicating that Dex alleviated the effects of I/R injury through other pathways. Inhibition of miR-17-3p in Dex-induced H9C2 cells during H/R increased the expression of inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-6, IL-1β and phosphorylated NFκB subunit p65, while Dex reduced the H/R-induced expression of these inflammatory mediators. Inhibition of TLR4 also attenuated H/R injury. In summary, the findings of the present study indicated that Dex reduced H/R injury in H9C2 cell via the modulation of inflammatory signaling pathways, and these inflammatory factors could be regulated by miR-17-3p.

Switch Off "Parallel Circuit": Insight of New Strategy of Simultaneously Suppressing Canonical and Noncanonical Inflammation Activation in Endotoxemic Mice.

Sepsis is a life-threatening inflammatory disease with a high mortality rate and huge implicative costs. Lipopolysaccharide (LPS) from gram-negative bacteria activates toll-like receptor 4 (TLR4) and may trigger septic shock. However, potent TLR4 inhibitors TAK-242 and Eritoran have been terminated in phase III clinical trials because of inadequate efficacy. Inspired by the recently discovered intracellular, noncanonical LPS receptors, it is considered that TLR4-mediated canonical and caspase-mediated noncanonical inflammation can be seen as a "parallel circuit" to induce sepsis and endotoxemia. Logically, it is proposed that the dual inhibition of caspase-4/5/11 and TLR4 can be a potential novel strategy to develop new therapeutics for sepsis. To verify the strategy, two potential compounds are found: Luteolin and Diacerein with substantial antiinflammatory activity in vitro and in vivo. The results show that the survival rate of endotoxemic mice treated by these compounds is increased remarkably. LPS-induced organ damage is also prevented. Moreover, these compounds result in physical and mental recovery for endotoxemic mice. Notably, Luteolin exhibits better antiinflammatory activity than TAK-242 at comparable TLR4-inhibitory levels. These findings indicate that simultaneous inhibition of TLR4 and caspase-4/5/11 can be an anticipative strategy defeating sepsis and endotoxemia, which can be translated into significant medical and economic benefits.

Interaction of M2 macrophages and endometrial cells induces downregulation of GRIM-19 in endometria of adenomyosis

Research questionDoes the aggregation of M2 macrophages affect the expression of gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) in adenomyosis? DesignEndometrial tissues were collected from patients with (n = 15) and without (n = 15) adenomyosis. Tissues were analysed for GRIM-19 and toll-like receptor 4 (TLR4) expression by immunohistochemistry and western blotting. Apoptosis was analysed by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. Human endometrial stromal cells (HESC) were transfected with GRIM-19 small interfering RNA (SiRNA) to knockdown GRIM-19 expression. The HESC were co-cultured with M2 macrophages to detect the influence of M2 macrophages in HESC cells. Analyses included GRIM-19, caspase-3 and TLR4 expression by western blotting, and GRIM-19 and TLR4 by quantitative real-time polymerase chain reaction. Apoptosis was measured by flow cytometry and TUNEL assay. Cell proliferation (Cell Counting Kit-8 assay) and migration assays were carried out. ResultsThe expression of GRIM-19 was significantly lower in adenomyosis lesions compared with controls (P < 0.001). Deficiency of GRIM-19 induced by siRNA decreased apoptosis and increased proliferation and migration in HESC. A significant decrease in GRIM-19 expression occurred in HESC after co-culture with M2 macrophages (P = 0.018). After co-culture with M2 macrophage, apoptosis decreased and proliferation and cell invasion in HESC increased. Protein (P = 0.006) and mRNA (P = 0.013) expression of TLR4 in HESC also reduced after this co-culture. Up-regulation of GRIM-19 occurred in HESC treated with the activator TLR4 (P = 0.016). Up-regulation of GRIM-19 was significantly reversed in cells treated with the TLR4 inhibitor (P = 0.011). ConclusionM2 macrophages may be involved in regulating the expression of GRIM-19 partly through the TLR4 signalling axis in adenomyosis.

Longifolioside A inhibits TLR4-mediated inflammatory responses by blocking PKCδ activation in LPS-stimulated THP-1 macrophages

Longifolioside A is an iridoid glucoside compound isolated from Pseudolysimachion rotundum var. subintegrum, which has been used in traditional herbal medicines to treat respiratory inflammatory diseases. Logifolioside A is a potent antioxidant; however, its underlying pharmacological mechanisms of action in inflammatory diseases are unknown.Here, we investigated the inhibitory effects of longifolioside A in lipopolysaccharide (LPS)-stimulated toll-like receptor 4 (TLR4) signal transduction systems using human THP-1 macrophages and HEK293 cells stably expressing human TLR4 protein (293/HA-hTLR4).Longifolioside A significantly reduced the release of inflammatory cytokines such as interleukin (IL)-6, -8, and tumor necrosis factor (TNF)-α in LPS-stimulated THP-1 macrophages. Furthermore, longifolioside A inhibited the expression of inflammatory mediator genes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 that produce nitric oxide (NO) and prostaglandin E2 (PGE2), respectively. Longifolioside A suppressed the phosphorylation of PKCδ, IRAK4, IKKα/β, IκBα, and mitogen-activated protein (MAP) kinases (ERK 1/2 and JNK, but not p38), thereby inactivating the nuclear localization of NF-κB and AP-1, and thus decreasing the expression of inflammatory response genes.Notably, longifolioside A disrupted the interaction between human TLR4 and the TIR domain-containing adaptor protein (TIRAP), an early step during TLR4 activation, thereby reducing IL-8 secretion in 293/HA-hTLR4 cells. This inhibitory effect was comparable to that of TAK-242 (a TLR4 inhibitor, or resatorvid). Our results indicate that longifolioside A prevents inflammatory response by suppressing TLR4 activation required for NF-κB and AP-1 activation.

MiR-201-5p alleviates lipopolysaccharide-induced renal cell dysfunction by targeting NOTCH3.

Lipopolysaccharide (LPS)-induced inflammation and dysfunction in the kidney may be the major risk factors for subsequent acute kidney injury (AKI). Previous studies have reported that up-regulation of notch receptor 3 (NOTCH3) expression is accompanied with renal epithelium and podocyte damage. Herein, we aimed to investigate whether NOTCH3 was involved in lipopolysaccharide (LPS)-induced AKI and renal cell dysfunction. Septic mice were established using LPS (20 mg/kg) intraperitoneally. mRNA and protein expression in the kidney and renal cell was performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting, respectively. Cell counting kit-8 (CCK8) and flow cytometry were used to measure cell viability and apoptosis, respectively. Bioinformatics algorithm and Luciferase reporter gene assay were performed to validate whether NOTCH3 was a direct target of miR-201-5p. Up-regulation of NOTCH3 and down-regulation of miR-201-5p were observed in the kidney of LPS-induced septic mice. LPS-stimulated TCMK-1 and MPC5 cells led to an increase in NOTCH3 and a decrease in miR-201-5p expression levels. Bioinformatics algorithm and experimental measurements validated that NOTCH3 was a direct target of miR-201-5p. Overexpression of miR-201-5p protected against LPS-induced renal cell growth inhibition, apoptosis and inflammatory response via the suppression of toll-like receptor 4 (TLR4)/NOTCH3 signaling pathway. The novel role of miR-201-5p via the inhibition of LPS-activated TLR4/NOTCH3 might provide a potential therapeutic strategy for the treatment of LPS-induced AKI.

Ischemic postconditioning attenuates the inflammatory response in ischemia/reperfusion myocardium by upregulating miR‑499 and inhibiting TLR2 activation.

Toll-like receptor 2 (TLR2)-mediated myocardial inflammation serves an important role in promoting myocardial ischemic/reperfusion (I/R) injury. Previous studies have shown that miR-499 is critical for cardioprotection after ischemic postconditioning (IPostC). Therefore, the present study evaluated the protective effect of IPostC on the myocardium by inhibiting TLR2, and also assessed the involvement of microRNA (miR)-499. Rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. The IPostC was 3 cycles of 30 sec of reperfusion and 30 sec of re-occlusion prior to reperfusion. In total, 90 rats were randomly divided into six groups (n=15 per group): Sham; I/R; IPostC; miR-499 negative control adeno-associated virus (AAV) vectors + IPostC; miR-499 inhibitor AAV vectors + IPostC; and miR-499 mimic AAV vectors + IPostC. It was identified that IPostC significantly decreased the I/R-induced cardiomyocyte apoptotic index (29.4±2.03% in IPostC vs. 42.64±2.27% in I/R; P<0.05) and myocardial infarct size (48.53±2.49% in IPostC vs. 66.52±3.1% in I/R; P<0.05). Moreover, these beneficial effects were accompanied by increased miR-499 expression levels (as demonstrated by reverse transcription-quantitative PCR) in the myocardial tissue and decreased TLR2, protein kinase C (PKC), interleukin (IL)-1β and IL-6 expression levels (as demonstrated by western blotting and ELISA) in the myocardium and serum. The results indicated that IPostC + miR-499 mimics significantly inhibited inflammation and the PKC signaling pathway and enhanced the anti-inflammatory and anti-apoptotic effects of IPostC. However, IPostC + miR-499 inhibitors had the opposite effect. Therefore, it was speculated that IPostC may have a miR-499-dependent cardioprotective effect. The present results suggested that miR-499 may be involved in IPostC-mediated ischemic cardioprotection, which may occur via local and systemic TLR2 inhibition, subsequent inhibition of the PKC signaling pathway and a decrease in inflammatory cytokine release, including IL-1β and IL-6. Moreover, these effects will ultimately lead to a decrease in the myocardial apoptotic index and myocardial infarct size via the induction of the anti-apoptotic protein Bcl-2, and inhibition of the pro-apoptotic protein Bax in myocardium.

Exenatide ameliorates experimental non-alcoholic fatty liver in rats via suppression of toll-like receptor 4/NFκB signaling: Comparison to metformin

AimsNon-alcoholic fatty liver disease (NAFLD) is a common liver disease. This study aimed to evaluate the role of exenatide compared with metformin in halting the progression of fatty liver stimulated by a high-fat diet (HiFD) in rats. Main methodsThirty male Wistar rats were allocated into 6 groups, 5 rats per each group. Group I: maintained on normal diet (normal group) for fourteen weeks. The other five groups were kept on HiFD throughout the experiment, HiFD was administered beside pharmacological treatments/or vehicle. Group II: (NAFLD control group), group III: received metformin (60 mg/kg/day, P.O.), group IV-VI: received exenatide (10, 20, and 40 μg/kg/day, S.C.) respectively for 7 weeks. At the end of the therapeutic period, fasting blood glucose was determined, and body weight was registered. Rats were sacrificed, and blood samples were taken to measure serum insulin, lipids, and liver enzymes. The liver index and homeostasis model of insulin resistance (HOMA-IR) index were calculated. Further, livers were dissected for histopathological examination and Western blot analysis. Key findingsNAFLD control group showed hyperglycemia, hyperinsulinemia, increased liver enzymes, hypertriglyceridemia, elevated hepatic lipid peroxides, and inflammatory mediators (interlukin 6, nuclear factor-κB, tumor necrosis factor-α and Toll-like receptor4) in addition to hepatic fatty degeneration. In a dose-dependent manner, exenatide significantly improved most of the above mentioned markers in comparsion with NAFLD at P≤0.05. SignificanceThe current results suggest that exenatide is equivalent to metformin in controlling insulin resistance, body weight gain, improving liver function, suppressing inflammation, and attenuating NAFLD progression in male rats.

Ortho-vanillin nanoparticle-doped glucan microspheresexacerbate the anti-arthritic effects of methotrexate in adjuvant-inducedarthritisin rats.

Methotrexate (MTX) commonly used in rheumatoidarthritis (RA)has severe adverse effects. Ortho-vanillin, an inhibitor of Toll-like receptors (TLR), can prevent inflammation. Glucan is a cereal fiber recognized by dectin-1 or β-glucan receptors of phagocytic macrophages. The purpose of the current project was to study the effect of co-administration of MTX and vanillin by targeted delivery to macrophages using β-glucan microspheres to reduce inflammation of RA. MTX and vanillin nanoparticles in bovine serum albumin (BSA) or gelatin were doped in glucan particles (GPs) and characterized for their physical properties. Twenty-four hours after induction of RA in paw of rats, they received normal saline (1mg/kg, ip), MTX (2mg/kg/week, ip), β-glucan (1mg/kg/week, ip), GPs-MTX (2mg/kg/week, ip), GPs-vanillin (200mg/kg/day, po), and GPs-MTX (2mg/kg/week, ip) plus GPs-vanillin (200mg/kg/day, po). The last group received free MTX ip and vanillin po for 14days. Then, joint diameters, TNF-α and IL-6, were evaluated in rats. The particle size of the GPs was 5.3µm. MTX loading efficiency in glucan microspheres was 64.5% and vanillin 44.2%. The microspheres released 88.7% of MTX and 95.1% of vanillin over 24h. The results of in vivo studies showed a significant reduction in paw volume, TNF-α and IL-6 (p < 0.05) in animals treated with combination of MTX and vanillin-doped glucan microspheres compared to the mixture of the two drugs in free form or each drug alone. Co-administration of MTX and vanillin-doped GPs may be more effective than MTX alone in RA.

Urolithin A, a Product of the Microbiome, Attenuates the Palmitate‐TLR4 Inflammatory Pathway in Renal Tubule Cells

Obesity is a major risk factor for cardiovascular disease and chronic renal injury. Tubulointerstial inflammation may reflect increased exposure of saturated fatty acids (sFA) to the renal epithelium and activation of the toll‐like receptor 4 (TLR4). Obesity‐induced inflammation may also facilitate gut microbiome dysbiosis and potentially alter the processing of exogenous polyphenolic compounds to metabolites with enhanced protective effects. Indeed, Urolithin A, the primary product of the microbiome metabolism of ellagitanins, promotes gut barrier function and attenuates colonic inflammation. However, the role of Urolithin A to attenuate tubular injury and inflammation has not been assessed. We propose that Urolithin A has anti‐inflammatory actions in rat proximal tubule cells (PTCs) chronically exposed to the sFA palmitate.PTCs (NRK‐52e cells) were exposed to palmitate [200 μM] for a 24 hours in the presence or absence of Urolithin A [50 μM], the TLR4 inhibitor TAK242 [1 μM], the EGFR inhibitor AG1478 [1 μM] and cSRC kinase inhibitor Dasatinib [10 μM] as both EGFR and cSRC may be integral to TLR4 activation. Cytokine expression in the PTCs was quantified by separate ELISAs.Palmitate exposure stimulated the robust release of PAI‐1 6‐fold [4.3 ± 0.3 vs. 24.2 ± 0.4 ng/ml; P&lt;0.001, n=3], CCL2 150‐fold [0.04 ± 0.01 vs. 6.2 ± 0.4 ng/ml; P&lt;0.001, n=3] and CCL5 10‐fold [23.4 ± 0.1 vs. 255 ± 11 pg/ml; P&lt;0.001, n=3]. The TLR4 inhibitor abolished the increase in CCL2 and CCL5, and markedly attenuated PAI‐1 release [70%, P&lt;0.01]. The EGFR inhibitor partially inhibited all three cytokines [40–50%, P&lt;0.01] while the cSRC inhibitor abolished PAI‐1 and partially inhibited CCL5, but failed to inhibit CCL2 release. Urolithin A essentially abolished the increase in PAI‐1 and CCL5, and markedly inhibited CCL2 release to palmitate [70%, P&lt;0.01].We conclude that Urolithin A may be renoprotective in obesity‐induced injury by targeting activation of TLR4 within the renal epithelium to attenuate the cytokine release. Moreover, a deficit in the microbiome‐dependent generation of Urolithin A may potentially promote renal inflammation.Support or Funding InformationChronic Disease Research Fund, AHA Transformative Grant 18TPA34170522; NIH R01HL146818, the Farley Hudson Foundation, the Hypertension &amp; Vascular Research Center

The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

Objective:Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2’-Fucosyllactose (2’-FL) and 6’-Sialyllactose (6’-SL), can reduce NEC through inhibition of TLR4 signaling.Design:NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2’-FL, 6’-SL or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, human tissue explants, and via in silico modeling.Results:Supplementation of infant formula with either 2’-FL and/or 6’-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2’-FL and 6’-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2’-FL and 6’-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling.Conclusion:2’-FL and 6’-SL, but not lactose, prevent NEC in mice and piglet models, and attenuate NEC-inflammation in human ileum, in part through TLR4 inhibition.

Heat Shock Protein 22 Attenuates Doxorubicin-Induced Cardiotoxicity via Regulating Inflammation and Apoptosis.

BackgroundThe antitumor effect of doxorubicin (DOX) is limited by its acute and chronic toxicity to the heart, which causes heart injury. Heat shock protein 22 (Hsp22) is a protein proved to exert anti-apoptosis and anti-inflammatory effects in other diseases and physical conditions. In this study, we aim to explore whether Hsp22 could exert a protective role during cardiac injury in response to DOX.MethodsThe overexpression of Hsp22 was mediated via adenovirus vector to clarify the role of Hsp22 in the cardiac injury caused by DOX. DOX-induced acute heart injury mouse model was established by single intraperitoneal injection of DOX (15 mg/kg). Subsequently, cardiac staining and molecular biological analysis were performed to analyze the morphological and biochemical effects of Hsp22 on cardiac injury. H9c2 cells were used for validation in vitro.ResultsAn increase in the expression level of Hsp22 was observed in DOX-treated heart tissue. Furthermore, cardiac-specific overexpression of Hsp22 showed reduced cardiac dysfunction, decrease in inflammatory response, and reduction in cell apoptosis in injury heart and cardiomyocytes induced by DOX in vivo and in vitro. Moreover, the suppression of Toll-like receptor (TLR)4/NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) was associated with the protective effect of Hsp22. Finally, the protective effect of Hsp22 cardiac function was almost abolished by overexpression of NLRP3 in DOX-treated mice.ConclusionIn summary, Hsp22 overexpression in the heart could suppress cardiac injury in response to DOX treatment through blocking TLR4/NLRP3 activation. Hsp22 may become a new therapeutic method for treating cardiac injury induced by DOX in cancer patients.

Oral Administration of Alpha Linoleic Acid Rescues Aβ-Induced Glia-Mediated Neuroinflammation and Cognitive Dysfunction in C57BL/6N Mice.

In this work, we evaluated the effects of alpha linoleic acid (ALA), an omega-3 polyunsaturated fatty acid, on amyloid-beta-induced glial-cell-mediated neuroinflammation, amyloidogenesis, and cognitive dysfunction in mice. After an infusion of Aβ1–42 (Aβ1–42, 5 μL/5 min/mouse, intracerebroventricular injection (i.c.v), and respective treatments of ALA (60 mg/kg per oral for six weeks), neuroinflammation, apoptotic markers, and synaptic markers were evaluated by Western blot and immunofluorescence analyses. According to our findings, the infusion of Aβ1–42 activated Toll-like receptor 4 (TLR4), glial fibrillary acidic protein (GFAP), and ionized calcium adaptor molecule 1 (Iba-1) in the frontal cortices and hippocampi of the Aβ1–42-injected mice to a greater extent than the Aβ1–42 + ALA-cotreated mice. Similarly, there was an elevated expression of phospho-c-Jun-N-terminal kinase (p-JNK), phospho-nuclear factor-kB p65 (p-NF-kB p65 (Ser536)), and tissue necrosis factor (TNF) in the Aβ1–42 infused mouse brains; interestingly, these markers were significantly reduced in the Aβ + ALA-cotreated group. The elevated expression of pro-apoptotic markers was observed during apoptotic cell death in the Aβ1–42-treated mouse brains, whereas these markers were markedly reduced in the Aβ + ALA-cotreated group. Moreover, Aβ1–42 infusion significantly increased amyloidogenesis, as assessed by the enhanced expression of the amyloid precursor proteins (APP) beta-amyloid cleaving enzyme-1 (BACE-1) and amyloid-beta (Aβ1–42) in the mouse brains, whereas these proteins were markedly reduced in the Aβ + ALA-cotreated group. We also checked the effects of ALA against Aβ-triggered synaptic dysfunction and memory dysfunction, showing that ALA significantly improved memory and synaptic functions in Aβ-treated mouse brains. These results indicated that ALA could be an applicable intervention in neuroinflammation, apoptotic cell loss, amyloidogenesis, and memory dysfunction via the inhibition of TLR4 and its downstream targets in Aβ + ALA-cotreated mouse brains.

Intestinal Dysbiosis and the Developing Lung: The Role of Toll-Like Receptor 4 in the Gut-Lung Axis.

BackgroundIn extremely premature infants, postnatal growth restriction (PNGR) is common and increases the risk of developing bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH). Mechanisms by which poor nutrition impacts lung development are unknown, but alterations in the gut microbiota appear to play a role. In a rodent model, PNGR plus hyperoxia causes BPD and PH and increases intestinal Enterobacteriaceae, Gram-negative organisms that stimulate Toll-like receptor 4 (TLR4). We hypothesized that intestinal dysbiosis activates intestinal TLR4 triggering systemic inflammation which impacts lung development.MethodsRat pups were assigned to litters of 17 (PNGR) or 10 (normal growth) at birth and exposed to room air or 75% oxygen for 14 days. Half of the pups were treated with the TLR4 inhibitor TAK-242 from birth or beginning at day 3. After 14 days, pulmonary arterial pressure was evaluated by echocardiography and hearts were examined for right ventricular hypertrophy (RVH). Lungs and serum samples were analyzed by western blotting and immunohistochemistry.ResultsPostnatal growth restriction + hyperoxia increased pulmonary arterial pressure and RVH with trends toward increased plasma IL1β and decreased IκBα, the inhibitor of NFκB, in lung tissue. Treatment with the TLR4 inhibitor attenuated PH and inflammation.ConclusionPostnatal growth restriction induces an increase in intestinal Enterobacteriaceae leading to PH. Activation of the TLR4 pathway is a promising mechanism by which intestinal dysbiosis impacts the developing lung.

Manipulation of macrophage polarization by peptide-coated gold nanoparticles and its protective effects on acute lung injury

BackgroundMacrophage polarization and reprogramming in the lung play a critical role in the initiation, development and progression of acute lung injury (ALI). Regulating the activation and differentiation of pulmonary macrophages may provide a potential therapeutic strategy to treat ALI. We previously developed a novel class of anti-inflammatory nanoparticles (P12) that can potently inhibit Toll-like receptor (TLR) signaling in macrophages. These bioactive nanodevices were made of gold nanoparticles (GNPs) coated with hexapeptides to not only ensure their physiological stability but also enable GNPs with TLR inhibitory activity.ResultsIn this study, using a lipopolysaccharide (LPS) induced ALI mouse model, we showed that P12 was able to alleviate lung inflammation and damage through reducing the infiltration of inflammatory cells and increasing the anti-inflammatory cytokine (IL-10) in the lung. These results prompted us to investigate possible macrophage polarization by P12. We first confirmed that P12 primarily targeted macrophages in the lung to exert anti-inflammatory activity. We then showed that P12 could drive the polarization of mouse bone marrow-derived macrophages (BMDMs) toward anti-inflammatory M2 phenotype. Interestingly, in the ALI mouse model, P12 was able to increase the alveolar M2 macrophages and reduce both the alveolar and interstitial M1 macrophages in the bronchoalveolar lavage fluid (BALF) and lung tissues.ConclusionThis study demonstrated that peptide-coated GNPs could induce M2 macrophage polarization in vitro and in vivo to effectively regulate lung inflammation, protect lung from injuries and promote inflammation resolution. The ability of regulating macrophage polarization together with TLR inhibition made such a bioactive nanodevice a new generation of potent therapeutics to treat ALI.

NEC-like intestinal injury is ameliorated by Lactobacillus rhamnosus GG in parallel with SIGIRR and A20 induction in neonatal mice.

Background:Exaggerated Toll-like receptor (TLR) signaling and intestinal dysbiosis are key contributors to necrotizing enterocolitis (NEC). Lactobacillus rhamnosus GG (LGG) decreases NEC in preterm infants, but underlying mechanisms of protection remain poorly understood. We hypothesized that LGG alleviates dysbiosis and upregulates TLR inhibitors to protect against TLR-mediated gut injury.Methods:Effects of LGG (low- and high-dose) on intestinal pro-inflammatory TLR signaling and injury in neonatal mice subjected to formula-feeding (FF) and NEC were determined. 16S sequencing of stool and expression of anti-TLR mediators Single Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) and A20 were analyzed.Results:FF induced mild intestinal injury with increased expression of interleukin-1 beta (IL-1β) and KC (mouse homolog of IL-8) compared to controls. LGG decreased IL-1β and KC in association with attenuated TLR signaling and increased SIGIRR and A20 expression in a dose-dependent manner. Low- and high-dose LGG had varying effects on gut microbiome despite both doses providing gut protection. Subsequent experiments of LGG on NEC revealed that pro-inflammatory TLR signaling and intestinal injury were also decreased, and SIGIRR and A20 expression increased, in a dose-dependent manner with LGG pre-treatment.Conclusion:LGG protects against intestinal TLR-mediated injury by upregulating TLR inhibitors without major changes in gut microbiome composition.

Clemastine Fumarate Protects Against Myocardial Ischemia Reperfusion Injury by Activating the TLR4/PI3K/Akt Signaling Pathway

Our pilot studies have shown that clemastine fumarate (CLE) can protect against myocardial ischemia-reperfusion injury (MIRI) through regulation of toll like receptor 4 (TLR4). However, the protective mechanism of CLE and related signaling pathways for MIRI remains unclear. The objective of this study is to determine the mechanism by which CLE relieves MIRI in cardiomyocytes and its relationship with the TLR4/PI3K/Akt signaling pathway. CCK8 analysis was used to test the optimal concentration of TLR4 inhibitor CLI-095 and TLR4 agonist lipopolysaccharide (LPS) on MIRI. The expression of inflammatory factors, oxidative stress response, cell damage, and intracellular calcium redistribution of cardiomyocytes were examined using the ELISA kits, Total Superoxide Dismutase Assay Kit with WST-8 and Lipid Peroxidation MDA Assay Kit, LDH Cytotoxicity Assay Kit, and laser scanning confocal microscope. The expression of TLR4/PI3K/Akt and cleaved caspase-3 were determined by Western blotting and immunofluorescent staining. Our results showed that MIRI aggravated the inflammatory response, oxidative stress, cellular damage of cardiomyocytes, and caused redistribution of intracellular calcium, upregulated the expression of TLR4 protein, cleaved caspase-3 protein, and down-regulated the expression of PI3K/Akt protein. After treatment with CLE, the inflammatory response, oxidative stress, and cellular damage of cardiomyocytes were alleviated, and intracellular calcium ion accumulation decreased. The expression of TLR4 protein, cleaved caspase-3 protein declined, but PI3K/Akt protein expression increased in cardiomyocytes treated with CLE. In addition, after treatment with the TLR4 inhibitor CLI-095, the results were similar to those of CLE treatment. The TLR4 agonist LPS aggravated the reactions caused by MIRI. The role of LPS was reversed after CLE treatment. These results suggested that CLE can attenuate MIRI by activating the TLR4/PI3K/Akt signaling pathway.

Up-regulation of miR-326 regulates pro-inflammatory cytokines targeting TLR-4 in buffalo granulosa cells

A genome-wide profiling of microRNA (miRNA) in endotoxin tolerant buffalo granulosa cells identified miR-326 amongst top-10 upregulated miRNAs. In this study, we have elucidated the role of miR-326 in granulosa cells in vitro. In-silico analysis revealed that miR-326 have binding site for 3′UTR of TLR-4 (Toll-like receptor 4). Transfection experiments showed that there was a significant inhibition of TLR-4 when miR-326 mimic was transfected to buffalo granulosa cells. Furthermore, when miR-326 transfected granulosa cells were exposed to LPS, followed by expression analysis of TLR-4 complex genes (TLR-4 and MyD88) and pro-inflammatory cytokines, we found a decreased expression of both TLR-4 complex and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β). We also found that the expression of anti-inflammatory (IL-10) gene was upregulated. To the best of our knowledge, this is the first study that showed the regulation of TLR-4 using miR-326. The present findings on regulation of TLR-4 are important and would help in understanding innate immunity regulation under different patho-physiology.