The MicroRNA network in sepsis: from biomarker discovery to novel targeted therapeutic strategies

Protecting developing and maturing spermatozoa and reproductive tissues from microbial damage is an emerging aspect of research in reproductive physiology. Bacterial, viral, and yeast infections of the testis and epididymis can hinder maturation and movement of spermatozoa, resulting in impaired fertility. Toll-like receptors (TLRs) are a broad family of innate immunity receptors that play critical roles in detecting and responding to invading pathogens. Objectives of this study were to determine if organs of the rat male reproductive tract express mRNAs for members of the TLR family, to characterize expression patterns for TLRs in different regions of the epididymis, and to determine if TLR adaptor and target proteins are present in the male reproductive tract. Messenger RNA for Tlr1-Tlr9 was abundantly expressed in testis, epididymis, and vas deferens, as determined by RT-PCR, while Tlr10 and Tlr11 were less abundantly expressed. Tlr mRNA expression showed no region-specific patterns in the epididymis. Immunoblot analysis revealed relatively equal levels of protein for TLRs 1, 2, 4, and 6 in testis, all regions of the epididymis and vas deferens, and lower levels of TLRs 3, 5, and 9-11. TLR7 was primarily detected in the testis. The TLR adapter proteins, myeloid differentiation primary response gene 88 and TLR adaptor molecule 1, as well as v-rel reticuloendotheliosis viral oncogene homolog and NFKBIA, were prominent in testis, epididymis, and vas deferens. The abundant expression of a majority of TLR family members together with expression of TLR adaptors and activation targets provides strong evidence that TLRs play important roles in innate immunity of the male reproductive tract.

Inflammatory signaling pathways in pancreatic β-cell: New insights into type 2 diabetes pathogenesis.

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

A bioinformatic approach to the identification of bacterial proteins interacting with Toll–interleukin 1 receptor–resistance (TIR) homology domains

The involvement of JAK/STAT signaling pathway in the treatment of Parkinson's disease

Activation of the Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway is known to play a key role in cardiogenesis and to afford cardioprotection against ischemia-reperfusion in adult. However, involvement of JAK2/STAT3 pathway and its interaction with other signaling pathways in developing heart transiently submitted to anoxia remains to be explored. Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30 min) and reoxygenation (80 min) with or without the antioxidant MPG, the JAK2/STAT3 inhibitor AG490 or the PhosphoInositide-3-Kinase (PI3K)/Akt inhibitor LY-294002. Time course of phosphorylation of STAT3α(tyrosine705) and Reperfusion Injury Salvage Kinase (RISK) proteins [PI3K, Akt, Glycogen Synthase Kinase 3beta (GSK3beta), Extracellular signal-Regulated Kinase 2 (ERK2)] was determined in homogenate and in enriched nuclear and cytoplasmic fractions of the ventricle. STAT3 DNA-binding was determined. The chrono-, dromo- and inotropic disturbances were also investigated by electrocardiogram and mechanical recordings. Phosphorylation of STAT3α(tyr705) was increased by reoxygenation, reduced (~50%) by MPG or AG490 but not affected by LY-294002. STAT3 and GSK3beta were detected both in nuclear and cytoplasmic fractions while PI3K, Akt and ERK2 were restricted to cytoplasm. Reoxygenation led to nuclear accumulation of STAT3 but unexpectedly without DNA-binding. AG490 decreased the reoxygenation-induced phosphorylation of Akt and ERK2 and phosphorylation/inhibition of GSK3beta in the nucleus, exclusively. Inhibition of JAK2/STAT3 delayed recovery of atrial rate, worsened variability of cardiac cycle length and prolonged arrhythmias as compared to control hearts. Thus, besides its nuclear translocation without transcriptional activity, oxyradicals-activated STAT3α can rapidly interact with RISK proteins present in nucleus and cytoplasm, without dual interaction, and reduce the anoxia-reoxygenation-induced arrhythmias in the embryonic heart.

BackgroundInhaled allergen challenges are often used to evaluate novel asthma treatments in early phase clinical trials. Current novel therapeutic targets in asthma include phosphoinositide 3-kinases (PI3K) delta and gamma, p38 mitogen-activated protein kinase (p38) and Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling pathways. The activation of these pathways following allergen exposure in atopic asthma patients it is not known.MethodsWe collected bronchial biopsies from 11 atopic asthma patients at baseline and after allergen challenge to investigate biomarkers of PI3K, p38 MAPK and JAK/STAT activation by immunohistochemistry. Cell counts and levels of eosinophil cationic protein and interleukin-5 were also assessed in sputum and bronchoalvelar lavage.ResultsBiopsies collected post-allergen had an increased percentage of epithelial cells expressing phospho-p38 (17.5 vs 25.6%, p = 0.04), and increased numbers of sub-epithelial cells expressing phospho-STAT5 (122.2 vs 540.6 cells/mm2, p = 0.01) and the PI3K marker phospho-ribosomal protein S6 (180.7 vs 777.3 cells/mm2,p = 0.005). Type 2 inflammation was increased in the airways post allergen, with elevated levels of eosinophils, interleukin-5 and eosinophil cationic protein.ConclusionsFuture clinical trials of novel kinase inhibitors could use the allergen challenge model in proof of concept studies, while employing these biomarkers to investigate pharmacological inhibition in the lungs.

BackgroundProbiotics have a beneficial effect on inflammatory responses and immune regulation, via Janus kinase/signal transduction and activator of transcription (JAK/STAT) and NF‐κB signaling pathways. To evaluate the precise effects of Lactobacillus spp. as a protective and therapeutic agent, we aimed to investigate the efficacy of Lactobacillus spp. in modulating JAK/STAT and nuclear factor kappa B (NF‐κB) inflammatory signaling pathways.MethodsA quantitative real‐time polymerase chain reaction (qPCR) assay was used to analyze the expression of JAK/STAT and inflammatory genes (TIR‑associated Protein [TIRAP], Interleukin 1 Receptor Associated Kinase[IRAK4], Nuclear factor‐kappa B Essential Modulator [NEMO], and receptor interacting protein [RIP]) followed by treatment of the HT‐29 cell line with sonicated pathogens before, after, and simultaneously with Lactobacillus spp. A cytokine assay was also used to evaluate interleukin (IL)‐6 and IL‐1β production after treatment with Lactobacillus spp.ResultsLactobacillus spp. downregulated JAK and TIRAP, IRAK4, NEMO, and RIP genes in the NF‐κB pathway compared to sonicate‐treated cells. The expression of STAT genes was different after treatment with probiotics. The production of IL‐6 and IL‐1β decreased after probiotic treatment.ConclusionsOur Lactobacillus spp. cocktail showed anti‐inflammatory effects on HT‐29 cells by modulating JAK/STAT and NF‐κB signaling pathways in all three treatment variants. Therefore, Lactobacillus spp. as a dietary supplement can both prevent and reduce inflammation‐related diseases such as inflammatory bowel disease.

A skewed ratio of pro-inflammatory to anti-inflammatory cytokines, elevated growth factor synthesis and T- and B-lymphocyte activation are 3 hallmarks of rheumatoid arthritis (RA) pathology. Interleukin-6 (IL-6), IL-7, IL-17, IL-12/IL-23 and growth factors, granulocyte macrophage-colony stimulating factor, IL-3, and erythropoietin activate the Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) pathway. Evidence showed that STAT protein phosphorylation (p-STAT) by activated JAKs is permissive for p-STAT to act as transcription factors by binding to STAT-responsive gene promoter sequences. This event is critical for perpetuating RA, in part, by up-regulating pro-inflammatory cytokine gene transcription. Activation of JAK/STAT by cytokines and growth factors can induce ‘cross-talk’ with other signaling pathways by which Stress-Activated Protein/Mitogen-Activated Protein Kinase (SAP/MAPK) and Phosphatidylinositide-3-Kinase (PI3K)-mediated signaling are also activated. JAK-specific small molecule inhibitors (SMIs) were developed to test whether JAK/STAT pathway blockade would regulate autoimmune-mediated inflammation. JAK-specific SMI blockade inhibited p-STAT induced by pro-inflammatory cytokines in vitro. Systemically administered JAK-specific SMI blockade also ameliorated biomarkers of inflammation in well-validated arthritis animal models. A few JAK-specific SMIs have made their way into RA clinical trials. In fact, the JAK3-specific SMI, CP-690,500 is the first JAK/STAT SMI to be assessed for clinical efficacy in a Phase III RA trial.

Cerebral ischemia is a cerebrovascular disease with symptoms caused by insufficient blood or oxygen supply to the brain. When blood supplied is restored after cerebral ischemia, secondary brain injury may occur, which is called cerebral ischemia-reperfusion injury (CIRI). In this process, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays an important role. It mediates neuroinflammation and participates in the regulation of physiological activities, such as cell proliferation, differentiation, and apoptosis. After CIRI, M1 microglia is activated and recruited by the damaged tissue. The inflammatory factors are produced by M1 microglia through the JAK/STAT pathway, eventually leading to cell apoptosis. Meanwhile, the JAK2/STAT3 signaling pathway and the expression of lipocalin-2 and caspase-3 could increase. In the pathway, phosphorylated JAK2 and phosphorylated STAT3 function of 2 ways. They not only promote the proliferation of neurons, but also affect the differentiation direction of neural stem cells by further acting on the Notch signaling pathway. Recently, traditional Chinese medicine (TCM) is a key player in CIRI, through JAK2, STAT3, STAT1 and their phosphorylation. Therefore, the review focuses on the JAK/STAT signaling pathway and its relationship with CIRI as well as the influence of the TCM on this pathway. It is aimed at providing the basis for future clinical research on the molecular mechanism of TCM in the treatment of CIRI.

Phosphatidylinositol (PI) 3-kinase is known to be activated by cytokine stimulation through different types of receptors to transduce intracellular responses. We have previously reported that leukemia inhibitory factor (LIF) induces the activation of Janus kinase signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein (MAP) kinase pathways through glycoprotein (gp) 130 in cardiac myocytes. However, whether PI 3-kinase is involved in regulation of gp130 signaling and the activation mechanisms by which it associates with other tyrosine-phosphorylated proteins remain unknown. We found that LIF induced the activation of PI 3-kinase in cardiac myocytes. Moreover, JAK1 binds to PI 3-kinase, and LIF stimulation increases the PI 3-kinase activity in JAK1 immunoprecipitates. Activation of MAP kinase and protein kinase B by LIF was attenuated by wortmannin. LIF-induced p70 S6 kinase activation, protein synthesis, and c-fos mRNA expression were inhibited by wortmannin and rapamycin. Both inhibitors failed to appreciably affect the phosphorylation of STAT3. In conclusion, PI 3-kinase is activated with LIF in cardiac myocytes, and JAK1 is found to associate with this enzyme. PI 3-kinase provides a crucial link between gp130, MAP kinase, protein kinase B, and p70 S6 kinase in cardiac myocytes.

Neuropathic pain is a very troublesome disease that seriously affects human life. Eriocalyxin B (EriB) has been revealed to attenuate various diseases through its anti-inflammatory effects, but its regulatory effects on neuropathic pain remains unclear. The paw withdrawal threshold and paw withdrawal thermal latency were detected through mechanical allodynia and thermal hyperalgesia tests. The spinal injury was assessed through hematoxylin and eosin staining. The cell apoptosis was measured through terminal deoxynucleotide transferase-mediated dUTP nick end-labeling assay. The protein expressions were examined through Western blot analysis. The mRNA expression was examined through reverse transcription-quantitative polymerase chain reaction. The ionized calcium-binding adaptor molecule 1 level in the spinal cord was evaluated through immunofluorescence assay. The levels of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 were measured through enzyme-linked-immunosorbent serologic assay. The chronic constriction injury (CCI) rat model was constructed for the study. Our results demonstrated that EriB relieved CCI-stimulated neuropathic pain and nerve damage. In addition, the enhanced neural apoptosis mediated by CCI induction was reduced after EriB treatment. In addition, EriB inhibited CCI-induced microglia activity and inflammation. At last, the Janus kinase 2–signal transducer and activator of transcription 3 (JAK2/STAT3) and nuclear factor kappa B (NF-κB) pathways were activated in CCI rat model, which were attenuated following EriB treatment. Importantly, EriB (10 mg/kg) had a strong effect that was similar to the positive control (1-μg/kg dexmedetomidine), suggesting that EriB may be an effective drug for neuropathic pain. This study demonstrated that EriB inhibited inflammation caused by CCI-induced microglia activation to relieve neuropathic pain through inhibition of JAK2/STAT3 and NF-κB pathways. This study may highlight the regulatory functions of EriB in the treatment of neuropathic pain.

Objective To evaluate the effects of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) signaling pathway on the brain injury induced by myocardial ischemia-reperfusion (I/R) in diabetic rats. Methods Pathogen-free male Sprague-Dawley rats, weighing 200-220 g, were used in this study.Diabetes mellitus was induced by intraperitoneal 1% streptozotocin 60 mg/kg and confirmed by blood glucose level ≥16.7 mmol/L 3 days later.Twenty-four rats with diabetes mellitus were randomly allocated into 3 groups (n=8 each) using a random number table: sham operation group (group S), I/R group, and myocardial I/R + AG490 (JAK inhibitor) group (group IA). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min, followed by 120 min of reperfusion in the rats anesthetized with pentobarbital sodium.AG490 3 mg/kg was injected intravenously at 20 min before reperfusion in group IA.The rats were sacrificed at 120 min of reperfusion, and the brains were removed for determination of caspase-3 and nuclear factor kappa B (NF-κB) activities (using colorimetric method), cell apoptosis (by TUNEL), and expression of interleukin-1 (IL-1), IL-6, IL-8, Bax, Bcl-2, cytochrome C (Cyt c), phosphorylated JAK2 (p-JAK2), and phosphorylated STAT3 (p-STAT3) (by Western blot). Apoptosis index was calculated. Results Compared with group S, the expression of Bax, Cyt c, IL-1, IL-6, IL-8, p-JAK2 and p-STAT3 was significantly up-regulated, the expression of Bcl-2 was down-regulated, and NF-κB and caspase-3 activities and apoptosis index were increased in I/R and IA groups (P<0.05). Compared with group I/R, the expression of Bax, Cyt c, IL-1, IL-6, IL-8, p-JAK2 and p-STAT3 was significantly down-regulated, the expression of Bcl-2 was up-regulated, and NF-κB and caspase-3 activities and apoptosis index were decreased in group IA (P<0.05). Conclusion Inflammatory responses mediated by JAK2/STAT3 signaling pathway are involved in the brain injury induced by myocardial I/R in diabetic rats. Key words: Janus kinase 2; STAT3 transcription factor; Diabetes mellitus; Myocardial reperfusion injury; Brain injuries

We previously reported that endothelial‐derived microparticles (EMP) could induce acute lung injury (ALI) and the mechanism is still not fully understood. Toll‐like receptor 4(TLR4) cascade has been considered to be involved in innate immune inflammation. As its downstream, janus kinases/signal transducers and activators of transcription (JAK/STAT), acts as a key signal pathway for immune responses. However, it remains unclear whether systemic circulating EMP could trigger TLR4‐pomoted JAK/STAT signaling contributing to ALI. EMP were isolated from human umbilical vein endothelial cells (HUVECs) stimulated with plasminogen activated inhibitor‐1. HUVECs were treated with/without different concentration of EMP. EMP induced inflammatory cytokines in cultured HUVECs. EMP could activated TLR4 and the downstream JAK3/STAT3 pathway. Silencing TLR4 inhibited the EMP‐induced JAK3/STAT3 pathway. EMP‐induced inflammatory response in plasma and bronchoalveolar lavage fluid, inflammatory cells infiltration. edema and JAK3/STAT3 pathway in the lung tissue, and ALI in C57BL6 mice. However, These effect induced by EMP were depressed in TLR4−/− mice. Our data demonstrated that EMP induce ALI by activating TLR4/JAK3/STAT3 pathway to promote inflammatory response. Our findings present the novel mechanisms by which EMP induce ALI.Support or Funding InformationThis research was financially supported by the National Natural Science Foundation of China (Grants 81670392, 81600382, 81770241, 81830013, 81970363 and Distinguished Young Scholar Grant 81325001), 973 project (2009CB522104) and International Cooperation project (2015DFA31070) from the Ministry of Science and Technology of China, Guangdong Natural Science Fund Committee (Grant 2015A030312009), the Changjiang Scholars Program from the Ministry of Education of China, the Sun Yat‐sen University Clinical Research 5010 Program.

The bidirectional role of the JAK2/STAT3 signaling pathway and related mechanisms in cerebral ischemia-reperfusion injury