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Network coupled cells, such as astrocytes, regulate their cellular homeostasis via Ca2+ signals spread between the cells through gap junctions. Intracellular Ca2+ release is controlled by different signaling pathways that can be stimulated by ATP, glutamate and serotonin (5-HT). Based on our findings, all these pathways are influenced by inflammatory agents and must be restored to fully recover the Ca2+ signaling network. An ultralow concentration of the local anesthetic agent bupivacaine reduced 5-HT-evoked intracellular Ca2+ release, and an ultralow concentration of the phosphodiesterase-5 inhibitor sildenafil in combination with vitamin D3 reduced ATP-evoked intracellular Ca2+ release. Combinations of these three substances downregulated 5-HT-, glutamate- and ATP-evoked intracellular Ca2+ release to a more normal Ca2+ signaling state. Furthermore, inflammatory Toll-like receptor 4 expression decreased with a combination of these three substances. Substance P receptor neurokinin (NK)-1 expression was reduced by ultralow concentrations of bupivacaine. Here, bupivacaine and sildenafil (at extremely low concentrations) combined with vitamin D3 have potential anti-inflammatory properties. According to the present study, drug combinations at the right concentrations, especially extremely low concentrations of bupivacaine and sildenafil, affect different cellular biochemical mechanisms and represent a potential solution for downregulating inflammatory parameters, thereby restoring cells or networks to normal physiological homeostasis. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is a newly emerged swine enteropathogenic coronavirus affecting pigs of all ages and causing diarrhea problems. Research findings indicate that PDCoV has evolved strategies to escape innate immune response in host cells, but mechanism of PDCoV in innate immune modulation is not well understood. In this study, we report our findings on identifying the alterations of host cell innate immune response affected by PDCoV infection and exploring the gene expression profiles of PK-15 cells at 0, 24, and 36 h PDCoV post infection by RNA sequencing. A total of 3,762 and 560 differentially expressed genes (DEGs) were screened by comparison of uninfected PK-15 cells and infected PK-15 cells at 24 h post infection (hpi) (INF_24h versus NC), and also comparison of infected PK-15 cells between 24 and 36 hpi (INF_36h versus INF_24h), which included 156 and 23 porcine innate immune-related genes in the DEGs of INF_24h versus NC and INF_36h versus INF_24h, respectively. Gene Ontology function classification and Kyoto Encyclopedia of Genes and Genomes signaling pathway enrichment analysis were performed based on the DEGs that exhibited the same expression tendencies with most of the innate immune-associated genes among these PK-15 cell samples described above. The enrichment results indicated that extensive gene functions and signaling pathways including innate immune-associated functions and pathways were affected by PDCoV infection. Particularly, 4 of 5 innate immune signaling pathways, which were primarily affected by PDCoV, played important roles in I-IFN’s antiviral function in innate immune response. Additionally, 16 of the host cell endogenous miRNAs were predicted as potential contributors to the modulation of innate immune response affected by PDCoV. Our research findings indicated that the innate immune-associated genes and signaling pathways in PK-15 cells could be modified by the infection of PDCoV, which provides a fundamental foundation for further studies to better understand the mechanism of PDCoV infections, so as to effectively control and prevent PDCoV-induced swine diarrheal disease outbreaks. [ABSTRACT FROM AUTHOR]

Neutrophils play a critical role in host defense against Pseudomonas aeruginosa infection. Mechanisms underlying the negative regulation of neutrophil function in bacterial clearance remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of P. aeruginosa clearance by neutrophils. PTP1B-deficient neutrophils display greatly enhanced bacterial phagocytosis and killing, which are accompanied by increased Toll-like receptor 4 (TLR4) signaling activation and nitric oxide (NO) production following P. aeruginosa infection. Interestingly, PTP1B deficiency mainly upregulates the production of IL-6 and IFN-β, leads to enhanced TLR4-dependent STAT1 activation and iNOS expression by neutrophils following P. aeruginosa infection. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a negative regulatory mechanism in neutrophil underlying the elimination of P. aeruginosa infection though a PTP1B-STAT1 interaction. [ABSTRACT FROM AUTHOR]

Background: Atrial fibrillation (AF) is the most common arrhythmia. Patients with valvular heart disease (VHD) frequently have AF. Growing evidence demonstrates that a specifically altered pattern of microRNA (miRNA) expression is related to valvular heart disease with atrial fibrillation (AF-VHD) processes. However, the combinatorial regulation by multiple miRNAs in inducing AF-VHD remains largely unknown. Methods: The work identified AF-VHD-specific miRNAs and their combinations through mapping miRNA expression profile into differential co-expression network. The expressions of some dysregulated miRNAs were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulations of signaling pathways by the combinatorial miRNAs were predicted by enrichment analysis tools. Results: Thirty-two differentially expressed (DE) miRNAs were identified to be AF-VHD-specific, some of which were new findings. These miRNAs interacted to form 5 combinations. qRT-PCR confirmed the different expression of several identified miRNAs, which illustrated the reliability and biomarker potentials of 32 dysregulation miRNAs. The biological characteristics of combinatorial miRNAs related to AF-VHD were highlighted. Twelve signaling pathways regulated by combinatorial miRNAs were predicted to be possibly associated with AF-VHD. Conclusions: The AF-VHD-related signaling pathways regulated by combinatorial miRNAs may play an important role in the occurrence of AF-VHD. The work brings new insights into biomarkers and miRNA combination regulation mechanism in AF-VHD as well as further biological experiments. [ABSTRACT FROM AUTHOR]

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection. [ABSTRACT FROM AUTHOR]

Necrosis is a form of cell death that results in rupture of the plasma membrane and the release of cellular contents, and it can give rise to sterile inflammation in the retina and other tissues. The secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells contributes to retinal homeostasis as well as to pathological angiogenesis. We have now examined the effect of a necrotic cell lysate prepared from human RPE cells (NLR) on the release of VEGF by healthy RPE cells. We found that NLR markedly increased the release of VEGF from RPE cells and that this effect was attenuated by nintedanib, a multiple receptor tyrosine kinase inhibitor, whereas it was unaffected by inhibitors of NF-κB signaling or of caspase-1. NLR also induced the phosphorylation of extracellular signal–regulated kinase (Erk) and signal transducer and activator of transcription 3 (Stat3) in a manner sensitive to inhibition by nintedanib, although inhibitors of Erk and Stat3 signaling pathways did not affect NLR-induced VEGF secretion. In addition, nintedanib attenuated the development of choroidal neovascularization in mice. Our results have thus shown that a necrotic lysate of RPE cells induced VEGF secretion from healthy RPE cells and that this effect was mediated by receptor tyrosine kinase signaling. They therefore suggest that VEGF secretion by healthy RPE cells is a potential therapeutic target for retinal diseases associated with sterile inflammation and pathological angiogenesis. [ABSTRACT FROM AUTHOR]

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a genetically heterogeneous subtype of B-cell ALL characterized by chromosomal rearrangements and mutations that result in aberrant cytokine receptor and kinase signaling. In particular, chromosomal rearrangements resulting in the overexpression of cytokine receptor-like factor 2 (CRLF2) occur in 50% of Ph-like ALL cases. CRLF2 overexpression is associated with particularly poor clinical outcomes, though the molecular basis for this is currently unknown. Glucocorticoids (GCs) are integral to the treatment of ALL and GC resistance at diagnosis is an important negative prognostic factor. Given the importance of GCs in ALL therapy and the poor outcomes for patients with CRLF2 overexpression, we hypothesized that the aberrant signal transduction associated with CRLF2 overexpression might mediate intrinsic GC insensitivity. To test this hypothesis, we exposed Ph-like ALL cells from patient-derived xenografts to GCs and found that CRLF2 rearranged (CRLF2R) leukemias uniformly demonstrated reduced GC sensitivity in vitro. Furthermore, targeted inhibition of signal transduction with the MEK inhibitor trametinib and the Akt inhibitor MK2206, but not the JAK inhibitor ruxolitinib, was sufficient to augment GC sensitivity. These data suggest that suboptimal GC responses may in part underlie the poor clinical outcomes for patients with CRLF2 overexpression and provide rationale for combination therapy involving GCs and signal transduction inhibitors as a means of enhancing GC efficacy. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling. [ABSTRACT FROM AUTHOR]

We have previously shown that endoplasmic reticulum stress (ER stress) represses the PTEN inducible kinase 1 (PINK1) in lung type II alveolar epithelial cells (AECII) reducing mitophagy and increasing the susceptibility to lung fibrosis. Although increased circulating mitochondrial DNA (mtDNA) has been reported in chronic lung diseases, the contribution of mitophagy in the modulation of mitochondrial DAMP release and activation of profibrotic responses is unknown. In this study, we show that ER stress and PINK1 deficiency in AECII led to mitochondrial stress with significant oxidation and damage of mtDNA and subsequent extracellular release. Extracellular mtDNA was recognized by TLR9 in AECII by an endocytic-dependent pathway. PINK1 deficiency-dependent mtDNA release promoted activation of TLR9 and triggered secretion of the profibrotic factor TGF-β which was rescued by PINK1 overexpression. Enhanced mtDNA oxidation and damage were found in aging and IPF human lungs and, in concordance, levels of circulating mtDNA were significantly elevated in plasma and bronchoalveolar lavage (BAL) from patients with IPF. Free mtDNA was found elevated in other ILDs with low expression of PINK1 including hypersensitivity pneumonitis and autoimmune interstitial lung diseases. These results support a role for PINK1 mediated mitophagy in the attenuation of mitochondrial damage associated molecular patterns (DAMP) release and control of TGF-β mediated profibrotic responses. [ABSTRACT FROM AUTHOR]

Macrophages (Mϕs) play a central role in mucosal immunity by pathogen sensing and instruction of adaptive immune responses. Prior challenge to endotoxin can render Mφs refractory to secondary exposure, suppressing the inflammatory response. Previous studies demonstrated a differential subset-specific sensitivity to endotoxin tolerance (ET), mediated by LPS from the oral pathogen, Porphyromonas gingivalis (PG). The aim of this study was to investigate ET mechanisms associated with Mφ subsets responding to entropathogenic E. coli K12-LPS. M1- and M2-like Mφs were generated in vitro from the THP-1 cell line by differentiation with PMA and Vitamin D3, respectively. This study investigated ET mechanisms induced in M1 and M2 Mφ subsets, by measuring modulation of expression by RT-PCR, secretion of cytokines by sandwich ELISA, LPS receptor, TLR4, as well as endogenous TLR inhibitors, IRAK-M and Tollip by Western blotting. In contrast to PG-LPS tolerisation, E. coli K12-LPS induced ET failed to exhibit a subset-specific response with respect to the pro-inflammatory cytokine, TNFα, whereas exhibited a differential response for IL-10 and IL-6. TNFα expression and secretion was significantly suppressed in both M1- and M2-like Mφs. IL-10 and IL-6, on the other hand, were suppressed in M1s and refractory to suppression in M2s. ET suppressed TLR4 mRNA, but not TLR4 protein, yet induced differential augmentation of the negative regulatory molecules, Tollip in M1 and IRAK-M in M2 Mφs. In conclusion, E. coli K12-LPS differentially tolerises Mφ subsets at the level of anti-inflammatory cytokines, associated with a subset-specific divergence in negative regulators and independent of TLR4 down-regulation. [ABSTRACT FROM AUTHOR]

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir. [ABSTRACT FROM AUTHOR]

Background: Toll-like receptors (TLRs) are sentinel receptors of the innate immune system. TLR4 detects bacterial lipopolysaccharide (LPS) and TLR5 detects bacterial flagellin. A common human nonsense polymorphism, TLR5:c.1174C>T, results in a non-functional TLR5 protein. Individuals carrying this variant have decreased mortality from melioidosis, infection caused by the flagellated Gram-negative bacterium Burkholderia pseudomallei. Although impaired flagellin-dependent signaling in carriers of TLR5:c.1174C>T is well established, this study tested the hypothesis that a functional effect of TLR5:c.1174C>T is flagellin-independent and involves LPS-TLR4 pathways. Methodology/Principal findings: Whole blood from two independent cohorts of individuals genotyped at TLR5:c.1174C>T was stimulated with wild type or aflagellated B. pseudomallei or purified bacterial motifs followed by plasma cytokine measurements. Blood from individuals carrying the TLR5:c.1174C>T variant produced less IL-6 and IL-10 in response to an aflagellated B. pseudomallei mutant and less IL-8 in response to purified B. pseudomallei LPS than blood from individuals without the variant. TLR5 expression in THP1 cells was silenced using siRNA; these cells were stimulated with LPS before cytokine levels in cell supernatants were quantified by ELISA. In these cells following LPS stimulation, silencing of TLR5 with siRNA reduced both TNF-α and IL-8 levels. These effects were not explained by differences in TLR4 mRNA expression or NF-κB or IRF activation. Conclusions/Significance: The effects of the common nonsense TLR5:c.1174C>T polymorphism on the host inflammatory response to B. pseudomallei may not be restricted to flagellin-driven pathways. Moreover, TLR5 may modulate TLR4-dependent cytokine production. While these results may have broader implications for the role of TLR5 in the innate immune response in melioidosis and other conditions, further studies of the mechanisms underlying these observations are required. [ABSTRACT FROM AUTHOR]

We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro, however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction. [ABSTRACT FROM AUTHOR]

α7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFκB). The α7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to α7 nAChR activation. However, mechanistic details of α7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NFκB-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NFκB signaling in GH4C1 cells expressing rat α7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides α7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing α7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NFκB inhibitor alpha (IκBα), an important intermediary in NFκB signaling. However, α7 antagonists methyllycaconitine and α-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking α7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of α7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context. [ABSTRACT FROM AUTHOR]

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection remains a major cause of hepatic inflammation and liver disease. HCV triggers NLRP3 inflammasome activation and interleukin-1β (IL-1β) production from hepatic macrophages, or Kupffer cells, to drive the hepatic inflammatory response. Here we examined HCV activation of the NLRP3 inflammasome signaling cascade in primary human monocyte derived macrophages and THP-1 cell models of hepatic macrophages to define the HCV-specific agonist and cellular processes of inflammasome activation. We identified the HCV core protein as a virion-specific factor of inflammasome activation. The core protein was both necessary and sufficient for IL-1β production from macrophages exposed to HCV or soluble core protein alone. NLRP3 inflammasome activation by the HCV core protein required calcium mobilization linked with phospholipase-C activation. Our findings reveal a molecular basis of hepatic inflammasome activation and IL-1β release triggered by HCV core protein. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are small non-coding RNA molecules that play an important role in the regulation of gene expression related to inflammatory responses. Human adipose stem cells are characterized by pluripotent differentiation potential and isolated from adipose tissues. These cells regulate inflammation mainly by interacting with immune cells and affecting the secretion of immune factors; details of this interaction are currently unknown. In the current study, we successfully established an acute inflammation model and a chronic inflammation model involving adipose stem cells. We used high-throughput miRNA microarray analysis to identify miRNAs that were significantly (p < 0.05) differentially expressed during both acute and chronic inflammation. Lipopolysaccharide (LPS) significantly (p < 0.05) reduced the expression of miR-223-3P and miR-2909, while promoting the production of pro-inflammatory cytokines, interleukin (IL) 6, IL-1β, and tumor necrosis factor (TNF)-α via the Toll-like receptor (TLR) 4/TLR2/nuclear factor (NF)-κB/signal transducer and activator of transcription (STAT) 3 signaling pathway in human adipose stem cells. Further, miR-223-3P expression was significantly (p < 0.05) reduced in human adipose stem cells during activation by IL-6 stimulation. The inducible down-regulation of miR-223-3P resulted in the activation of STAT3, which was directly targeted by miR-223-3P. STAT3 directly targeted TLR4 and TLR2, promoting the production of the pro-inflammatory cytokine, IL-6, and formed a positive feedback loop to regulate IL-6 levels. Similarly, TNF-α significantly (p < 0.05) increased the expression of miR-223-3p, with LPS and TLR4/TLR2/NF-κB/STAT3 forming a negative feedback loop to regulate TNF-α levels. In addition, miR-2909, which depends on NF-κB, targeted Krueppel-like factor (KLF) 4 to regulate the levels of pro-inflammatory cytokines, IL-6, IL-1β, and TNF-α. We conclude that miR-223-3p and miR-2909 form a complex regulatory network with pro-inflammatory factors and signaling pathways in adipose stem cells stimulated by LPS. These findings will inform the development of therapies against autoimmune and inflammatory diseases. [ABSTRACT FROM AUTHOR]

Viral-bacterial coinfections, such as with influenza A virus and Streptococcus pneumoniae (S.p.), are known to cause severe pneumonia. It is well known that the host response has an important role in disease. Interleukin-1β (IL-1β) is an important immune signaling cytokine responsible for inflammation and has been previously shown to contribute to disease severity in numerous infections. Other studies in mice indicate that IL-1β levels are dramatically elevated during IAV-S.p. coinfection. However, the regulation of IL-1β during coinfection is unknown. Here, we report the NLRP3 inflammasome is the major inflammasome regulating IL-1β activation during coinfection. Furthermore, elevated IL-1β mRNA expression is due to enhanced TLR2-MYD88 signaling, which increases the amount of pro-IL-1β substrate for the inflammasome to process. Finally, NLRP3 and high IL-1β levels were associated with increased bacterial load in the brain. Our results show the NLRP3 inflammasome is not protective during IAV-S.p. coinfection. [ABSTRACT FROM AUTHOR]

Mammalian microRNAs (miRNAs) play a critical role in modulating the response of immune cells to stimuli. Cannabinoids are known to exert beneficial actions such as neuroprotection and immunosuppressive activities. However, the underlying mechanisms which contribute to these effects are not fully understood. We previously reported that the psychoactive cannabinoid Δ9–tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD) differ in their anti-inflammatory signaling pathways. Using lipopolysaccharide (LPS) to stimulate BV-2 microglial cells, we examined the role of cannabinoids on the expression of miRNAs. Expression was analyzed by performing deep sequencing, followed by Ingenuity Pathway Analysis to describe networks and intracellular pathways. miRNA sequencing analysis revealed that 31 miRNAs were differentially modulated by LPS and by cannabinoids treatments. In addition, we found that at the concentration tested, CBD has a greater effect than THC on the expression of most of the studied miRNAs. The results clearly link the effects of both LPS and cannabinoids to inflammatory signaling pathways. LPS upregulated the expression of pro-inflammatory miRNAs associated to Toll-like receptor (TLR) and NF-κB signaling, including miR-21, miR-146a and miR-155, whereas CBD inhibited LPS-stimulated expression of miR-146a and miR-155. In addition, CBD upregulated miR-34a, known to be involved in several pathways including Rb/E2f cell cycle and Notch-Dll1 signaling. Our results show that both CBD and THC reduced the LPS-upregulated Notch ligand Dll1 expression. MiR-155 and miR-34a are considered to be redox sensitive miRNAs, which regulate Nrf2-driven gene expression. Accordingly, we found that Nrf2-mediated expression of redox-dependent genes defines a Mox-like phenotype in CBD treated BV-2 cells. In summary, we have identified a specific repertoire of miRNAs that are regulated by cannabinoids, in resting (surveillant) and in LPS-activated microglia. The modulated miRNAs and their target genes are controlled by TLR, Nrf2 and Notch cross-talk signaling and are involved in immune response, cell cycle regulation as well as cellular stress and redox homeostasis. [ABSTRACT FROM AUTHOR]

Problem: Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact. Method of study: We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot. Results: qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect. Conclusion: Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch. [ABSTRACT FROM AUTHOR]

Neuroinflammation and neurodegeneration are common during prion infection, but the mechanisms that underlie these pathological features are not well understood. Several components of innate immunity, such as Toll-like receptor (TLR) 4 and Complement C1q, have been shown to influence prion disease. To identify additional components of innate immunity that might impact prion disease within the central nervous system (CNS), we screened RNA from brains of pre-clinical and clinical 22L-infected mice for alterations in genes associated with innate immunity. Transcription of several genes encoding damage-associated molecular pattern (DAMP) proteins and receptors were increased in the brains of prion-infected mice. To investigate the role of some of these proteins in prion disease of the CNS, we infected mice deficient in DAMP receptor genes Tlr2, C3ar1, and C5ar1 with 22L scrapie. Elimination of TLR2 accelerated disease by a median of 10 days, while lack of C3aR1 or C5aR1 had no effect on disease tempo. Histopathologically, all knockout mouse strains tested were similar to infected control mice in gliosis, vacuolation, and PrPSc deposition. Analysis of proinflammatory markers in the brains of infected knockout mice indicated only a few alterations in gene expression suggesting that C5aR1 and TLR2 signaling did not act synergistically in the brains of prion-infected mice. These results indicate that signaling through TLR2 confers partial neuroprotection during prion infection. [ABSTRACT FROM AUTHOR]

Schistosomiasis is endemic to many regions of the world and affects approximately 200 million people. Conventional adaptive T cell responses are considered to be the primary contributors to the pathogenesis of Schistosoma japonicum infection, leading to liver granuloma and fibrosis. However, the functional polarization of macrophages and the associated underlying molecular mechanisms during the pathogenesis of schistosomiasis remains unknown. In the present study, we found that excretory-secretory (ES) antigens derived from S. japonicum eggs can activate macrophages, which exhibit an M2b polarization. Furthermore, ES antigen-induced M2b polarization was found to be dependent on enhanced NF-κB signaling mediated by the MyD88/MAPK pathway in a TLR2-dependent manner. In addition, the cytokine profile of the liver macrophages from wild-type-infected mice are quite distinct from those found in TLR2 knockout-infected mice by quantitative PCR analysis. More importantly, the size of granuloma and the severity of the fibrosis in the livers of TLR2-/- mice were significantly reduced compared to that in WT mice. Our findings reveal a novel role for M2b polarization in the pathogenesis of schistosome infection. [ABSTRACT FROM AUTHOR]

The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens. [ABSTRACT FROM AUTHOR]

Higher vertebrates have evolved innate and adaptive immune systems to defend against foreign substances and pathogens. Sophisticated regulatory circuits are needed to avoid inappropriate immune responses and inflammation. GPR108 is a seven-transmembrane family protein that activates NF-κB strongly when overexpressed. Surprisingly, its action in a physiological context is that of an antagonist of Toll-like receptor (TLR)-mediated signaling. Cells from Gpr108-null mice exhibit enhanced cytokine secretion and NF-κB and IRF3 signaling, whereas Gpr108-null macrophages reconstituted with GPR108 exhibit blunted signaling. Co-expression of TLRs and GPR108 reduces NF-κB and IFNβ promoter activation compared to expression of either TLRs or GPR108 alone. Upon TLR stimulation GPR108 abundance increases and the protein engages TLRs and their partners to reduce MyD88 expression and interfere with its binding to TLR4 through blocking MyD88 ubiquitination. In turn GPR108 is antagonized by TIRAP, an adaptor protein for TLR and MyD88. The interrelationships between GPR108 and innate immune signaling components are multifactorial and point to a membrane-associated signaling structure of significant complexity. [ABSTRACT FROM AUTHOR]

Ocular dryness is a characteristic feature of primary Sjögren’s syndrome (pSS). This may result in dry eye disease (DED), leading to damage of the ocular surface. Additional, non-invasive diagnostic techniques are needed when evaluating pSS patients. Hence, screening for disease-specific biomarkers in biological fluid could be promising. We have previously examined the proteome of tear fluid from pSS patients through Liquid chromatography-mass spectrometry (LC-MS), and conducted a thorough ocular evaluation of patients with pSS. In this study we further explored the association between dry eye manifestations and protein expression in tear fluid of pSS patients. Medical history of 27 patients and 32 healthy controls was gathered. Subjective complaints were registered through questionnaires. Objective findings including tear osmolarity, tear film break up time (TFBUT), Schirmer’s test, and ocular and corneal surface staining were also recorded. LC-MS was conducted formerly on tear fluid from all subjects in order to generate proteomic biomarker profiles. Scaffold was employed to analyse the LC-MS data for quantitative differences between patient and control groups, and the mean spectral counts were calculated for the five most upregulated proteins in relation to DED manifestations. Dysregulated cellular processes were identified in pSS patients using FunRichv3 enrichment analysis. The five most upregulated proteins previously identified in pSS patients were DNA (apurinic or apyrimidinic site) lyase (APEX1), thioredoxin-dependent peroxidase reductase (PRDX3), copine (CPNE1), aconitate hydratase (ACO2), and LIM domain only protein 7 (LMO7), in descending order. A significant increase in mean spectral counts for these proteins were observed in pSS patients with pathological DED manifestations compared to healthy controls (p<0.0001). Consequently, dysregulated cellular pathways involving innate and adaptive immunity were also detected. In conclusion, our observations suggest a relationship between presence of dry eye signs and upregulated proteins in tear fluid from patients with pSS. Further studies are needed in order to replicate the concepts explored and analyses performed in a greater cohort of pSS patients, where sensitivity and specificity of the methods conducted can also be verified further. [ABSTRACT FROM AUTHOR]

Herbal extracts represent an ample source of natural compounds, with potential to be used in improving human health. There is a growing interest in using natural extracts as possible new treatment strategies for inflammatory diseases. We therefore aimed at identifying herbal extracts that affect inflammatory signaling pathways through toll-like receptors (TLRs), TLR2 and TLR4. Ninety-nine ethanolic extracts were screened in THP-1 monocytes and HeLa-TLR4 transfected reporter cells for their effects on stimulated TLR2 and TLR4 signaling pathways. The 28 identified anti-inflammatory extracts were tested in comparative assays of stimulated HEK-TLR2 and HEK-TLR4 transfected reporter cells to differentiate between direct TLR4 antagonistic effects and interference with downstream signaling cascades. Furthermore, the ten most effective anti-inflammatory extracts were tested on their ability to inhibit nuclear factor-κB (NF-κB) translocation in HeLa-TLR4 transfected reporter cell lines and for their ability to repolarize M1-type macrophages. Ethanolic extracts which showed the highest anti-inflammatory potential, up to a complete inhibition of pro-inflammatory cytokine production were Castanea sativa leaves, Cinchona pubescens bark, Cinnamomum verum bark, Salix alba bark, Rheum palmatum root, Alchemilla vulgaris plant, Humulus lupulus cones, Vaccinium myrtillus berries, Curcuma longa root and Arctostaphylos uva-ursi leaves. Moreover, all tested extracts mitigated not only TLR4, but also TLR2 signaling pathways. Seven of them additionally inhibited translocation of NF-κB into the nucleus. Two of the extracts showed impact on repolarization of pro-inflammatory M1-type to anti-inflammatory M2-type macrophages. Several promising anti-inflammatory herbal extracts were identified in this study, including extracts with previously unknown influence on key TLR signaling pathways and macrophage repolarization, serving as a basis for novel lead compound identification. [ABSTRACT FROM AUTHOR]

Different computational approaches are employed to efficiently identify novel repositioning possibilities utilizing different sources of information and algorithms. It is critical to propose high-valued candidate-repositioning possibilities before conducting lengthy in vivo validation studies that consume significant resources. Here we report a novel multi-methodological approach to identify opportunities for drug repositioning. We performed analyses of real-world data (RWD) acquired from the United States Food and Drug Administration’s Adverse Event Reporting System (FAERS) and the claims database maintained by the Japan Medical Data Center (JMDC). These analyses were followed by cross-validation through bioinformatics analyses of gene expression data. Inverse associations revealed using disproportionality analysis (DPA) and sequence symmetry analysis (SSA) were used to detect potential drug-repositioning signals. To evaluate the validity of the approach, we conducted a feasibility study to identify marketed drugs with the potential for treating inflammatory bowel disease (IBD). Primary analyses of the FAERS and JMDC claims databases identified psycholeptics such as haloperidol, diazepam, and hydroxyzine as candidates that may improve the treatment of IBD. To further investigate the mechanistic relevance between hit compounds and disease pathology, we conducted bioinformatics analyses of the associations of the gene expression profiles of these compounds with disease. We identified common biological features among genes differentially expressed with or without compound treatment as well as disease-perturbation data available from open sources, which strengthened the mechanistic rationale of our initial findings. We further identified pathways such as cytokine signaling that are influenced by these drugs. These pathways are relevant to pathologies and can serve as alternative targets of therapy. Integrative analysis of RWD such as those available from adverse-event databases, claims databases, and transcriptome analyses represent an effective approach that adds value to efficiently identifying potential novel therapeutic opportunities. [ABSTRACT FROM AUTHOR]

Countless neurodegenerative diseases are associated with perverse multiple targets of cyclic nucleotide signalling, hastening neuronal death. Cilostazol, a phosphodiesterase-III inhibitor, exerts neuroprotective effects against sundry models of neurotoxicity, however, its role against Huntington’s disease (HD) has not yet been tackled. Hence, its modulatory effect on several signalling pathways using the 3-nitropropionic acid (3-NP) model was conducted. Animals were injected with 3-NP (10 mg/kg/day, i.p) for two successive weeks with or without the administration of cilostazol (100 mg/kg/day, p.o.). Contrary to the 3-NP effects, cilostazol largely preserved striatal dopaminergic neurons, improved motor coordination, and enhanced the immunohistochemical reaction of tyrosine hydroxylase enzyme. The anti-inflammatory effect of cilostazol was documented by the pronounced reduction of the toll like receptor-4 (TLR-4) protein expression and the inflammatory cytokine IL-6, but with a marked elevation in IL-10 striatal contents. As a consequence, cilostazol reduced IL-6 downstream signal, where it promoted the level of suppressor of cytokine signalling 3 (SOCS3), while abated the phosphorylation of Janus Kinase 2 (JAK-2) and Signal transducers and activators of transcription 3 (STAT-3). Phosphorylation of the protein kinase B/glycogen synthase kinase-3β/cAMP response element binding protein (Akt/GSK-3β/CREB) cue is another signalling pathway that was modulated by cilostazol to further signify its anti-inflammatory and antiapoptotic capacities. The latter was associated with a reduction in the caspase-3 expression assessed by immunohistochemical assay. In conclusion the present study provided a new insight into the possible mechanisms by which cilostazol possesses neuroprotective properties. These intersecting mechanisms involve the interference between TLR-4, IL-6-IL-10/JAK-2/STAT-3/SOCS-3, and Akt/GSK-3β/CREB signalling pathways. [ABSTRACT FROM AUTHOR]

Innate immune cells, such as macrophages, respond to pathogen-associated molecular patterns, such as a lipopolysaccharide (LPS), to secrete various inflammatory mediators. Recent studies have suggested that damage-associated molecular patterns (DAMPs), released extracellularly from damaged or immune cells, also play a role in the activation of inflammatory responses. In this study, to prevent excess inflammation, we focused on DAMPs-mediated signaling that promotes LPS-stimulated inflammatory responses, especially adenosine 5’-triphosphate (ATP)-triggered signaling through the ionotropic purinergic receptor 7 (P2X7R), as a potential new anti-inflammatory target of natural polyphenols. We focused on the phenomenon that ATP accelerates the production of inflammatory mediators, such as nitric oxide, in LPS-stimulated J774.1 mouse macrophages. Using an siRNA-mediated knockdown and specific antagonist, it was found that the ATP-induced enhanced inflammatory responses were mediated through P2X7R. We then screened 42 polyphenols for inhibiting the ATP/P2X7R-induced calcium influx, and found that several polyphenols exhibited significant inhibitory effects. Especially, a flavonoid baicalein significantly inhibited ATP-induced inflammation, including interleukin-1β secretion, through inhibition of the ATP/P2X7R signaling. These findings suggest that ATP/P2X7R signaling plays an important role in excess inflammatory responses and could be a potential anti-inflammatory target of natural polyphenolic compounds. [ABSTRACT FROM AUTHOR]

Background: Pulmonary vascular remodeling of pulmonary arterial hypertension (PAH) is characterized by an inappropriate increase of vascular cells. The receptor for advanced glycation end products (RAGE) is a type I single-pass transmembrane protein belonging to the immunoglobulin superfamily and is involved in a broad range of hyperproliferative diseases. RAGE is also implicated in the etiology of PAH and is overexpressed in pulmonary artery smooth muscle cells (PASMCs) in patients with PAH. We examined the role of RAGE in the inappropriate increase of PASMCs in patients with PAH. Methods and results: PASMCs were obtained from 12 patients with PAH including 9 patients with idiopathic PAH (IPAH) and 3 patients with heritable PAH (HPAH) (2 patients with BMPR2 mutation and one patient with SMAD9 mutation) who underwent lung transplantation. Western blot analysis and immunofluorescence staining revealed that RAGE and S100A8 and A9, ligands of RAGE, were overexpressed in IPAH and HPAH-PASMCs in the absence of any external growth stimulus. PDGF-BB (10 ng/mL) up-regulated the expression of RAGE in IPAH and HPAH-PASMCs. PAH-PASMCs are hyperplastic in the absence of any external growth stimulus as assessed by 3H-thymidine incorporation. This result indicates overgrowth characterized by continued growth under a condition of no growth stimulation in PAH-PASMCs. PDGF-BB stimulation caused a higher growth rate of PAH-PASMCs than that of non-PAH-PASMCs. AS-1, an inhibitor of TIR domain-mediated RAGE signaling, significantly inhibited overgrowth characterized by continued growth under a condition of no growth stimulation in IPAH and HPAH-PASMCs (P<0.0001). Furthermore, AS-1 significantly inhibited PDGF-stimulated proliferation of IPAH and HPAH-PASMCs (P<0.0001). Conclusions: RAGE plays a crucial role in the inappropriate increase of PAH-PASMCs. Inhibition of RAGE signaling may be a new therapeutic strategy for PAH. [ABSTRACT FROM AUTHOR]

Toll like receptors (TLRs) are important pattern recognition receptors that can detect pathogen and danger associated molecular patterns to initiate an innate immune response. TLR1 and 2 heterodimerize at the plasma membrane upon binding to triacylated lipopeptides from bacterial cell walls, or to the synthetic ligand Pam3CSK4. TLR1/2 dimers interact with adaptor molecules TIRAP and MyD88 to initiate a signalling cascade that leads to activation of key transcription factors, including NF-kB. Despite TLRs being extensively studied over the last two decades, the real-time kinetics of ligand binding and receptor activation remains largely unexplored. We aimed to study the kinetics of TLR activation and recruitment of adaptors, using TLR1/2 dimer interactions with adaptors MyD88 and TIRAP. Bioluminescence resonance energy transfer (BRET) allows detection of real-time protein-protein interactions in living cells, and was applied to study adaptor recruitment to TLRs. Energy transfer showed interactions between TLR2 and TIRAP, and between TLR2 and MyD88 only in the presence of TIRAP. Quantitative BRET and confocal microscopy confirmed that TIRAP is necessary for MyD88 interaction with TLR2. Furthermore, constitutive proximity between the proteins in the absence of Pam3CSK4 stimulation was observed with BRET, and was not abrogated with lowered protein expression, changes in protein tagging strategies, or use of the brighter NanoLuc luciferase. However, co-immunoprecipitation studies did not demonstrate constitutive interaction between these proteins, suggesting that the interaction observed with BRET likely represents artefacts of protein overexpression. Thus, caution should be taken when utilizing protein overexpression in BRET studies and in investigations of the TLR pathway. [ABSTRACT FROM AUTHOR]

NLRC3, a member of the NLR family, has been reported as a negative regulator of inflammatory signaling pathways in innate immune cells. However, the direct role of NLRC3 in modulation of CD4+ T-cell responses in infectious diseases has not been studied. In the present study, we showed that NLRC3 plays an intrinsic role by suppressing the CD4+ T cell phenotype in lung and spleen, including differentiation, activation, and proliferation. NLRC3 deficiency in CD4+ T cells enhanced the protective immune response against Mycobacterium tuberculosis infection. Finally, we demonstrated that NLRC3 deficiency promoted the activation, proliferation, and cytokine production of CD4+ T cells via negatively regulating the NF-κB and MEK-ERK signaling pathways. This study reveals a critical role of NLRC3 as a direct regulator of the adaptive immune response and its protective effects on immunity during M. tuberculosis infection. Our findings also suggested that NLRC3 serves as a potential target for therapeutic intervention against tuberculosis. [ABSTRACT FROM AUTHOR]

Gamma interferon (IFN-γ) is known to negatively regulate murine gammaherpesvirus-68 (MHV-68 or γHV-68) replication. This process involves the suppression of the viral gene replication and transcription activator (RTA) promoter, as well as activation of signal transducers and activators of transcription (STAT1). Notably, this effect is gradually attenuated during MHV-68 infection of bone marrow-derived macrophages (BMMs), which raised the possibility that the virus may utilize a mechanism that counteracts the antiviral effect of IFN-γ. By identifying the cellular factors that negatively regulate JAK-STAT1 signaling, we revealed that the infection of BMMs by MHV-68 induces the expression of suppressor of cytokine signaling 1 (SOCS1) and that depletion of SOCS1 restores the inhibitory effect of IFN-γ on virus replication. Moreover, we demonstrated that the expression of SOCS1 was induced as a result of the Toll-like receptor 3 (TLR3) mediated activation of the NF-κB signaling cascade. In conclusion, we report that TLR3-TRAF-NF-κB signaling pathway play a role in the induction of SOCS1 that counteracts the antiviral effect of IFN-γ during MHV-68 infection. This process is cell type-specific: it is functional in macrophages, but not in epithelial cells or fibroblasts. Our study reveals a mechanism that balances the immune responses and the escape of a gamma-herpesvirus in some antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Objective: Periodontitis is a microbe-induced chronic inflammatory disease. Previous exposure of the host to bacteria or their virulence factors leads to refractory responses to further stimuli, which is called tolerance. Porphyromonas gingivalis (P. gingivalis) is one of the most important pathogenic microorganisms associated with periodontitis, and is a potent inducer of pro- and anti-inflammatory cytokines. The aim of this study was to explore the roles and possible mechanisms of tolerance induced by P. gingivalis. Methods: THP-1-derived macrophages were pretreated with 1x108 colony-forming units/ml P. gingivalis ATCC 33277 or 21 clinical isolates from moderate to severe chronic periodontitis patients (24 h), washed (2 h) and treated with P. gingivalis ATCC 33277 or the same clinical isolates again (24 h). Levels of pro-inflammatory cytokines TNF-α and IL-1β and anti-inflammatory cytokine IL-10 in supernatants were detected by ELISA. Moreover, to identify the possible mechanisms for the changes in cytokine secretion, Toll-like receptor 2 (TLR2) and TLR4 protein expressions were explored in these cells by flow cytometry. Results: After repeated challenge with P. gingivalis ATCC 33277 or clinical isolates, production of TNF-α and IL-1β in macrophages was decreased significantly compared with that following a single stimulation (p<0.05), while only comparable levels of IL-10 were detected in P. gingivalis ATCC 33277 or clinical isolate-tolerized cells (p>0.05). In addition, there was interstrain variability in the ability to induce IL-1β and IL-10 production after repeated P. gingivalis stimulation. However, no significant changes in TLR2 or TLR4 were detected in macrophages that were repeatedly treated with P. gingivalis ATCC 33277 or clinical isolates compared with those stimulated with P. gingivalis only once (p>0.05). Conclusions: Repeated P. gingivalis stimulation triggered tolerance, which might contribute to limiting periodontal inflammation. However, tolerance induced by P. gingivalis might develop independently of TLR2 and TLR4 and be related to molecules in signaling pathways downstream of TLR2 and TLR4. [ABSTRACT FROM AUTHOR]

Macrophages (MØs) are sentinels of the immune system that use pattern recognition receptors such as Toll-like receptors (TLR) to detect invading pathogens and immune receptors such as FcγR to sense the host’s immune state. Crosstalk between these two signaling pathways allows the MØ to tailor the cell’s overall response to prevailing conditions. However, the molecular mechanisms underlying TLR-FcγR crosstalk are only partially understood. Therefore, we employed an immunologically-relevant MØ stimulus, an nactivated gram-negative acterium that bears agonists but no TLR4 agonist (iBTLR2) opsonized with a monoclonal antibody (mAb-iBTLR2), as a tool to study FcγR regulation of TLR2-driven production of IL-6, a key inflammatory cytokine. We chose this particular agonist as an investigational tool because MØ production of any detectable IL-6 in response to mAb-iBTLR2 requires both TLR2 and FcγR signaling, making it an excellent system for the study of receptor synergy. Using genetic, pharmacological and immunological approaches, we demonstrate that the murine MØ IL-6 response to mAb-iBTLR2 requires activation of both the TLR/NF-κB and FcγR/ITAM signaling pathways. mAb-iBTLR2 engagement of TLR2 drives NF-κB activation and up-regulation of IL-6 mRNA but fails to result in IL-6 cytokine production/release. Here, Src family kinase-driven FcγR ITAM signaling is necessary to enable IL-6 mRNA incorporation into polysomes and translation. These results reveal a novel mechanism by which FcγR ITAM signaling synergizes with TLR signaling, by “licensing” cytokine mRNA ribosome binding/translation to drive a strong murine MØ cytokine response. [ABSTRACT FROM AUTHOR]

Toll like receptors (TLRs) share a conserved structure comprising the N-terminal ectodomain, a transmembrane segment and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. Proper assembly of the TIR domain is crucial for signal transduction; however, the contribution of individual motifs within the TIR domain to TLR trafficking and signaling remains unclear. We targeted a highly conserved tyrosine (Y870) located in the box 1 region of the TIR domain of most TLRs, including TLR9, previously described to be a critical site of phosphorylation in TLR4. We reconstituted bone marrow-derived dendritic cells (BMDC) from Tlr9-/- mice WT TLR9 or Y870F or Y870A mutants. Despite normal interactions with the luminal chaperones GRP94 and UNC93B1, Y870F conferred only partial responsiveness to CpG, and Y870A had no activity and functioned as a dominant negative inhibitor when coexpressed with endogenous TLR9. This loss of function correlated with reduction or absence, respectively, of the 80 kDa mature form of TLR9. In Y870F-expressing cells, CpG-dependent signaling correlated directly with levels of the mature form, suggesting that signaling did not require tyrosine phosphorylation but rather that the Y870F mutation conferred reduced receptor levels due to defective processing or trafficking. Microscopy revealed targeting of the mutant protein to an autophagolysosome-like structure for likely degradation. Collectively we postulate that the conserved Y870 in the TIR domain does not participate in phosphorylation-induced signaling downstream of ligand recognition, but rather is crucial for proper TIR assembly and ER egress, resulting in maturation-specific stabilization of TLR9 within endolysosomes and subsequent pro-inflammatory signaling. [ABSTRACT FROM AUTHOR]

Leptospirosis is a bacterial zoonosis, caused by Leptospira spp., that leads to significant morbidity and mortality worldwide. Despite considerable advances, much is yet to be discovered about disease pathogenicity. The influence of epigenetic mechanisms, particularly RNA-mediated post-transcriptional regulation of host immune response has been described following a variety of bacterial infections. The current study examined the microtranscriptome of macrophages J774A.1 following an 8h infection with virulent, attenuated and saprophyte strains of Leptospira. Microarray analysis revealed that 29 miRNAs were misregulated following leptospiral infection compared to control macrophages in a strain and virulence-specific manner. Pathway analysis for targets of these differentially expressed miRNAs suggests that several processes involved in immune response could be regulated by miRNAs. Our data provides the first evidence that host miRNAs are regulated by Leptospira infection in macrophages. A number of the identified miRNA targets participate in key immune response processes. We suggest that post-transcriptional regulation by miRNAs may play a role in host response to infection in leptospirosis. [ABSTRACT FROM AUTHOR]

The activity of Cullin-RING ubiquitin E3 ligases (CRL) is regulated by NEDD8 modification. DCN-like proteins promote Cullin neddylation as scaffold-like E3s. One DCNL, DCNL5, is highly expressed in immune tissue. Here, we provide evidence that DCNL5 may be involved in innate immunity, as it is a direct substrate of the kinase IKKα during immune signalling. We find that upon activation of Toll-like receptors, DCNL5 gets rapidly and transiently phosphorylated on a specific N-terminal serine residue (S41). This phosphorylation event is specifically mediated by IKKα and not IKKβ. Our data for the first time provides evidence that DCNL proteins are post-translationally modified in an inducible manner. Our findings also provide the first example of a DCNL member as a kinase substrate in a signalling pathway, indicating that the activity of at least some DCNLs may be regulated. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) are the first to identify disturbances in the immune system, recognizing pathogens such as bacteria, fungi, and viruses. Since the inflammation process plays an important role in several diseases, TLRs have been considered potential therapeutic targets, including treatment for cancer. However, TLRs’ role in cancer remains ambiguous. This study aims to analyze the expression levels of plasmatic cell membrane TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) in human astrocytomas the most prevalent tumors of CNS different grades (II-IV). We demonstrated that TLR expressions were higher in astrocytoma samples compared to non-neoplastic brain tissue. The gene and protein expressions were observed in GBM cell lines U87MG and A172, proving their presence in the tumor cells. Associated expressions between the known heterodimers TLR1-TLR2 were found in all astrocytoma grades. In GBMs, the mesenchymal subtype showed higher levels of TLR expressions in relation to classical and proneural subtypes. A strong association of TLRs with the activation of cell cycle process and signaling through canonical, inflammasome and ripoptosome pathways was observed by in silico analysis, further highlighting TLRs as interesting targets for cancer treatment. [ABSTRACT FROM AUTHOR]

Recognition of Pathogen-associated Molecular Patterns (PAMPs) by Toll-like receptors is central to innate immunity. Many bacterial PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid have amphiphilic properties. The hydrophobicity of amphiphilic PAMPs contributes to increasing entropy and causes these molecules to self-aggregate or bind host carrier proteins in aqueous physiological environments. The goal of this work was to determine how innate immune signaling is impacted by physical presentation and association of amphiphilic PAMPs with serum carrier proteins, using LPS as an example molecule. Specifically, we measured LPS-induced cytokine profiles in murine macrophages when the antigen was presented associated with the various serum carrier proteins in serum versus a serum-depleted system. Our study demonstrates that the observed cytokine profiles are dramatically different when LPS is presented in buffer, versus in serum when it is associated with proteins, specifically with respect to inhibition of pro-inflammatory cytokines in the latter. These studies suggest that LPS-mediated cytokine expression is dependent on its presentation in physiological systems. The amphiphilicity of bacterial PAMPs and consequent association with lipoproteins is a feature, which should be taken into account in the design of in vitro experiments. Further studies of the interdependencies of different serum carriers can identify pathways for drug delivery and diagnostics. [ABSTRACT FROM AUTHOR]

Macrophages exhibit diverse effector phenotypes depending on the stimuli and their microenvironment. Classically activated macrophages are primed with interferon (IFN)γ and stimulated with pathogen-associated molecular patterns. They produce inflammatory mediators and inflammatory cytokines, such as IL-12. In the presence of immune complexes (ICs), activated macrophages have decreased IL-12 production and increased IL-10 production and presumably act as regulatory macrophages. Notch signaling has been shown to regulate the effector functions of classically activated macrophages. In this study, we investigated whether Notch signaling is active in lipopolysaccharide (LPS)-stimulated macrophages in the presence of ICs. LPS/IC stimulation increased the level of cleaved Notch1 in murine macrophages, while IC stimulation alone did not. Delta-like 4, but not Jagged1, was responsible for generating cleaved Notch1. The activation of Notch signaling by LPS/ICs depended upon NF-κB and MEK/Erk pathway activation. Macrophages with the targeted deletion of Rbpj, which encodes a DNA-binding protein central to canonical Notch signaling, produced significantly less IL-10 upon LPS/IC stimulation. A similar impact on IL-10 production was observed when Notch signaling was inhibited with a gamma-secretase inhibitor (GSI). Defects in NF-κB p50 nuclear localization were observed in GSI-treated macrophages and in Rbpj-/- macrophages, suggesting cross-regulation between the Notch and NF-κB pathways. Transcriptomic analysis revealed that Notch signaling regulates the transcription of genes involved in the cell cycle, macrophage activation, leukocyte migration and cytokine production in LPS/IC-stimulated macrophages. Taken together, these results suggest that the Notch signaling pathway plays an important role in regulating the functions of macrophages activated by LPS and ICs. [ABSTRACT FROM AUTHOR]

Endothelial cells play an essential role in regulating an immune response through promoting leukocyte adhesion and cell migration and production of cytokines such as TNFα. Regulation of endothelial cell immune function is tightly regulated and recent studies suggest that extracellular vesicles (EVs) are prominently involved in this process. However, the importance of EVs in regulating endothelial activation in the context of a bacterial infection is poorly understood. To begin addressing this knowledge gap we characterized the endothelial cell response to EVs released from Mycobacterium tuberculosis (Mtb) infected macrophages. Our result showed increased macrophage migration through the monolayer when endothelial cells were pretreated with EVs isolated from Mtb-infected macrophages. Transcriptome analysis showed a significant upregulation of genes involved in cell adhesion and the inflammatory process in endothelial cells treated with EVs. These results were validated by quantitative PCR and flow cytometry. Pathway analysis of these differentially expressed genes indicated that several immune response-related pathways were up-regulated. Endothelial cells were also treated with EVs isolated from the serum of Mtb-infected mice. Interestingly, EVs isolated 14 days but not 7 or 21 days post-infection showed a similar ability to induce endothelial cell activation suggesting a change in EV function during the course of an Mtb infection. Immunofluorescence microscopy result indicated that NF-κB and the Type 1 interferon pathways were involved in endothelial activation by EVs. In summary, our data suggest that EVs can activate endothelial cells and thus may play an important role in modulating host immune responses during an Mtb infection. [ABSTRACT FROM AUTHOR]

Toll-like receptor 3 (TLR3) senses dsRNA intermediates produced during RNA virus replication to activate innate immune signaling pathways through adaptor protein TRIF. Many viruses have evolved strategies to block TLR3-mediated interferon signaling via targeting TRIF. Here we studied how hepatitis C virus (HCV) antagonizes the TLR3-mediated interferon signaling. We found that HCV-encoded NS4B protein inhibited TLR3-mediated interferon signaling by down-regulating TRIF protein level. Mechanism studies indicated that the downregulation of TRIF by NS4B was dependent on caspase8. NS4B transfection or HCV infection can activate caspase8 to promote TRIF degradation, leading to suppression of TLR3-mediated interferon signaling. Knockout of caspase8 can prevent TRIF degradation triggered by NS4B, thereby enhancing the TLR3-mediated interferon signaling activation in response to HCV infection. In conclusion, our work revealed a new mechanism for HCV to evade innate immune response by blocking the TLR3-mediated interferon signaling via NS4B-induced TRIF degradation. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing pancreatic beta cells. While the role of adaptive immunity has been extensively studied, the role of innate immune responses and particularly of Toll- like Receptor (TLR) signaling in T1D remains poorly understood. Here we show that myeloid cell-specific MyD88 deficiency considerably protected mice from the development of streptozotocin (STZ)-induced diabetes. The protective effect of MyD88 deficiency correlated with increased expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in pancreatic lymph nodes from STZ-treated mice and in bone marrow-derived dendritic cells (BMDC) stimulated with apoptotic cells. Mice with myeloid cell specific TIR-domain-containing adapter-inducing interferon-β (TRIF) knockout showed a trend towards accelerated onset of STZ-induced diabetes, while TRIF deficiency resulted in reduced IDO expression in vivo and in vitro. Moreover, myeloid cell specific MyD88 deficiency delayed the onset of diabetes in Non-Obese Diabetic (NOD) mice, whereas TRIF deficiency had no effect. Taken together, these results identify MyD88 signaling in myeloid cells as a critical pathogenic factor in autoimmune diabetes, which is antagonized by TRIF-dependent responses. This differential function of MyD88 and TRIF depends at least in part on their opposite effects in regulating IDO expression in phagocytes exposed to apoptotic cells. [ABSTRACT FROM AUTHOR]

Increased consumption of Western-type diets and environmental insults lead to wide-spread increases in the plasma levels of saturated fatty acids and lipoprotein oxidation. The aim of this study is to examine whether palmitate and minimally modified low-density lipoprotein (mmLDL) exert an additive effect on macrophage activation. We found that CXCL2 and TNF-α secretion as well as ERK and p38 phosphorylation were additively increased by co-treatment of J774 macrophages with palmitate and mmLDL in the presence of lipopolysaccharide (LPS). Furthermore, the analysis of differentially expressed genes using the KEGG database revealed that several pathways, including cytokine-cytokine receptor interaction, and genes were significantly altered. These results were validated with real-time PCR, showing upregulation of Il-6, Csf3, Il-1β, and Clec4d. The present study demonstrated that palmitate and mmLDL additively potentiate the LPS-induced activation of macrophages. These results suggest the existence of synergistic mechanisms by which saturated fatty acids and oxidized lipoproteins activate immune cells. [ABSTRACT FROM AUTHOR]

DNA damage observed during plant immune responses is reported to be an intrinsic component of plant immunity. However, other immune responses may suppress DNA damage to maintain host genome integrity. Here, we show that immunity-related DNA damage can be abrogated by preventing cell death triggered by Nucleotide-binding, Leucine-rich-repeat immune Receptors (NLRs). SNI1 (suppressor of npr1-1, inducible 1), a subunit of the structural maintenance of chromosome (SMC) 5/6 complex, was reported to be a negative regulator of systemic acquired resistance (SAR) and to be necessary for controlling DNA damage. We find that cell death and DNA damage in sni1 loss-of-function mutants are prevented by mutations in the NLR signaling component EDS1. Similar to sni1, elevated DNA damage is seen in other autoimmune mutants with cell death lesions, including camta3, pub13 and vad1, but not in dnd1, an autoimmune mutant with no visible cell death. We find that as in sni1, DNA damage in camta3 is EDS1-dependent, but that it is also NLR-dependent. Using the NLR RPM1 as a model, we also show that extensive DNA damage is observed when an NLR is directly triggered by effectors. We also find that the expression of DNA damage repair (DDR) genes in mutants with cell death lesions is down regulated, suggesting that degraded DNA that accumulates during cell death is a result of cellular dismantling and is not sensed as damaged DNA that calls for repair. Our observations also indicate that SNI1 is not directly involved in SAR or DNA damage accumulation. [ABSTRACT FROM AUTHOR]

The article discusses the decoy mechanisms in the interaction of host-pathogen. Topics mention including the antagonistic interactions between the pathogens and the hosts, different types of decoys with classification and examples and the term decoy in the host-pathogen interactions which describes the molecules.