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Aims Phagocytic and autophagic responses are critical for effective host defense against bacterial infection. Bacterial DNA which contains unmethylated Cytosine-phosphate-Guanine (CpG) motifs can trigger a variety of defense mechanisms via Toll-like receptor 9 (TLR9). Here, we aimed to investigate the underlying mechanism of TLR9-mediated phagocytosis and autophagy in Staphylococcus aureus ( S. aureus )-stimulated macrophages. Main methods The macrophage cell line RAW264.7 or primary peritoneal macrophage was pretreated with CpG-ODN and then stimulated by S. aureus , where some of them were pretreated with SP600125 or SB203580 simultaneously. The protein expressions of TLR9, MyD88, SR-A, CD36, LC3, Beclin-1, and phosphorylated level of c-Jun N-terminal kinase (JNK), P38 and extracellular-regulated protein kinase (ERK) were detected by western blotting. The phagocytosis and LC3 punctate-structures of macrophage were observed by confocal laser scanning microscope. Key findings CpG-ODN significantly amplified S. aureus- induced phagocytosis and autophagy of RAW264.7 and TLR9 +/+ primary peritoneal macrophage as compared to that of Non-CpG treated cells, while such effect was abolished in TLR9 −/− primary peritoneal macrophages. Meanwhile, CpG-ODN significantly enhanced S. aureus -induced phosphorylation of JNK and P38 but not ERK in RAW264.7. Specific inhibition of JNK or P38 by SP600125 or SB203580, dramatically down-regulated CpG-induced phagocytosis and autophagy in S. aureus -stimulated RAW264.7 and TLR9 +/+ primary peritoneal macrophage, while they showed no further down-regulation of phagocytosis and autophagy in TLR9 −/− primary peritoneal macrophages. Significance Our data indicated that CpG-ODN activates TLR9-JNK/P38 signaling to promote phagocytosis and autophagy in S. aureus- stimulated macrophages, these findings provide novel insights into how innate immune cells defend bacterial infection via TLR9. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) play pivotal role in the pathogenesis of allergic airway diseases such as asthma. TLR9 is one of the most extensively studied TLRs as an approach to treat asthma. In this study, we investigated the role of TLR9 in the allergic airway inflammation and the underlying mechanism. Wild-type (WT) mice and TLR9−/− mice were sensitized and challenged with OVA to establish allergic airway disease model. We found that the expression of TLR9 was elevated concomitantly with airway inflammation post-OVA challenge, and TLR9 deficiency effectively inhibited airway inflammation, including serum OVA-specific immunoglobulin E (IgE), pulmonary inflammatory cell recruitment, mucus secretion, and bronchoalveolar lavage fluid (BALF) inflammatory cytokine production. Meanwhile, the protein expression of hydroxyindole-o-methyltransferase (HIOMT) in lung tissues, the level of melatonin in serum, and BALF were reduced in OVA-challenged WT mice, while these reductions were significantly restored by TLR9 deficiency. Additionally, we showed that although TLR9 deficiency had no effect on OVA-induced phosphorylation of JNK, inhibition of JNK by specific inhibitor SP600125 significantly decreased OVA-induced expression of TLR9, suggesting that JNK is the upstream signal molecular of TLR9. Furthermore, SP600125 treatment promoted resolution of allergic airway inflammation in OVA-challenged WT mice, but not further ameliorated allergic airway inflammation in OVA-challenged TLR9−/− mice. Similarly, SP600125 significantly restored the protein expression of HIOMT and the level of melatonin in OVA-challenged WT mice, while such effect was not further enhanced by TLR9 deficiency. Collectively, our results indicated that JNK-TLR9 signal pathway mediates allergic airway inflammation through suppressing melatonin biosynthesis. [ABSTRACT FROM AUTHOR]

Background c-Jun N-terminal kinase (JNK) relays extracellular stimuli through phosphorylation cascades that lead to various cell responses. In the present study, we aimed to investigate the effect of the JNK inhibitor SP600125 on the resolution of airway inflammation, and the underlying mechanism using a murine acute asthma model. Methods Female C57BL/6 mice were sensitized with saline or ovalbumin (OVA) on day 0, and challenged with OVA on day 14–20. Meanwhile, some of the mice were treated with SP600125 (30 mg/kg) intraperitoneally 2 h before each challenge. The airway inflammation was evaluated by counting the numbers of various types of inflammatory cells in bronchoalveolar lavage fluid (BALF), histopathology, cytokines production and mucus secretion in individual mouse. In addition, we analyzed the protein levels of phosphorylated JNK and TLR9 in the lung tissues. Results SP600125 markedly reduced the invasion of inflammatory cells into the peribronchial regions, and decreased the numbers of eosinophils, monocytes, neutrophils and lymphocytes in BALF. SP600125 also reduced the level of plasma OVA-specific IgE, lowered the production of pro-inflammatory cytokines in BALF and alleviated mucus secretion. Meanwhile, SP600125 inhibited OVA-induced, increased expression of p-JNK and TLR9 in the lung tissues. Conclusions Collectively, our data demonstrated that SP600125 promoted resolution of allergic airway inflammation via TLR9 in an OVA-induced murine acute asthma model. The JNK-TLR9 pathway may be a new therapeutic target in the treatment for the allergic asthma. [ABSTRACT FROM AUTHOR]

The rational of neural stem cells ( NSCs) in the therapy of neurological disease is either to replace dead neurons or to improve host neuronal survival, the latter of which has got less attention and the underlying mechanism is as yet little known. Using a transwell co-culture system, we reported that, in organotypic brain slice cultures, NSCs significantly improved host neuronal viability. Interestingly, this beneficial effect of NSCs was abrogated by a microglial inhibitor minocycline, while it was mimicked by a microglial agonist, Toll-like receptor 9 ( TLR9) ligand CpG- ODN, which supports the pro-vital mediation by microglia on this NSCs-improved neuronal survival. Moreover, we showed that NSCs significantly induced host microglial movement and higher expression of a microglial marker IBA-1, the latter of which was positively correlated with TLR9 or extracellular-regulated protein kinases 1/2 ( ERK1/2) activation. Real-time PCR revealed that NSCs inhibited the expression of pro-inflammatory molecules, but significantly increased the expression of molecules associated with a neuroprotective phenotype such as CX3 CR1, triggering receptor expressed on myeloid cells-2 ( TREM2) and insulin growth factor 1 ( IGF-1). Similarly, in the microglia cells, NSCs induced the same microglial response as that in the slices. Further treatment with TLR9 ligand CpG- ODN, TLR9 inhibitor chloroquine ( CQ) or ERK1/2 inhibitor U0126 demonstrated that TLR9- ERK1/2 pathway was involved in the NSCs-induced microglial activation. Collectively, this study indicated that NSCs improve host neuronal survival by switching microglia from a detrimental to a neuroprotective phenotype in adult mouse brain, and the microglial TLR9- ERK1/2 pathway seems to participate in this NSCs-mediated rescue action. [ABSTRACT FROM AUTHOR]

Abstract: Toll-like receptor 2 (TLR2) is involved in phagocytosis and autophagy to enhance host innate immune response to bacterial infection. TLR2 has been reported to participate in the recognition of Staphylococcus aureus (S. aureus). However, the role of TLR2 in phagocytosis and autophagy in S. aureus-stimulated macrophages and the underlying mechanisms as yet remain unclear. In the present study, stimulation of mouse macrophage cell line RAW264.7 with S. aureus activated multiple signaling pathways including mitogen-activated protein kinases (MAPKs), myeloid differentiation factor 88 (MyD88), phosphatidylinositide 3-kinase (PI3K) and Rac1 and triggered autophagy process. Knockdown of TLR2 by siRNA significantly reduced phagocytosis and autophagy of macrophages upon S. aureus infection. Interestingly, TLR2 siRNA markedly attenuated S. aureus-induced phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 or extracellular regulated protein kinase (ERK) in macrophages. Similarly, SP600125, a JNK inhibitor, also down-regulated phagocytosis and autophagy in S. aureus-stimulated macrophages. Furthermore, TLR2 siRNA and SP600125 simultaneous treatment showed similar phagocytosis and autophagy compared to that in TLR2 siRNA treatment alone. Collectively, our results indicate that TLR2 may be critical for phagocytosis and autophagy through JNK signaling pathway, and provide an underlying mechanistic link between innate immune receptor and induction of phagocytosis and autophagy in S. aureus-stimulated macrophages. [Copyright &y& Elsevier]

As a pattern recognition receptor which activates innate immune system, toll-like receptor 2 (TLR2) has been reportedly mediates allergic airway inflammation (AAI), yet the underlying mechanism remains elusive. Here, in a murine AAI model, TLR2−/− mice showed decreased airway inflammation, pyroptosis and oxidative stress. RNA-sequencing revealed that allergen-induced hif1 signaling pathway and glycolysis were significantly downregulated when TLR2 was deficient, which were confirmed by lung protein immunoblots. Glycolysis inhibitor 2-Deoxy- d -glucose (2-DG) inhibited allergen-induced airway inflammation, pyroptosis, oxidative stress and glycolysis in wild type (WT) mice, while hif1α stabilizer ethyl 3,4-dihydroxybenzoate (EDHB) restored theses allergen-induced changes in TLR2−/− mice, indicating TLR2-hif1α-mediated glycolysis contributes to pyroptosis and oxidative stress in AAI. Moreover, upon allergen challenge, lung macrophages were highly activated in WT mice but were less activated in TLR2−/− mice, 2-DG replicated while EDHB reversed such effect of TLR2 deficiency on lung macrophages. Likewise, both in vivo and ex vivo WT alveolar macrophages (AMs) exhibited higher TLR2/hif1α expression, glycolysis and polarization activation in response to ovalbumin (OVA), which were all inhibited in TLR2−/− AMs, suggesting AMs activation and metabolic switch are dependent on TLR2. Finally, depletion of resident AMs in TLR2−/− mice abolished while transfer of TLR2−/− resident AMs to WT mice replicated the protective effect of TLR2 deficiency on AAI when administered before allergen challenge. Collectively, we suggested that loss of TLR2-hif1α-mediated glycolysis in resident AMs ameliorates allergic airway inflammation that inhibits pyroptosis and oxidative stress, therefore the TLR2-hif1α-glycolysis axis in resident AMs may be a novel therapeutic target for AAI. [Display omitted] • TLR2-hif1α-glycolysis axis is necessary for pyroptosis and oxidative stress in AAI. • TLR2-hif1α-glycolysis axis promotes lung macrophage activation in AAI. • Loss of TLR2-hif1α-glycolysis in resident AMs ameliorates AAI. [ABSTRACT FROM AUTHOR]

Activation of toll-like receptor (TLR) signaling that initiates an innate immune response to pathogens must be strictly regulated to prevent excessive inflammatory damage in the host. Here, we demonstrate that Mer receptor tyrosine kinase (MerTK) is a negative regulatory molecule in the lipoteichoic acid (LTA)-induced inflammatory response. LTA that activated TLR2 signaling concomitantly induced activation of MerTK signaling in RAW264.7 macrophages, including phosphoinositide 3-kinase (PI3K)/Akt and suppressor of cytokine signaling 3 (SOCS3). Moreover, LTA induced MerTK activation in a time-dependent manner, and LTA-induced MerTK activation was dependent on the ligand Gas6. Additionally, pretreatment with a specific Mer-blocking antibody significantly inhibited LTA-induced phosphorylation of MerTK, while further enhancing LTA-induced phosphorylation of IκB-α and NF-κBp65 as well as production of TNF-α and IL-6. Meanwhile, the antibody blockade of MerTK markedly prevented LTA-induced Akt phosphorylation and SOCS3 expression, both of which were crucial for the inhibition of TLR2-mediated immune response. Collectively, these results suggest, for the first time, that MerTK is an intracellular negative feedback regulator that inhibits the inflammatory response of LTA-stimulated macrophages through the PI3K/Akt pathway and SOCS3 protein. [ABSTRACT FROM AUTHOR]

• TLR9 servers as a pro-inflammatory regulator of OVA-induced airway inflammation. • OVA-induced activation of NLRP3 inflammasome was dependent on TLR9 signal. • OVA-induced oxidative stress was mediated by TLR9 signal. • TLR9 mediates the activation of NLRP3 inflammasome and oxidative stress in S.aureus -stimulated Raw264.7 cells. Toll-like receptor 9 (TLR9) has been reported to mediate airway inflammation, however, the underlying mechanism is poorly understood. In the present study, our objective was to reveal whether TLR9 regulates NLRP3 inflammasome and oxidative stress in murine allergic airway inflammation and Raw264.7 cells. Female wild type(WT)and TLR9−/−mice on C57BL/6 background were used to induce allergic airway inflammation by challenge of OVA, and Raw264.7 cells with or without TLR9 knockdown by small interfering RNA (siRNA) were stimulated by S.aureus. The results demonstrated that deletion of TLR9 effectively attenuated OVA-induced allergic airway inflammation including inflammatory cells infiltration and goblet cell hyperplasia. Meanwhile, OVA-induced protein expression of NLRP3, caspase-1(p20) and mature IL-1β, as well as secretion of IL-1β and IL-18 in wild type mice (WT) was obviously suppressed by TLR9 deficiency. Concomitantly, the expression of oxidative markers 8-OhDG and nitrotyrosine was increased in OVA-challenged WT mice, while TLR9 deficiency significantly inhibited such increase. Similarly, in the in vitro study, we found that knockdown of TLR9 markedly suppressed S.aureus- induced activation of NLRP3 inflammasome and oxidative stress in Raw264.7 cells. Collectively, our findings indicated that TLR9 may mediate allergic airway inflammation via activating NLRP3 inflammasome and oxidative stress. [ABSTRACT FROM AUTHOR]