Tanreqing Injection Attenuates Macrophage Activation and the Inflammatory Response via the lncRNA-SNHG1/HMGB1 Axis in Lipopolysaccharide-Induced Acute Lung Injury.

The etiology of acute lung injury (ALI) is not clear, and the treatment of ALI presents a great challenge. This study aimed to investigate the pathogenesis and potential therapeutic targets of ALI and to define the target gene of Tanreqing (TRQ), which is a traditional Chinese medicine formula composed of five medicines, scutellaria baicalensis, bear bile powder, goat horn powder, honeysuckle and forsythia. Macrophage activation plays a critical role in many pathophysiological processes, such as inflammation. Although the regulation of macrophage activation has been extensively investigated, there is little knowledge of the role of long noncoding RNAs (lncRNAs) in this process. In this study, we found that lncRNA-SNHG1 expression is distinctly regulated in differently activated macrophages in that it is upregulated in LPS. LncRNA-SNHG1 knockdown attenuates LPS-induced M1 macrophage activation. The SNHG1 promoter was bound by NF-κB subunit p65, indicative of SNHG1 being a direct transcriptional target of LPS-induced NF-κB activation. SNHG1 acts as a proinflammatory driver that leads to the production of inflammatory cytokines and the activation of macrophages and cytokine storms by physically interacting with high-mobility group box 1 (HMGB1) in ALI. TRQ inhibited NF-κB signaling activation and binding of NF-κB to the SNHG1 promoter. In conclusion, this study defined TRQ target genes, which can be further elucidated as mechanism(s) of TRQ action, and provides insight into the molecular pathogenesis of ALI. The lncRNA-SNHG1/HMGB1 axis is an ideal therapeutic for ALI treatment.

Extracellular vesicles from M1-polarized macrophages promote inflammation in the temporomandibular joint via miR-1246 activation of the Wnt/ß-catenin pathway.

Macrophage-mediated regulation of chondrocytes plays an important role in promoting temporomandibular joint (TMJ) inflammation. We investigated whether extracellular vesicles (EVs) derived from M1 macrophages (M1-EVs) have a proinflammatory effect on TMJ inflammation and what the associated mechanisms are. In vitro, purified THP-1 cell-derived M1-EVs were applied to human TMJ condylar chondrocytes, and in vivo M1-EVs derived from bone marrow-derived macrophages (BMDMs) were injected into rat TMJs. The levels of IL-6, IL-8, IL-1ß, and matrix metalloproteinase were then evaluated and found to be upregulated in the chondrocytes and rat TMJs. MicroRNA sequencing analysis was performed to identify differential expression of miRNAs, including miR-1246. High expression of miR-1246 in M1-EVs from synovial fluid of patients with TMJ osteoarthritis and synovitis was verified by RT-PCR. We then identified miR-1246 targets GSK3ß and Axin2 and found that miR-1246 inhibits GSK3ß and Axin2 expression, causing activation of the Wnt/ß-catenin pathway and inflammation in condylar chondrocytes. Our study found that M1-EVs promote inflammation by transfer of miR-1246 to condylar chondrocytes, thus providing new insight into one mechanism that can promote TMJ inflammation.

Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1ß Primed Tendon Cells.

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.

Tumor-Treating Fields Induce RAW264.7 Macrophage Activation Via NK-κB/MAPK Signaling Pathways.

OBJECTIVE: Tumor-treating fields are currently used to successfully treat various cancers; however, the specific pathways associated with its efficacy remain unknown in the immune responses. Here, we evaluated tumor-treating fields-mediated initiation of the macrophage-specific immune response. MATERIALS AND METHODS: We subjected RAW 264.7 mouse macrophages to clinically relevant levels of tumor-treating fields (0.9 V/cm, 150 kHz) and evaluated alterations in cytokine expression and release, as well as cell viability. Additionally, we investigated the status of immunomodulatory pathways to determine their roles in tumor-treating fields-mediated immune activation. RESULTS AND DISCUSSION: Our results indicated that tumor-treating fields treatment at 0.9 V/cm decreased cell viability and increased cytokine messenger RNA/protein levels, as well as levels of nitric oxide and reactive oxygen species, relative to controls. The levels of tumor necrosis factor α, interleukin 1ß, and interleukin 6 were markedly increased in tumor-treating fields-treated RAW 264.7 cells cocultured with 4T1 murine mammary carcinoma cells compared with those in 4T1 or RAW 264.7 cells with or without tumor-treating fields treatment. Moreover, the viability of 4T1 cells treated with the conditioned medium of tumor-treating fields-stimulated RAW 264.7 cells decreased, indicating that macrophage activation by tumor-treating fields effectively killed the tumor cells. Moreover, tumor-treating fields treatment activated the nuclear factor κB and mitogen-activated protein kinase pathways involved in immunomodulatory signaling. CONCLUSION: These results provide critical insights into the mechanisms through which tumor-treating fields affect macrophage-specific immune responses and the efficacy of this method for cancer treatment.

Impaired microglia process dynamics post-stroke are specific to sites of secondary neurodegeneration.

Stroke induces tissue death both at the site of infarction and at secondary sites connected to the primary infarction. This latter process has been referred to as secondary neurodegeneration (SND). Using predominantly fixed tissue analyses, microglia have been implicated in regulating the initial response at both damage sites post-stroke. In this study, we used acute slice based multiphoton imaging, to investigate microglia dynamic process movement in mice 14 days after a photothrombotic stroke. We evaluated the baseline motility and process responses to locally induced laser damage in both the peri-infarct (PI) territory and the ipsilateral thalamus, a major site of post-stroke SND. Our findings show that microglia process extension toward laser damage within the thalamus is lost, yet remains robustly intact within the PI territory. However, microglia at both sites displayed an activated morphology and elevated levels of commonly used activation markers (CD68, CD11b), indicating that the standardly used fixed tissue metrics of microglial "activity" are not necessarily predictive of microglia function. Analysis of the purinergic P2 Y12 receptor, a key regulator of microglia process extension, revealed an increased somal localization on nonresponsive microglia in the thalamus. To our knowledge, this is the first study to identify a non-responsive microglia phenotype specific to areas of SND post-stroke, which cannot be identified by the classical assessment of microglia activation but rather the localization of P2 Y12 to the soma.

Chamomile Flower, Myrrh, and Coffee Charcoal, Components of a Traditional Herbal Medicinal Product, Diminish Proinflammatory Activation in Human Macrophages.

A traditional herbal medicinal product, containing myrrh, chamomile flower, and coffee charcoal, has been used in Germany for the relief of gastrointestinal complaints for decades. Clinical studies suggest its use in the maintenance therapy of inflammatory bowel disease. However, the pharmacological mechanisms underlying the clinical effects are not yet fully understood.The present study aims to elucidate immunopharmacological activities of myrrh, chamomile flower, and coffee charcoal by studying the influence of each plant extract on gene expression and protein release of activated human macrophages.The plant extracts effect on gene and protein expression of activated human monocyte-derived macrophages was investigated by microarray gene expression analysis and assessment of the release of pro- and anti-inflammatory mediators (TNFα, chemokine CXCL13, and interleukin-10) using an ELISA test system.The extracts of myrrh, chamomile flower, and coffee charcoal influenced gene expression of activated human macrophages within the cytokine/chemokine signaling pathway. Particularly, chemokine gene expression was suppressed. Subsequently, the production of CXCL13 and, to a minor extent, cytokine TNFα was inhibited by all herbal extracts. Chamomile flower and coffee charcoal extracts enhanced interleukin-10 release from activated macrophages. The observed effects on protein release were comparable to the effect of budesonide, which decreased TNFα and CXCL13 and enhanced interleukin-10 release.The components of the herbal medicinal product influence the activity of activated human macrophages on both gene and protein level. The induced alterations within chemokine/cytokine signaling could contribute to a positive effect on the immunological homeostasis, which is disturbed in patients with chronic intestinal inflammation.

N-acylethanolamine-hydrolyzing acid amidase and fatty acid amide hydrolase inhibition differentially affect N-acylethanolamine levels and macrophage activation.

N-acylethanolamines (NAEs) such as N-palmitoylethanolamine and anandamide are endogenous bioactive lipids having numerous functions, including the control of inflammation. Their levels and therefore actions can be controlled by modulating the activity of two hydrolytic enzymes, N-acylethanolamine-hydrolyzing acid amidase (NAAA) and fatty acid amide hydrolase (FAAH). As macrophages are key to inflammatory processes, we used lipopolysaccharide-activated J774 macrophages, as well as primary mouse alveolar macrophages, to study the effect of FAAH and NAAA inhibition, using PF-3845 and AM9053 respectively, on macrophage activation and NAE levels measured by HPLC-MS. Markers of macrophage activation were measured by qRT-PCR and ELISA. Activation of macrophages decreased NAAA expression and NAE hydrolytic activity. FAAH and NAAA inhibition increased the levels of the different NAEs, although with different magnitudes, whether in control condition or following LPS-induced macrophage activation. Both inhibitors reduced several markers of macrophage activation, such as mRNA expression of inflammatory mediators, as well as cytokine and prostaglandin production, with however some differences between FAAH and NAAA inhibition. Most of the NAEs tested - including N-docosatetraenoylethanolamine and N-docosahexaenoylethanolamine - also reduced LPS-induced mRNA expression of inflammatory mediators, again with differences depending on the marker and the NAE, thus offering a potential explanation for the differential effect of the inhibitors on macrophage activation markers. In conclusion, we show different and complementary effects of NAE on lipopolysaccharide-induced macrophage activation. Our results support an important role for inhibition of NAE hydrolysis and NAAA inhibition in particular in controlling macrophage activation, and thus inflammation.

Lotus leaf (Nelumbo nucifera) and its active constituents prevent inflammatory responses in macrophages via JNK/NF-κB signaling pathway.

Inflammation is a serious health issue worldwide that induces many diseases, such as inflammatory bowel disease (IBD), sepsis, acute pancreatitis and lung injury. Thus, there is a great deal of interest in new methods of limiting inflammation. In this study, we investigated the leaves of Nelumbo nucifera Gaertn, an aquatic perennial plant cultivated in eastern Asia and India, in anti-inflammatory pharmacological effects in the murine macrophage cell line RAW264.7. Results showed that lipopolysaccharide (LPS) increased the protein expression of inducible nitric oxide synthase (iNOS) and COX-2, as well as the mRNA expression and level of IL-6 and TNF-α, while NNE significantly reduced these effects of LPS. LPS also induced phospho-JNK protein expression. The JNK-specific inhibitor SP600125 decreased the proteins expression of phospho-JNK, iNOS, COX-2, and the mRNAs expression and levels of IL-6 and TNF-α. Further, NNE reduced the protein expression of phospho-JNK. LPS was also found to promote the translocation of NF-κB from the cytosol to the nucleus and to decrease the expression of cytosolic IκB. NNE and SP600125 treatment recovered the LPS-induced expression of NF-κB and IκB. While phospho-ERK and phospho-p38 induced by LPS, could not be reversed by NNE. To further investigate the major components of NNE in anti-inflammatory effects, we determined the quercetin and catechin in inflammatory signals. Results showed that quercetin and catechin significantly decreased the proteins expression of iNOS, COX-2 and phospho-JNK. Besides, the mRNAs and levels of IL-6 and TNF-α also decreased by quercetin and catechin treatment in LPS-induced RAW264.7 cells. These results showed that NNE and its major components quercetin and catechin exhibit anti-inflammatory activities by inhibiting the JNK- and NF-κB-regulated pathways and could therefore be an useful anti-inflammatory agent.

Anti-inflammatory effect of Lycii radicis in LPS-stimulated RAW 264.7 macrophages.

The root bark of Lycium barbarum (Lycii radicis cortex, LRC) is used as a cooling agent for fever and night sweats in East Asian traditional medicine. The inhibitory effect of LRC water extract on inflammation is unknown. In this study, the anti-inflammatory effect of LRC was investigated in lipopolysaccharide (LPS)-stimulated mouse macrophage, RAW 264.7 cells. LRC extract significantly decreased the LPS-induced production of inflammatory mediators, nitric oxide (NO), prostaglandin (PG) E2 and pro-inflammatory cytokines, interleukin (IL)-1ß and IL-6 in the cells. In addition, LRC extract inhibited the LPS-induced expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 mRNA and protein, and inflammatory cytokines mRNA in the cells. The action mechanism of LRC underlies the blocking of LPS-mediated p38 and Jun N-terminal kinase (JNK), mitogen-activated protein kinases (MAPKs), and the nuclear factor (NF)-κB signaling pathway. These results indicate that LRC extract inhibits the inflammatory response in activated macrophages by down-regulating the transcription levels of inflammatory mediators and blocking the MAPKs and NF-κB pathway.

Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK.

BACKGROUND & AIMS: Mechanisms underlying synergistic liver injury caused by alcohol and obesity are not clear. We have produced a mouse model of synergistic steatohepatitis by recapitulating the natural history of the synergism seen in patients for mechanistic studies. METHODS: Moderate obesity was induced in mice by 170% overnutrition in calories using intragastric overfeeding of high fat diet. Alcohol (low or high dose) was then co-administrated to determine its effects. RESULTS: Moderate obesity plus alcohol intake causes synergistic steatohepatitis in an alcohol dose-dependent manner. A heightened synergism is observed when a high alcohol dose (32g/kg/d) is used, resulting in plasma ALT reaching 392±28U/L, severe steatohepatitis with pericellular fibrosis, marked M1 macrophage activation, a 40-fold induction of iNos, and intensified nitrosative stress in the liver. Hepatic expression of genes for mitochondrial biogenesis and metabolism are significantly downregulated, and hepatic ATP level is decreased. Synergistic ER stress evident by elevated XBP-1, GRP78 and CHOP is accompanied by hyperhomocysteinemia. Despite increased caspase 3/7 cleavage, their activities are decreased in a redox-dependent manner. Neither increased PARP cleavage nor TUNEL positive hepatocytes are found, suggesting a shift of apoptosis to necrosis. Surprisingly, the synergism mice have increased plasma adiponectin and hepatic p-AMPK, but adiponectin resistance is shown downstream of p-AMPK. CONCLUSIONS: Nitrosative stress mediated by M1 macrophage activation, adiponectin resistance, and accentuated ER and mitochondrial stress underlie potential mechanisms for synergistic steatohepatitis caused by moderate obesity and alcohol.

Activation of murine macrophages via TLR2 and TLR4 is negatively regulated by a Lyn/PI3K module and promoted by SHIP1.

Src family kinases are involved in a plethora of aspects of cellular signaling. We demonstrate in this study that the Src family kinase Lyn negatively regulates TLR signaling in murine bone marrow-derived macrophages (BMM Phis) and in vivo. LPS-stimulated Lyn(-/-) BMM Phis produced significantly more IL-6, TNF-alpha, and IFN-alpha/beta compared with wild type (WT) BMM Phis, suggesting that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMM Phis in response to the TLR2 ligand FSL-1, but not to ligands for TLR3 (dsRNA) or TLR9 (CpG 1668). In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than did WT mice after i. v. injection of LPS or FSL-1. Although Lyn clearly acted as a negative regulator downstream of TLR4 and TLR2, it did not, different from what was proposed previously, prevent the induction of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after subsequent stimulation with a high dose of LPS in both WT and Lyn(-/-) BMM Phis, as well as in vivo. Mechanistically, Lyn interacted with PI3K; in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1(-/-) BMM Phis, exerting enhanced PI3K-pathway activation, produced fewer cytokines than did WT BMM Phis. The data suggest that the Lyn-mediated negative regulation of TLR signaling proceeds, at least in part, via PI3K.

Gene expression profiling of macrophages following mice treatment with an immunomodulator medication.

Canova (CA) is a complex homeopathic medication used in diseases where the immune system is depressed. Previous studies demonstrated that it is neither toxic nor mutagenic and activates macrophages. We now evaluate CA effects on cytokine production and gene expression from mice macrophages. The global view of changes in expression of genes with known functions can provide a vivid picture of the way in which cell adapts to a changing environment or a challenge. We found a decrease in IL-2 and IL-4 production and a differential expression in 147 genes from CA group. These genes are mainly involved in transcription/translation, cell structure and dynamics, immune response, cytoprotection, enzymatic process, and receptors/ligands. With gene expression analysis we state that this medication provokes a reaction that involves alterations in gene expression profile mainly in the ones involved with macrophages activation, corroborating the laboratorial research and the clinical data.

IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype.

BACKGROUND: "Alternatively-activated" macrophages are found in Th2-mediated inflammatory settings such as nematode infection and allergic pulmonary inflammation. Due in part to a lack of markers, these cells have not been well characterized in vivo and their function remains unknown. RESULTS: We have used murine macrophages elicited by nematode infection (NeM(phi)) as a source of in vivo derived alternatively activated macrophages. Using three distinct yet complementary molecular approaches we have established a gene expression profile of alternatively activated macrophages and identified macrophage genes that are regulated in vivo by IL-4. First, genes abundantly expressed were identified by an expressed sequence tag strategy. Second, an array of 1176 known mouse genes was screened for differential expression between NeM(phi) from wild type or IL-4 deficient mice. Third, a subtractive library was screened to identify novel IL-4 dependent macrophage genes. Differential expression was confirmed by real time RT-PCR analysis. CONCLUSIONS: Our data demonstrate that alternatively activated macrophages generated in vivo have a gene expression profile distinct from any macrophage population described to date. Several of the genes we identified, including those most abundantly expressed, have not previously been associated with macrophages and thus this study provides unique new information regarding the phenotype of macrophages found in Th2-mediated, chronic inflammatory settings. Our data also provide additional in vivo evidence for parallels between the inflammatory processes involved in nematode infection and allergy.

Isolation of nine gene sequences induced by silica in murine macrophages.

Macrophage activation by silica is the initial step in the development of silicosis. To identify genes that might be involved in silica-mediated activation, RAW 264.7 mouse macrophages were treated with silica for 48 h, and a subtracted cDNA library enriched for silica-induced genes (SIG) was constructed and differentially screened. Nine cDNA clones (designated SIG-12, -14, -20, -41, -61, -81, -91, -92, and -111) were partially sequenced and compared with sequences in GenBank/EMBL databases. SIG-12, -14, and -20 corresponded to the genes for ribosomal proteins L13a, L32, and L26, respectively. SIG-61 is the mouse homologue of p21 RhoC. SIG-91 is identical to the 67-kDa high-affinity laminin receptor. Four genes were not identified and are novel. All of the mRNAs corresponding to the nine cloned cDNAs were inducible by silica. Steady-state levels of mRNAs in RAW 264.7 cells treated with various macrophage activators and inducers of signal transduction pathways were determined. A complex pattern of induction and repression was found, indicating that upon phagocytosis of silica particles, many regulatory mechanisms of gene expression are simultaneously triggered.

Molecular biology of macrophage activation: a pathway whereby psychosocial factors can potentially affect health.

Over the past decade, numerous links between the mind, the immune system, and the nervous system have been clearly elucidated in regard to the lymphoid elements of the immune system. Recently, attention has been given to the suggestion that such links exist to mononuclear phagocytes. The fundamental biology and physiology of these cells are clearly consonant with such a role. Mononuclear phagocytes bear receptors on their surface for numerous ligands, including neuroendocrine peptides and hormones. Furthermore, macrophages can be modulated by a variety of life-style choices such as smoking, ingestion of alcohol, and dietary lipid. Mononuclear phagocytes are key cells in the development of atherosclerosis and play significant roles in host protection against neoplasia and in the development of certain autoimmune diseases. In regard to atherogenesis, stress can potentiate the effects of a high lipid diet in initiating formation of the macrophage-laden lesions of early atherosclerosis. We have been able to show that oxidized lipids, acting in concert with stress as modeled pharmacologically in terms of catecholamines, induces a macrophage phenotype which would likely promote the development of atherosclerosis but lower host resistance to neoplasia. We are currently testing this hypothesis critically. Over the coming decade, it will be important for numerous investigators to examine the broad hypothesis that mononuclear phagocytes are one key element in the induction of psychosocially produced diseases. A clear understanding of the biology and molecular biology of these cells will provide a potent tool for analyzing these problems.

Molecular cloning and characterization of a murine LPS-inducible cDNA.

Murine macrophages respond to endotoxins by inducing a vast array of genes that play a major role in the host's response to infection and tumor growth. We have isolated and characterized a 1.8-kb cDNA, designated IRG2, from a cDNA library prepared from RNA isolated from the murine cell line, RAW 264.7, after bacterial LPS stimulation. The cDNA encodes a protein of 47 kDa that is the murine homologue of a small family of proteins described from IFN-induced human cells. The IRG2 message does not appear until 3 h after LPS exposure and its induction is dependent on new protein synthesis. IRG2 induction by LPS is slightly inhibited by the anti-inflammatory steroid, dexamethasone. Increasing cytosolic cAMP with either forskolin, dibutyryl cAMP, or 8-(4-chlorophenylthio)-cAMP caused marked inhibition of the LPS induction of IRG2. In contrast, activation of PKC with phorbol ester potentiated the LPS response. Removing extracellular Ca2+ with EGTA inhibited IRG2 induction; increasing intracellular calcium with the calcium ionophore A23187 led to enhanced levels of the IRG2 transcript. These data suggest that the induction of IRG2 occurs via a PKC pathway.