Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Apoptotic neutrophils and nitric oxide regulate cytokine production by IFN-γ-stimulated macrophages

Early apoptotic neutrophils but not secondary necrotic ones down-regulate LPS-induced proinflammatory cytokine production of macrophages, thereby contributing to the resolution of inflammation. IFN-γ is also a well-known stimulant of macrophages, but how the apoptotic neutrophils affect IFN-γ-stimulated macrophages remains largely unexplored. Since IFN-γ induces the expression of inducible nitric oxide (NO) synthase, we examined the production of NO and various cytokines, including MIP-2, TNF-α, IL-12p40, IL-6, IL-10, and TGF-β, by IFN-γ-stimulated murine macrophages, the effect of coculturing the macrophages with early apoptotic or secondary necrotic neutrophils, and the regulatory role of NO in such cocultures. IFN-γ induced significant production of NO, IL-12p40, and IL-6 by macrophages, but not other cytokines. Early apoptotic neutrophils but not secondary necrotic ones promoted NO production, whereas secondary necrotic ones and their supernatants promoted TNF-α production. In contrast, both early apoptotic and secondary necrotic neutrophils suppressed IL-12p40 and IL-6 production. Furthermore, macrophages from inducible NO synthase-deficient mice produced significantly higher levels of MIP-2 than those from wild-type mice. Consistent with this, treatment of macrophages with l-NAME, an NO synthase inhibitor, also induced the production of a large amount of MIP-2. In conclusion, this study suggests that early apoptotic neutrophils are critical in the resolution of inflammation, but that secondary necrotic neutrophils may not cause an inflammatory response. Apoptotic neutrophils, however, appear not to modulate cytokine production via NO.     

The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures

Background

Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-β1. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an α-smooth muscle actin-Cre transgenic mouse (α-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-β1 stimulation in vitro.

Methods

We generated the α-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse α-smooth muscle actin (α-SMA) promoter. α-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain α-SMA-Cre/R26R. β-galactosidase (βgal) staining, α-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in α-SMA-Cre/R26R mice was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-β1 and examined the localization of the myofibroblast markers α-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence.

Results

βgal staining in organs of healthy α-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by α-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. In vitro, addition of TGF-β1 to 16HBE cells could also stimulate the expression of α-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT.

Conclusion

We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.     

Picrasma quassioides inhibits LPS- and IFN-γ-stimulated nitric oxide production and inflammatory response in RAW264.7 macrophage cells

The present study was designed to investigate the anti-inflammatory effects of Picrasma quassioides (P. quassioides) in lipopolysaccharide (LPS)- and interferon (IFN)-γ-stimulated RAW 264.7 cells. P. quassioides has been used as a traditional medicine for the treatment of gastro-enteritis, eczema, and snakebite. P. quassioides significantly decreased LPS- and IFN-γ-stimulated nitric oxide (NO) production in a concentration-dependent manner. Real-time PCR or Western blotting confirmed that the expression of the extra-cellular signal-regulated kinase (ERK), p42 and, p38 mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mediated MAPK signaling pathways in LPS- and IFN-γ-stimulated RAW264.7 cells. Mechanistic studies revealed the activities of nuclear factor kappa B (NF-κB). As P. quassioides regulated the gene expression of iNOS and COX-2 in RAW264.7 cells, it might be a promising agent for the prevention and/or treatment of various inflammatory diseases.     

Suppression of nitric oxide production on LPS/IFN-γ-stimulated RAW264.7 macrophages by a novel catechin, pilosanol N, from Agrimonia pilosa Ledeb

A novel catechin, pilosanol N (1), was isolated from Agrimonia pilosa Ledeb and its structure was determined by (1)H, (13)C NMR and HRESI-MS analyses. Compound 1 inhibited nitric oxide (NO) production in LPS/IFN-γ -induced RAW264.7 macrophages, and then the iNOS gene expression and its protein production with LPS/IFN-γ treatment cells were also suppressed in the presence of 1. In addition, compound 1 scavenged NO or nitrogen radicals generated from NOR3 (4-ethyl-2-hydroxyamino-5-nitro-3-hexenamide) as an NO donor. These results indicated that pilosanol N can decrease the level of NO through a mechanism that involved both a decrease in the NO production and NO scavenging.     

Suppression of iNOS expression by fucoidan is mediated by regulation of p38 MAPK,JAK/STAT,AP-1 and IRF-1, and depends on up-regulation of scavenger receptor B1 expression in TNF-α- and IFN-γ-stimulated C6 glioma cells

In neurodegenerative disorders, activated glial cells overproduce nitric oxide (NO), which causes neurotoxicity. Inducible NO synthase (iNOS) is a potential therapeutic target in neurodegenerative diseases. Here, we examined the action of fucoidan, a high-molecular-weight sulfated polysaccharide, on tumor necrosis factor-α (TNF-α)- and interferon-γ (IFN-γ)-induced NO production in C6 glioma cells. Fucoidan suppressed TNF-α- and IFN-γ-induced NO production and iNOS expression. In addition, fucoidan inhibited TNF-α- and IFN-γ-induced AP-1, IRF-1, JAK/STAT and p38 mitogen-activated protein kinase (MAPK) activation and induced scavenger receptor B1 (SR-B1) expression. Blocking of SR-B1 did not reverse the inhibitory effect of fucoidan on TNF-α- and IFN-γ- stimulated NO production. However, inhibition of SR-B1 expression by siRNA increased iNOS expression and p38 phosphorylation in TNF-α- and IFN-γ-stimulated C6 cells.Overall, p38 MAPK, AP-1, JAK/STAT and IRF-1 play an important role in the inhibitory effect of fucoidan on TNF-α- and IFN-γ-stimulated NO production, and intracellular SR-B1 expression may be related to the inhibition of iNOS expression by fucoidan via regulation of p38 phosphorylation. The present results also suggest that fucoidan could be a potential therapeutic agent for treating inflammatory-related neuronal injury in neurological disorders.     

Regulation of chemokine and chemokine receptor expression by PPARγ in adipocytes and macrophages

Background

PPARγ plays a key role in adipocyte biology, and Rosiglitazone (Rosi), a thiazolidinedione (TZD)/PPARγ agonist, is a potent insulin-sensitizing agent. Recent evidences demonstrate that adipose tissue inflammation links obesity with insulin resistance and that the insulin-sensitizing effects of TZDs result, in part, from their anti-inflammatory properties. However the underlying mechanisms are unclear.

Methodology and Principal Findings

In this study, we establish a link between free fatty acids (FFAs) and PPARγ in the context of obesity-associated inflammation. We show that treatment of adipocytes with FFAs, in particular Arachidonic Acid (ARA), downregulates PPARγ protein and mRNA levels. Furthermore, we demonstrate that the downregulation of PPARγ by ARA requires the activation the of Endoplamsic Reticulum (ER) stress by the TLR4 pathway. Knockdown of adipocyte PPARγ resulted in upregulation of MCP1 gene expression and secretion, leading to enhanced macrophage chemotaxis. Rosi inhibited these effects. In a high fat feeding mouse model, we show that Rosi treatment decreases recruitment of proinflammatory macrophages to epididymal fat. This correlates with decreased chemokine and decreased chemokine receptor expression in adipocytes and macrophages, respectively.

Conclusions and Significance

In summary, we describe a novel link between FAs, the TLR4/ER stress pathway and PPARγ, and adipocyte-driven recruitment of macrophages. We thus both describe an additional potential mechanism for the anti-inflammatory and insulin-sensitizing actions of TZDs and an additional detrimental property associated with the activation of the TLR4 pathway by FA.     

Suppression of LPS-induced inflammatory and NF-κB responses by anomalin in RAW 264.7 macrophages

The treatment of inflammatory diseases today is largely based on interrupting the synthesis or action of the mediators that drive the host's response to injury. It is on the basis of this concept that most of the anti-inflammatory drugs have been developed. In our continuous search for novel anti-inflammatory agents from traditional medicinal plants, Saposhnikovia divaricata has been a focus of our investigations. Anomalin, a pyranocoumarin constituent of S. divaricata, exhibits potent anti-inflammatory activity. To clarify the cellular signaling mechanisms underlying the anti-inflammatory action of anomalin, we investigated the effect of anomalin on the production of inflammatory molecules in LPS-stimulated murine macrophages. The anomalin dose-dependently inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression in LPS-stimulated RAW 264.7 macrophage. Molecular analysis using quantitative real time polymerase chain reaction (qRT-PCR) revealed that several pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced by anomalin, and this reduction correlated with the down-regulation of the NF-κB signaling pathway. In addition, anomalin suppressed the LPS-induced phosphorylation and degradation of IκBα. To further study the mechanisms underlying its anti-inflammatory activity, an electrophoretic mobility shift assay (EMSA) using a (32) P-labeled NF-κB probe was conducted. LPS-induced NF-κB DNA binding was drastically abolished by anomalin. The present data suggest that anomalin is a major anti-inflammatory agent and may be a potential therapeutic candidate for the treatment of inflammatory disorders.     

oxLDL-Induced Lipid Accumulation in Glomerular Podocytes: Role of IFN-γ, CXCL16, and ADAM10

Previous studies have shown that lipid accumulation plays an important role in the pathogenesis and development of glomerular sclerosis. oxLDL caused damage in renal mesangial cells, endothelial cells, and podocytes, and podocytes might be the major victim of oxLDL insult. However, the regulatory mechanism of how oxLDL induces the damage of podocytes remains to be elucidated. In this study, oil red staining was used to investigate the lipid accumulation in podocytes. Moreover, the effects of CXCL16 antibody, IFN-γ, and ADAM10 inhibitor on oxLDL intake and CXCL16 expression were also explored to elucidate the regulatory factors of lipid accumulation in podocytes.     

PRSS14/Epithin is induced in macrophages by the IFN-γ/JAK/STAT pathway and mediates transendothelial migration

PRSS14/Epithin (also known as matriptase and ST14), a member of the type II transmembrane serine proteases, is primarily found in a subpopulation of normal epithelial cells and in epithelial cancers. Its known functions include maintaining the epithelial barrier, thymic development, and cancer progression. In this study, we show that several macrophage cell lines and activated bone marrow-derived macrophages also express PRSS14/Epithin. Surface expression, as well as cytoplasmic expression, was detectable upon activation by IFN-γ, but not TNF-α or TGF-β. Induction of the protein appeared to be restricted to macrophages. IFN-γ showed a biphasic regulation in RAW264.7 cells, and upregulated expression was sustained for several days. This induction by IFN-γ was partially through the increase of PRSS14/Epithin mRNA production, which is downstream of the JAK pathway, shown by the inhibition by tyrphostin AG490. Using chromatin immunoprecipitation, we verified that two sites among six putative STAT1 binding sites in the PRSS14/Epithin promoter were occupied by STAT1 upon activation. Treatment with IFN-γ enhanced the serum-triggered transendothelial migration of RAW264.7 cells, but not that of PRSS14/Epithin knock-down RAW264.7 cells, although they express multiple markers such as ICAM1, CD80, and CD40 at normal levels. These data strongly suggest that PRSS14/Epithin plays an important role in the transendothelial migration of activated macrophages in the inflammatory microenvironment, and the mode of action is similar to the events in cancer metastasis.     

Improved survival in tumor-bearing SCID mice treated with interferon-γ-inducible protein 10 (IP-10/CXCL10)

Tumor growth requires angiogenesis, which in turn requires an imbalance in the presence of angiogenic and angiostatic factors. We have shown that the CXC chemokine family, consisting of members that are either angiogenic or angiostatic, is a major determinant of tumor-derived angiogenesis in non-small-cell lung cancer (NSCLC). Intratumor injection of interferon-inducible protein 10 (IP-10, or CXCL10), an angiostatic CXC chemokine, led to reduced tumor growth in a SCID mouse model of NSCLC. In this study, we hypothesized that treatment with CXCL10 would, by restoring the angiostatic balance, improve long-term survival in NSCLC-bearing SCID mice. To test this hypothesis, A549 NSCLC cells were injected in the subcutis of the flank, followed by intratumor injections with CXCL10 continuously (group I), or for ten weeks (group II), or a control group (human serum albumin). Median survival was 169, 130, and 86 days respectively (P<0.0001). We extended these studies to examine the mechanism of prolonged survival in CXCL10-treated mice. CXCL10 treatment inhibited lung metastases, but was dependent upon continued treatment, and was associated with an increased rate of apoptosis in the primary tumor, with no direct effect on the proliferation of the NSCLC cells. Furthermore, the inhibition of lung metastases was due to the angiostatic effect of CXCL10 on the primary tumor, since the rate of apoptosis within lung metastases was unaffected. These data suggest that anti-angiogenic therapy of human lung cancer should be continued indefinitely to realize persistent benefit, and confirms the anti-metastatic capacity of localized angiostatic therapy.     

Suppression of IRF4 by IRF1, 3, and 7 in Noxa expression is a necessary event for IFN-γ-mediated tumor elimination

IFN-γ plays a critical role in tumor immunosurveillance by affecting either immune cells or tumor cells; however, IFN-mediated effects on tumor elimination are largely unknown. In this study, we showed that IFN regulatory factors (IRF) modulated by IFNs up- and downregulated Noxa expression, a prodeath BH3 protein, in various cancer cells. Inhibition of Noxa expression using short hairpin RNA in tumor cells leads to resistance against lipopolysaccharide (LPS)-induced tumor elimination, in which IFN-γ is known as a critical effecter in mice. Chromatin immunoprecipitation analysis in both CT26 cells and SP2/0 cells, sensitive and resistant to LPS-induced tumor elimination, respectively, revealed that the responsiveness of IRF1, 3, 4, and 7 in the Noxa promoter region in response to IFN-γ might be crucial in LPS-induced tumor elimination. IRF1, 3, and 7 were upregulated by IFN-γ and activated Noxa expression, leading to the death of Noxa wild-type baby mouse kidney (BMK) cells but not of Noxa-deficient BMK cells. In contrast, IRF4 acts as a repressor for Noxa expression and inhibits cell death induced by IRF1, 3, or 7. Therefore, although IFN-γ alone are not able to induce cell death in tumor cells in vitro, Noxa induction by IFN-γ, which is regulated by the balance between its activators (IRF1, 3, and 7) and its repressor (IRF4), is crucial to increasing the susceptibility of tumor cells to immune cell-mediated cytotoxicity.     

Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF

The GG₂EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferationin vitro have recently been performed. We observed that the combination of 5–25 U/ml recombinant mouse interferon- (rmIFN-) plus 0.03 – 0.3 µg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG₂EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0–10%, respectively. Subsequent studies established that biologically active ILI-like (2–4 U/ml) and TNF-like (50–100 U/ml) activities were released into the supernatants of LPS-treated GG₂EE cells. The combination of IFN- + LPS induced more (6–8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG₂EE cells by IFN- + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 at a concentration of 10 U/ml inhibited GG₂EE proliferation by 25–30%, while rmIFN- (25 U/ml) + rhIL1 (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 could completely replace LPS in the LPS + rmIFN- combination. Further, the combination of low doses of rhIL1 (0.1 to 1 U/ml) plus rmTNF (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN- to inhibit proliferation of GG₂EE cells by 98–99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN- to inhibit the oncogene-driven proliferation of GG₂EE cells.     

Brucella abortus inhibits IFN-γ-induced FcγRI expression and FcγRI-restricted phagocytosis via toll-like receptor 2 on human monocytes/macrophages

The strategies that allow Brucella abortus to persist for years inside macrophages subverting host immune responses are not completely understood. Immunity against this bacterium relies on the capacity of IFN-γ to activate macrophages, endowing them with the ability to destroy intracellular bacteria. We report here that infection with B. abortus down-modulates the expression of the type I receptor for the Fc portion of IgG (FcγRI, CD64) and FcγRI-restricted phagocytosis regulated by IFN-γ in human monocytes/macrophages. Both phenomena were not dependent on bacterial viability, since they were also induced by heat-killed B. abortus (HKBA), suggesting that they were elicited by a structural bacterial component. Accordingly, a prototypical B. abortus lipoprotein (L-Omp19), but not its unlipidated form, inhibited both CD64 expression and FcγRI-restricted phagocytosis regulated by IFN-γ. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited CD64 expression, indicating that any Brucella lipoprotein could down-modulate CD64 expression and FcγRI-restricted phagocytosis. Pre-incubation of monocytes/macrophages with anti-TLR2 mAb blocked the inhibition of the CD64 expression mediated by HKBA and L-Omp19. These results, together with our previous observations establish that B. abortus utilizes its lipoproteins to inhibit the monocytes/macrophages activation mediated by IFN-γ and to subvert host immunonological responses.