Syntenin-1-mediated arthritogenicity is advanced by reprogramming RA metabolic macrophages and Th1 cells.

OBJECTIVES: Syntenin-1, a novel endogenous ligand, was discovered to be enriched in rheumatoid arthritis (RA) specimens compared with osteoarthritis synovial fluid and normal synovial tissue (ST). However, the cellular origin, immunoregulation and molecular mechanism of syntenin-1 are undescribed in RA. METHODS: RA patient myeloid and lymphoid cells, as well as preclinical models, were used to investigate the impact of syntenin-1/syndecan-1 on the inflammatory and metabolic landscape. RESULTS: Syntenin-1 and syndecan-1 (SDC-1) co-localise on RA ST macrophages (MΦs) and endothelial cells. Intriguingly, blood syntenin-1 and ST SDC-1 transcriptome are linked to cyclic citrullinated peptide, erythrocyte sedimentation rate, ST thickness and bone erosion. Metabolic CD14+CD86+GLUT1+MΦs reprogrammed by syntenin-1 exhibit a wide range of proinflammatory interferon transcription factors, monokines and glycolytic factors, along with reduced oxidative intermediates that are downregulated by blockade of SDC-1, glucose uptake and/or mTOR signalling. Inversely, IL-5R and PDZ1 inhibition are ineffective on RA MΦs-reprogrammed by syntenin-1. In syntenin-1-induced arthritis, F4/80+iNOS+RAPTOR+MΦs represent glycolytic RA MΦs, by amplifying the inflammatory and glycolytic networks. Those networks are abrogated in SDC-1-/- animals, while joint prorepair monokines are unaffected and the oxidative metabolites are moderately replenished. In RA cells and/or preclinical model, syntenin-1-induced arthritogenicity is dependent on mTOR-activated MΦ remodelling and its ability to cross-regulate Th1 cells via IL-12 and IL-18 induction. Moreover, RA and joint myeloid cells exposed to Syntenin-1 are primed to transform into osteoclasts via SDC-1 ligation and RANK, CTSK and NFATc1 transcriptional upregulation. CONCLUSION: The syntenin-1/SDC-1 pathway plays a critical role in the inflammatory and metabolic landscape of RA through glycolytic MΦ and Th1 cell cross-regulation (graphical abstract).

Saga of monokines in shaping tumour-immune microenvironment: Origin to execution.

Cellular communication mediated by cytokines is an important mechanism dictating immune responses, their cross talk and final immune output. Cytokines play a major role in dictating the immune outcome to cancer by regulating the events of development, differentiation and activation of innate immune cells. Cytokines are pleiotropic in nature, hence understanding their role individually or as member of network cytokines is critical to delineate their role in tumour immunity. Tumour systemically manipulates the immune system to evade and escape immune recognition for their uncontrollable growth and metastasis. The developing tumour comprise a large and diverse set of myeloid cells which are vulnerable to manipulation by the tumour-microenvironment. The innate immune cells of the monocytic lineage skew the fate of the adaptive immune cells and thus dictating cancer elimination or progression. Targeting cells at tumour cite is preposterous owing to their tight network, poor reach and abundance of immunosuppressive mechanisms. Monocytic lineage-derived cytokines (monokines) play crucial role in tumour regression or progression by either directly killing the tumour cells with TNFα or promoting its growth by TGFß. In addition, the monokines like IL-12, IL-1ß, IL-6, IL-10 and TGFß direct the adaptive immune cells to secrete anti-tumour cytokines, TNFα, IFNγ, perforin and granzyme or pro-tumour cytokines, IL-10 and TGFß. In this review, we elucidate the roles of monokines in dictating the fate of tumour by regulating responses at various stages of generation, differentiation and activation of immune cells along with the extensive cross talk. We have attempted to delineate the synergy and antagonism of major monokines among themselves or with tumour-derived or adaptive immune cytokines. The review provides an update on the possibilities of placing monokines to potential practical use as cytokine therapy against cancer.

Dynamic changes in maternal immune biomarkers during labor in nulliparous vs multiparous women.

BACKGROUND: Immunomodulation is observed in human parturition. However, data from longitudinal studies for the prelabor phase and the active phase of labor are lacking, and no study had compared the immune responses during labor between nulliparous and multiparous women. OBJECTIVE: This study aimed to investigate the temporal changes of immune biomarkers in maternal blood from the prelabor phase to the latent and active phases of labor and to compare the dynamic changes between nulliparous and multiparous women. STUDY DESIGN: A prospective case-control study was conducted on women who had induction of labor at term followed by vaginal delivery. Maternal blood was serially collected at 3 consecutive time points: (1) before the onset of labor, (2) during the latent phase of labor, and (3) during the active phase of labor. Peripheral immune cells were measured by 4-color flow cytometry, and the plasma concentrations of cytokines and chemokines were measured by cytometric bead arrays. A longitudinal comparison was made to assess the dynamic changes in inflammatory parameters over 3 time points in nulliparous and multiparous women, respectively, and a cross-sectional comparison was made between nulliparous and multiparous women. RESULTS: A total of 40 women, including 20 nulliparous and 20 multiparous, were included in the study. Prelabor circulating levels of macrophage inflammatory protein-1ß, monokine induced by gamma interferon, and interferon gamma-induced protein-10 were higher in multiparous women than in nulliparous women. In the latent phase of labor, the innate immune system in both groups responded with increases in neutrophils and interleukin 6, and the nulliparous women showed a more pronounced response. During the active phase of labor, such innate immune response continued with both groups, with additional increases in natural killer cells, monocyte chemoattractant protein-1, interleukin 8, and interleukin 10. Conversely, the adaptive immune system in nulliparous women showed a reduction in both cytotoxic and helper T cells, whereas the adaptive immune system in multiparous women only had a reduction in helper T cells, showing a smaller reduction. CONCLUSION: Innate and adaptive immune responses partake in immunomodulation during human parturition. Nulliparous and multiparous women showed different responses in their blood levels of immune cells and biomarkers during the different phases of labor.

Inhaled particulate matter affects immune responsiveness of human lung phagocytes to mycobacteria.

The influence of smoke-derived or air pollution-derived cytoplasmic particulate matter (PM) can be detrimental and can lead to failed lung immunity. We investigated mycobacterial uptake, intracellular replication, and soluble immune-mediator responses of human bronchoalveolar lavage cells (BALCs) loaded with/without PM, to infection with mycobacterial strains. We observed that only BALCs containing PM display an ex vivo phenotypic profile dominated by spontaneous interleukin (IL)-10 production. PM-loaded BALCs retained the ability to phagocytose both Mycobacterium bovis Bacille Calmette Guérin (BCG) and Mycobacterium tuberculosis (M.tb) ΔleuDΔpanCD at equal efficacy as clear non-PM-loaded BALCs. However, immune responsiveness, such as the production of IL-6 (P = 0.015) and tumor necrosis factor-α (TNF)-α (P = 0.0172) immediately post M. bovis BCG infection, were dramatically lower in black BALCs loaded with PM versus clear non-PM-loaded BALCs. By 24 h post infection, differential immune responses to M. bovis BCG between black versus clear BALC waned, and instead, production of IL-6 (P = 0.03) and IL-1α (P = 0.04) by black BALCs was lower versus clear BALCs following M.tb ΔleuDΔpanCD infection. Considering that TNF-α and IL-6 are characterized as critical to host protection against mycobacteria, our findings suggest that BALCs loaded with inhaled PM, display lower levels of antimycobacterial mediators and that the response magnitude differs according to infective mycobacterial strain. Even though this did not translate into altered mycobacterial killing at early time points post infection, the long-term impact of such changes remains to be established.

Ethyl ferulate contributes to the inhibition of the inflammatory responses in murine RAW 264.7 macrophage cells and acute lung injury in mice.

BACKGROUND: Ethyl ferulate (EF) is a derivative of ferulic acid (FA), which is a monomeric component purified from the traditional medicinal herb Ferula, but its effects have not been clear yet. The purpose of this study was to evaluate whether EF can reduce inflammation levels in macrophages by regulating the Nrf2-HO-1 and NF-кB pathway. METHODS: The LPS-induced raw 264.7 macrophage cells model was used to determine the anti-inflammatory and anti-oxidative stress effects of EF. The levels of IL-1ß, IL-6, TNF-α and PGE2 were analyzed by ELISA. The mRNA and protein of COX-2, iNOS, TNF-α, IL-6, HO-1 and Nrf2 were identified by RT-PCR analysis and western blotting. Intracellular ROS levels were assessed with DCFH oxidation staining. The expressions of NF-кB p-p65 and Nrf2 were analyzed by immunofluorescence assay. The inhibitory effect of Nrf2 inhibitor ML385 (2µM) on mediatation of antioxidant activity by raw 264.7 macrophage cells was evaluated. The effect of EF was confirmed in acute lung injury mice model. RESULTS: In our research, EF reduced the expression of iNOS, COX2 and the production of PGE2. EF could inhibit the production of pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in lipopolysaccharide (LPS) stimulated macrophages and decreased expression of IL-6 and TNF-α in LPS stimulated macrophages. Furthermore, EF inhibited NF-кB p65 from transporting to the nucleus, decreased the expression of p-IкBα, significantly decreased the level of intracellular reactive oxygen species (ROS) and activated Nrf2/HO-1 pathways. EF could attenuate the degree of leukocyte infiltration, reduced MPO activity, mRNA levels and secretion of TNF-α and IL-6 in vivo. EF exhibited potent protective effects against LPS-induced acute lung injury in mice. CONCLUSIONS: Collectively, our data showed that EF relieved LPS-induced inflammatory responses by inhibiting NF-κB pathway and activating Nrf2/HO-1 pathway, known to be involved in the regulation of inflammatory responses by Nrf2.

Anti-Inflammatory Activity and Mechanism of Isookanin, Isolated by Bioassay-Guided Fractionation from Bidens pilosa L.

Bidens pilosa L. (Asteraceae) has been used historically in traditional Asian medicine and is known to have a variety of biological effects. However, the specific active compounds responsible for the individual pharmacological effects of Bidens pilosa L. (B. pilosa) extract have not yet been made clear. This study aimed to investigate the anti-inflammatory phytochemicals obtained from B. pilosa. We isolated a flavonoids-type phytochemical, isookanin, from B. pilosa through bioassay-guided fractionation based on its capacity to inhibit inflammation. Some of isookanin's biological properties have been reported; however, the anti-inflammatory mechanism of isookanin has not yet been studied. In the present study, we evaluated the anti-inflammatory activities of isookanin using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We have shown that isookanin reduces the production of proinflammatory mediators (nitric oxide, prostaglandin E2) by inhibiting the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated macrophages. Isookanin also inhibited the expression of activator protein 1 (AP-1) and downregulated the LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun NH2-terminal kinase (JNK) in the MAPK signaling pathway. Additionally, isookanin inhibited proinflammatory cytokines (tumor necrosis factor-a (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1ß (IL-1ß)) in LPS-induced THP-1 cells. These results demonstrate that isookanin could be a potential therapeutic candidate for inflammatory disease.

Kefiran ameliorates malfunctions in primary and functional immune cells caused by lipopolysaccharides.

Kefiran is a water-soluble polysaccharide well recognized as a bioactive ingredient to enhance nutritional and health-promoting features. Also, some therapeutic properties have made this macromolecule an active ingredient in ointments and oral anti-inflammatory drugs. However, the details of the molecular and cellular aspects of these effects have not been addressed. In this study, lipopolysaccharides (LPS)-induced monocytes, lymphocytes, and monocyte-derived dendritic cells (MDDCs) as representative cells for both innate and adaptive immunity were treated with kefiran for 2 h. Kefiran had an anti-inflammatory effect on monocytes to reduce pro-inflammatory cytokines, interleukin 1 ß (IL-1ß) & tumor necrosis factor α (TNF-α), as well as nuclear factor kappa b (NF-kb). However, it did not affect lymphocytes. Overexpression of Toll-like receptor 4 (TLR4) in LPS-induced cells was not reduced after kefiran treatment. Kefiran balanced MDDCs secretion of pro/anti-inflammatory cytokines by reducing and enhancing the expression of IL-1ß and interleukin 10 (IL-10), respectively. Also, kefiran decreased the number of apoptotic immature MDDCs and promoted dose-dependent phagocytosis capacity of MDDCs. According to the results of the current study, it may be concluded that the immunomodulatory effects of kefiran are due to antagonist against innate immune receptors especially TLR4. The results of this study can be used as a guide to developing kefiran-based non-aggressive anti-inflammatory drugs. Furthermore, understanding the immunobiological effects of kefiran on monocytes and lymphocytes was another outcome of this study.

Structural characterization and immunostimulatory activity of a glucan from Cyclina sinensis.

Cyclina sinensis is an edible clam widely distributed along the coastal waters of Asia. In the present study, a polysaccharide (CSP-1) isolated from C. sinensis was purified by a DEAE-Sepharose Fast Flow column, and it had an average molecular weight of 3.8 × 105 Da and a prevalent component monosaccharide of Glc. The results of methylation analysis and 1D/2D NMR indicated that CSP-1 was a glycogen constructed with α-1,4-Glc and branched at C-6 every 9 Glc residues. In addition, Cong red test suggests CSP-1 was not a helical conformation, and irregular and spherical lumps were observed by AFM. Moreover, CSP-1 was found to possess potent immunostimulatory activity on the basis of its significant abilities to enhance NO production and cytokines (TNF-α, IL-1ß and IL-6) secretion in RAW 264.7 macrophages.

Pristine graphene induces innate immune training.

Graphene-based materials are of increasing interest for their potential use in biomedical applications. However, there is a need to gain a deeper understanding of how graphene modulates biological responses before moving towards clinical application. Innate immune training is a recently described phenomenon whereby cells of the innate immune system are capable of being programmed to generate an increased non-specific response upon subsequent challenge. This has been well established in the case of certain microbes and microbial products. However, little is known about the capacity of particulate materials, such as pristine graphene (pGr), to promote innate immune training. Here we report for the first time that while stimulation with pGr alone does not directly induce cytokine secretion by bone-marrow derived macrophages (BMDMs), it programs them for enhanced secretion of proinflammatory cytokines (IL-6, TNF-α) and a concomitant decrease in production of the regulatory cytokine, IL-10 after Toll-like receptor (TLR) ligand stimulation. This capacity of pGr to program cells for enhanced inflammatory responses could be overcome if the nanomaterial is incorporated in a collagen matrix. Our findings thus demonstrate the potential of graphene to modulate innate immunity over long timescales and have implications for the design and biomedical use of pGr-based materials.

Effect of Herbal Formulation on Immune Response Enhancement in RAW 264.7 Macrophages.

Immune response is a necessary self-defense mechanism that protects the host from infectious organisms. Many medicinal plants are popularly used in Asian folk medicine to increase body resistance. An herbal formulation named KM1608 was prepared from three medicinal plants: Saussurea lappa, Terminalia chebula, and Zingiber officinale. In this study, we evaluated the immune stimulatory effect of KM1608 on RAW 264.7 murine macrophages. Network pharmacological analyses were used to predict potential immune response pathways of major compounds from KM1608. The cytotoxicity and immuno-stimulating effect of KM1608 were determined using cell viability and nitric oxide assays. The underlying mechanism of immunomodulatory activity was evaluated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) of pro-inflammatory cytokines. The results of network pharmacological analysis suggested that major compounds from KM1608 possess anticancer potential via immune signaling pathways. After treatment with KM1608 at 25-100 µg/mL for 24 h, the level of nitric oxide was increased in the dose-dependent manner. The results of quantitative real-time PCR showed that KM1608 stimulates the expression of immune cytokines (interferon (IFN)-α, -ß, IL-1ß, -6, IL-10, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)) in macrophages. KM1608 extract is a potential agent for immune response enhancement.

Men Have a Stronger Monocyte-Derived Cytokine Production Response upon Stimulation with the Gram-Negative Stimulus Lipopolysaccharide than Women: A Pooled Analysis Including 15 Study Populations.

The incidence of bacterial infections and sepsis, as well as the mortality risk from sepsis, is sex specific. These clinical findings have been attributed to sex differences in immune responsiveness. The aim of the present study was to investigate sex differences in monocyte-derived cytokine production response upon stimulation with the gram-negative stimulus lipopolysaccharide (LPS) using cytokine data from 15 study populations. Individual data on ex vivo cytokine production response upon stimulation with LPS in whole blood were available for 4,020 subjects originating from these 15 study populations, either from the general population or from patient populations with specific diseases. Men had a stronger cytokine production response than women to LPS for tumour necrosis factor-α, interleukin (IL)-6, IL-12, IL-1ß, IL-1RA, and IL-10, but not for interferon-γ. The granulocyte-macrophage colony-stimulating factor production response was lower in men than in women. These sex differences were independent of chronological age. As men had higher monocyte concentrations, we normalized the cytokine production responses for monocyte concentration. After normalization, the sex differences in cytokine production response to LPS disappeared, except for IL-10, for which the production response was lower in men than in women. A sex-based approach to interpreting immune responsiveness is crucial.

Macrophage-derived nanovesicles exert intrinsic anti-inflammatory properties and prolong survival in sepsis through a direct interaction with macrophages.

Despite numerous advances in medical treatment, sepsis remains one of the leading causes of death worldwide. Sepsis is characterized by the involvement of all organs and tissues as a consequence of blood poisoning, resulting in organ failure and eventually death. Effective treatment remains an unmet need and novel approaches are urgently needed. The growing evidence of clinical and biological heterogeneity of sepsis suggests precision medicine as a possible key for achieving therapeutic breakthroughs. In this scenario, biomimetic nanomedicine represents a promising avenue for the treatment of inflammatory diseases, including sepsis. We investigated the role of macrophage-derived biomimetic nanoparticles, namely leukosomes, in a lipopolysaccharide-induced murine model of sepsis. We observed that treatment with leukosomes was associated with significantly prolonged survival. In vitro studies elucidated the potential mechanism of action of these biomimetic vesicles. The direct treatment of endothelial cells (ECs) with leukosomes did not alter the gene expression profile of EC-associated cell adhesion molecules. In contrast, the interaction of leukosomes with macrophages induced a decrease of pro-inflammatory genes (IL-6, IL-1b, and TNF-α), an increase of anti-inflammatory ones (IL-10 and TGF-ß), and indirectly an anti-inflammatory response on ECs. Taken together, these results showed the ability of leukosomes to regulate the inflammatory response in target cells, acting as a bioactive nanotherapeutic.

Chlamydia trachomatis ct143 stimulates secretion of proinflammatory cytokines via activating the p38/MAPK signal pathway in THP-1 cells.

Chlamydia trachomatis (Ct) infections can cause bacterial sexually-transmitted and preventable blindness. The Ct infections induced excessive cytokines generation which attributed to pathologic changes in host cells. However, the precise mechanisms of Ct-induced cytokines production are still unclear.CT143 protein was identified as a novel Ct specific protein with high immunogenicity. In the present study. The CT143 fusion protein was recombined and purified. The mice immune serum was prepared by immunizing BALB/c mice with the purified fusion protein. The specificity of the antibody was confirmed using Immunoblotting. Indirect immunoflurescence assay (IFA) and Immunoblotting assays were performed to detect the temporal and spatial characteristics of CT143 in Ct infected cells. ELISA was performed to analyze the secretion of proinflammatory cytokines IL-1ß, IL-8 and TNF-α by human macrophages under the stimulation of CT143 protein. Finally, the involvement of p38 signaling in CT143-induced cytokine secretion was validated. CT143 protein was located in the inclusion body and represented an Elementary body (EB)-related protein, which may be encoded by the mid- and late-stage expressing genes. CT143 protein could stimulate the secretion of inflammatory cytokines in macrophages which differentiated from THP-1 This induction may be mediated by the activation of p38 signaling. In summary, CT143 protein is involved in inflammatory processes during Ct infection.

Effects of pelargonidin-3-O-glucoside and its metabolites on lipopolysaccharide-stimulated cytokine production by THP-1 monocytes and macrophages.

Epidemiological evidence suggests cardioprotective effects of anthocyanin consumption. This study examined the predominant strawberry anthocyanin, pelargonidin-3-O-glucoside (Pg-3-glc), and three of its plasma metabolites (protocatechuic acid [PCA], 4-hydroxybenzoic acid, and phloroglucinaldehyde [PGA]) for effects on the production of selected cytokines by lipopolysaccharide-stimulated THP-1 monocytes and macrophages. Concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8 and IL-10 were determined using a cytometric bead array kit. PCA at 0.31, 1.25 and 20 µM and PGA at 5 and 20 µM decreased the concentration of IL-6 in the monocyte cultures, but there were no effects on TNF-α, IL-1ß, IL-8 and IL-10 and there were no effects of the other compounds. In the macrophage cultures, PGA at 20 µM decreased the concentrations of IL-6 and IL-10, but there was no effect on TNF-α, IL-1ß and IL-8 and there were no effects of the other compounds. In conclusion, while the effects of PGA were only observed at the higher, supraphysiological concentration and are thus considered of limited physiological relevance overall, the anti-inflammatory properties of PCA were observed at both the lower, physiologically relevant, and the higher concentrations; however, effects were modest and limited to IL-6 and monocytes. These preliminary data suggest potential for physiologically attainable PCA concentrations to modulate IL-6 production by monocytes.

Instructing Human Macrophage Polarization by Stiffness and Glycosaminoglycan Functionalization in 3D Collagen Networks.

Dynamic alterations of composition and mechanics of the extracellular matrix are suggested to modulate cellular behavior including plasticity of macrophages (MPhs) during wound healing. In this study, engineered 3D fibrillar matrices based on naturally occurring biopolymers (collagen I, glycosaminoglycans (GAGs)) are used to mimic matrix stiffening as well as modification by sulfated and nonsulfated GAGs at different stages of wound healing. Human MPhs are found to sensitively respond to these microenvironmental cues in terms of polarization toward proinflammatory or wound healing phenotypes over 6 days in vitro. MPhs exhibit a wound healing phenotype in stiffer matrices as determined by protein and gene expression of relevant cytokines (IL10, IL12, and TNFα). Presence of sulfated and nonsulfated GAGs inhibits this polarization effect. Furthermore, control experiments on 2D matrices stress the relevance of using stiffness-controlled 3D matrices, as MPhs show a reciprocal polarization behavior depending on GAG presence. Hence, the results indicate a strong influence of dimensionality, stiffness, and GAG presence of the biomaterial scaffold on MPh polarization and emphasize the need for matrices closely mimicking the 3D in vivo context with a variable stiffness and GAG composition in in vitro studies.

High dilutions of antimony modulate cytokines production and macrophage - Leishmania (L.) amazonensis interaction in vitro.

BACKGROUND: In previous results mice treated with high dilutions of antimony presented reduction of monocyte migration to the site of infection with increase in B lymphocytes population in the local lymph node. AIMS: To know the mechanisms involved, a series of in vitro studies was done, using co-cultures of macrophages (RAW 264.7) and Leishmania (L.) amazonensis treated with different dilutions of antimony (Antimonium crudum or AC), in different times. METHODOLOGY: Spreading, phagocytosis, the oxidative activity of macrophages, the viability of free promastigotes and the cytokines/chemokines concentration in the supernatant were evaluated. The assays were performed in quadruplicate. RESULTS: Cells treated with AC 30cH (10-58M) and AC 200cH (10-398M) presented a temporary reduction of the spreading after 02h of incubation, followed by increase after 48h, being the most significant increase observed after the AC 200cH treatment. However, the percentage of internalized parasites at 48, 96 and 120h of incubation was also higher in cells treated with AC 200cH. It is suggested that the AC 200cH improves the ability of phagocytes to internalize the parasites, but not to digest them. The cytokines-chemokines panel corroborated these results. Both dilutions potentiated the parasite-induced reduction of cytokines production, especially IL-6, IL 12 p40 and γ-IFN, after 48h of incubation. In addition, the production of MIP-1 beta (CCL4), a chemokine involved in chronic inflammation, was also reduced after 120h. A specific effect of AC 30cH was seen by the inhibition of two peaks of CCL2 (MCP-1) observed in infected macrophages, at 24 and 120h. Since this cytokine is an important chemokine for monocytes, it explains the results obtained formerly in vivo. The morphology of macrophages after acridine orange staining revealed that the treatment with AC 30cH reduced substantially the acid vacuoles in the cytoplasm, indicating a certain inability of these cells to digest the parasites. On the other hand, a large peak of VEGF-A, associated with increase of internalized parasites was observed after 120h of treatment with AC 200cH, which could be associated to the regulation of the chronic inflammation events by M1-M2 polarization. There was no statistical difference among groups regarding the production of TNF, NO and H2O2, showing that the drugs do not alter macrophage cytotoxic activity. A clear quantitative and qualitative variation of the modulatory effects of AC 30cH and 200cH was seen, in function of time. CONCLUSIONS: Both dilutions were able to potentiate the decrease of most of cytokines and chemokines induced by the parasite infection in vitro, which explains the clinical improvement seen previously in vivo, however, the mechanisms involved and the epidemiological significance of these findings are still under discussion.

3-Methoxy-catalposide inhibits inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

Pseudolysimachion rotundum var. subintegrum is utilized as a traditional herbal remedy to treat cough, bronchitis, and asthma in Korea, Russia, China, and Europe. Here, we show that 3-methoxy-catalposide, a novel iridoide glycoside isolated from P. rotundum var. subintegrum has the anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated macrophages. The chemical structure of 3-methoxy-catalposide was determined by NMR, optical rotation and HRESIMS. In in vitro experiment, RAW264.7 cells were treated with 3-methoxy-catalposide for 2h before exposure to LPS for different times. Inflammatory gene and protein expressions were assayed using RT-PCR and ELISA. Activities of signal proteins were examined using western analysis. Our results demonstrated that 3-methoxy-catalposide significantly inhibits the expression of cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in RAW264.7 cells stimulated by LPS, thereby suppressing the release of prostaglandin E2 (PGE2) and nitric oxide (NO). Moreover, 3-methoxy-catalposide markedly reduced the LPS-induced expression of pro-inflammatory genes, such as interleukin (IL)-6, IL-1ß, and TNF-α. Further, 3-methoxy-catalposide inhibited both LPS-induced activation of three MAP kinases (ERK 1/2, JNK, and p38) and the nuclear translocation of NF-κB and AP-1. These results support that 3-methoxy-catalposide may be a promising candidate for inflammation treatment.

A Potential Mechanism for the Anti-Apoptotic Property of Koumine Involving Mitochondrial Pathway in LPS-Mediated RAW 264.7 Macrophages.

Koumine is a kind of alkaloid extracted from Gelsemium elegans (G. elegans). Benth, which has shown promise as an anti-tumor, anxiolytic, and analgesic agent. In our present study, the effect of koumine on lipopolysaccharide (LPS)-mediated RAW 264.7 cell apoptosis was evaluated. MTT assays showed that koumine obviously increased cell viability in LPS-mediated RAW 264.7 macrophages. Preincubation with koumine ameliorated LPS-medicated apoptosis by decreasing reactive oxygen species (ROS) production, which resulted in a significant decrease in the levels of nitric oxide (NO) and inducible nitric oxide synthase (iNOS). In addition, koumine-pretreated RAW 264.7 macrophages exhibited reduction of LPS-induced levels of TNF-α, IL-1ß, and IL-6 mRNA. Furthermore, pretreatment with koumine suppressed LPS-mediated p53 activation, loss of mitochondrial membrane potential, caspase-3 activation, decrease of Bcl-2 expression, and elevation of Bax and caspase-3 expressions, suggesting that koumine might act directly on RAW 264.7 cells to inhibit LPS-induced apoptosis. It seems as though the mechanism that koumine possesses is the anti-apoptotic effect mediated by suppressing production of ROS, activation of p53, and mitochondrial apoptotic pathways in RAW 264 cells. Koumine could potentially serve as a protective effect against LPS-induced apoptosis.