Murine Peritoneal Cells Research Articles

Abstract TMEM-040: TUMOR ASSOCIATED MACROPHAGES MEDIATES THE ONCOGENIC EFFECT OF FGF18 IN OVARIAN CANCER

Abstract Epithelial ovarian cancer is the fifth leading cause of cancer-related death among women and has the highest case-fatality rate among gynecologic cancers. Effective management of this deadly disease still remains scanty due to the inadequate understanding of the molecular mechanisms associated with the tumor progression and prognosis. We have previously reported the tumor-promoting role of Fibroblast growth factor 18 (FGF18), which have been identified through genomic analysis of ovarian tumors. Initial studies suggested FGF18 promotes ovarian tumor progression through NF-κB pathway mediated elevation of proinflammatory cytokines, which mediate the increased tumor burden, angiogenesis and infiltration of macrophages in FGF18 expressing xenografts. Based on these findings, the present study aims to delineate the mechanism by which FGF18 modulates the phenotype of tumor associated macrophages (TAMs) as well as the reciprocal tumor-promoting feedback from the TAMs which potentially contributes FGF18 mediated ovarian tumorigenesis. Immunohistochemical staining of a tissue array comprising 216 archived high-grade advanced stage serous ovarian tumor specimens revealed a significant correlation between the FGF18 expression and infiltration of M2-polarized macrophages (by CD163). In addition, the density of intratumoral M2 TAMs inversely associated with patients' overall survival as a negative prognostic factor independent to age, tumor grade and debulking status. These findings suggest intratumoral TAMs may play an important role in ovarian tumorigenesis. In vitro co-culture studies further demonstrated that human monocyte cell line THP-1 or murine peritoneal macrophage cell line IC-21 could significantly activate the NF-κB pathway and increase the production of various proinflammatory cytokines in ovarian cancer cells. Co-culture also increased the migratory, adhesive and angiogenic potential of ovarian cancer cells cells. Addition of an IKKβ inhibitor into the co-culture system nullified the cytokine production and oncogenic changes of tumor cells promoted by monocytes/macrophages, implicating the involvement of NF-κB signaling in the crosstalk. Conversely, liposome clodronate mediated in vivo macrophage depletion significantly shrunk the tumor burden of the intraperitoneal xenograft derived from FGF18 overexpressing SKOV3 cells, while minimal effect was observed over the control RFP overexpressing xenograft. Besides, macrophage depletion also markedly decreased the intratumoral microvessel density induced by FGF18 overexpression. Taken together, our data potentially suggest TAMs may mediate the FGF18 related ovarian tumorigenesis by augmenting the proinflammatory status of the tumors. Citation Format: Wei Wei and Michael J. Birrer. TUMOR ASSOCIATED MACROPHAGES MEDIATES THE ONCOGENIC EFFECT OF FGF18 IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-040.

IL-17A and complement contribute to killing of pneumococci following immunization with a pneumococcal whole cell vaccine

The pneumococcal whole cell vaccine (PWCV) has been investigated as an alternative to polysaccharide-based vaccines currently in use. It is a non-encapsulated killed vaccine preparation that induces non-capsular antibodies protecting mice against invasive pneumococcal disease (IPD) and reducing nasopharyngeal (NP) carriage via IL-17A activation of mouse phagocytes. Here, we show that PWCV induces antibody and IL-17A production to protect mice against challenge in a fatal aspiration-sepsis model after only one dose. We observed protection even with a boiled preparation, attesting to the stability and robustness of the vaccine. PWCV antibodies were shown to bind to different encapsulated strains, but complement deposition on the pneumococcal surface was observed only on serotype 3 strains; using flow cytometer methodology, variations in PWCV quality, as in the boiled vaccine, were detected. Moreover, anti-PWCV induces phagocytosis of different pneumococcal serotypes by murine peritoneal cells in the presence of complement or IL-17A. These findings suggest that complement and IL-17A may participate in the process of phagocytosis induced by PWCV antibodies. IL-17A can stimulate phagocytic cells to kill pneumococcus and this is enhanced in the presence of PWCV antibodies bound to the bacterial cell surface. Our results provide further support for the PWCV as a broad-range vaccine against all existing serotypes, potentially providing protection for humans against NP colonization and IPD. Additionally, we suggest complement deposition assay as a tool to detect subtle differences between PWCV lots.

Coxiella burnetii Avirulent Nine Mile Phase II Induces Caspase-1-Dependent Pyroptosis in Murine Peritoneal B1a B Cells.

Our recent study demonstrated that virulent Coxiella burnetii Nine Mile phase I (NMI) is capable of infecting and replicating within peritoneal B1a cells and that B1a cells play an important role in host defense against C. burnetii infection in mice. However, it remains unknown if avirulent Nine Mile phase II (NMII) can infect and replicate in B1a cells and whether NMI and NMII can differentially interact with B1a cells. In this study, we examined if NMI and NMII can differentially modulate host cell apoptotic signaling in B1a cells. The results showed that NMII induced dose-dependent cell death in murine peritoneal B1a cells but NMI did not, suggesting that NMI and NMII may differentially activate host cell apoptotic signaling in B1a cells. Western blotting indicated that NMII-induced B1a cell death was not dependent on either caspase-3 or PARP-1 cleavage, but cleavage of caspase-1 was detected in NMII-infected B1a cells. In addition, inhibition or deficiency of caspase-1 activity blocked NMII-induced B1a cell death. These results suggest that NMII induces a caspase-1-dependent pyroptosis in murine peritoneal B1a cells. We also found that heat-killed NMII and type 4 secretion system (T4SS) mutant NMII were unable to induce B1a cell death and that NMII infection did not induce cell death in peritoneal B1a cells from Toll-like receptor 2 (TLR-2)- or NLRP3 inflammasome-deficient mice. These data suggest that NMII-induced caspase-1-dependent pyroptosis may require its T4SS and activation of the TLR-2 and NLRP3 signaling pathways.

Abstract 4083: Tumor associated macrophages mediates the oncogenic effect of FGF18 in ovarian cancer

Abstract Epithelial ovarian cancer is the fifth leading cause of cancer-related death among women and has the highest case-fatality rate among gynecologic cancers. Effective management of this deadly disease still remains scanty due to the inadequate understanding of the molecular mechanisms associated with the tumor progression and prognosis. We have previously reported the tumor-promoting role of Fibroblast growth factor 18 (FGF18), which have been identified through genomic analysis of ovarian tumors. Initial studies suggested FGF18 promotes ovarian tumor progression through NF-κB pathway mediated elevation of proinflammatory cytokines, which mediate the increased tumor burden, angiogenesis and infiltration of macrophages in FGF18 expressing xenografts. Based on these findings, the present study aims to delineate the mechanism by which FGF18 modulates the phenotype of tumor associated macrophages (TAMs) as well as the reciprocal tumor-promoting feedback from the TAMs which potentially contributes FGF18 mediated ovarian tumorigenesis. Immunohistochemical staining of a tissue array comprising 216 archived high-grade advanced stage serous ovarian tumor specimens revealed a significant correlation between the FGF18 expression and infiltration of M2-polarized macrophages (by CD163). In addition, the density of intratumoral M2 TAMs inversely associated with patients’ overall survival as a negative prognostic factor independent to age, tumor grade and debulking status. These findings suggest intratumoral TAMs may play an important role in ovarian tumorigenesis. In vitro co-culture studies further demonstrated that human monocyte cell line THP-1 or murine peritoneal macrophage cell line IC-21 could significantly activate the NF-κB pathway and increase the production of various proinflammatory cytokines in A224 ovarian cancer cells. Co-culture also increased the migratory potential of A224 cells. Addition of an IKKβ inhibitor into the co-culture system nullified the cytokine production and migration of tumor cells promoted by monocytes/macrophages, implicating the involvement of canonical NF-κB signaling in the crosstalk. Conversely, liposome clodronate mediated in vivo macrophage depletion significantly shrunk the tumor burden of the intraperitoneal xenograft derived from FGF18 overexpressing SKOV3 cells, while minimal effect was observed over the control RFP overexpressing xenograft. Besides, macrophage depletion also markedly decreased the intratumoral microvessel density induced by FGF18 overexpression. Taken together, our data potentially suggest TAMs may mediate the FGF18 related ovarian tumorigenesis by augmenting the proinflammatory status of the tumors. Citation Format: Wei Wei, Michael Birrer. Tumor associated macrophages mediates the oncogenic effect of FGF18 in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4083.

Accelerated transformation of macrophage-derived foam cells in the presence of collagen-induced arthritis mice serum is associated with dyslipidemia

Objective: Atherosclerosis characterized by accumulation of foam cells in the arterial intimal layer is accelerated in rheumatoid arthritis (RA) patients. We and others have previously demonstrated that serum from RA patients and collagen-induced arthritis (CIA) mice had proatherogenic features that might lead to progression of atherosclerosis. Here we further examined the effects of serum from CIA mice on the transformation of macrophage-derived foam cells, and investigated potential mechanism. Methods: DBA/1j mice were used to establish CIA model. Murine peritoneal macrophages and macrophage cell line RAW264.7 were treated with different dilute concentrations of mice serum. Results: CIA mice serum increased cholesterol influx and accumulation in murine macrophages, and markedly up-regulated scavenger receptor CD36 expression in the cells, but had no effect on intracellular lipid efflux. Neutralizing monocyte chemotactic protein (MCP)-1, the most significant altered cytokine we observed between normal and CIA mice serum to CIA mice could not reverse these effects. However, administering simvastatin to CIA mice could lower high-density lipoprotein-cholesterol (HDL-C) level and elevate oxidized low-density lipoprotein (ox-LDL) level in CIA mice serum, with attendant decreased lipid accumulation as well as CD36 expression in murine macrophages. Conclusion: Accelerated transformation of macrophage-derived foam cells via up-regulated CD36 expression is related to dyslipidemia rather than elevated inflammatory factor MCP-1 level in CIA mice serum. Decreased HDL-C and higher ox-LDL levels in CIA mice serum may link RA to atherosclerosis.

Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF) mediates innate immune responses in murine peritoneal mesothelial cells through TLR3 and TLR4 stimulation

Mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. Although mesothelial cells are known to express multiple Toll-like receptors (TLRs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. In the present study, we investigated the role of Toll/IL-1 domain-containing adaptor inducing IFN-β (TRIF), one of the two major TLRs–adaptor molecules, on innate immune response induced by TLR3 and TLR4 stimulation in murine peritoneal mesothelial cells (PMCs). TRIF was strongly expressed in PMCs and its deficiency led to impaired production of cytokines and chemokines by poly I:C and LPS in the cells. Activation of NF-κB or MAPKs through poly I:C and LPS stimulation was reduced in TRIF-deficient PMCs as compared to the WT cells. TRIF was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (iNOS) and IFN-β in PMCs in response to poly I:C and LPS. Furthermore, both Escherichia coli and Pseudomonas aeruginosa induced high level of IL-6, CXCL1, and CCL2 production in PMCs, which was significantly impaired by TRIF deficiency. These results demonstrated that TRIF is required for optimal activation of innate immune responses in mesothelial cells against microbial infections.

녹차 효소 처리 다당의 화학적 특성 및 면역증진 활성

In order to develop new immuno-stimulating ingredients from mature leaves of green tea, crude polysaccharides were isolated from pectinase digests of tea leaves (green tea enzyme digestion, GTE-0), after which their immuno-stimulating activities and chemical properties were examined. GTE-0 mainly contained neutral sugars (54.9%) such as glucose (14.2%), arabinose (12.2%), rhamnose (11.1%), and galacturonic acid (45.1%), which are characteristic of pectic polysaccharides. The anti-complementary activity of GTE-0 was similar to that of polysaccharide K (used as positive control). Number of morphologically activated macrophages was significantly increased in the GTE-0-treated group. GTE-0 significantly augmented H₂O₂ and reactive oxygen species production by murine peritoneal macrophage cells in a dose-dependent manner, whereas production of nitric oxide showed the highest activity at a dose of 100 μg/mL among all tested concentrations. Murine peritoneal macrophages stimulated with GTE-0 showed enhanced production of various cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factors-α in a dose-dependent manner. Further, GTE-0 induced higher phagocytic activity in a dose-dependent manner. In ex vivo assay for cytolytic activity of murine peritoneal macrophages, GTE-0-treated group showed significantly higher activity compared to the untreated group at an effector-to-target cell ratio of 20. The above results lead us to conclude that polysaccharides from leaves of green tea have a potent immuno-stimulating effect on murine peritoneal macrophage cells.

Differential induction of inflammatory cytokines and reactive oxygen species in murine peritoneal macrophages and resident fresh bone marrow cells by acute staphylococcus aureus infection: contribution of toll-like receptor 2 (TLR2).

Among the known Toll-like receptors (TLRs), Toll-like receptor 2 (TLR2) is a key sensor for detecting Staphylococcus aureus invasion. But the function of TLR2 during S. aureus infection in different cell populations is unclear. Two different cell subtypes were chosen to study the interaction of S. aureus with TLR2 because macrophages are extremely different from one compartment to another and their capacity to respond to live bacteria or bacterial products differs from one site to another. The contribution of TLR2 to the host innate response against acute live S. aureus infection and heat-killed S. aureus (HKSA) using anti-TLR2 antibody in murine peritoneal macrophages and resident fresh bone marrow cells has been investigated here. TLR2 blocking before infection induces the release of interleukin (IL)-10 by macrophages thereby inhibiting excessive production of oxidants by activating antioxidant enzymes. TLR2-blocked peritoneal macrophages showed impaired release of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and IL-6 in response to both live and heat-killed S. aureus infection except bone marrow cells. TLR2-mediated free radical production and killing of S. aureus were modulated by TLR2 blocking in peritoneal macrophages and resident bone marrow cells. This study supported that S. aureus persists in resident bone marrow cells in a state of quiescence.

The receptor for advanced glycation end-products (RAGE) plays a key role in the formation of nanotubes (NTs) between peritoneal mesothelial cells and in murine kidneys.

The receptor for advanced glycation end-products (RAGE), a multiligand receptor of the immunoglobulin superfamily, takes part in various inflammatory processes. The role of this receptor in the context of intercellular communication, like nanotube (NT)-mediated interaction, is largely unknown. Here, we use cell cultures of human and murine peritoneal mesothelial cells as well as murine kidneys from wild-type and RAGE knockout mouse models to assess the role of RAGE in NT formation and function. We show that loss of RAGE function results in reduced NT numbers under physiological conditions and demonstrate the involvement of MAP kinase signaling in NT formation. Additionally, we show for the first time the existence of NTs in murine kidney tissue and confirm the correlation of RAGE expression and NT numbers. Under elevated oxidative stress conditions like renal ischemia or peritoneal dialysis, we demonstrate that RAGE absence does not prevent NT formation. Rather, increased NT numbers and attenuated kidney tissue damage could be observed, indicating that, depending on the predominant conditions, RAGE affects NT formation with implications for cellular communication.

Interleukin-33 requires CMRF35-like molecule-1 expression for induction of myeloid cell activation.

IL-33 is a potent activator of various cells involved in allergic inflammation, including eosinophils and mast cells. Despite its critical role in Th2 disease settings, endogenous molecular mechanisms that may regulate IL-33-induced responses remain to be defined. We have recently shown that eosinophils express CMRF35-like molecule (CLM)-1. Yet, the role of CLM-1 in regulating eosinophil functions is still elusive. CLM-1 and CLM-8 expression and cellular localization were assessed in murine bone marrow-derived and/or peritoneal cells at baseline and following IL-33 stimulation (flow cytometry, western blot). IL-33-induced mediator release and signaling were assessed in wild-type (wt) and Clm1(-/-) cells and mice. BM-derived eosinophils express high levels of glycosylated CLM-1. IL-33 induced a rapid, specific, concentration- and time-dependent upregulation of CLM-1 in eosinophils (in vitro and in vivo). Clm1(-/-) eosinophils secreted less IL-33-induced mediators than wt eosinophils. CLM-1 co-localized to ST2 following IL-33 stimulation and was required for IL-33-induced NFκB and p38 phosphorylation. Th2 cytokine (e.g., IL-5, IL-13) and chemokine (e.g., eotaxins, CCL2) secretion was markedly attenuated in IL-33-treated Clm1(-/-) mice. Subsequently, IL-33-challenged mice displayed reduced infiltration of mast cells, macrophages, neutrophils, and B cells. Despite the markedly impaired IL-33-induced eotaxin expression in Clm1(-/-) mice, eosinophil accumulation was similar in wt and Clm1(-/-) mice, due to hyperchemotactic responses of Clm1(-/-) eosinophils. CLM-1 is a novel regulator of IL-33-induced eosinophil activation. These data contribute to the understanding of endogenous molecular mechanisms regulating IL-33-induced responses and may ultimately lead to receptor-based tools for future therapeutic intervention in IL-33-associated diseases.

Molecular Cloning and Function Characterization of a New Macrophage-Activating Protein from Tremella fuciformis

Silver ear mushroom ( Tremella fuciformis ) is an edible fungus with health benefits. In this study, we purified a new T. fuciformis protein (TFP) and demonstrated its ability to activate primary murine macrophages. The isolation procedure involved ammonium sulfate fractionation and ion exchange chromatography. TFP naturally formed a 24 kDa homodimeric protein and did not contain glycan residues. The TFP gene was cloned using the rapid amplification of cDNA ends method, and the cDNA sequence of TFP was composed of 408 nucleotides with a 336 nucleotide open reading frame encoding a 112 amino acid protein. TFP was capable of stimulating TNF-α, IL-1β, IL-1ra, and IL-12 production in addition to CD86/MHC class II expression, mRNA expression of M1-type chemokines, and nuclear NF-κB accumulation in murine peritoneal macrophage cells. Furthermore, TFP failed to stimulate TLR4-neutralized and TLR4-knockout macrophages, suggesting that TLR4 is a required receptor for TFP signaling on macrophages. Taken together, these results indicate that TFP may be an important bioactive compound from T. fuciformis that induces M1-polarized activation through a TLR4-dependent NF-κB signaling pathway.

Inhibition of macrophage functions by the C-terminus of murine S100A9 is dependent on B-1 cells.

The protein S100A9 plays a key role in the control of inflammatory response. The C-terminus of the murine S100A9 protein (mS100A9p) downregulates the spreading and phagocytic activity of adherent peritoneal cells. Murine peritoneal cells are constituted by macrophages and B-1 cells, and the latter exert an inhibitory effect on macrophage functions by secreting interleukin- (IL-) 10. Here, we investigated the influence of B-1 cells on the inhibitory effect evoked by mS100A9p on macrophages. mS100A9p did not alter spreading and phagocytosis either by peritoneal macrophages obtained from mice deprived of B-1 cells or by bone marrow-derived macrophages (BMDMϕ). Nevertheless, when BMDMϕ were cocultivated by direct or indirect contact with B-1 cells treated with mS100A9p, the phagocytosis by BMDMϕ was decreased, showing that the effect of mS100A9p on macrophages was modulated by B-1 cells and/or their secretory compounds. Furthermore, the inhibitory action of mS100A9p on phagocytosis by adherent peritoneal cells was abolished in cells obtained from IL-10 knockout mice. Taken together, the results show that mS100A9p has no direct inhibitory effect on macrophages; however, mS100A9p modulates B-1 cells, which in turn downregulates macrophages, at least in part, via IL-10. These data contribute to the characterization of S100A9 functions involving B-1 cells in the regulation of the inflammatory process.

An Intrinsic Propensity of Murine Peritoneal B1b Cells to Switch to IgA in Presence of TGF-β and Retinoic Acid

AimsIn the present study we have investigated the comparative switching propensity of murine peritoneal and splenic B cell subpopulations to IgA in presence of retinoic acid (RA) and TGF-β.Methods and ResultsTo study the influence of RA and TGF-β on switching of B cell subpopulations to IgA, peritoneal (B1a, B1b and B2 cells) and splenic (B1a, marginal zone, and B2) B cells from normal BALB/c mice were FACS purified, cultured for 4 days in presence of RA and TGF-β and the number of IgA producing cells was determined by ELISPOT assay or FACS analysis. In presence of TGF-β, peritoneal B1b cells switched to IgA more potently than other peritoneal B cell subpopulations. When TGF-β was combined with retinoic acid (RA), switching to IgA was even more pronounced. Under these conditions, “innate” B cells like peritoneal and splenic B1 cells and MZ B cells produced IgA more readily than B2 cells. Additionally, high frequency of nucleotide exchanges indicating somatic hypermutation in VH regions was observed. Besides IgA induction, RA treatment of sorted PEC and splenic B cells led to expression of gut homing molecules - α4β7 and CCR9. Intraperitoneal transfer of RA-treated B1 cells into Rag1-/- recipients resulted in IgA in serum and gut lavage, most efficiently amongst B1b cell recipients.ConclusionPresent study demonstrates the differential and synergistic effect of RA and TGF-β on switching of different B cell subpopulations to IgA and establishes the prominence of peritoneal B1b cells in switching to IgA under the influence of these two factors. Our study extends our knowledge about the existing differences among B cell subpopulations with regards to IgA production and indicates towards their differential contribution to gut associated humoral immunity.

β-defensins activate human mast cells via Mas-related Gene-X2: cross-regulation by LPS (P4179)

Abstract Human β-defensins (hBDs) stimulate degranulation in rat peritoneal mast cells and cause increased vascular permeability in wild-type but not mast cell-deficient rats. Here, we sought to determine if hBDs activate murine and human mast cells and to delineate the mechanisms of their regulation. hBD2 and hBD3 did not induce degranulation in murine peritoneal or bone marrow-derived mast cells in vitro and had no effect on vascular permeability in vivo. By contrast, they induced sustained Ca2+ mobilization and substantial degranulation in human mast cells with hBD3 being more potent. While human mast cells endogenously express G protein coupled receptor (GPCR); Mas-related gene X2 (MrgX2), murine mast cells and rat basophilic leukemia, RBL-2H3 cells do not. Silencing the expression of MrgX2 in human mast cells inhibited hBD2/3-induced degranulation and ectopic expression of MrgX2 in RBL-2H3 cells restored these responses. Interestingly, lipopolysaccharide (LPS) inhibited hBD3 but not hBD2-induced Ca2+ mobilization and degranulation. Thus, hBDs activate human mast cells via MrgX2 and that the resistance of murine mast cells likely reflects the absence of this GPCR. Furthermore, the ability of LPS to inhibit hBD3/MrgX2-mediated mast cell activation provides a novel complexity of the role of mast cells in host defense and inflammation.

Intracellular localization of myeloperoxidase in murine peritoneal B-lymphocytes and macrophages

Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions.

Oxidised dextrans influence on reactive oxygen species generation by murine peritoneal exudate phagocytic cells

The effects of oxidized dextrans of different molecular weight on reactive oxygen species production and transmembrane mitochondrial potential of macrophages and neutrophils have been studied in vivo and in vitro. Oxidised dextrans demonstrated moderate direct antioxidant ability but induced intracellular oxidative stress through the increase of oxygen radical generation. This effect of the investigated compounds amplifies the cytotoxic and bactericidal potential of phagocytes and can influence isoniazid metabolism, thus increasing its efficiency in therapy of infectious diseases.

Critical role of Toll‐like receptor 2 in Bacteroides fragilis‐mediated immune responses in murine peritoneal mesothelial cells

In this study, the role of Toll-like receptor 2 (TLR2) in immune responses of murine peritoneal mesothelial cells against Bacteroides fragilis was investigated. Enzyme linked immunosorbent assay was used to measure cytokines and chemokines. Activation of nuclear factor κB (NF-κB-α) and mitogen-activated protein kinases (MAP kinases) was investigated by western blot analysis. B. fragilis induced production of interleukin-6, chemokine (C-X-C motif) ligand 1 (CXCL1) and chemokine (C-C motif) ligand 2 (CCL2) in wild type peritoneal mesothelial cells; this was impaired in TLR2-deficient cells. In addition, in response to B. fragilis, phosphorylation of inhibitory NF-κB-α and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) was induced in wild type mesothelial cells, but not in TLR2-deficient cells,. Inhibitor assay revealed that NF-κB and MAPKs are essential for B. fragilis-induced production of CXCL1 and CCL2 in mesothelial cells. These findings suggest that TLR2 mediates immune responses in peritoneal mesothelial cells in response to B. fragilis.

Effects of sterigmatocystin on TNF-α, IL-6 and IL-12 expression in murine peripheral blood mononuclear cells and peritoneal macrophages in vivo

Sterigmatocystin (ST) is a toxic metabolite mainly produced by the fungi Aspergillus nidulans and Aspergillus versicolor. ST is considered a potent carcinogen, mutagen and teratogen. However, over the past few years, it has been demonstrated that it is less acutely toxic to rodents in vivo. In this study, we evaluated the putative effects of ST on the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-12 at mRNA levels in murine peripheral blood mononuclear cells (mPBMCs) and peritoneal macrophage cells and on the serum TNF-α and IL-6 levels in BALB/c mice. Our results show the downregulation of TNF-α, IL-6 and IL-12 mRNA expression in mPBMCs and peritoneal macrophage cells using semi-quantitative reverse transcription-polymerase chain reaction following ST treatment by intraperitoneal injection. Additionally, serum TNF-α and IL-6 levels were also decreased as shown by enzyme-linked immunosorbent assay (ELISA). These results suggest that ST contamination has negative immunomodulatory effects through the downregulation of cytokine expression and secretion.

Immunostimulatory properties of the major protein from the stem of the Ayurvedic medicinal herb, guduchi (Tinospora cordifolia)

Ethnopharmacological relevanceGuduchi (Tinospora cordifolia), a widely used plant in folk and Ayurvedic systems of medicine is well known for its immunomodulatory activity; however, the presence of an immunomodulatory protein (ImP) in guduchi has not been investigated. Materials and methodsGuduchi ImP was purified from dry stem powder extract by anion-exchange chromatography on Q-Sepharose. Characterization of guduchi ImP was performed by SDS-PAGE, periodic acid-Schiff staining, HPLC, and immunochemical analyses. Immunostimulatory activity was assessed by lymphocyte proliferation and macrophage activation assays. Fresh guduchi stem/leaf, guduchi satwa and guduchi capsules were also analyzed for the presence of guduchi ImP. ResultsGuduchi ImP was purified to homogeneity from dry stem powder extract (∼150mg protein per 100g guduchi stem powder) as a single chain acidic protein (25kDa) without glycans; it was noticeably absent in guduchi leaf. Guduchi satwa and guduchi capsule preparations also lacked this protein. Guduchi ImP showed ∼3-fold mitogenic activity compared to untreated murine splenocytes in the 1–10μg/mL concentration range; 5–7-fold increase in mitogenic activity was seen in the case of murine thymocytes vs. control. The purified protein also induced nitric oxide production from macrophages present in isolated murine peritoneal exudates cells. Guduchi ImP displays enhanced phagocytosis of yeast cells by macrophages. Guduchi ImP does not possess hemagglutination activity (towards rabbit and human erythrocytes of all blood groups) indicating that the immunomodulatory protein is not a lectin. ConclusionsThe confirmation of an immunomodulatory protein in guduchi stem showing lymphoproliferative and macrophage-activating properties reinforces the rationale of the use of guduchi preparations in several Ayurvedic medicines for immunomodulation. To our knowledge, this is the first report of an immunomodulatory protein isolated from guduchi.

Carbon tetrachloride: A hepatotoxin causes oxidative stress in murine peritoneal macrophage and peripheral blood lymphocyte cells

Context: Carbon tetrachloride (CCl4) is frequently used as a chemical inducer of tissue damage. Their effects on mouse peritoneal macrophages and also in peripheral blood lymphocytes are still unknown.Objective: Therefore we tried to focus on intracellular oxidative stress produced by CCl4 in mouse macrophage and lymphocyte cells.Methods: Intraperitoneal administration of CCl4 induces intracellular superoxide anions production in mouse macrophages and peripheral blood lymphocytes and leads a subsequent lipid peroxidation and protein oxidation. N-acetyl cystein (NAC) and vitamin C were administered intraperitoneally at a dose of 150 mg/kg and their effect on demodulating the oxidative stress is also checked.Result and discussion: Several in vitro approaches have already been established as a free radical scavenging models, but this free radical screening models is not always correlated with the in vivo screening models. NAC and vitamin C were administered intraperitoneally and significant reduction of the oxidative stress in term of scavenging of toxic superoxide anion observed in both the macrophages and lymphocytes.Conclusion: Therefore we are hopeful that our work will light a new insight into the screening of in vivo free radical scavenging model for evaluating anti-inflammatory compounds.