Genome-Wide Transcriptional Analysis Reveals Novel AhR Targets That Regulate Dendritic Cell Function during Influenza A Virus Infection.

Activation of the ligand inducible aryl hydrocarbon receptor (AhR) during primary influenza A virus infection diminishes host responses by negatively regulating the ability of dendritic cells (DC) to prime naive CD8+ T cells, which reduces the generation of CTL. However, AhR-regulated genes and signaling pathways in DCs are not fully known. In this study, we used unbiased gene expression profiling to identify differentially expressed genes and signaling pathways in DCs that are modulated by AhR activation in vivo. Using the prototype AhR agonist TCDD, we identified the lectin receptor Cd209a (DC-SIGN) and chemokine Ccl17 as novel AhR target genes. We further show the percentage of DCs expressing CD209a on their surface was significantly decreased by AhR activation during infection. Whereas influenza A virus infection increased CCL17 protein levels in the lung and lung-draining lymph nodes, this was significantly reduced following AhR activation. Targeted excision of AhR in the hematopoietic compartment confirmed AhR is required for downregulation of CCL17 and CD209a. Loss of AhR's functional DNA-binding domain demonstrates that AhR activation alone is necessary but not sufficient to drive downregulation. AhR activation induced similar changes in gene expression in human monocyte-derived DCs. Analysis of the murine and human upstream regulatory regions of Cd209a and Ccl17 revealed a suite of potential transcription factor partners for AhR, which may coregulate these genes in vivo. This study highlights the breadth of AhR-regulated pathways within DCs, and that AhR likely interacts with other transcription factors to modulate DC functions during infection.

Targeted deletion of the aryl hydrocarbon receptor in dendritic cells prevents thymic atrophy in response to dioxin.

In nearly every species examined, administration of the persistent environmental pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) causes profound immune suppression and thymic atrophy in an aryl hydrocarbon receptor (AhR) dependent manner. Moreover, TCDD alters the development and differentiation of thymocytes, resulting in decreases in the relative proportion and absolute number of double positive (DP, CD4+CD8+) thymocytes, as well as a relative enrichment in the relative proportion and absolute number of double negative (DN, CD4-CD8-) and single-positive (SP) CD4+CD8- and CD4-CD8+ thymocytes. Previous studies suggested that the target for TCDD-induced thymic atrophy resides within the hemopoietic compartment and implicated apoptosis, proliferation arrest of thymic progenitors, and emigration of DN thymocytes to the periphery as potential contributors to TCDD-induced thymic atrophy. However, the precise cellular and molecular mechanisms involved remain largely unknown. Our results show that administration of 10 µg/kg TCDD and 8 mg/kg 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) induced AhR-dependent thymic atrophy in mice on day 7, whereas 100 mg/kg indole 3-carbinol (I3C) did not. Though our studies demonstrate that TCDD triggers a twofold increase in the frequency of apoptotic thymocytes, TCDD-induced thymic atrophy is not dependent on Fas-FasL interactions, and thus, enhanced apoptosis is unlikely to be a major mechanistic contributor. Finally, our results show that activation of the AhR in CD11c+ dendritic cells is directly responsible for TCDD-induced alterations in the development and differentiation of thymocytes, which results in thymic atrophy. Collectively, these results suggest that CD11c+ dendritic cells play a critical role in mediating TCDD-induced thymic atrophy and disruption of T lymphocyte development and differentiation in the thymus.

Dendritic Cell Assays.

Generation of dendritic cells from both mouse and human tissues is a valuable technique for downstream immunotoxicological applications. Here, we describe methods for generation of four subsets of dendritic cells from murine bone marrow and three subsets of dendritic cells from human peripheral blood mononuclear cells.

Aryl hydrocarbon receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells.

Currently, it is not well understood how ligands of the aryl hydrocarbon receptor (AhR) modify inflammatory responses triggered by Toll-like receptor (TLR) agonists in human dendritic cells (DCs). Here, we show that AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the tryptophan derivatives 6-formylindolo[3,2-b] carbazole (FICZ), kynurenine (kyn), and the natural dietary compound indole-3-carbinol (I3C) differentially modify cytokine expression in human monocyte-derived DCs (MoDCs). The results show that TLR-activated MoDCs express higher levels of AhR and are more sensitive toward the effects of AhR ligands. Depending on the cytokine, treatment with AhR ligands led to a synergistic or antagonistic effect of the TLR-triggered response in MoDCs. Thus, activation of AhR increased the expression of interleukin (IL)-1ß, but decreased the expression of IL-12A in TLR-activated MoDCs. Furthermore, TCDD and FICZ may have opposite effects on the expression of cytochrome P4501A1 (CYP1A1) in TLR8-activated MoDCs indicating that the effect of the specific AhR ligand may depend on the presence of the specific TLR agonist. Gene silencing showed that synergistic effects of AhR ligands on TLR-induced expression of IL-1ß require a functional AhR and the expression of NF-κB RelB. On the other hand, repression of IL-12A by TCDD and FICZ involved the induction of the caudal type homeobox 2 (CDX2) transcription factor. Additionally, the levels of DC surface markers were decreased in MoDCs by TCDD, FICZ and I3C, but not by kyn. Overall, these data demonstrate that AhR modulates TLR-induced expression of cytokines and DC-specific surface markers in MoDCs involving NFκB RelB and the immune regulatory factor CDX2.

Environmental Immunology: Lessons Learned from Exposure to a Select Panel of Immunotoxicants.

Exposure to environmental contaminants can produce profound effects on the immune system. Many classes of xenobiotics can significantly suppress or enhance immune responsiveness depending on the levels (i.e., dose) and context (i.e., timing, route) of exposure. Although defining the effects that toxicants can have on the immune system is a valuable component to improving public health, environmental immunology has greatly enhanced our understanding of how the immune system functions and has provided innovative avenues to explore new immunotherapies. This Brief Review focuses on three examples of how immunotoxicology has benefitted the field of immunology, presenting information on the aryl hydrocarbon receptor signaling pathway, the immunomodulatory effects of nanomaterials, and the impact of xenobiotic exposure on the developing immune system. Collectively, contributions from immunotoxicology have significantly enhanced public health and spurred seminal advances in both basic and applied immunology.

Acute Exposure to Crystalline Silica Reduces Macrophage Activation in Response to Bacterial Lipoproteins.

Numerous studies have examined the relationship between alveolar macrophages (AMs) and crystalline silica (SiO2) using in vitro and in vivo immunotoxicity models; however, exactly how exposure to SiO2 alters the functionality of AM and the potential consequences for immunity to respiratory pathogens remains largely unknown. Because recognition and clearance of inhaled particulates and microbes are largely mediated by pattern recognition receptors (PRRs) on the surface of AM, we hypothesized that exposure to SiO2 limits the ability of AM to respond to bacterial challenge by altering PRR expression. Alveolar and bone marrow-derived macrophages downregulate TLR2 expression following acute SiO2 exposure (e.g., 4 h). Interestingly, these responses were dependent on interactions between SiO2 and the class A scavenger receptor CD204, but not MARCO. Furthermore, SiO2 exposure decreased uptake of fluorescently labeled Pam2CSK4 and Pam3CSK4, resulting in reduced secretion of IL-1ß, but not IL-6. Collectively, our data suggest that SiO2 exposure alters AM phenotype, which in turn affects their ability to uptake and respond to bacterial lipoproteins.

In vitro exposure to the herbicide atrazine inhibits T cell activation, proliferation, and cytokine production and significantly increases the frequency of Foxp3+ regulatory T cells.

The herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-s-triazine) is the most common water contaminant in the United States. Atrazine is a phosphodiesterase inhibitor and is classified as an estrogen disrupting compound because it elevates estrogen levels via induction of the enzyme aromatase. Previous studies have shown that atrazine exposure alters the function of innate immune cells such as NK cells, DC, mast cells, and macrophages. In this study we have examined the impact of in vitro atrazine exposure on the activation, proliferation, and effector cytokine production by primary murine CD4(+) T lymphocytes. We found that atrazine exposure significantly inhibited CD4(+) T cell proliferation and accumulation as well as the expression of the activation markers CD25 and CD69 in a dose-dependent manner. Interestingly, the effects were more pronounced in cells from male animals. These effects were partially mimicked by pharmacological reagents that elevate intracellular cAMP levels and addition of exogenous rmIL-2 further inhibited proliferation and CD25 expression. Consistent with these findings, atrazine exposure during T cell activation resulted in a 2- to 5-fold increase in the frequency of Foxp3(+) CD4(+) T cells.

Role of the aryl hydrocarbon receptor (AhR) in lung inflammation.

Millions of individuals worldwide are afflicted with acute and chronic respiratory diseases, causing temporary and permanent disabilities and even death. Oftentimes, these diseases occur as a result of altered immune responses. The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, acts as a regulator of mucosal barrier function and may influence immune responsiveness in the lungs through changes in gene expression, cell-cell adhesion, mucin production, and cytokine expression. This review updates the basic immunobiology of the AhR signaling pathway with regards to inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and silicosis following data in rodent models and humans. Finally, we address the therapeutic potential of targeting the AhR in regulating inflammation during acute and chronic respiratory diseases.

Aryl hydrocarbon receptor (AhR) regulates silica-induced inflammation but not fibrosis.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is responsible for mediating a variety of pharmacological and toxicological effects caused by halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, recent evidence has revealed that the AhR also has numerous physiological roles aside from xenobiotic metabolism, including regulation of immune and inflammatory signaling as well as normal development and homeostasis of several organs. To investigate the role of the AhR in crystalline silica (SiO(2))-induced inflammation and fibrosis, C57Bl/6 and AhR(-/)(-) mice were exposed to SiO(2) or vehicle. Similarly, C57Bl/6 mice were exposed to SiO(2) and TCDD either simultaneously or sequentially to assess whether AhR activation alters inflammation and fibrosis. SiO(2)-induced acute lung inflammation was more severe in AhR(-)(/-) mice; however, the fibrotic response of AhR(-)(/-) mice was attenuated compared with C57Bl/6 mice. In a model of chronic SiO(2) exposure, AhR activation by TCDD in C57Bl/6 mice resulted in reduced inflammation; however, the fibrotic response was not affected. Bone marrow-derived macrophages (BMM) from AhR(-)(/-) mice also produced higher levels of cytokines and chemokines in response to SiO(2). Analysis of gene expression revealed that BMM derived from AhR(-)(/-) mice exhibit increased levels of pro-interleukin (IL)-1ß, IL-6, and Bcl-2, yet decreased levels of signal transducers and activators of transcription (STAT)2, STAT5a, and serpin B2 (Pai-2) in response to SiO(2).

Inhibition of TLR ligand- and interferon gamma-induced murine microglial activation by Panax notoginseng.

Among the many products which influence microglial activation and resulting neuroinflammation, herbal medicine has recently drawn much attention due to its immunomodulatory and neuroprotective activities. The purpose of the current study was to investigate the effects of an extract of Panax notoginseng (NotoG™) on TLR ligand- and IFNγ-induced activation in N9 and EOC20 microglial cells lines. NotoG suppressed microglial activation as measured by reduced expression of accessory molecules (CD40 and CD86), decreased production of inflammatory mediators (IL-6 and TNFα), and diminished release of antibacterial products (nitric oxide). Furthermore, this immunosuppressive activity was neither dependent on the glucocorticoid receptor, nor the result of a single ginsenosides (Rb1, Rg1, or Re), which are the major active constituents of the whole extract. NotoG and select ginsenosides may therefore be of therapeutic benefit in treating or preventing neurodegenerative diseases such as multiple sclerosis and parkinson's disease.

Dietary ligands of the aryl hydrocarbon receptor induce anti-inflammatory and immunoregulatory effects on murine dendritic cells.

Activation of the aryl hydrocarbon receptor (AhR) in immune cells, such as dendritic cells (DCs), can lead to suppressed immune responses. Although AhR activation is most recognized for mediating the effects of its prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), many compounds existing in dietary sources can also bind the AhR. Because the immunomodulatory effects of indole-3-carbinol (I3C) and indirubin-3'-oxime (IO) have yet to be investigated in DCs, we evaluated the potential immunomodulatory effects of these compounds on murine DCs. We hypothesized that I3C and IO suppress immune and inflammatory responses in DCs. We found that both I3C and IO decreased the expression of CD11c, CD40, and CD54 while they increased expression of MHC2 and CD80. Following lipopolysaccharide (LPS)-activation, I3C and IO suppressed the production of pro-inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-12, and nitric oxide but increased IL-10 levels. These effects of I3C and IO were partially mediated by the AhR. Additionally, immunoregulatory genes, such as ALDH1A, IDO and TGFB, were upregulated following treatment with I3C or IO. Both I3C and IO decreased basal levels of nuclear factor-kappa B p65, but only I3C suppressed the LPS-induced activity of RelB. Finally, when cultured with naïve T cells, bone marrow-derived dendritic cells treated with the dietary AhR ligands increased the frequency of Foxp3+ Tregs in an antigen-specific manner. Taken together, these results indicate that I3C and IO exhibit immunosuppressive and anti-inflammatory effects on DCs. Because I3C and IO are significantly less toxic than TCDD, these natural products may ultimately become useful therapeutics for the treatment of autoimmune and inflammatory diseases.

Anti-inflammatory effects of natural product formulations on murine dendritic cells.

The popularity and availability of herbal extracts has increased dramatically over the last decade, providing an inexpensive manner of self-medication. Although the efficacy of individual extracts is currently being studied intensively, research regarding complex mixtures is limited. Therefore, we evaluated the effects of three complex formulations, including BRC-301, a polyherbal extract; BRC-304, a mixture of vitamins, minerals, antioxidant enzymes, botanical extracts, and carotenoids; and BRC-306, a proprietary blend of Uncaria tomentosa (cat's claw) and Phytolens(®) on murine dendritic cells (DCs). We hypothesized that these formulations would decrease the inflammatory responsiveness and innate function of DCs. In order to address this hypothesis, we evaluated the effects of BRC-301, BRC-304, and BRC-306 on DC2.4 cells and assessed the effects of BRC-301 on bone marrow-derived DCs (bmDCs). Lipopolysaccharide (LPS) stimulation of DC2.4 cells and bmDCs induced production of nitric oxide (NO), TNF-α, and IL-6, a response that was modulated by concomitant treatment with non-cytotoxic concentrations of BRC-301. In contrast, only the production of NOor IL-6 by LPS-activated DC2.4 cells was affected by BRC-304 or BRC-306, respectively. Flow cytometric evaluation following concurrent BRC-301 and LPS treatment revealed an increased relative expression of CD11c, CD86, and CD54 on bmDCs and an increased frequency of bmDCs expressing MHC II. Finally, BRC-301 enhanced the uptake of fluorescein isothiocyanate-conjugated ovalbumin by bmDCs. Taken together, these results suggest that these commercially available formulations modulate the innate responsiveness of murine DCs and may enhance their ability to initiate T cell-mediated immunity.

Aryl hydrocarbon receptor activation by TCDD reduces inflammation associated with Crohn's disease.

Crohn's disease results from a combination of genetic and environmental factors that trigger an inappropriate immune response to commensal gut bacteria. The aryl hydrocarbon receptor (AhR) is well known for its involvement in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental contaminant that affects people primarily through the diet. Recently, TCDD was shown to suppress immune responses by generating regulatory T cells (Tregs). We hypothesized that AhR activation dampens inflammation associated with Crohn's disease. To test this hypothesis, we utilized the 2,4,6-trinitrobenzenesulfonic acid (TNBS) murine model of colitis. Mice were gavaged with TCDD prior to colitis induction with TNBS. Several parameters were examined including colonic inflammation via histological and flow cytometric analyses. TCDD-treated mice recovered body weight faster and experienced significantly less colonic damage. Reduced levels of interleukin (IL) 6, IL-12, interferon-gamma, and tumor necrosis factor-α demonstrated suppression of inflammation in the gut following TCDD exposure. Forkhead box P3 (Foxp3)(egfp) mice revealed that TCDD increased the Foxp3+ Treg population in gut immune tissue following TNBS exposure. Collectively, these results suggest that activation of the AhR by TCDD decreases colonic inflammation in a murine model of colitis in part by generating regulatory immune cells. Ultimately, this work may lead to the development of more effective therapeutics for the treatment of Crohn's disease.

Consequences of AhR activation in steady-state dendritic cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical aryl hydrocarbon receptor (AhR) ligand and a potent immunotoxicant. However, the mechanisms underlying TCDD-induced immunomodulation remain to be defined. Dendritic cells are professional antigen-presenting cells that constitutively express the AhR and are sensitive to TCDD-induced AhR activation. We hypothesized that AhR activation alters the differentiation and function of steady-state bone marrow-derived dendritic cells (BMDCs). To test this hypothesis, steady-state BMDCs from C57BL/6 mice were grown in the presence of TCDD or vehicle. TCDD-treated steady-state BMDCs (TCDD-BMDCs) displayed decreased expression of CD11c and CD11a, whereas increasing the frequency of major histocompatibility complex class II, CD86, CD80, and CD54. Similar phenotypic alterations were observed with the AhR ligands 6-formylindolo[3,2-b]carbazole and 2-(1H-indole-3'-carbonyl)-thiazole-4-carboxylic acid (ITE). TCDD-BMDCs from AhR(-/-) mice were refractory to TCDD-induced surface marker alterations, whereas TCDD-BMDCs from AhR(dbd/dbd) mice displayed similar phenotypic alterations as AhR(+/+) TCDD-BMDCs. Following lipopolysaccharide (LPS), cytosine-phosphate-guanine (CpG), or Imiquimod stimulation, TCDD-BMDCs secreted less interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-10, and IL-12. TCDD also altered NF-κB family member-binding activity in unstimulated and LPS- or CpG-stimulated steady-state BMDCs. The internalization of the soluble antigens, ovalbumin, and acetylated low-density lipoprotein was decreased, whereas internalization of latex beads was increased in TCDD-BMDCs when compared with vehicle-BMDCs. TCDD-BMDCs displayed increased messenger RNA expression of the regulatory gene IDO2 and following LPS stimulation upregulated IDO1, IDO2, TGFß1, and TGFß3 gene expression. Additionally, TCDD-BMDCs increased the generation of CD4(+) CD25(+) FoxP3(+) Tregs in vitro in an IDO-dependent fashion. However, TCDD-treated BMDCs did not alter antigen-specific T-cell activation in vivo. Overall, TCDD-induced AhR activation alters the differentiation, activation, innate, and immunoregulatory function but not the T cell-activating capacity of steady-state BMDCs.

Functional and phenotypic effects of AhR activation in inflammatory dendritic cells.

Aryl hydrocarbon receptor (AhR) activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces immune suppression. Dendritic cells (DCs) are key antigen presenting cells governing T cell activation and differentiation. However, the consequences of AhR activation in DCs are not fully defined. We hypothesized that AhR activation alters DC differentiation and generates dysfunctional DCs. To test this hypothesis, inflammatory bone marrow-derived DCs (BMDCs) from C57Bl/6 mice were generated in the presence of vehicle or TCDD. TCDD decreased CD11c expression but increased MHC class II, CD86 and CD25 expression on the BMDCs. The effects of TCDD were strictly AhR-dependent but not exclusively DRE-mediated. Similar effects were observed with two natural AhR ligands, 6-formylindolo[3,2-b]carbazole (FICZ) and 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid (ITE). TCDD increased LPS- and CpG-induced IL-6 and TNF-alpha production by BMDCs but decreased their NO production. TCDD decreased CpG-induced IL-12p70 production by BMDCs but did not affect their secretion of IL-10. TCDD downregulated LPS- and CpG-induced NF-kB p65 levels and induced a trend towards upregulation of RelB levels in the BMDCs. AhR activation by TCDD modulated BMDC uptake of both soluble and particulate antigens. Induction of indoleamine-2,3-dioxygenase (IDO) and TGF-beta3 has been implicated in the generation of regulatory T cells following AhR activation. TCDD increased IDO1, IDO2 and TGF-beta3 mRNA levels in BMDCs as compared to vehicle. Despite the induction of regulatory mediators, TCDD-treated BMDCs failed to suppress antigen-specific T cell activation. Thus, AhR activation can directly alter the differentiation and innate functions of inflammatory DCs without affecting their ability to successfully interact with T cells.

Effects of TCDD on the fate of naive dendritic cells.

The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes immune suppression via activation of the aryl hydrocarbon receptor. Dendritic cells (DCs), the professional antigen-presenting cells in the immune system, are adversely affected by TCDD. We hypothesized that TCDD alters DC homeostasis, resulting in a loss of DCs in naive mice. To test this hypothesis, C57Bl/6 mice were gavaged with either vehicle or an immunosuppressive dose of TCDD (15 microg/kg). TCDD exposure decreased the frequency and number of splenic CD11c(high) DCs on day 7 when compared with vehicle-treated controls. TCDD increased the expression of CD86 and CD54, while decreasing the frequency of splenic CD11c(high) DCs expressing CD11a and major histocompatibility complex (MHC) class II. Moreover, TCDD selectively decreased the CD11c(high)CD8alpha(-)33D1(+) splenic DCs specialized at activating CD4(+) T cells but did not affect the regulatory CD11c(high)CD8alpha(+)DEC205(+) splenic DCs. TCDD did not alter the number or frequency of CD11c(low) splenic DCs but decreased their MHC class II and CD11a expression. Loss of splenic CD11c(high) DCs was independent of Fas-mediated apoptosis and was not due to alterations in the numbers of common DC precursors in the bone marrow or their ability to generate steady-state DCs in vitro. Instead, increased CCR7 expression on CD11c(high) DCs suggested involvement of a migratory event. Popliteal and brachial lymph node CD11c(+) cells showed elevated levels of MHC class II and CD40 following TCDD exposure. Collectively, this study shows the presence of a TCDD-sensitive splenic DC subpopulation in naive mice, suggesting that TCDD may induce suppression of T-cell-mediated immunity by disrupting DC homeostasis.

Echinacea purpurea extracts modulate murine dendritic cell fate and function.

Echinacea is a top-selling herbal remedy that purportedly acts as an immunostimulant. However, the specific immunomodulatory effects of Echinacea remain to be elucidated. We focused on defining the effects of Echinacea purpurea extracts in dendritic cells (DCs), which generate innate and adaptive immune responses. We hypothesized that E. purpurea extracts would enhance murine bone marrow-derived DC (BMDC) activation leading to increased immune responses. The fate and function of DCs from C57Bl/6 mice was evaluated following 48h exposure to E. purpurea root and leaf extracts. Flow cytometry revealed that the polysaccharide-rich root extract increased the expression of MHC class II, CD86, and CD54 surface biomarkers whereas the alkylamide-rich leaf extract inhibited expression of these molecules. Production of IL-6 and TNF-alpha increased in a concentration-dependent manner with exposure to the root, but not leaf, extract. In contrast, the leaf but not root extract inhibited the enzymatic activity of cyclooxygenase-2. While both extracts decreased the uptake of ovalbumin by BMDCs, the leaf but not root extract inhibited the antigen-specific activation of naïve CD4(+) T cells from OT II/Thy1.1 mice. Collectively, these results suggest that E. purpurea can be immunostimulatory, immunosuppressive, and/or anti-inflammatory depending on the portion of the plant and extraction method.

Anti-inflammatory effects of natural product formulations on murine macrophages.

The popularity of herbal supplements, especially those with purported anti-inflammatory effects, has drastically increased in recent years as more people have turned to natural therapeutics. As the supplement industry is loosely regulated, the safety and efficacy of these products is poorly understood. In the present study, we examined the effects of natural product formulations prepared by the Biotics Research Corporation (BRC) on cyclooxygenase (COX) enzyme activity. We also evaluated the immune responsiveness of RAW264.7 macrophages, a key cell population involved in the inflammation, to those formulations. As a result, three supplements, BRC-301, BRC-304, and BRC-306, selectively inhibited COX-2, the inducible isoform involved in inflammation. Further evaluation of these three products indicated that BRC-304 and BRC-306 produced minimal effects on the production of inflammatory mediators by lipopolysaccharide (LPS)-stimulated macrophages. BRC-301 decreased the LPS-induced production of nitric oxide and IL-6, as well as CD40 expression. Collectively, these results suggest that the BRC-301 extract, comprising several polyphenolic natural products, may have a protective effect in chronic inflammatory disorders.