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Aims: Activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway in normal cells inhibits carcinogenesis, whereas constitutive activation of Nrf2 in cancer cells promotes tumor growth and chemoresistance. However, the effects of Nrf2 activation in immune cells during lung carcinogenesis are poorly defined and could either promote or inhibit cancer growth. Our studies were designed to evaluate tumor burden and identify immune cell populations in the lungs of Nrf2 knockout (KO) versus wild-type (WT) mice challenged with vinyl carbamate. Results: Nrf2 KO mice developed lung tumors earlier than the WT mice and exhibited more and larger tumors over time, even at late stages. T cell populations were lower in the lungs of Nrf2 KO mice, whereas tumor-promoting macrophages and myeloid-derived suppressor cells were elevated in the lungs and spleen, respectively, of Nrf2 KO mice relative to WT mice. Moreover, 34 immune response genes were significantly upregulated in tumors from Nrf2 KO mice, especially a series of cytokines (Cxcl1, Csf1, Ccl9, Cxcl12, etc.) and major histocompatibility complex antigens that promote tumor growth. Innovation: Our studies discovered a novel immune signature, characterized by the infiltration of tumor-promoting immune cells, elevated cytokines, and increased expression of immune response genes in the lungs and tumors of Nrf2 KO mice. A complementary profile was also found in lung cancer patients, supporting the clinical significance of our findings. Conclusion: Overall, our results confirmed a protective role for Nrf2 in late-stage carcinogenesis and, unexpectedly, suggest that activation of Nrf2 in immune cells may be advantageous for preventing or treating lung cancer.

Tert-butylhydroquinone (tBHQ) is a commonly used food preservative with known immunomodulatory activity; however, there is little information regarding its role on natural killer (NK) cell activation and function. tBHQ is a known activator of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which results in induction of cytoprotective genes. Activation of Nrf2 has been shown to modulate immune responses in a number of different models. In addition, studies in our laboratory have shown that tBHQ inhibits numerous early events following T cell activation. In the current study, we investigated whether activated NK cells are impacted by tBHQ, since many signaling cascades that control NK cell effector function also contribute to T cell function. Splenocytes were isolated from female, wild-type C57Bl/6J mice and treated with 1 μM or 5 μM tBHQ. NK cell function was assessed after activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 24 h. Activation of NK cells in the presence of tBHQ decreased total NK cell percentage, production of intracellular interferon gamma (IFNƔ), granzyme B, and perforin, and induction of the cell surface proteins CD25 and CD69, which are markers of NK cell activation. In addition to NK cell effector function, NK cell maturation was also altered in response to tBHQ. Notably, this is the first study to demonstrate that the Nrf2 activator, tBHQ, negatively impacts effector function and maturation of NK cells.

Kupffer cells and monocyte-derived macrophages are critical for liver repair after acetaminophen (APAP) overdose. These cells produce promitogenic cytokines and growth factors, and they phagocytose dead cell debris, a process that is critical for resolution of inflammation. The factors that regulate these dynamic functions of macrophages after APAP overdose, however, are not fully understood. We tested the hypothesis that the fibrinolytic enzyme, plasmin, is a key regulator of macrophage function after APAP-induced liver injury. In these studies, inhibition of plasmin in mice with tranexamic acid delayed up-regulation of proinflammatory cytokines after APAP overdose. In culture, plasmin directly, and in synergy with high-mobility group B1, stimulated Kupffer cells and bone marrow–derived macrophages to produce cytokines by a mechanism that required NF-κB. Inhibition of plasmin in vivo also prevented trafficking of monocyte-derived macrophages into necrotic lesions after APAP overdose. This prevented phagocytic removal of dead cells, prevented maturation of monocyte-derived macrophages into F4/80-expressing macrophages, and prevented termination of proinflammatory cytokine production. Our studies reveal further that phagocytosis is an important stimulus for cessation of proinflammatory cytokine production as treatment of proinflammatory, monocyte-derived macrophages, isolated from APAP-treated mice, with necrotic hepatocytes decreased expression of proinflammatory cytokines. Collectively, these studies demonstrate that plasmin is an important regulator of macrophage function after APAP overdose.

Perivascular adipose tissue (PVAT) is protective and reduces contraction of blood vessels in health. PVAT is composed of adipocytes, multiple types of immune cells and stromal cells. Interleukin (IL)-10, an anti-inflammatory cytokine usually produced by T cells, B cells and macrophages, was identified as one of the highly expressed (mRNA) cytokines in the mesenteric PVAT of healthy rats. One report suggested that exogenous IL-10 causes relaxation of mouse mesenteric arteries, also suggesting that IL-10 maybe a potential anti-contractile factor. Hence, we hypothesized that PVAT-derived IL-10 causes vasorelaxation and/or reduces vasoconstriction, thus contributing to the anti-contractile nature of PVAT in health. Mesenteric arteries from rats and mice expressed the receptor for IL-10 (in tunica intima and media) as determined by immunohistochemistry. Mesenteric resistance arteries for rats and superior mesenteric artery for mice were used for isometric contractility studies. Increasing concentrations [0.4– 100 ng/mL] of recombinant rat/ mouse (rr/ mr) IL-10 or vehicle was directly added to half-maximally constricted (phenylephrine, PE) vessels (without PVAT, with endothelium). IL-10 did not cause a direct vasorelaxation. Further, the ability of rrIL-10 to cause a rightward or downward shift of a vasoconstriction-response curve was tested in the rat. The vessels were incubated with rrIL-10 [100 ng/mL or 10 ng/mL] or vehicle for 1.5 hours in the tissue bath followed by a cumulative PE [10(−8)–10(−4) M] or U46619 [10(−10)–10(−5) M] response curve. The maximal contractions and EC(50) values were similar in IL-10 incubated vessels vs vehicle. Thus, acute exposure of exogenous IL-10 did not reduce local vasoconstriction. To further test if endogenous IL-10 from PVAT was anti-contractile, superior mesenteric arteries from IL-10 WT and KO mice, with and without PVAT, were subjected to increasing concentrations of PE. The anti-contractile nature of PVAT was preserved with both short-term and prolonged depletion (using younger and older mice, respectively) of endogenous IL-10 in males and females. Contrary to our hypothesis, PVAT-derived IL-10 neither caused vasorelaxation nor reduced local vasoconstriction directly/ indirectly. Therefore, IL-10 does not contribute to the anti-contractile nature of PVAT in healthy rodents.

Perfluorononanoic acid (PFNA) is a perfluoroalkyl substance (PFAS) that is structurally related to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Whereas PFOA and PFOS are known immunotoxicants, PFNA is less well characterized. Our previous study showed that PFNA has immunomodulatory effects on leukocyte populations and immune function. The present studies sought to determine whether, and to what degree, the immune system recovered 28 days after PFNA exposure. None of the parameters measured had fully recovered. A few parameters had partially recovered, including decreased spleen size and the decreased ratio of the CD4+/CD8+ double-positive population in thymus. The majority of effects of PFNA remained unchanged 28 days after exposure, including decreased proportion of intact thymocytes (as determined by FSC vs SSC), alterations in the ratios of immune cell populations in spleen and the CD4+, CD8+ and double-negative populations in thymus. Notably, PFNA markedly increased the TNFα response to LPS in vivo, and no recovery was evident 28 days after exposure. The effect of PFNA on CD4+ T cells, CD8+ T cells and CD19+ cells was more pronounced in females. The current study demonstrates that a single high dose exposure to PFNA (e.g. as might occur accidentally in an occupational setting) has long-lasting effects on the immune system.

Melanoma is the most dangerous form of skin cancer with the majority of deaths arising from metastatic disease. Evidence implicates Rho-activated gene transcription in melanoma metastasis mediated by the nuclear localization of the transcriptional coactivator, myocardin-related transcription factor (MRTF). Here, we highlight a role for Rho and MRTF signaling and its reversal by pharmacologic inhibition using in vitro and in vivo models of human melanoma growth and metastasis. Using two cellular models of melanoma, we clearly show that one cell type, SK-Mel-147, is highly metastatic, has high RhoC expression, and MRTF nuclear localization and activity. Conversely, SK-Mel-19 melanoma cells have low RhoC expression, and decreased levels of MRTF-regulated genes. To probe the dependence of melanoma aggressiveness to MRTF transcription, we use a previously developed small-molecule inhibitor, CCG-203971, which at low micromolar concentrations blocks nuclear localization and activity of MRTF-A. In SK-Mel-147 cells, CCG-203971 inhibits cellular migration and invasion, and decreases MRTF target gene expression. In addition, CCG-203971–mediated inhibition of the Rho/MRTF pathway significantly reduces cell growth and clonogenicity and causes G1 cell-cycle arrest. In an experimental model of melanoma lung metastasis, the RhoC-overexpressing melanoma cells (SK-Mel-147) exhibited pronounced lung colonization compared with the low RhoC–expressing SK-Mel-19. Furthermore, pharmacologic inhibition of the MRTF pathway reduced both the number and size of lung metastasis resulting in a marked reduction of total lung tumor burden. These data link Rho and MRTF-mediated signaling with aggressive phenotypes and support targeting the MRTF transcriptional pathway as a novel approach to melanoma therapeutics. Mol Cancer Ther; 16(1); 193–204. ©2016 AACR.

Coagulation cascade activation and fibrin deposits have been implicated or observed in diverse forms of liver damage. Given that fibrin amplifies pathological inflammation in several diseases through the integrin receptor αMβ2, we tested the hypothesis that disruption of the fibrin(ogen)-αMβ2 interaction in Fibγ(390-396A) mice would reduce hepatic inflammation and fibrosis in an experimental setting of chemical liver injury. Contrary to our hypothesis, α-naphthylisothiocyanate (ANIT)-induced liver fibrosis increased in Fibγ(390-396A) mice, whereas inflammatory cytokine expression and hepatic necrosis were similar to ANIT-challenged wild-type (WT) mice. Increased fibrosis in Fibγ(390-396A) mice appeared to be independent of coagulation factor 13 (FXIII) transglutaminase, as ANIT challenge in FXIII-deficient mice resulted in a distinct pathological phenotype characterized by increased hepatic necrosis. Rather, bile duct proliferation underpinned the increased fibrosis in ANIT-exposed Fibγ(390-396A) mice. The mechanism of fibrin-mediated fibrosis was linked to interferon (IFN)γ induction of inducible nitric oxide synthase (iNOS), a gene linked to bile duct hyperplasia and liver fibrosis. Expression of iNOS messenger RNA was significantly increased in livers of ANIT-exposed Fibγ(390-396A) mice. Fibrin(ogen)-αMβ2 interaction inhibited iNOS induction in macrophages stimulated with IFNγ in vitro and ANIT-challenged IFNγ-deficient mice had reduced iNOS induction, bile duct hyperplasia, and liver fibrosis. Further, ANIT-induced iNOS expression, liver fibrosis, and bile duct hyperplasia were significantly reduced in WT mice administered leukadherin-1, a small molecule that allosterically enhances αMβ2-dependent cell adhesion to fibrin. These studies characterize a novel mechanism whereby the fibrin(ogen)-integrin-αMβ2 interaction reduces biliary fibrosis and suggests a novel putative therapeutic target for this difficult-to-treat fibrotic disease.

Nrf2 is a transcription factor that regulates cytoprotective cellular responses to oxidative and electrophilic stress. Nrf2 is potently activated by the synthetic food additive, tert-butylhydroquinone (tBHQ), which is widely used as a preservative in oils and processed foods. Previously published studies have established that tBHQ has numerous effects on T cell function. The purpose of this study was to determine the effect of tBHQ on B cell function and the role of Nrf2 in these effects. Specifically, we investigated T cell-independent B cell activation, differentiation, and IgM antibody production. Murine wild-type and Nrf2-null splenocytes were isolated, treated with tBHQ (0.25–2.5 μm), and activated by lipopolysaccharide (LPS), a T cell-independent B cell activator. Our findings indicate that tBHQ significantly enhanced IgM production in activated wild-type, but not Nrf2-null, B cells, suggesting this effect is Nrf2-dependent. In contrast, tBHQ significantly decreased the induction of CD69, CD25, CD22, and CD138 in both wild-type and Nrf2-null splenocytes. These findings indicate that the tBHQ-mediated increase in IgM is Nrf2-dependent, whereas the inhibition of CD69, CD25, CD22 and CD138 is Nrf2-independent. Overall, this study demonstrates that in addition to its effects on T cells, tBHQ also has potent effects on T cell-independent B cell function.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces hepatic oxidative stress following activation of the aryl hydrocarbon receptor (AhR). Our recent studies showed TCDD induced pyruvate kinase muscle isoform 2 (Pkm2) as a novel antioxidant response in normal differentiated hepatocytes. To investigate cooperative regulation between nuclear factor, erythroid derived 2, like 2 (Nrf2) and the AhR in the induction of Pkm2, hepatic chromatin immunoprecipitation sequencing (ChIP-seq) analyses were integrated with RNA sequencing (RNA-seq) time-course data from mice treated with TCDD for 2–168 hours. ChIP-seq analysis 2 hours after TCDD treatment identified genome-wide NRF2 enrichment. Approximately 842 NRF2-enriched regions were located in the regulatory region of differentially expressed genes (DEGs), whereas 579 DEGs showed both NRF2 and AhR enrichment. Sequence analysis of regions with overlapping NRF2 and AhR enrichment showed over-representation of either antioxidant or dioxin response elements, although 18 possessed both motifs. NRF2 exhibited negligible enrichment within a closed Pkm chromatin region, whereas the AhR was enriched 29-fold. Furthermore, TCDD induced Pkm2 in primary hepatocytes from wild-type and Nrf2-null mice, indicating NRF2 is not required. Although NRF2 and AhR cooperate to regulate numerous antioxidant gene expression responses, the induction of Pkm2 by TCDD is independent of reactive oxygen species–mediated NRF2 activation.

Bile acids (BAs) are known to regulate BA synthesis and transport by the farnesoid X receptor in the liver (FXR-SHP) and intestine (FXR-Fgf15). However, the relative importance of individual BAs in regulating these processes is not known. Therefore, mice were fed various doses of five individual BAs, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxoycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) in their diets at various concentrations for one week to increase the concentration of one BA in the enterohepatic circulation. The mRNA of BA synthesis and transporting genes in liver and ileum were quantified. In the liver, the mRNA of SHP, which is the prototypical target gene of FXR, increased in mice fed all concentrations of BAs. In the ileum, the mRNA of the intestinal FXR target gene Fgf15 was increased at lower doses and to a higher extent by CA and DCA than by CDCA and LCA. Cyp7a1, the rate-limiting enzyme in BA synthesis, was decreased more by CA and DCA than CDCA and LCA. Cyp8b1, the enzyme that 12-hydroxylates BAs and is thus responsible for the synthesis of CA, was decreased much more by CA and DCA than CDCA and LCA. Surprisingly, neither a decrease in the conjugated BA uptake transporter (Ntcp) nor increase in BA efflux transporter (Bsep) was observed by FXR activation, but an increase in the cholesterol efflux transporter (Abcg5/Abcg8) was observed with FXR activation. Thus in conclusion, CA and DCA are more potent FXR activators than CDCA and LCA when fed to mice, and thus they are more effective in decreasing the expression of the rate limiting gene in BA synthesis Cyp7a1 and the 12-hydroxylation of BAs Cyp8b1, and are also more effective in increasing the expression of Abcg5/Abcg8, which is responsible for biliary cholesterol excretion. However, feeding BAs do not alter the mRNA or protein levels of Ntcp or Bsep, suggesting that the uptake or efflux of BAs is not regulated by FXR at physiological and pharmacological concentrations of BAs.

We previously demonstrated that activation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) promotes CD4+ Th2 differentiation. In the current study, we assessed the role of Nrf2 in early events following T cell activation. The Nrf2 activators, tBHQ (tert-butylhydroquinone) and CDDO-Im (the imidazolide derivative of the triterpenoid CDDO), were used in conjunction with splenocytes derived from wild-type and Nrf2-null mice to distinguish between Nrf2-specific and off-target effects. CDDO-Im inhibited early IFNγ and TNFα production in a largely Nrf2-dependent manner. In contrast, tBHQ and CDDO-Im had little effect on expression of CD25 or CD69. Furthermore, tBHQ inhibited GM-CSF and IL-2 production in both wild-type and Nrf2-null T cells, suggesting this effect is Nrf2-independent. Conversely, CDDO-Im caused a concentration-dependent increase in IL-2 secretion in wild-type, but not Nrf2-null, splenocytes, suggesting that Nrf2 promotes IL-2 production. Interestingly, both compounds inhibit NFκB DNA binding, where the suppression by tBHQ is Nrf2-independent and CDDO-Im is Nrf2-dependent. Surprisingly, as compared to wild-type splenocytes, Nrf2-null splenocytes showed lower nuclear accumulation of c-Jun, a member of the AP-1 family of transcription factors, which have been shown to drive multiple immune genes, including IL-2. Both Nrf2 activators caused a Nrf2-dependent trend towards increased nuclear accumulation of c-Jun. These data suggest that modulation of cytokine secretion by tBHQ likely involves multiple pathways, including AP-1, NFκB, and Nrf2. Overall, the data suggest that Nrf2 activation inhibits secretion of the Th1 cytokine IFNγ, and increases early production of IL-2, which has been shown to promote Th2 differentiation, and may support the later occurrence of Th2 polarization.

SummaryBackground The prevalence of asthma in the elderly is increasing and associated with higher mortality than in children or young adults. However, the effects of age on the development and character of allergic asthma have been understudied. It has been suggested that mixed Th2/Th17 responses cause more severe forms of asthma, but the role of Th17 response in allergic airway disease and aging is not well understood. Objective To investigate age-dependent characteristics and Th17 immune response in allergic airway disease in a murine house dust mite (HDM)-allergen model. Methods Twelve-week-old and 15-month-old male BALB/c mice were sensitized and challenged with HDM. Bronchoalveolar lavage fluid (BALF), airway inflammation and hyperresponsiveness (AHR), serum immunoglobulin and splenic T cells were assessed. Age-related T cell activation was analyzed in a co-culture with bone marrow-derived dendritic cells (BMDC) and splenic CD4+T cells from young and old mice. Results Features of allergic airway disease such as mucous cell hyperplasia, infiltration of airway eosinophils and lymphocytes, Th2 cytokine expression and serum IgG1 levels were greater in old compared to young mice. In contrast to the more marked inflammatory/immune responses to HDM in old mice, AHR was greater in young HDM-treated mice. Only the old mice developed airway neutrophil infiltration and a Th17 immune response upon HDM exposure, with increases in BALF cytokines IL-17A and KC, and Th17 cytokine producing T cells in the spleen. Stimulation of CD4+T cells and BMDC co-cultures with HDM, resulted in an enhanced Th17 cytokine response in cells isolated from old mice. Conclusions and Clinical Relevance Our findings in mice suggest that the severity and character of allergic airway disease are age dependent, with a bias towards a Th17 immune response with aging. Elderly, asthmatics may be prone to develop severe allergic airway inflammation with a mixed Th2/Th17 immune response.

Background and Purpose: The proteasome is a multi-subunit complex that proteolytically cleaves proteins. The replacement of the constitutive proteasome subunits β1, β2, and/or β5 with the IFNγ-inducible subunits LMP2, MECL1, and/or LMP7 results in the ‘immunoproteasome’. The inducible subunits change the cleavage specificities of the proteasome, but it is unclear whether they have functions in addition to this. The purpose of the present study was to determine the role of the proteasome in general, as well as LMP7 and MECL1 specifically, with regard to cytokine production by activated primary splenocytes. Methods: A LMP7/MECL1-null mouse was engineered to determine the roles of these subunits in cytokine production. Isolated splenocytes from wild-type and LMP7/MECL1–/– mice were treated with lactacystin and activated with PMA and ionomycin and subsequently cytokine mRNA levels were quantified. Results: The present study demonstrates that LMP7/MECL1 regulates the expression of IFNγ, IL4, IL10, IL2Rβ, GATA3, and t-bet. In contrast, the regulation of IL2, IL13, TNFα, and IL2Rα by the proteasome appears to occur independently of LMP7/MECL1. Conclusions: Collectively, the present study demonstrates that LMP7 and MECL1 regulate cytokine expression, suggesting this system represents a novel mechanism for the regulation of cytokines and cytokine signaling.

The pregnane X receptor (PXR) is a key regulator of xenobiotic metabolism and disposition in liver. However, little is known about the PXR DNA-binding signatures in vivo, or how PXR regulates novel direct targets on a genome-wide scale. Therefore, we generated a roadmap of hepatic PXR bindings in the entire mouse genome [chromatin immunoprecipitation (ChIP)-Seq]. The most frequent PXR DNA-binding motif is the AGTTCA-like direct repeat with a 4 bp spacer [direct repeat (DR)-4)]. Surprisingly, there are also high motif occurrences with spacers of a periodicity of 5 bp, forming a novel DR-(5 n+4) pattern for PXR binding. PXR-binding overlaps with the epigenetic mark for gene activation (histone-H3K4-di-methylation), but not with epigenetic marks for gene suppression (DNA methylation or histone-H3K27-tri-methylation) (ChIP-on-chip). After administering a PXR agonist, changes in mRNA of most PXR-direct target genes correlate with increased PXR binding. Specifically, increased PXR binding triggers the trans-activation of critical drug-metabolizing enzymes and transporters. The mRNA induction of these genes is absent in PXR-null mice. The current work provides the first in vivo evidence of PXR DNA-binding signatures in the mouse genome, paving the path for predicting and further understanding the multifaceted roles of PXR in liver.

Our recent studies with lactacystin, a prototype proteasome inhibitor, have suggested that the proteasome is a key regulator of LPS-induced signaling pathways contributing to the inflammatory process. Moreover, lactacystin protects animals from LPS-induced shock. Therefore, we sought to identify other less toxic compounds that would block the chymotrypsin-like activity of the proteasome or LPS-induced nitric oxide (NO). After screening over 100 natural compounds (based on chemistry and inhibition of LPS-induced biological activities), we now report for the first time that quercetin, like lactacystin (the prototype proteasome inhibitor), and mevinolin are also inhibitors of the chymotrypsin-like activity of the cellular proteasome within living cells. In addition, this study also suggests that mevinolin and quercetin both have relatively potent anti-inflammatory effects on LPS-treated macrophages in vitro. Interestingly, both of these compounds behave like lactacystin in that they block LPS-induced NO to a greater extent than TNF-α. The results of our experiments clearly suggest that mevinolin, in combination with the antibiotic imipenem, can provide protection against polymicrobial septic lethality induced by cecal-ligation and puncture in mice. Collectively, these studies strongly support the conclusion that therapeutic targeting of cellular proteasomes, in conjunction with standard antimicrobial therapy, may be of considerable survival benefit in the treatment of septic shock.

Previous studies from this laboratory have demonstrated that a COX-2 metabolite of the endogenous cannabinoid, 2-arachidonyl glycerol (2-AG), inhibits IL-2 secretion in activated T cells through PPARgamma activation independent of the cannabinoid receptors, CB1/CB2. Because numerous cyclooxygenase (COX) products have been shown to activate PPARgamma, the primary purpose of the present studies was to determine the role of COX metabolism in the inhibition of IL-2 secretion by 2-AG. Pretreatment with nonselective and COX-2-specific inhibitors completely abrogated 2-AG-mediated suppression of IL-2 secretion. In contrast, pretreatment with COX-1-specific inhibitors had no effect upon 2-AG-mediated inhibition of IL-2 secretion. Interestingly, the current studies also demonstrate that while the potency of 2-AG is comparable between human Jurkat T cells and murine splenocytes, anandamide (AEA) is markedly more potent in suppressing IL-2 production in Jurkat T cells compared to murine splenocytes. Additionally, the present studies also demonstrate that COX-2 protein is readily detectable in resting Jurkat T cells, which is in contrast to resting murine splenocytes in which COX-2 protein is virtually undetectable. Furthermore, COX-2 protein and mRNA levels are significantly increased over basal levels by 2h following activation of Jurkat cells, whereas increases in COX-2 protein in murine splenocytes are not observed until 4h after cellular activation. These studies suggest that the potency of AEA in the suppression of IL-2 secretion may correlate with COX-2 protein levels in different T cell models. The present studies are also significant in that they demonstrate 2-AG-mediated inhibition of IL-2 secretion is dependent upon COX-2 metabolism.

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative that binds cannabinoid receptors CB1 and CB2 and is hence termed an endocannabinoid. 2-AG also modulates a variety of immunological responses, including expression of the autocrine/paracrine T cell growth factor interleukin (IL)-2. The objective of the present studies was to determine the mechanism responsible for IL-2 suppression by 2-AG. Because of the labile properties of 2-AG, 2-AG ether, a nonhydrolyzable analog of 2-AG, was also used. Both 2-AG and 2-AG ether suppressed IL-2 expression independently of CB1 and CB2, as demonstrated in leukocytes derived from CB1/CB2-null mice. Moreover, we demonstrated that both 2-AG and 2-AG ether treatment activated peroxisome proliferator-activated receptor gamma (PPARgamma), as evidenced by forced differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPARgamma-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPARgamma in IL-2 suppression by 2-AG and 2-AG ether was examined in Jurkat T cells. Concordant with PPARgamma involvement, the PPARgamma-specific antagonist 2-chloro-5-nitro-N-(4-pyridyl)-benzamide (T0070907) blocked 2-AG- and 2-AG ether-mediated IL-2 suppression. Likewise, 2-AG suppressed the transcriptional activity of two transcription factors crucial for IL-2 expression, nuclear factor of activated T cells and nuclear factor kappaB, in the absence but not in the presence of T0070907. 2-AG treatment also induced PPARgamma binding to a PPAR response element in activated Jurkat T cells. Together, the aforementioned studies identify PPARgamma as a novel intracellular target of 2-AG, which mediates the suppression of IL-2 by 2-AG in a manner that is independent of CB1 and/or CB2.

Arachidonyl ethanolamine, which is commonly known as anandamide, was the first endogenous compound to be identified that binds to the cannabinoid receptors. Anandamide mimics many of the physiological effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), including hypothermia, antinociception, immobility, catalepsy, and immune modulation. In the present studies, we show that anandamide caused a concentration-dependent inhibition of interleukin-2 in primary splenocytes. The CB1 and CB2 antagonists, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-H-pyrazole-3 carboxyamidehydrochloride] and SR144528 [N-[(1S)-endo-1,3,3,-trimethylbicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide], when used in combination, did not antagonize the inhibition of interleukin-2 by anandamide. Additionally, neither UCM707 [N-(3-furanylmethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide], the inhibitor of the putative anandamide membrane transporter (AMT), nor methyl arachidonoyl fluorophosphonate (MAFP), the inhibitor of fatty acid amidohydrolase (FAAH), were able to affect the inhibitory activity of anandamide upon interleukin-2. Interestingly, arachidonic acid caused a concentration-dependent inhibition of interleukin-2 secretion (IC(50) = 10.3 microM), which was similar to that of structurally related anandamide (IC(50) = 11.4 microM). The inhibition of interleukin-2 by anandamide and arachidonic acid was partially reversed by pretreatment with the nonspecific cyclooxygenase inhibitors, flurbiprofen and piroxicam. Moreover, NS398 [N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide], a cyclooxygenase-2-specific inhibitor, also attenuated the inhibitory effects of anandamide and arachidonic acid upon interleukin-2 secretion. Finally, pretreatment with a peroxisome proliferator-activated receptor gamma (PPARgamma)-specific antagonist, T0070907 [2-chloro-5-nitro-N-4-pyridinyl-benzamide], partially antagonized anandamide-mediated suppression of IL-2 secretion. Collectively, the aforementioned studies suggest that inhibition of interleukin-2 secretion by anandamide is independent of CB1/CB2 and the AMT/FAAH system. Additionally, these studies also suggest that inhibition of interleukin-2 is mediated by a PPARgamma, which is activated by a cyclooxygenase-2 metabolite of anandamide.

Cannabinoids exhibit immunosuppressive actions that include inhibition of interleukin-2 production in response to a variety of T cell activation stimuli. Traditionally, the effects of these compounds have been attributed to cannabinoid receptors CB1 and CB2, both of which are expressed in mouse splenocytes. Therefore, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorphenyl)-4-methyl-H-pyrazole-3 carboxyamidehydrochloride (SR141716A), a CB1 antagonist, and N-[(1S)-endo-1,3,3,-trimethyl-bicyclo[2,2,1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), a CB2 antagonist, were used to investigate the role of cannabinoid receptors in the cannabinoid-induced inhibition of phorbol ester plus calcium ionophore (PMA/Io)-stimulated interleukin-2 production by mouse splenocytes. PMA/Io-stimulated interleukin-2 production was inhibited by cannabinol, cannabidiol, and both WIN 55212-2 stereoisomers with a rank order potency of R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl) methanone mesylate (WIN 55212-2) approximately cannabidiol > S-(-)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-napthanlenyl) methanone mesylate (WIN 55212-3) approximately cannabinol. Cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2 was not attenuated by the presence of both SR144528 and SR141716A. Using pertussis toxin to address the role of G protein-coupled receptors in this response, it was determined that pertussis toxin treatment did not attenuate cannabinol-induced inhibition of PMA/Io-stimulated interleukin-2. With the demonstration that cannabinoid-induced inhibition of PMA/Io-stimulated interleukin-2 was not mediated via CB1 or CB2, alternative targets of cannabinoids in T cells were examined. Specifically, it was demonstrated that cannabinoids elevated intracellular calcium concentration in resting splenocytes and that the cannabinol-induced elevation in intracellular calcium concentration was attenuated by treatment with both SR144528 and SR141716A. Interestingly, pretreatment of splenocytes with agents that elevate intracellular calcium concentration inhibited PMA/Io-stimulated interleukin-2 production, suggesting that an elevation in intracellular calcium concentration might be involved in the mechanism of interleukin-2 inhibition. These studies suggest that immune modulation produced by cannabinoids involves multiple mechanisms, which might be both cannabinoid receptor-dependent and -independent.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor that induces a variety of cytoprotective genes. Nrf2 also mediates immunosuppressive effects in multiple inflammatory models. Upon activation, Nrf2 dissociates from its repressor protein, Keap1, and translocates to the nucleus where it induces Nrf2 target genes. The Nrf2-Keap1 interaction is disrupted by the environmental toxicant and chemotherapeutic agent arsenic trioxide (ATO). The purpose of the present study was to determine the effects of ATO on early events of T cell activation and the role of Nrf2 in those effects. The Nrf2 target genes Hmox-1, Nqo-1, and Gclc were all upregulated by ATO (1-2 μM) in splenocytes derived from wild-type, but not Nrf2-null, mice, suggesting that Nrf2 is activated by ATO in splenocytes. ATO also inhibited IFNγ, IL-2, and GM-CSF mRNA and protein production in wild-type splenocytes activated with the T cell activator, anti-CD3/anti-CD28. However, ATO also decreased production of these cytokines in activated splenocytes from Nrf2-null mice, suggesting the inhibition is independent of Nrf2. Interestingly, ATO inhibited TNFα protein secretion, but not mRNA expression, in activated splenocytes suggesting the inhibition is due to post-transcriptional modification. In addition, c-Fos DNA binding was significantly diminished by ATO in wild-type and Nrf2-null splenocytes activated with anti-CD3/anti-CD28, consistent with the observed inhibition of cytokine production by ATO. Collectively, this study suggests that although ATO activates Nrf2 in splenocytes, inhibition of early T cell cytokine production by ATO occurs independently of Nrf2 and may instead be due to impaired AP-1 DNA binding.

Hepatocyte (HPC) apoptosis occurs in association with hepatotoxic responses and chronic liver disease, and is coupled to activation of the blood coagulation cascade. HPCs have been shown to express tissue factor (TF), the primary activator of blood coagulation, in a form that lacks procoagulant activity. In this study, we determined the effect of inducing HPC apoptosis on the procoagulant activity of TF. Treatment of primary mouse HPCs with the Fas death receptor agonist (anti-CD95 antibody, Jo2) triggered apoptosis as shown by cleavage of caspase-3, increased caspase-3 proteolytic activity, and cell surface exposure of phosphatidylserine (PS). Jo2-induced apoptosis significantly increased TF-dependent factor Xa generation by HPCs. Moreover, Jo2 treatment was associated with increased levels of microparticle-associated TF procoagulant activity in the culture medium. Pretreatment with a caspase-3 inhibitor significantly reduced Jo2-induced HPC TF activity and prevented the increase in microparticle-associated TF procoagulant activity. Application of the high-affinity PS-binding protein lactadherin inhibited TF-dependent factor Xa generation by Jo2-treated HPCs and dramatically reduced microparticle-associated TF procoagulant activity. Treatment of wild-type mice with a sublethal dose of Jo2 was associated with a robust increase in the activation of coagulation as measured by plasma thrombin-antithrombin (TAT) levels; whereas mice with liver-specific TF deficiency had significantly lower TAT levels. Overall, the results indicate that Fas-initiated, caspase-3-dependent HPC apoptosis increases TF procoagulant activity through a mechanism involving PS externalization. This suggests that activation of liver TF likely contributes to the procoagulant state associated with HPC apoptosis in liver toxicity and disease.

During obstructive cholestasis, increased concentrations of bile acids activate ERK1/2 in hepatocytes, which up-regulates early growth response factor 1, a key regulator of proinflammatory cytokines, such as macrophage inflammatory protein 2 (MIP-2), which, in turn, exacerbates cholestatic liver injury. Recent studies have indicated that IL-17A contributes to hepatic inflammation during obstructive cholestasis, suggesting that bile acids and IL-17A may interact to regulate hepatic inflammatory responses. We treated mice with an IL-17A neutralizing antibody or control IgG and subjected them to bile duct ligation. Neutralization of IL-17A prevented up-regulation of proinflammatory cytokines, hepatic neutrophil accumulation, and liver injury, indicating an important role for IL-17A in neutrophilic inflammation during cholestasis. Treatment of primary mouse hepatocytes with taurocholic acid (TCA) increased the expression of MIP-2. Co-treatment with IL-17A synergistically enhanced up-regulation of MIP-2 by TCA. In contrast to MIP-2, IL-17A did not affect up-regulation of Egr-1 by TCA, indicating that IL-17A does not affect bile acid–induced activation of signaling pathways upstream of early growth response factor 1. In addition, bile acids increased expression of IL-23, a key regulator of IL-17A production in hepatocytes in vitro and in vivo. Collectively, these data identify bile acids as novel triggers of the IL-23/IL-17A axis and suggest that IL-17A promotes hepatic inflammation during cholestasis by synergistically enhancing bile acid–induced production of proinflammatory cytokines by hepatocytes.

In this study, we characterized tissue factor (TF) expression in mouse hepatocytes (HPCs) and evaluated its role in mouse models of HPC transplantation and acetaminophen (APAP) overdose. TF expression was significantly reduced in isolated HPCs and liver homogenates from TF(flox/flox)/albumin-Cre mice (HPC(ΔTF) mice) compared with TF(flox/flox) mice (control mice). Isolated mouse HPCs expressed low levels of TF that clotted factor VII-deficient human plasma. In addition, HPC TF initiated factor Xa generation without exogenous factor VIIa, and TF activity was increased dramatically after cell lysis. Treatment of HPCs with an inhibitory TF antibody or a cell-impermeable lysine-conjugating reagent prior to lysis substantially reduced TF activity, suggesting that TF was mainly present on the cell surface. Thrombin generation was dramatically reduced in APAP-treated HPC(ΔTF) mice compared with APAP-treated control mice. In addition, thrombin generation was dependent on donor HPC TF expression in a model of HPC transplantation. These results suggest that mouse HPCs constitutively express cell surface TF that mediates activation of coagulation during hepatocellular injury.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of obesity and metabolic syndrome and contributes to increased risk of cardiovascular disease and liver-related morbidity and mortality. Indeed, obese patients with metabolic syndrome generate greater amounts of thrombin, an indication of coagulation cascade activation. However, the role of the coagulation cascade in Western diet–induced NAFLD has not been investigated. Using an established mouse model of Western diet–induced NAFLD, we tested whether the thrombin receptor protease-activated receptor 1 (PAR-1) and hematopoietic cell–derived tissue factor (TF) contribute to hepatic steatosis. In association with hepatic steatosis, plasma thrombin-antithrombin levels and hepatic fibrin deposition increased significantly in C57Bl/6J mice fed a Western diet for 3 months. PAR-1 deficiency reduced hepatic inflammation, particularly monocyte chemoattractant protein-1 expression and macrophage accumulation. In addition, PAR-1 deficiency was associated with reduced steatosis in mice fed a Western diet, including reduced liver triglyceride accumulation and CD36 expression. Similar to PAR-1 deficiency, hematopoietic cell TF deficiency was associated with reduced inflammation and reduced steatosis in livers of low-density lipoprotein receptor–deficient mice fed a Western diet. Moreover, hematopoietic cell TF deficiency reduced hepatic fibrin deposition. These studies indicate that PAR-1 and hematopoietic cell TF are required for liver inflammation and steatosis in mice fed a Western diet.

Previous studies have demonstrated that the proteasome inhibitor, lactacystin, suppresses cytokine production and induction of other inflammatory mediators by LPS-stimulated macrophages. The purpose of the present studies was to determine the effect of lactacystin upon the function of activated human Jurkat T cells and murine splenocytes. Lactacystin treatment suppressed interleukin (IL)-2, interferon (IFN)gamma, and IL-13 production similarly in both activated Jurkat cells and primary splenocytes. Interestingly, lactacystin produced differential effects on IL-4 transcription in the two models. While lactacystin inhibited IL-4 mRNA transcription in primary splenocytes, it induced IL-4 mRNA in a concentration-dependent manner in Jurkat cells. The increase in IL-4 mRNA levels by lactacystin did not correlate with increases in T(H2)-specific transcription factors, avian musculoaponeurotic fibrosarcoma AS42 oncogene homolog (c-maf) or GATA binding protein 3 (GATA-3). In addition, the binding of both GATA-3 and t-bet to their respective response elements was essentially unchanged by lactacystin treatment in both splenocytes and Jurkat T cells, suggesting the induction of IL-4 is due to other mechanisms. Collectively, the current studies suggest proteasomal activity has differential effects on IL-4 transcription in activated Jurkat cells and primary splenocytes.

The use of the chemotherapeutic drug cisplatin is limited in part by nephrotoxicity. Cisplatin causes renal DNA adducts and oxidative stress in rodents. The transcription factor Nrf2 (nuclear factor E2-related factor 2) induces expression of cytoprotective genes, including Nqo1 (NADPH:quinone oxidoreductase 1), Ho-1 (heme oxygenase-1), and Gclc (glutamate cysteine ligase catalytic subunit), in response to electrophilic and oxidative stress. In the present study, plasma and kidneys from wild-type and Nrf2-null mice were collected after receiving cisplatin for evaluation of renal injury, inflammation, mRNA, and protein expression. Compared with wild types, more extensive nephrotoxicity was observed in Nrf2-null mice after cisplatin treatment. Kidneys from Nrf2-null mice treated with cisplatin had more neutrophil infiltration accompanied by increased p65 nuclear factor κB binding and elevated inflammatory mediator mRNA levels. Cisplatin increased renal mRNA and protein expression of cytoprotective genes (Nqo1, Ho-1, Gclc) and transporters Mrp2 and Mrp4 in wild-type but not in Nrf2-null mice. Lastly, the Nrf2 activator, CDDO-Im [2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide], increased Nrf2 signaling in kidneys from wild-type mice and protected them from cisplatin toxicity. Collectively, these data indicate that the absence of Nrf2 exacerbates cisplatin renal damage and that pharmacological activation of Nrf2 may represent a novel therapy to prevent kidney injury. Coordinated regulation of detoxification enzymes and drug transporters and suppression of inflammation by Nrf2 during cisplatin nephrotoxicity are probable defense mechanisms to eliminate toxic mediators and promote proximal tubule recovery. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.

The term "tolerance" from an immunological perspective, broadly encompasses a number of phenomena, but generally refers to a diminished responsiveness to LPS and/or other microbial products. With the discovery that many of the immunological, physiological and/or pathophysiological effects of LPS can be attributed to the lipid A moiety of the LPS molecule, a number of different lipid A analogs were synthesized with the goal of developing a drug that could be used clinically to treat cancer. In many instances, the development of tolerance to the lipid A congeners confounded the utility of these analogs as cancer therapeutics. In certain circumstances, however, the development of tolerance in patients has been utilized therapeutically to protect immunosuppressed patients from sepsis. Although numerous studies have been designed to investigate the development of tolerance, the underlying molecular mechanism remains unclear. This may be due, in part, to differences in the experimental models used, the sources and types of microbes and microbial products studied, kinetics of responses, and/or other experimental conditions. Nonetheless, a number of different signaling pathways have been identified as potentially modulating and/or triggering the development of tolerance. Though complex and incompletely understood, the capacity of tolerance to impact lipid A-based therapeutics, either positively or negatively, is inarguable, thus underscoring the necessity for further investigation toward elucidating the mechanisms contributing to the development of tolerance to lipid A and its analogs.

2-Arachidonoyl-glycerol (2-AG), an endogenous ligand for cannabinoid receptor types 1 and 2 (CB1 and CB2), has previously been demonstrated to modulate immune functions including suppression of interleukin-2 expression and nuclear factor of activated T cells (NFAT) activity. The objective of the present studies was to investigate the effect of 2-AG on interferon-γ (IFN-γ) expression and associated upstream signaling events. Pretreatment of splenocytes with 2-AG markedly suppressed phorbol 12-myristate 13-acetate plus calcium ionophore (PMA/Io)-induced IFN-γ secretion. In addition, 2-AG suppressed IFN-γ steady-state mRNA expression in a concentration-dependent manner. To unequivocally determine the putative involvement of CB1 and CB2, splenocytes derived from CB1−/−/CB2−/− knockout mice were used. No difference in the magnitude of IFN-γ suppression by 2-AG in wild-type versus CB1/CB2 null mice was observed. Time-of-addition studies revealed that 2-AG treatment up to 12 h post-cellular activation resulted in suppression of IFN-γ, which was consistent with a time course conducted with cyclosporin A, an inhibitor of NFAT activity. Coincidentally, 2-AG perturbed the nuclear translocation of NFAT protein and blocked thapsigargin-induced elevation in intracellular calcium, suggesting that altered calcium regulation might partly explain the suppression of NFAT nuclear translocation and subsequent IFN-γ production. Indeed, Io partially attenuated the 2-AG-induced suppression of PMA/Io-stimulated IFN-γ production. Taken together, these data demonstrate that 2-AG suppresses IFN-γ expression in murine splenocytes in a CB receptor-independent manner and that the mechanism partially involves suppression of intracellular calcium signaling and perturbation of NFAT nuclear translocation.