Korean red ginseng extract prevents bone loss in an oral model of glucocorticoid induced osteoporosis in mice.

The gut microbiota and barrier function play important roles in bone health. We previously demonstrated that chronic glucocorticoid (GC)-induced bone loss in mice is associated with significant shifts in gut microbiota composition and impaired gut barrier function. Korean Red Ginseng (KRG, Panax Ginseng Meyer, Araliaceae) extract has been shown to prevent glucocorticoid-induced osteoporosis (GIO) in a subcutaneous pellet model in mice, but its effect on gut microbiota and barrier function in this context is not known. The overall goal of this study was to test the effect of KRG extract in a clinically relevant, oral model of GIO and further investigate its role in modulating the gut-bone axis. Growing male mice (CD-1, 8 weeks) were treated with 75 µg/mL corticosterone (∼9 mg/kg/day) or 0.4% ethanol vehicle in the drinking water for 4 weeks. During this 4-week period, mice were treated daily with 500 mg/kg/day KRG extract dissolved in sterile water or an equal amount of sterile water via oral gastric gavage. After 4 weeks of treatment, we assessed bone volume, microbiota composition, gut barrier integrity, and immune cells in the bone marrow (BM) and mesenteric lymph nodes (MLNs). 4 weeks of oral GC treatment caused significant distal femur trabecular bone loss, and this was associated with changes in gut microbiota composition, impaired gut barrier function and altered immune cell composition. Importantly, KRG extract prevented distal femur trabecular bone loss and caused significant alterations in gut microbiota composition but had only modest effects on gut barrier function and immune cell populations. Taken together, these results demonstrate that KRG extract significantly modulates the gut microbiota-bone axis and prevents glucocorticoid-induced bone loss in mice.

A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Arsenic trioxide inhibits the response of primary human B cells to influenza virus A in vitro.

Arsenic compounds are common environmental toxicants worldwide and particularly enriched in the Northeast and the Southwestern United States, the Alps, and Bangladesh. Exposure to arsenic is linked with various detrimental health outcomes, including cancer, cognitive decline, and kidney damage. Our group has previously shown that arsenic trioxide alters T cell cytokine production. In the current study, we demonstrate that exposure to arsenic compounds alters B cell function in an in vitro influenza model. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood and cultured with arsenic trioxide (As3O2) and subsequently challenged with Influenza A virus. B cells showed decreased expression of CD267, surface IgG and CD80 when treated with As3O2. Taken together, the data suggest that As3O2 affects the activation and surface antibody expression of human peripheral B cells. Overall, this suggests that As3O2 exposure could cause impaired humoral immunity.

Interleukin-10 disrupts liver repair in acetaminophen-induced acute liver failure.

Introduction: Systemic levels of the anti-inflammatory cytokine interleukin 10 (IL-10) are highest in acetaminophen (APAP)-induced acute liver failure (ALF) patients with the poorest prognosis. The mechanistic basis for this counterintuitive finding is not known, as induction of IL-10 is hypothesized to temper the pathological effects of immune cell activation. Aberrant production of IL-10 after severe liver injury could conceivably interfere with the beneficial, pro-reparative actions of immune cells, such as monocytes. Methods: To test this possibility, we determined whether IL-10 levels are dysregulated in mice with APAP-induced ALF and further evaluated whether aberrant production of IL-10 prevents monocyte recruitment and/or the resolution of necrotic lesions by these cells. Results: Our studies demonstrate that in mice challenged with 300 mg/kg acetaminophen (APAP), a hepatotoxic dose of APAP that fails to produce ALF (i.e., APAP-induced acute liver injury; AALI), Ly6Chi monocytes were recruited to the liver and infiltrated the necrotic lesions by 48 hours coincident with the clearance of dead cell debris. At 72 hours, IL-10 was upregulated, culminating in the resolution of hepatic inflammation. By contrast, in mice treated with 600 mg/kg APAP, a dose that produces clinical features of ALF (i.e., APAP-induced ALF; AALF), IL-10 levels were markedly elevated by 24 hours. Early induction of IL-10 was associated with a reduction in the hepatic numbers of Ly6Chi monocytes resulting in the persistence of dead cell debris. Inhibition of IL-10 in AALF mice, beginning at 24 hours after APAP treatment, increased the hepatic numbers of monocytes which coincided with a reduction in the necrotic area. Moreover, pharmacologic elevation of systemic IL-10 levels in AALI mice reduced hepatic myeloid cell numbers and increased the area of necrosis. Discussion: Collectively, these results indicate that during ALF, aberrant production of IL-10 disrupts the hepatic recruitment of monocytes, which prevents the clearance of dead cell debris. These are the first studies to document a mechanistic basis for the link between high IL-10 levels and poor outcome in patients with ALF.

A Cell Atlas of Thoracic Aortic Perivascular Adipose Tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. To examine the cell-specificity of recognized mechanotransducers we used single nuclei RNA sequencing (snRNAseq) of the thoracic aorta PVAT (taPVAT) from male Dahl SS rats compared to subscapular brown adipose tissue (BAT). Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNAseq, identifying 8 major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. Presence of PIEZO1 in the PVAT was confirmed by RNAscope® and IHC; antagonism of PIEZO1 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.

The protective effect of butylated hydroxytoluene and 3-hydroxytyrosol on food allergy in mice.

OBJECTIVE: To test the effect of two dietary antioxidants: butylated hydroxytoluene (BHT) and 3-hydroxytyrosol (3-HT) in experimental food allergy. METHODS: BALB/c mice maintained on control diet or diet with BHT or 3-HT were sensitized with ovalbumin (OVA) or saline through transdermal exposure. Plasma OVA-specific IgE (OVA-IgE) and IgG1 (OVA-IgG1) antibody levels were determined using ELISA. Sensitized mice were challenged by oral gavage with OVA. Rectal temperature (RT) was measured before and after challenge. Mast cell degranulation was quantified by measuring the plasma levels of mouse mucosal mast cell protease-1 (mMCP-1). Flow cytometry was carried out to evaluate the percentage Th2 cells from the spleen. RESULTS: Mice on either a 3-HT or BHT diet showed a significantly decreased IgE response to OVA sensitization and less severe anaphylaxis, as evidenced by a diminished drop in body temperature, attenuated clinical signs, a more rapid recovery and decreased mast cell degranulation (as determined by lower plasma mMCP-1 levels). CONCLUSION: The present study indicates two dietary antioxidants: BHT and 3-HT may be protective against experimental food allergy. These results suggest 3-HT and BHT could potentially be useful for prevention of food allergy.

Trivalent arsenic impairs the effector response of human CD4+ and CD8+ T cells to influenza A virus ex vivo.

Arsenic is a persistent environmental contaminant that humans are exposed to primarily through contaminated water supplies. Arsenic has been shown to have numerous immunomodulatory effects, including deleterious effects on T cell function. However, the effect of arsenic on human T cell function in the context of influenza infection remains poorly characterized. The goal of this study was to determine the effects of arsenic on T cell activation and effector function in a human-relevant ex vivo model with influenza challenge. Flow cytometric analysis of T cells following the treatment of primary human peripheral blood mononuclear cells with environmentally relevant concentrations of arsenic trioxide and subsequent challenge with influenza A virus showed reduced viability, alterations in activation, a reduction in the population of memory cells, and reduced effector function evidenced by decreased IFNγ and granzyme B production. Overall, these studies suggest that arsenic impairs the human T cell response to influenza which corroborates epidemiological findings and could have further implications for antiviral immunity and vaccine efficacy.

The Complicated Role of Nuclear Factor Erythroid-Derived 2-Like 2 in Allergy and Asthma.

Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a stress-activated transcription factor that is highly responsive to oxidative stress and electrophilic stimuli. Upon activation, Nrf2 upregulates a battery of cytoprotective genes meant to prevent cell death or damage. In many models of inflammation, Nrf2 protects against the immune response and decreases injury, including in the context of asthma and allergy. However, in some models of asthma and allergy, Nrf2 either does not play a role or can even exacerbate inflammation. In general, the reasons behind these discrepancies are not clear and the mechanisms by which Nrf2 modulates immune response are largely uncharacterized. The aim of this review is to highlight current literature assessing the role of Nrf2 in allergy and asthma to understand Nrf2 as a potential therapeutic target. SIGNIFICANCE STATEMENT: Nuclear factor erythroid-derived 2-like 2 (Nrf2) is an important immune mediator that modulates numerous immune cell types in various inflammatory diseases, including allergy and asthma. There is considerable interest in Nrf2 as a drug target in inflammation, which is complicated by the complex nature of Nrf2 in the immune system. This review focuses on the role of Nrf2 in asthma and allergy, including in regulating immune cell function and in detoxifying xenobiotics that exacerbate these diseases.

The role of Nrf2 in autoimmunity and infectious disease: Therapeutic possibilities.

Nrf2 is a cytoprotective transcription factor which is involved in ameliorating oxidative stress and toxic insults. Recently, an immunomodulatory role for Nrf2 has gained appreciation as it has been shown to protect cells and hosts alike in a variety of immune and inflammatory disorders. However, Nrf2 utilizes numerous distinct pathways to elicit its immunomodulatory effects. In this review, we summarize the literature discussing the roles of Nrf2 in autoimmunity and infectious diseases with a goal of understanding the potential to therapeutically target Nrf2.

Phenotypic Changes in T Cell and Macrophage Subtypes in Perivascular Adipose Tissues Precede High-Fat Diet-Induced Hypertension.

Perivascular adipose tissue (PVAT) may connect adiposity to hypertension because of its vasoactive functions and proximity to blood vessels. We hypothesized that immune cell changes in PVATs precede the development of high fat diet (HFD)-induced hypertension. Both sexes of Dahl S rat become equally hypertensive when fed a HFD. Further, both sexes would have similar immune cell composition in PVATs with the development and progression of hypertension. Male and female Dahl S rats were fed a regular (10% calories from fat; CD) diet or a HFD (60%) from weaning. PVATs from around the thoracic aorta (APVAT) and small mesenteric vessels (MRPVAT) were harvested at 10 weeks (pre-hypertensive), 17 weeks (onset), or 24 (hypertensive) weeks on diet. RNA-sequencing in MRPVAT at 24 weeks indicated sex-differences with HFD (>CD) and diet-differences in males (>females). The top 2 out of 7 immune processes with the maximum number of differentially expressed genes (DEGs) were associated with immune effector processes and leukocyte activation. Macrophages and T cells (and their activation status), neutrophils, mast, B and NK cells were measured by flow cytometry. Sex-specific changes in the number of CD4 memory T cells (males > females) and M2-like macrophages (females > males) in PVATs occur with a HFD before hypertension developed. Sex-differences became more prominent with the development and progression of hypertension, driven by the diet (HFD > CD). These findings suggest that though the magnitudes of increased blood pressure were equivalent in both sexes, the associated phenotypic changes in the immune subsets within the PVATs were different in the male vs. the female with the development and progression of hypertension.

Nrf2-dependent and -independent effects of tBHQ in activated murine B cells.

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.

Naïve, Regulatory, Activated, and Memory Immune Cells Co-exist in PVATs That Are Comparable in Density to Non-PVAT Fats in Health.

Perivascular adipose tissue (PVAT), the fat surrounding peripheral blood vessels, is protective and reduces the contraction of blood vessels in health. PVAT is composed of adipocytes, stromal cells, and immune cells. Recent work supports eosinophils as one of the cell types key to the anti-contractile nature of PVAT in health. Hence, we hypothesized that there exists a basally activated immune cell community in healthy PVAT that is distinctly different from non-PVAT fats. PVATs were from around mesenteric resistance vessels (MRPVAT - white fat) and thoracic aorta (APVAT - brown fat). Non-PVATs included retroperitoneal (RP fat - white fat) and subscapular (SS fat - brown fat) while the spleen was a positive control. Tissues were harvested from adult male and female Sprague Dawley rats. Six primary immune cell types were identified in PVATs. T cells (CD4 and CD8), B cells, natural killer (NK) cells, macrophages, mast cells, and neutrophils in the stromal vascular fraction of each fat were identified using nine-color flow cytometry. PVATs contained a higher number of total immune cells vs. their respective non-PVAT fats in females. Females had a higher number of T cells in MRPVAT vs. males. Females also had a greater number of T cells and total immune cells in APVAT vs. males. Further, activation, differentiation, and/or polarization of various immune cell types were similarly determined by flow cytometry. PVATs were similar to their respective non-PVAT fats in density of recently activated B cells (B220+ CD25+). However, MRPVAT in females had a higher number of naïve CD4 T cells vs. MRPVAT in males and APVAT in females. MRPVAT also had denser naïve CD8 T cells vs. APVAT in females. Overall, this research for the first time has identified a community of discrete populations of immune cells (naive/recently activated/regulatory/memory) in healthy PVATs. Contrary to our hypothesis, PVATs are more similar than different in density to their respective non-PVAT fats.

Differential Sensitivity of Kupffer Cells and Hepatic Monocyte-Derived Macrophages to Bacterial Lipopolysaccharide.

The liver contains two distinct populations of macrophages, monocyte-derived macrophages (MDMs), which primarily reside proximal to the Glisson's capsule and Kupffer cells, which reside within the sinusoids. Kupffer cells infiltrate the liver during embryogenesis and are replenished from local proliferation of mature Kupffer cells. By contrast MDMs arise from hematopoietic stem cells in the bone marrow and are replenishedfrom circulating monocytes. Studies have revealed that these two hepatic macrophage populations possess distinct transcriptomic profiles, suggesting that they may be functionally distinct. In the present study, we tested the hypothesis that MDMs and Kupffer cells are differentially sensitive to bacterial lipopolysaccharide (LPS). MDMs and Kupffer cells were purified to greater than 90% from the livers of mice by using magnetic beads labeled with Cx3cr1 antibody for MDMs and F4/80 antibody for Kupffer cells. Basal levels of tumor necrosis factor-α (TNF-α) mRNA were higher in MDMs when compared to Kupffer cells. After treatment with LPS, mRNA levels of TNF-α, Cxcll, and Cxcl2 were increased to a greater extent in MDMs when compared to Kupffer cells. To confirm these findings, Kupffer cells and MDMs were isolated from mice in which bone marrow transplantation was used to selectively tag cells arising from hematopoietic stem cells in adult mice. Similar to above, treatment of MDMs with LPS increased TNF-α, Cxcll, and Cxcl2 to a greater extent when compared to Kupffer cells. Collectively, these results indicate that MDMs exhibit a greater pro-inflammatory phenotype in the liver when exposed to LPS.

Dichotomous Role of Plasmin in Regulation of Macrophage Function after Acetaminophen Overdose.

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.

Chronic low-level cadmium exposure in rats affects cytokine production by activated T cells.

Cadmium is a toxic metal and common environmental contaminant. Chronic cadmium exposure results in kidney, bone, reproductive, and immune toxicity as well as cancer. Cadmium induces splenomegaly and affects the adaptive immune system, but specific effects vary depending on the dose, model, and endpoint. This study investigates the effects of subchronic, oral, and low-dose cadmium exposure (32 ppm cadmium chloride in drinking water for 10 weeks) on the rat immune system, focusing on T cell function. Cadmium-exposed animals demonstrated slight increases in the spleen-to-body weight ratios, and decreases in overall splenic cell numbers and markers of oxidative stress. The relative ratios of splenic cell populations remained similar, except for modest increases in regulatory T cells in the cadmium-exposed animals. Cadmium exposure also significantly increased the production of IFNγ, a pro-inflammatory cytokine, and IL-10, a cytokine produced by multiple T cell subsets that typically inhibits IFNγ expression, by activated T cells. The increase in IFNγ and IL-10 suggests that cadmium exposure may affect multiple T cell subsets. Collectively, this study suggests that subchronic, low-dose cadmium exposure impacts both immune cell function and cellularity, and may enhance inflammatory responses.

The Nrf2 activator tBHQ inhibits the activation of primary murine natural killer cells.

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 (IFNÉ£), 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.

Comparison of Hepatic NRF2 and Aryl Hydrocarbon Receptor Binding in 2,3,7,8-Tetrachlorodibenzo-p-dioxin-Treated Mice Demonstrates NRF2-Independent PKM2 Induction.

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.

Identification of an Unfavorable Immune Signature in Advanced Lung Tumors from Nrf2-Deficient Mice.

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. Antioxid. Redox Signal.

Differential effects of the Nrf2 activators tBHQ and CDDO-Im on the early events of T cell activation.

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 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 toward 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.