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Background and AimsIn severe cases of acetaminophen (APAP) overdose, acute liver injury rapidly progresses to acute liver failure (ALF), producing life-threatening complications including, hepatic encephalopathy (HE) and multi-organ failure (MOF). Systemic levels of interleukin-6 (IL-6) and IL-10 are highest in ALF patients with the most severe complications and the poorest prognosis. The mechanistic basis for dysregulation of these cytokines, and their association with outcome in ALF, remain poorly defined.MethodsTo investigate the impact of IL-6 and IL-10 in ALF, we used an experimental setting of failed liver repair after APAP overdose in which a high dose of APAP is administered (i.e., 500-600 mg/kg). Mice were treated with neutralizing antibodies to block IL-6 and IL-10.ResultsIn mice with APAP-induced ALF, high levels of IL-10 reduced monocyte recruitment and trafficking in the liver resulting in impaired clearance of dead cell debris. Kupffer cells in these mice, displayed features of myeloid-derived suppressor cells, including high level expression of IL-10 and PD-L1, which were increased in an IL-6-dependent manner. Similar to ALF patients with HE, cerebral blood flow was reduced in mice with APAP-induced ALF. Remarkably, although IL-6 is hepatoprotective in mice treated with low doses of APAP (i.e., 300 mg/kg), IL-6 neutralization in mice with APAP-induced ALF fully restored cerebral blood flow and reduced mortality.ConclusionCollectively, these studies demonstrate that exaggerated production of IL-6 in APAP-induced ALF triggers immune suppression (i.e., high levels of IL-10 and PD-L1), reduces cerebral blood flow (a feature of hepatic encephalopathy), disrupts liver repair (i.e., failed clearance of dead cells), and increases mortality.

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., Graphical Abstract Sex-differences in immune cell activation status (numbers/mean fluorescence intensity) in MRPVAT (A) and APVAT (B). (Key: e.g., at 10 weeks, higher density/MFI of M1-like macrophages occur in CD females vs. CD males in MRPVAT.) Differences in mean arterial pressure between HFD and respective CD-fed rats are presented as mm Hg [Supplementary Figure 1 and as measured by radiotelemetry (Fernandes et al., 2018)] with 10, 17, and 24 weeks on diet.

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.

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.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert-butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NFκB activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NFκB activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NFκB activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NFκB and Nrf2.

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.

Perivascular adipose tissue (PVAT) is protective and reduces the contraction of blood vessels in health. Eosinophils are one of the key players to the anti-contractile nature of PVAT in health. This finding led to our hypothesis that an ‘active’ immune complement exists in healthy PVAT. Male and female Sprague Dawley rats (12-14 weeks age) were used. PVATs from the thoracic aorta (brown APVAT), mesenteric resistance vessels (white MRPVAT), non-PVATs: subscapular (brown SS fat), retroperitoneal (white RP fat) and spleen (positive control) were harvested from each animal. T cells (CD4 and CD8), B cells, NK cells, macrophages, mast cells and neutrophils in the stromal vascular fraction were quantified using 9-color flow cytometry. Activation status (effector/ memory/ regulatory) of macrophages, T and B cells were also determined by flow cytometry. The number of immune cells are normalized to tissue weight (table). MRPVAT has denser total immune population (both sexes) and active macrophage pool (females) vs RP fat. APVAT homes a denser immune niche (females) and active T cell, B cell pools (both sexes) vs SS fat. This is the first study to characterize ‘active’ immune populations in healthy PVATs vs non-PVATs in male and female rats. Further studies to understand the functions of basally active immune cells in healthy PVATs are warranted.

OBJECTIVES: We examined the effects of the food preservative, tert-Butylhydroquinone (tBHQ), on natural killer (NK) cells when infected with influenza in C57Bl/6 mice. Previously, we found tBHQ to negatively impact splenic NK cell effector function in vitro. Therefore, we hypothesized that the consumption of tBHQ would impair the NK cell response against a primary influenza virus infection. METHODS: Female, C57Bl/6 mice were fed either a diet containing 0.0014% tBHQ or a control diet two weeks prior to infection. Mice were instilled intranasally with 21.78 HAU of influenza A/PR/8/34 (H1N1) and were monitored for two days. Body weight was recorded as a measurement for infection. After day two of infection, lungs were collected and processed for analysis by flow cytometry, enzyme-linked immunosorbent assay (ELISA), and total antioxidant capacity (TAC). RESULTS: The NK cell activation markers, CD25 and CD69, were not affected by tBHQ consumption. Additionally, tBHQ did not have an effect on maturation of NK cells or interferon (IFN)γ production. Notably, NK cell expression of granzyme B was significantly decreased with tBHQ in infected mice. Furthermore, mice on the tBHQ diet showed decreased TAC after two days of infection compared to mice on the control diet, suggesting increased oxidative stress in the tBHQ group. CONCLUSIONS: tBHQ had no effect on NK cell activation, maturation, and IFNγ production after two days of influenza infection. However, decreased expression of granzyme B by NK cells suggests tBHQ may impair NK cell cytoxicity during the infection. Moreover, the decrease in TAC suggests increased oxidative stress in the tBHQ-treated mice. FUNDING SOURCES: This study was funded by National Institute of Environmental Health Sciences (ES024966).

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.

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.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates a battery of antioxidant, detoxification, and cell stress genes. It is activated by oxidative stress and a number of exogenous compounds, one of which is tert-butylhydroquinone (tBHQ), a widely used food preservative. Nrf2 modulates immune responses in numerous rodent models of inflammation, but its effects on human immune cells are not well characterized. The purpose of these studies was to evaluate the effects of the Nrf2 activator tBHQ on early events of T cell activation in primary human cells. Treatment with tBHQ induced mRNA expression of the Nrf2 target genes HMOX-1, GCLC, and NQO1, and also increased NRF2 mRNA expression, albeit to a lesser extent than the other target genes. tBHQ decreased production of the cytokines IL-2 and IFN-γ at both the protein and mRNA levels after stimulation with anti-CD3/anti-CD28 in human peripheral blood mononuclear cells and to an even greater extent in isolated CD4 T cells. Likewise, tBHQ decreased induction of CD25 and CD69 in peripheral blood mononuclear cells (PBMCs) and this decrease was even more marked in isolated CD4 T cells. In addition, tBHQ inhibited induction of NFκB DNA binding in anti-CD3/anti-CD28-activated PBMCs. Collectively, these data suggest that tBHQ inhibits activation of primary human CD4 T cells, which correlates with activation of Nrf2 and inhibition of NFκB DNA binding. Although these studies suggest the food additive tBHQ negatively impacts T cell activation, further studies will be needed to fully elucidate the effect of tBHQ on human immune responses.

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.

Activation of Nrf2 and its downstream target genes serves as a master defense mechanism, protecting cells against oxidative and electrophilic stress. Numerous activators of Nrf2 inhibit carcinogenesis. However, genetic alterations resulting in constitutive Nrf2 activation in human lung tumors promotes tumor growth and chemoresistance. The dual role of Nrf2 in cancer has generated a high level of interest, but little is known regarding the effect of Nrf2 on immune cells during lung carcinogenesis. In this study, we used the potent carcinogen vinyl carbamate to induce lung tumors in mice. Tumor burden, the immunophenotype, and genetic signatures were compared in the lungs of Nrf2 knockout (-/-, KO) vs. wildtype (+/+, WT) mice. Nrf2 KO mice were more susceptible to vinyl carbamate; these mice developed tumors earlier than the WT mice and exhibited more and larger tumors over times ranging from 20-32 weeks after initiation. By 32 weeks, WT mice have an average of 6.5 ± 0.95 tumors per lung compared to 12.25 ± 2.6 tumors in the KO mice. Immune cell populations including T cells (CD4+ and CD8+), B cells, macrophages and myeloid derived suppressor cells were investigated in the lungs and spleens of Nrf2 WT and KO mice challenged with vinyl carbamate. T cell populations were significantly (P < 0.05) reduced in the lungs of Nrf2 KO mice, while tumor-promoting macrophages and myeloid derived suppressor cells were elevated in the lung and spleen, respectively, of Nrf2 KO mice compared to WT mice. Moreover, immune response genes, especially a series of cytokines (Cxcl1, Cxcl12, Csf1, and Ccl9) and MHC antigens that promote tumor growth, were significantly upregulated in tumors from Nrf2-/- mice. Importantly, this expression profile is conserved in patients with lung cancer. Overall, our results confirmed a tumor protective role of Nrf2 in carcinogenesis and suggest an important regulatory role for Nrf2 in immune cells in both mouse and human lung tumors. Citation Format: Di Zhang, Jonathan Rennhack, Eran Andrechek, Cheryl Rockwell, Karen Liby. Different immune signatures in advanced lung tumors of Nrf2 wildtype vs. knockout mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4051.

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.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor activated by cell stresses, such as reactive oxygen species and electrophilic stimuli. Upon activation Nrf2 accumulates within the nucleus, where it induces transcription of its target genes. In mice a growing number of studies indicate a role for Nrf2 in the development of immune mediated diseases, including inflammatory diseases such as allergy and autoimmunity. Recently, we have shown that the Nrf2 activator, tBHQ, modulates early events of T cell activation in human Jurkat T cells. This is evidenced by a marked inhibition of the cytokine IL-2, as well as a reduction in the expression of CD25, the high-affinity subunit of the IL-2 receptor. The suppression of IL-2 by tBHQ correlates with a concurrent suppression of the transcription factor NFκB, a transcription factor known to have multiple binding sites within the IL-2 promoter. Although we have shown that the synthetic food additive tBHQ modulates the early events of Jurkat ...

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.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that is activated by cellular stresses, such as oxidative compounds. After activation, Nrf2 induces transcription of its target genes, many of which have cytoprotective functions. Previously, we have shown that activation of Nrf2 by tert-butylhydroquinone (tBHQ) skews murine CD4⁺ T-cell differentiation. Although the role of Nrf2 in murine T cells is somewhat characterized, it is largely uncharacterized in human T cells. Therefore, the aim of the current studies was to characterize the effects of the Nrf2 activator, tBHQ, on the early events of human CD4⁺ T-cell activation. Pretreatment of Jurkat T cells with tBHQ, prior to activation with anti-CD3/anti-CD28, diminished the production of interleukin-2 (IL-2) at both the transcript and protein levels. Similarly, the expression of CD25 also diminished, albeit to a lesser degree than IL-2, after pretreatment with tBHQ. The decrease in IL-2 production was not due to decreased nuclear translocation of c-fos or c-jun. Although tBHQ caused both a delay and a decrease in Ca²⁺ influx in activated Jurkat cells, no decrease in nuclear factor of activated T cells (NFAT) DNA binding or transcriptional activity was observed. In contrast to NFAT, tBHQ significantly decreased NFκB transcriptional activity. Collectively, our studies show that the Nrf2 activator, tBHQ, inhibits IL-2 and CD25 expression, which correlates with decreased NFκB transcriptional activity in activated Jurkat cells. Overall, our studies suggest that Nrf2 represents a novel mechanism for the regulation of both human and mouse T cell function.

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.

otrganic perfluorochemicals (PFCs) have become an environmental concern due to widespread detection in human blood and experimental evidence for immune, developmental, and liver toxicity. Whereas the blood concentrations of many PFCs are declining, blood levels of Perfluorononanoic Acid (PFNA) are rising in the United States. The purpose of the present studies was to determine the effects of PFNA on lymphoid organs and immune cells of C57BL/6 mice. The present study demonstrates that PFNA produces immunotoxic effects in both male and female C57BL/6 mice as evidenced by splenic atrophy, decreased splenocyte numbers, and a marked reduction in thymocyte viability. The current study also demonstrates that the effects of PFNA on different leukocyte populations are not uniform. The CD4+CD8+ double-positive thymocytes were particularly sensitive to PFNA in which the proportion of this population was >95% decreased relative to the entire CD4+ thymocyte population in PFNA-treated mice. Interestingly, PFNA also markedly increased serum levels of TNFα in response to LPS in mice. Collectively, the present studies demonstrate that PFNA decreases lymphocyte viability and alters the immune response to LPS in C57BL/6 mice.

Nrf2 is a transcription factor that is activated by cell stress, such as the presence of oxidative compounds. After activation and translocation to the nucleus, Nrf2 binds to and induces the transc...

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.