Gene Trap Mice Reveal an Essential Function of Dual Specificity Phosphatase Dusp16/MKP-7 in Perinatal Survival and Regulation of Toll-like Receptor (TLR)-induced Cytokine Production

MAPK activity is negatively regulated by members of the dual specificity phosphatase (Dusp) family, which differ in expression, substrate specificity, and subcellular localization. Here, we investigated the function of Dusp16/MKP-7 in the innate immune system. The Dusp16 isoforms A1 and B1 were inducibly expressed in macrophages and dendritic cells following Toll-like receptor stimulation. A gene trap approach was used to generate Dusp16-deficient mice. Homozygous Dusp16tp/tp mice developed without gross abnormalities but died perinatally. Fetal liver cells from Dusp16tp/tp embryos efficiently reconstituted the lymphoid and myeloid compartments with Dusp16-deficient hematopoietic cells. However, GM-CSF-induced proliferation of bone marrow progenitors in vitro was impaired in the absence of Dusp16. In vivo challenge with Escherichia coli LPS triggered higher production of IL-12p40 in mice with a Dusp16-deficient immune system. In vitro, Dusp16-deficient macrophages, but not dendritic cells, selectively overexpressed a subset of TLR-induced genes, including the cytokine IL-12. Dusp16-deficient fibroblasts showed enhanced activation of p38 and JNK MAPKs. In macrophages, pharmacological inhibition and siRNA knockdown of JNK1/2 normalized IL-12p40 secretion. Production of IL-10 and its inhibitory effect on IL-12 production were unaltered in Dusp16tp/tp macrophages. Altogether, the Dusp16 gene trap mouse model identifies an essential role in perinatal survival and reveals selective control of differentiation and cytokine production of myeloid cells by the MAPK phosphatase Dusp16.     

Nrf2-dependent repression of interleukin-12 expression in human dendritic cells exposed to inorganic arsenic

Inorganic arsenic, a well-known Nrf2 inducer, exerts immunosuppressive properties. In this context, we recently reported that the differentiation of human blood monocytes into immature dendritic cells (DCs), in the presence of low and noncytotoxic concentrations of arsenic, represses the ability of DCs to release key cytokines in response to different stimulating agents. Particularly, arsenic inhibits the expression of human interleukin-12 (IL-12, also named IL-12p70), a major proinflammatory cytokine that controls the differentiation of Th1 lymphocytes. In the present study, we determined if Nrf2 could contribute to these arsenic immunotoxic effects. To this goal, human monocyte-derived DCs were first differentiated in the absence of metalloid and then pretreated with arsenic just before DC stimulation with lipopolysaccharide (LPS). Under these experimental conditions, arsenic rapidly and stably activates Nrf2 and increases the expression of Nrf2 target genes. It also significantly inhibits IL-12 expression in activated DCs, at both mRNA and protein levels. Particularly, arsenic reduces mRNA levels of IL12A and IL12B genes which encodes the p35 and p40 subunits of IL-12p70, respectively. tert-Butylhydroquinone (tBHQ), a reference Nrf2 inducer, mimics arsenic effects and potently inhibits IL-12 expression. Genetic inhibition of Nrf2 expression markedly prevents the repression of both IL12 mRNA and IL-12 protein levels triggered by arsenic and tBHQ in human LPS-stimulated DCs. In addition, arsenic significantly reduces IL-12 mRNA levels in LPS-activated bone marrow-derived DCs from Nrf2^+/+ mice but not in DCs from Nrf2^−/− mice. Finally, we show that, besides IL-12, arsenic significantly reduces the expression of IL-23, another heterodimer containing the p40 subunit. In conclusion, our study demonstrated that arsenic represses IL-12 expression in human-activated DCs by specifically stimulating Nrf2 activity.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 μM CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFNγ through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPARγ receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2α, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2α induced by LPS/IFNγ. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the ‘oligoprotective'' effects of CBD during inflammation.