NLRP6 negatively regulates type 2 immune responses in mice.

BACKGROUND: Inflammasomes are large protein complexes that assemble in the cytosol in response to danger such as tissue damage or infection. Following activation, inflammasomes trigger cell death and the release of biologically active forms of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome is required for IL-18 secretion by intestinal epithelial cells, macrophages, and T cells, contributing to homeostasis and self-defense against pathogenic microbes. However, the involvement of NLRP6 in type 2 lung inflammation remains elusive. METHODS: Wild-type (WT) and Nlrp6-/- mice were used. Birch pollen extract (BPE)-induced allergic lung inflammation, eosinophil recruitment, Th2-related cytokine and chemokine production, airway hyperresponsiveness, and lung histopathology, Th2 cell differentiation, GATA3, and Th2 cytokines expression, were determined. Nippostrongylus brasiliensis (Nb) infection, worm count in intestine, type 2 innate lymphoid cell (ILC2), and Th2 cells in lungs were evaluated. RESULTS: We demonstrate in Nlrp6-/- mice that a mixed Th2/Th17 immune responses prevailed following birch pollen challenge with increased eosinophils, ILC2, Th2, and Th17 cell induction and reduced IL-18 production. Nippostrongylus brasiliensis infected Nlrp6-/- mice featured enhanced early expulsion of the parasite due to enhanced type 2 immune responses compared to WT hosts. In vitro, NLRP6 repressed Th2 polarization, as shown by increased Th2 cytokines and higher expression of the transcription factor GATA3 in the absence of NLRP6. Exogenous IL-18 administration partially reduced the enhanced airways inflammation in Nlrp6-/- mice. CONCLUSIONS: In summary, our data identify NLRP6 as a negative regulator of type 2 immune responses.

Neutralization of either IL-17A or IL-17F is sufficient to inhibit house dust mite induced allergic asthma in mice.

T helper (Th)17 immune response participates in allergic lung inflammation and asthma is reduced in the absence of interleukin (IL)-17 in mice. Since IL-17A and IL-17F are induced and bind the shared receptor IL-17RA, we asked whether both IL-17A and IL-17F contribute to house dust mite (HDM) induced asthma. We report that allergic lung inflammation is attenuated in absence of either IL-17A or IL-17F with reduced airway hyperreactivity, eosinophilic inflammation, goblet cell hyperplasia, cytokine and chemokine production as found in absence of IL-17RA. Furthermore, specific antibody neutralization of either IL-17A or IL-17F given during the sensitization phase attenuated allergic lung inflammation and airway hyperreactivity. In vitro activation by HDM of primary dendritic cells revealed a comparable induction of CXCL1 and IL-6 expression and the response to IL-17A and IL-17F relied on IL-17RA signaling via the adaptor protein act1 in fibroblasts. Therefore, HDM-induced allergic respiratory response depends on IL-17RA via act1 signaling and inactivation of either IL-17A or IL-17F is sufficient to attenuate allergic asthma in mice.

Protein kinase Cθ controls type 2 innate lymphoid cell and TH2 responses to house dust mite allergen.

BACKGROUND: Protein kinase C (PKC) θ, a serine/threonine kinase, is involved in TH2 cell activation and proliferation. Type 2 innate lymphoid cells (ILC2s) resemble TH2 cells and produce the TH2 cytokines IL-5 and IL-13 but lack antigen-specific receptors. The mechanism by which PKC-θ drives innate immune cells to instruct TH2 responses in patients with allergic lung inflammation remains unknown. OBJECTIVES: We hypothesized that PKC-θ contributes to ILC2 activation and might be necessary for ILC2s to instruct the TH2 response. METHODS: PRKCQ gene expression was assessed in innate lymphoid cell subsets purified from human PBMCs and mouse lung ILC2s. ILC2 activation and eosinophil recruitment, TH2-related cytokine and chemokine production, lung histopathology, interferon regulatory factor 4 (IRF4) mRNA expression, and nuclear factor of activated T cells (NFAT1) protein expression were determined. Adoptive transfer of ILC2s from wild-type mice was performed in wild-type and PKC-θ-deficient (PKC-θ-/-) mice. RESULTS: Here we report that PKC-θ is expressed in both human and mouse ILC2s. Mice lacking PKC-θ had reduced ILC2 numbers, TH2 cell numbers and activation, airway hyperresponsiveness, and expression of the transcription factors IRF4 and NFAT1. Importantly, adoptive transfer of ILC2s restored eosinophil influx and IL-4, IL-5 and IL-13 production in lung tissue, as well as TH2 cell activation. The pharmacologic PKC-θ inhibitor (Compound 20) administered during allergen challenge reduced ILC2 numbers and activation, as well as airway inflammation and IRF4 and NFAT1 expression. CONCLUSIONS: Therefore our findings identify PKC-θ as a critical factor for ILC2 activation that contributes to TH2 cell differentiation, which is associated with IRF4 and NFAT1 expression in allergic lung inflammation.

Caspase-1 activation by NLRP3 inflammasome dampens IL-33-dependent house dust mite-induced allergic lung inflammation.

The cysteine protease caspase-1 (Casp-1) contributes to innate immunity through the assembly of NLRP3, NLRC4, AIM2, and NLRP6 inflammasomes. Here we ask whether caspase-1 activation plays a regulatory role in house dust mite (HDM)-induced experimental allergic airway inflammation. We report enhanced airway inflammation in caspase-1-deficient mice exposed to HDM with a marked eosinophil recruitment, increased expression of IL-4, IL-5, IL-13, as well as full-length and bioactive IL-33. Furthermore, mice deficient for NLRP3 failed to control eosinophil influx in the airways and displayed augmented Th2 cytokine and chemokine levels, suggesting that the NLPR3 inflammasome complex controls HDM-induced inflammation. IL-33 neutralization by administration of soluble ST2 receptor inhibited the enhanced allergic inflammation, while administration of recombinant IL-33 during challenge phase enhanced allergic inflammation in caspase-1-deficient mice. Therefore, we show that caspase-1, NLRP3, and ASC, but not NLRC4, contribute to the upregulation of allergic lung inflammation. Moreover, we cannot exclude an effect of caspase-11, because caspase-1-deficient mice are deficient for both caspases. Mechanistically, absence of caspase-1 is associated with increased expression of IL-33, uric acid, and spleen tyrosine kinase (Syk) production. This study highlights a critical role of caspase-1 activation and NLPR3/ASC inflammasome complex in the down-modulation of IL-33 in vivo and in vitro, thereby regulating Th2 response in HDM-induced allergic lung inflammation.

Thymic Stromal Lymphopoietin Enhances Th2/Th22 and Reduces IL-17A in Protease-Allergen-Induced Airways Inflammation.

Background. Thymic stromal lymphopoietin (TSLP) is induced in allergic skin and lung inflammation in man and mice. Methods. Allergic lung inflammation induced by two proteases allergens HDM and papain and a classical allergen ovalbumin was evaluated in vivo in mice deficient for TSLPR. Eosinophil recruitment, Th2 and Th17 cytokine and chemokine levels were determined in bronchoalveolar lavage fluid, lung homogenates and lung mononuclear cells ex vivo. Results. Here we report that mice challenged with house dust mite extract or papain in the absence of TSLPR have a drastic reduction of allergic inflammation with diminished eosinophil recruitment in BAL and lung and reduced mucus overproduction. TSLPR deficient DCs displayed diminished OVA antigen uptake and reduced capacity to activate antigen specific T cells. TSLPR deficient mice had diminished proinflammatory IL-1 ß , IL-13, and IL-33 chemokines production, while IL-17A, IL-12p40 and IL-10 were increased. Together with impaired Th2 cytokines, IL-17A expressing TCR ß (+) T cells were increased, while IL-22 expressing CD4(+) T cells were diminished in the lung. Conclusion. Therefore, TSLPR signaling is required for the development of both Th2 and Th22 responses and may restrain IL-17A. TSLP may mediate its effects in part by increasing allergen uptake and processing by DCs resulting in an exacerbated asthma.

Brain phenotype of transgenic mice overexpressing cystathionine ß-synthase.

BACKGROUND: The cystathionine ß-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line. CONCLUSION/SIGNIFICANCE: We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.

PCP4 (PEP19) overexpression induces premature neuronal differentiation associated with Ca(2+) /calmodulin-dependent kinase II-δ activation in mouse models of Down syndrome.

Pcp4/pep19 is a modulator of Ca(2+) -CaM, a key molecule for calcium signaling, expressed in postmitotic neuroectoderm cells during mouse embryogenesis. The PCP4 gene is located on human chromosome 21 and is present in three copies in Down syndrome (DS). To evaluate the consequences of three copies of this gene on the development of these cells in the nervous system, we constructed a transgenic (TgPCP4) mouse model, with one copy of human PCP4, and investigated the effects in this model and in the Ts1Cje, a mouse model of DS. During embryogenesis, we analyzed 1) the level of pcp4 transcript and protein in the two models; 2) the extent of colabeling for markers of neuronal differentiation (ßIII-tubulin, Map2c, calbindin, and calretinin) and pcp4 by immunofluorescence analysis and overall protein levels of these markers by Western blotting; and 3) the rate of activation of CaMKII, a Ca(2+) -CaM target, to evaluate the impact of pcp4 overexpression on the Ca(2+) -CaM signaling pathway. We showed that three copies of the pcp4 gene induced the overexpression of transcripts and proteins during embryogenesis. Pcp4 overexpression 1) induced precocious neuronal differentiation, as shown by the distribution and levels of early neuronal markers; and 2) was associated with an increase in CaMKIIδ activation, confirming involvement in neuronal differentiation in vivo via a Pcp4-Ca(2+) -CaM pathway. TgPCP4 and Ts1Cje mice developed similar modifications, demonstrating that these mechanisms may account for abnormal neuronal development in DS.

Green tea polyphenols rescue of brain defects induced by overexpression of DYRK1A.

Individuals with partial HSA21 trisomies and mice with partial MMU16 trisomies containing an extra copy of the DYRK1A gene present various alterations in brain morphogenesis. They present also learning impairments modeling those encountered in Down syndrome. Previous MRI and histological analyses of a transgenic mice generated using a human YAC construct that contains five genes including DYRK1A reveal that DYRK1A is involved, during development, in the control of brain volume and cell density of specific brain regions. Gene dosage correction induces a rescue of the brain volume alterations. DYRK1A is also involved in the control of synaptic plasticity and memory consolidation. Increased gene dosage results in brain morphogenesis defects, low BDNF levels and mnemonic deficits in these mice. Epigallocatechin gallate (EGCG) - a member of a natural polyphenols family, found in great amount in green tea leaves - is a specific and safe DYRK1A inhibitor. We maintained control and transgenic mice overexpressing DYRK1A on two different polyphenol-based diets, from gestation to adulthood. The major features of the transgenic phenotype were rescued in these mice.

Increased dosage of DYRK1A and brain volumetric alterations in a YAC model of partial trisomy 21.

A yeast artificial chromosome (YAC) transgenic murine model of partial trisomy 21 overexpressing five human genes -- including DYRK1A, which encodes a serine threonine kinase involved in cell cycle control -- has been shown to present an increase in brain weight. We analyzed this new phenotype by measuring total and regional brain volumes at different ages, using a 7 Tesla magnetic resonance imaging volumetric approach. Volumetric measurements showed a total volume increase of 13.6% in adult mice. Changes in brain morphogenesis were already visible at a very early postnatal stage (postnatal days 2-7). Region-specific changes were characterized from postnatal day 15 to 5 months. These results, made it possible to define region-specific effects of DYRK1A overexpression, with the strongest increase seen in the thalamus-hypothalamus area (24%).

Effects of catechin on homocysteine metabolism in hyperhomocysteinemic mice.

We have recently focused on the interaction between hyperhomocysteinemia, defined by high plasma homocysteine levels, and paraoxonase-1 expression and found a reduced activity of paraoxonase-1 associated with a reduced gene expression in the liver of cystathionine beta synthase (CBS) deficient mice, a murine model of hyperhomocysteinemia. As it has been demonstrated that polyphenolic compounds could modulate the expression level of the paraoxonase-1 gene in vitro, we have investigated the possible effect of flavonoid supplementation on the impaired paraoxonase-1 gene expression and activity induced by hyperhomocysteinemia and have evaluated the link with homocysteine metabolism. High-methionine diet significantly increased serum homocysteine levels, decreased hepatic CBS activity, and down-regulated paraoxonase-1 mRNA and its activity. However, chronic administration of catechin but not quercetin significantly reduced plasma homocysteine levels, attenuated the reduction of the hepatic CBS activity, and restored the decreased paraoxonase-1 gene expression and activity induced by chronic hyperhomocysteinemia. These data suggest that catechin could act on the homocysteine levels by increasing the rate of catabolism of homocysteine.

Thiol compounds metabolism in mice, rats and humans: comparative study and potential explanation of rodents protection against vascular diseases.

BACKGROUND: Rodents are often used as animal models to dissect mechanisms underlying hyperhomocysteinemia atherogenicity in humans. However, neither wild-type rodents nor cystathionine beta-synthase deficient mice develop spontaneous atherosclerosis. We investigated whether species-specific differences in thiols metabolism may explain the respective sensitivity of rodents and humans to hyperhomocysteinemia. METHODS: Thiols and vitamins B levels were determined in normohomocysteinemic humans and rodents, and in hyperhomocysteinemic mice. RESULTS: In basal status, although plasma homocysteine, cysteine and cysteinylglycine levels were lower, glutathione levels were higher in mice than in humans (4.0+/-1.6 vs. 7.9+/-2.2, P<0.0005; 147.4+/-40.3 vs. 278.5+/-50.0, P<0.0001; 2.3+/-0.7 vs. 36.6+/-7.3, P<0.0001; and 70.9+/-20.1 vs. 4.6+/-1.6, P<0.0001). Serum vitamin B12 and folate levels were 2.5- and 7.7-fold higher in rats than in humans. In wild-type mice, the increase in plasma Hcy levels induced by methionine-enriched diet was accompanied by a proportional increase in GSH levels. CONCLUSION: GSH levels are enough to modulate Hcy effects in normo- and hyperhomocysteinemic mice but not in humans. This rodents characteristic, likely supported by species differences in the relative contribution of remethylation and transsulfuration pathways, may partly explain their protection against atherosclerosis.

Hyperkeratosis in cystathionine beta synthase-deficient mice: an animal model of hyperhomocysteinemia.

Cystathionine beta synthase (CBS) is a crucial regulator of plasma concentrations of homocysteine. Severe hyperhomocysteinemia due to CBS deficiency confers diverse clinical manifestations. Patients with severe hyperhomocysteinemia have fine hair and thin skin, but it is unclear whether these changes are related to CBS deficiency or are coincidental. To investigate these aspects of hyperhomocysteinemia, we characterized skin abnormalities of CBS-deficient mice, a murine model of severe hyperhomocysteinemia. Histological and histomorphometric analyses revealed that CBS-deficient mice have wrinkled skin with hyperkeratinosis of the epidermis and thinning of the dermis.