Image_4.tif

<p>Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.</p

Image_1.tif

<p>Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.</p

Image_3.tif

<p>Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.</p

Image_2.tif

<p>Natural killer T (NKT) cells are prominent innate-like lymphocytes in the liver with critical roles in immune responses during infection, cancer, and autoimmunity. Interferon gamma (IFN-γ) and IL-4 are key cytokines rapidly produced by NKT cells upon recognition of glycolipid antigens presented by antigen-presenting cells (APCs). It has previously been reported that the transcriptional coactivator β-catenin regulates NKT cell differentiation and functionally biases NKT cell responses toward IL-4, at the expense of IFN-γ production. β-Catenin is not only a central effector of Wnt signaling but also contributes to other signaling networks. It is currently unknown whether Wnt ligands regulate NKT cell functions. We thus investigated how Wnt ligands and β-catenin activity shape liver NKT cell functions in vivo in response to the glycolipid antigen, α-galactosylceramide (α-GalCer) using a mouse model. Pharmacologic targeting of β-catenin activity with ICG001, as well as myeloid-specific genetic ablation of Wntless (Wls), to specifically target Wnt protein release by APCs, enhanced early IFN-γ responses. By contrast, within several hours of α-GalCer challenge, myeloid-specific Wls deficiency, as well as pharmacologic targeting of Wnt release using the small molecule inhibitor IWP-2 impaired α-GalCer-induced IFN-γ responses, independent of β-catenin activity. These data suggest that myeloid cell-derived Wnt ligands drive early Wnt/β-catenin signaling that curbs IFN-γ responses, but that, subsequently, Wnt ligands sustain IFN-γ expression independent of β-catenin activity. Our analyses in ICG001-treated mice confirmed a role for β-catenin activity in driving early IL-4 responses by liver NKT cells. However, neither pharmacologic nor genetic perturbation of Wnt production affected the IL-4 response, suggesting that IL-4 production by NKT cells in response to α-GalCer is not driven by released Wnt ligands. Collectively, these data reveal complex temporal roles of Wnt ligands and β-catenin signaling in the regulation of liver NKT cell activation, and highlight Wnt-dependent and -independent contributions of β-catenin to NKT cell functions.</p

SNPs within <i>MERTK</i> define both risk and protective haplotypes associated with MS susceptibility.

<p>28 SNPs within the <i>MERTK</i> gene form a single block of very high LD (D'>0.99, LOD≥2). The five most frequent haplotypes (population frequency >1%) are shown in this schematic, along with the <i>p</i>-value of association of each haplotype with MS susceptibility as determined using a Chi-square test. Arrowhead indicates the haplotype-tagging allele of rs13414207 in haplotype 5. The alleles presented for rs17174870 are inferred from data obtained from sequencing the opposite strand but are presented as CT to maintain consistency with the remainder of the study.</p

Top 10 SNPs within <i>MERTK</i> associated with Multiple Sclerosis susceptibility.

<p>Top 10 SNPs within <i>MERTK</i> associated with Multiple Sclerosis susceptibility.</p

Gene model association of rs13414207 with Multiple Sclerosis.

<p>Gene model association of rs13414207 with Multiple Sclerosis.</p

Results of the association testing for variants identified in <i>MERTK</i>.

<p>Results of the association testing for variants identified in <i>MERTK</i>.</p

The haplotype structure of variants identified in <i>MERTK</i>.

<p>The 52 variants in <i>MERTK</i> genotyped for association with MS susceptibility fall into 4 separate blocks. Haplotype blocks are connected with thick lines if connections are observed in >10% samples and thin lines if connections are observed in >1% samples. A schematic of the <i>MERTK</i> gene is shown underneath indicating the relationship of the blocks to the physical structure of <i>MERTK</i>. The haplotypes coloured in red are significantly associated with MS susceptibility (p<0.05) and the haplotype coloured in blue is associated with protection (p<0.05). The <i>p</i>-value of association was determined using a Chi-square test. #This variant represents a tri-nucleotide in-del (T/- = TGG; -)</p

Summary of variants identified in <i>MERTK</i>.

<p>Summary of variants identified in <i>MERTK</i>.</p