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LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-γ production by repressing β-catenin activation in dendritic cells.

Orr SJ, Burg AR, Chan T, Quigley L, Jones GW, Ford JW, Hodge D, Razzook C, Sarhan J, Jones YL, Whittaker GC, Boelte KC, Lyakh L, Cardone M, O'Connor GM, Tan C, Li H, Anderson SK, Jones SA, Zhang W, Taylor PR, Trinchieri G, McVicar DW - PLoS Pathog. (2013)

Bottom Line: The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses.Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels.In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Inflammation Program, National Cancer Institute-Frederick, Frederick, Maryland, United States of America.

ABSTRACT
Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

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LAB is required for efficient Th1 responses.(A–C) Purified wild-type naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of conditioned medium from BMDCs cultured with 1×105 heat-killed C. albicans yeast or 1 µg/ml LPS. (A–B) The cells were restimulated with PMA and Ionomycin and IFN-γ and IL-17A levels were analyzed by flow cytometry. Flow plots are representative of three replicates. (B) Graphs display mean +/− s.e.m. % cells expressing IFN-γ or IL-17A from three replicates analyzed by flow cytometry. Black bars represent WT, white bars represent Lat2−/−. (C) IFN-γ levels in the supernatants from three replicates were measured after 4 days. Data are representative of 3 independent experiments. *p<0.05 (1-way ANOVA, Bonferroni's post-test).
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ppat-1003357-g005: LAB is required for efficient Th1 responses.(A–C) Purified wild-type naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of conditioned medium from BMDCs cultured with 1×105 heat-killed C. albicans yeast or 1 µg/ml LPS. (A–B) The cells were restimulated with PMA and Ionomycin and IFN-γ and IL-17A levels were analyzed by flow cytometry. Flow plots are representative of three replicates. (B) Graphs display mean +/− s.e.m. % cells expressing IFN-γ or IL-17A from three replicates analyzed by flow cytometry. Black bars represent WT, white bars represent Lat2−/−. (C) IFN-γ levels in the supernatants from three replicates were measured after 4 days. Data are representative of 3 independent experiments. *p<0.05 (1-way ANOVA, Bonferroni's post-test).

Mentions: C. albicans contains ligands for many receptors (including Dectin-1, Dectin-2 and TLRs 2 and 4) and systemic infection with C. albicans involves both Th1 and Th17 responses [5]. In vitro however, the T cell response to C. albicans is dominated by Th17 polarization [6]. In order to assess the capacity of Lat2−/− DC to direct both Th17 and Th1 responses in the context of reduced IL-12 production, heat-killed C. albicans and the alternate TLR4 selective ligand, LPS, were used respectively. Supernatants from WT and Lat2−/− BMDCs cultured in the presence of heat-killed C. albicans yeast or LPS were added to naïve WT CD4+ T cells stimulated with anti-CD3 and anti-CD28. IFN-γ and IL-17A production were measured by flow cytometry and ELISA following 4 days of culture. As expected, conditioned media from BMDCs stimulated with heat-killed yeast induced a robust Th17 response while stimulation with LPS induced a Th1 response (Fig. 5A-B). HKY-stimulated WT and Lat2−/− BMDCs induced comparable Th17 responses (Fig. 5A–B), likely due to the production of similar levels of IL-1β and only a minimal defect in IL-23 production from Lat2−/− BMDCs. Interestingly, the proportion of IFN-γ-secreting CD4+ T cells arising from LPS stimulated Lat2−/− BMDCs was significantly reduced compared to WT BMDCs (Fig. 5A–C), which could be attributed to the significant reduction in IL-12p70 production from Lat2−/− BMDCs (Fig. 4E–F). These data indicate that LAB-facilitated cytokine production is important for inducing Th1 responses but not Th17 responses in vitro. As both Th1 and Th17 responses are important for in vivo protection against C. albicans infections [6], [9], our data suggests that defective Th1 responses in Lat2−/− mice may be responsible for the increased susceptibility of these mice to C. albicans infection.


LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-γ production by repressing β-catenin activation in dendritic cells.

Orr SJ, Burg AR, Chan T, Quigley L, Jones GW, Ford JW, Hodge D, Razzook C, Sarhan J, Jones YL, Whittaker GC, Boelte KC, Lyakh L, Cardone M, O'Connor GM, Tan C, Li H, Anderson SK, Jones SA, Zhang W, Taylor PR, Trinchieri G, McVicar DW - PLoS Pathog. (2013)

LAB is required for efficient Th1 responses.(A–C) Purified wild-type naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of conditioned medium from BMDCs cultured with 1×105 heat-killed C. albicans yeast or 1 µg/ml LPS. (A–B) The cells were restimulated with PMA and Ionomycin and IFN-γ and IL-17A levels were analyzed by flow cytometry. Flow plots are representative of three replicates. (B) Graphs display mean +/− s.e.m. % cells expressing IFN-γ or IL-17A from three replicates analyzed by flow cytometry. Black bars represent WT, white bars represent Lat2−/−. (C) IFN-γ levels in the supernatants from three replicates were measured after 4 days. Data are representative of 3 independent experiments. *p<0.05 (1-way ANOVA, Bonferroni's post-test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649983&req=5

ppat-1003357-g005: LAB is required for efficient Th1 responses.(A–C) Purified wild-type naïve CD4+ T cells were stimulated with anti-CD3 and anti-CD28 for 4 days in the presence of conditioned medium from BMDCs cultured with 1×105 heat-killed C. albicans yeast or 1 µg/ml LPS. (A–B) The cells were restimulated with PMA and Ionomycin and IFN-γ and IL-17A levels were analyzed by flow cytometry. Flow plots are representative of three replicates. (B) Graphs display mean +/− s.e.m. % cells expressing IFN-γ or IL-17A from three replicates analyzed by flow cytometry. Black bars represent WT, white bars represent Lat2−/−. (C) IFN-γ levels in the supernatants from three replicates were measured after 4 days. Data are representative of 3 independent experiments. *p<0.05 (1-way ANOVA, Bonferroni's post-test).
Mentions: C. albicans contains ligands for many receptors (including Dectin-1, Dectin-2 and TLRs 2 and 4) and systemic infection with C. albicans involves both Th1 and Th17 responses [5]. In vitro however, the T cell response to C. albicans is dominated by Th17 polarization [6]. In order to assess the capacity of Lat2−/− DC to direct both Th17 and Th1 responses in the context of reduced IL-12 production, heat-killed C. albicans and the alternate TLR4 selective ligand, LPS, were used respectively. Supernatants from WT and Lat2−/− BMDCs cultured in the presence of heat-killed C. albicans yeast or LPS were added to naïve WT CD4+ T cells stimulated with anti-CD3 and anti-CD28. IFN-γ and IL-17A production were measured by flow cytometry and ELISA following 4 days of culture. As expected, conditioned media from BMDCs stimulated with heat-killed yeast induced a robust Th17 response while stimulation with LPS induced a Th1 response (Fig. 5A-B). HKY-stimulated WT and Lat2−/− BMDCs induced comparable Th17 responses (Fig. 5A–B), likely due to the production of similar levels of IL-1β and only a minimal defect in IL-23 production from Lat2−/− BMDCs. Interestingly, the proportion of IFN-γ-secreting CD4+ T cells arising from LPS stimulated Lat2−/− BMDCs was significantly reduced compared to WT BMDCs (Fig. 5A–C), which could be attributed to the significant reduction in IL-12p70 production from Lat2−/− BMDCs (Fig. 4E–F). These data indicate that LAB-facilitated cytokine production is important for inducing Th1 responses but not Th17 responses in vitro. As both Th1 and Th17 responses are important for in vivo protection against C. albicans infections [6], [9], our data suggests that defective Th1 responses in Lat2−/− mice may be responsible for the increased susceptibility of these mice to C. albicans infection.

Bottom Line: The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses.Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels.In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Inflammation Program, National Cancer Institute-Frederick, Frederick, Maryland, United States of America.

ABSTRACT
Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

Show MeSH
Related in: MedlinePlus