Final Report Summary - LUNG DCS UNFOLD (Involvement of the endoplasmic reticulum stress response in lung dendritic cell function and inflammatory lung diseases.)
Lung DCs unfold
Fabiola Osorio and Bart N Lambrecht
Unit Immunoregulation and Mucosal Immunology, VIB Inflammation Research Center, Belgium
GROUP-ID Consortium, Ghent University and University Hospital, Belgium
Department of Respiratory Medicine, Ghent University, Belgium
Dendritic cells (DCs) are a heterogeneous population of leukocytes that upon activation translate innate into adaptive immunity. In tissues such as the lung, DCs are key coordinators of the immune response to several inflammatory diseases including asthma. To perform this task, DCs are equipped with a broad variety of signalling proteins specialized in detecting various sources of stress. Whereas DC activation has been archetypically studied in response to microbes, the mechanisms by which DCs sense stress originated by perturbations of intracellular compartments remains largely unexplored. In fact, various triggers of inflammation including oxidative stress, starvation and chronic excess of metabolic factors induce improper protein folding. Still, it remains unclear whether accumulation of misfolded proteins is a danger signal sufficient to activate DCs for initiation of adaptive immunity.
This project plans to elucidate whether signalling proteins involved in sensing of misfolded proteins contribute to DC homeostasis and to the development of inflammatory lung diseases.
The accumulation of misfolded proteins within the endoplasmic reticulum (ER) is a process known as ER stress. To prevent the detrimental effects of ER stress, higher eukaryotic cells possess a three-branched signal transduction pathway known as unfolded-protein response (UPR). The UPR sensors inositol requiring enzyme (IRE)1, activating transcription factor (ATF)6, and PKR-like ER kinase (PERK) induce the transcription factors XBP1(X-box binding protein 1), ATF6 and ATF4 respectively, resulting in activation of genes involved in the ER biosynthetic machinery and protein folding.
The most conserved UPR sensor is IRE-1α, a protein that possesses endonuclease and kinase activity. Upon activation, IRE-1α cleaves the mRNA for XBP-1 causing a shift in its reading frame yielding a potent transcription factor (named XBP-1s) involved in ER biogenesis. In addition to XBP-1 mRNA, the RNase domain of IRE1α cleave certain mRNA species that encode for proteins of diverse nature. The identification of novel mRNA targets of IRE-1α is today subject of extensive research. In the immune system, the role of the IRE-1α/XBP-1 axis has been extended beyond the conventional UPR. The pathway was shown to be required for the differentiation of immune cells including plasma cells and DCs.
The contribution of the UPR to inflammatory lung diseases is yet to be elucidated. Notably, single nucleotide polymorphisms (SNP) at chromosome 17q21 near the ORMDL3 gene are associated to the development of childhood asthma. ORMDLs are an evolutionary conserved family of ER-resident proteins and their link with the UPR emerges from studies in ORMDL-deficient yeast, which display constitutive ER stress. In addition, it has been proposed that ORMDL3 modulates calcium signalling leading to reduced PERK and ATF6 activation. These data suggest that ORMDL proteins might be homeostatic sensors of membrane components and it opens new avenues for investigating the role ER stress and asthma.
Based on the aspects mentioned above, we hypothesize that activation of the UPR in DCs acts as an endogenous danger signal that plays a fundamental role in the development and function of DCs and their involvement in inflammatory lung diseases.
Objectives of this project:
1. Effect of the IRE-1α/XBP1 signalling pathway on DC development and function
To develop this objective, we made use of two transgenic mice lines. First, the ER stress reporter mice (ERAI) that reports XBP-1s activity fused to Venus fluorescent protein (Venus FP) allowing for reporting the IRE-1α arm of the UPR. Second, we crossed the conditional xbp1fl/fl mice with cd11cCre mice (thereafter called DC-XBP-1 mice) in order to ablate XBP-1 in DCs and evaluate the contribution of this transduction pathway in DC function.
Flow cytometry analysis of ERAI mice revealed that DCs were the predominant subset activating IRE-1α in steady state over several immune cell types analyzed. Remarkably, within conventional DC subsets, those of the CD8α subtype constituted the brightest cell expressing Venus FP in the whole spleen. Notably, IRE-1α activation did not reflect a generalized UPR as cells did not activate ATF6 or ATF4-dependent genes. Furthermore, expression of Venus FP was detected in the precursor cell that originates CD8α DC indicating that preferential induction of IRE-1α activity is intrinsic to CD8α+ cDC development.
To assess the role of the IRE-1α/XBP-1 axis in DC function, DC-XBP-1 mice were analyzed. Phenotypic studies revealed that there was a differential effect of XBP-1 deficiency in DC subsets. Loss of XBP-1 resulted in reduced expression of the surface marker CD11c in the CD8α+ subtype of DCs and not in the CD11b+ DC counterpart. Furthermore, XBP-1 loss affected several aspects of DC biology in CD8α DC exclusively. These include the organization of the endoplasmic reticulum and the ability to present and cross-present antigens to cytotoxic T cells. Thus, our findings indicate that the effects of IRE-1α/XBP-1 are unique to the “CD8α-like” lineage of DCs.
Microarray analysis of DC subsets from DC-XBP-1 mice versus wild type littermates confirmed that the transcriptional control of IRE-1α/XBP-1 operates only in CD8α DCs. Several genes found in this study were reported XBP-1 targets and genes involved in ER biogenesis and/or function. More importantly, the IRE-1α/XBP-1 arm of the UPR also controlled genes involved in antigen uptake and processing. These include tapasin, which is involved in antigen presentation via MHC-I, the integrin itgb2 and ER-Golgi intermediate compartment (ERGIC)-3. Further studies in IRE-1α deficient mice revealed that those targets constituted direct substrates of IRE-1α in DCs, and thus, identified novel mRNAs targeted by this pathway in the immune system.
Overall, we have unveiled a precisely regulated feedback circuit involving IRE-1α and XBP-1 that controls the homeostasis of CD8α+ DCs. Future studies will elucidate whether interfering with the activity of IRE-1α could be employed as a strategy to improve vaccination. This is an attractive scenario, enforced by the finding that a systems biology approach to predict vaccine responses to seasonal influenza identified XBP-1 as a central hub predicting early immune responses after vaccination. This work has been recently submitted for publication and is currently under revision.
2. Induction of ER stress pathways in DC subsets in response to allergens and environmental pollutants.
Induction of the UPR in DCs was evaluated in response to the environmental inducers: house dust mite allergen (HDM), lipopolysaccharide (LPS), cigarette smoke extract (CS), and diesel exhaust particles (DEP). Of those, HDM and LPS triggered induction of the UPR chaperone Bip and activation of XBP-1. This is a very interesting finding given that HDM and LPS are relevant triggers of asthma in physiology. Experiments designed to determine the contribution of XBP-1 signalling to cytokine production and function in response to allergen triggering are planned. These experiments also include the induction of HDM-driven asthma in DC-XBP-1 mice.
3. Involvement of the asthma-related ER stress modifier ORMDL3 in lung inflammatory diseases
ORMDL3 is the first component of the UPR that has been directly associated with asthma. To address the functional relevance of ORMDL3 in vitro and in vivo, the host laboratory successfully developed the ormdl3fl/fl mice (targeted by the EUCOMM consortium, Nr. 240408) during the course of this project. Mice are already available and have been crossed to a ubiquitous Cre deletor strain (sox2-Cre). At the moment, mice are ready to use and the colony is being expanded in order to obtain enough mice for asthma experiments. We will focus our studies on the development and function of DC subsets of ORMDL3 deficient mice. The elucidation of the role of ORMDL3 will determine whether ORMDL3 deficiency affects ER stress pathways and furthermore, how this factor contributes to the development of asthma.
Fabiola Osorio and Bart N Lambrecht
Unit Immunoregulation and Mucosal Immunology, VIB Inflammation Research Center, Belgium
GROUP-ID Consortium, Ghent University and University Hospital, Belgium
Department of Respiratory Medicine, Ghent University, Belgium
Dendritic cells (DCs) are a heterogeneous population of leukocytes that upon activation translate innate into adaptive immunity. In tissues such as the lung, DCs are key coordinators of the immune response to several inflammatory diseases including asthma. To perform this task, DCs are equipped with a broad variety of signalling proteins specialized in detecting various sources of stress. Whereas DC activation has been archetypically studied in response to microbes, the mechanisms by which DCs sense stress originated by perturbations of intracellular compartments remains largely unexplored. In fact, various triggers of inflammation including oxidative stress, starvation and chronic excess of metabolic factors induce improper protein folding. Still, it remains unclear whether accumulation of misfolded proteins is a danger signal sufficient to activate DCs for initiation of adaptive immunity.
This project plans to elucidate whether signalling proteins involved in sensing of misfolded proteins contribute to DC homeostasis and to the development of inflammatory lung diseases.
The accumulation of misfolded proteins within the endoplasmic reticulum (ER) is a process known as ER stress. To prevent the detrimental effects of ER stress, higher eukaryotic cells possess a three-branched signal transduction pathway known as unfolded-protein response (UPR). The UPR sensors inositol requiring enzyme (IRE)1, activating transcription factor (ATF)6, and PKR-like ER kinase (PERK) induce the transcription factors XBP1(X-box binding protein 1), ATF6 and ATF4 respectively, resulting in activation of genes involved in the ER biosynthetic machinery and protein folding.
The most conserved UPR sensor is IRE-1α, a protein that possesses endonuclease and kinase activity. Upon activation, IRE-1α cleaves the mRNA for XBP-1 causing a shift in its reading frame yielding a potent transcription factor (named XBP-1s) involved in ER biogenesis. In addition to XBP-1 mRNA, the RNase domain of IRE1α cleave certain mRNA species that encode for proteins of diverse nature. The identification of novel mRNA targets of IRE-1α is today subject of extensive research. In the immune system, the role of the IRE-1α/XBP-1 axis has been extended beyond the conventional UPR. The pathway was shown to be required for the differentiation of immune cells including plasma cells and DCs.
The contribution of the UPR to inflammatory lung diseases is yet to be elucidated. Notably, single nucleotide polymorphisms (SNP) at chromosome 17q21 near the ORMDL3 gene are associated to the development of childhood asthma. ORMDLs are an evolutionary conserved family of ER-resident proteins and their link with the UPR emerges from studies in ORMDL-deficient yeast, which display constitutive ER stress. In addition, it has been proposed that ORMDL3 modulates calcium signalling leading to reduced PERK and ATF6 activation. These data suggest that ORMDL proteins might be homeostatic sensors of membrane components and it opens new avenues for investigating the role ER stress and asthma.
Based on the aspects mentioned above, we hypothesize that activation of the UPR in DCs acts as an endogenous danger signal that plays a fundamental role in the development and function of DCs and their involvement in inflammatory lung diseases.
Objectives of this project:
1. Effect of the IRE-1α/XBP1 signalling pathway on DC development and function
To develop this objective, we made use of two transgenic mice lines. First, the ER stress reporter mice (ERAI) that reports XBP-1s activity fused to Venus fluorescent protein (Venus FP) allowing for reporting the IRE-1α arm of the UPR. Second, we crossed the conditional xbp1fl/fl mice with cd11cCre mice (thereafter called DC-XBP-1 mice) in order to ablate XBP-1 in DCs and evaluate the contribution of this transduction pathway in DC function.
Flow cytometry analysis of ERAI mice revealed that DCs were the predominant subset activating IRE-1α in steady state over several immune cell types analyzed. Remarkably, within conventional DC subsets, those of the CD8α subtype constituted the brightest cell expressing Venus FP in the whole spleen. Notably, IRE-1α activation did not reflect a generalized UPR as cells did not activate ATF6 or ATF4-dependent genes. Furthermore, expression of Venus FP was detected in the precursor cell that originates CD8α DC indicating that preferential induction of IRE-1α activity is intrinsic to CD8α+ cDC development.
To assess the role of the IRE-1α/XBP-1 axis in DC function, DC-XBP-1 mice were analyzed. Phenotypic studies revealed that there was a differential effect of XBP-1 deficiency in DC subsets. Loss of XBP-1 resulted in reduced expression of the surface marker CD11c in the CD8α+ subtype of DCs and not in the CD11b+ DC counterpart. Furthermore, XBP-1 loss affected several aspects of DC biology in CD8α DC exclusively. These include the organization of the endoplasmic reticulum and the ability to present and cross-present antigens to cytotoxic T cells. Thus, our findings indicate that the effects of IRE-1α/XBP-1 are unique to the “CD8α-like” lineage of DCs.
Microarray analysis of DC subsets from DC-XBP-1 mice versus wild type littermates confirmed that the transcriptional control of IRE-1α/XBP-1 operates only in CD8α DCs. Several genes found in this study were reported XBP-1 targets and genes involved in ER biogenesis and/or function. More importantly, the IRE-1α/XBP-1 arm of the UPR also controlled genes involved in antigen uptake and processing. These include tapasin, which is involved in antigen presentation via MHC-I, the integrin itgb2 and ER-Golgi intermediate compartment (ERGIC)-3. Further studies in IRE-1α deficient mice revealed that those targets constituted direct substrates of IRE-1α in DCs, and thus, identified novel mRNAs targeted by this pathway in the immune system.
Overall, we have unveiled a precisely regulated feedback circuit involving IRE-1α and XBP-1 that controls the homeostasis of CD8α+ DCs. Future studies will elucidate whether interfering with the activity of IRE-1α could be employed as a strategy to improve vaccination. This is an attractive scenario, enforced by the finding that a systems biology approach to predict vaccine responses to seasonal influenza identified XBP-1 as a central hub predicting early immune responses after vaccination. This work has been recently submitted for publication and is currently under revision.
2. Induction of ER stress pathways in DC subsets in response to allergens and environmental pollutants.
Induction of the UPR in DCs was evaluated in response to the environmental inducers: house dust mite allergen (HDM), lipopolysaccharide (LPS), cigarette smoke extract (CS), and diesel exhaust particles (DEP). Of those, HDM and LPS triggered induction of the UPR chaperone Bip and activation of XBP-1. This is a very interesting finding given that HDM and LPS are relevant triggers of asthma in physiology. Experiments designed to determine the contribution of XBP-1 signalling to cytokine production and function in response to allergen triggering are planned. These experiments also include the induction of HDM-driven asthma in DC-XBP-1 mice.
3. Involvement of the asthma-related ER stress modifier ORMDL3 in lung inflammatory diseases
ORMDL3 is the first component of the UPR that has been directly associated with asthma. To address the functional relevance of ORMDL3 in vitro and in vivo, the host laboratory successfully developed the ormdl3fl/fl mice (targeted by the EUCOMM consortium, Nr. 240408) during the course of this project. Mice are already available and have been crossed to a ubiquitous Cre deletor strain (sox2-Cre). At the moment, mice are ready to use and the colony is being expanded in order to obtain enough mice for asthma experiments. We will focus our studies on the development and function of DC subsets of ORMDL3 deficient mice. The elucidation of the role of ORMDL3 will determine whether ORMDL3 deficiency affects ER stress pathways and furthermore, how this factor contributes to the development of asthma.