MicroRNA-155 Protects Group 2 Innate Lymphoid Cells From Apoptosis to Promote Type-2 Immunity.

Group-2 innate lymphoid cells (ILC2) play critical roles in the initiation and maintenance of type-2 immune responses, predominantly through their production of the type-2 cytokines IL-5, IL-9, and IL-13. ILC2 are essential for the efficient elimination of helminth parasites, but also contribute to the detrimental type-2 immune responses that underlie diseases such as asthma and allergy. While several transcription factors have been identified that regulate the development and function of ILC2, less is known about the post-transcriptional mechanisms that regulate these processes. We identified micro-RNAs (miRNAs) that are co-ordinately regulated in ILC2 from mice exposed to two different stimuli, namely IL-33 "alarmin" administration or Nippostrongylus brasiliensis parasitic worm infection. miR-155 is upregulated in ILC2 in response to both stimuli and miR-155-/- mice had impaired IL-33-driven ILC2 responses. Using mixed bone marrow chimeras, we demonstrate that this deficit is intrinsic to ILC2 and that miR-155 protects ILC2 from apoptosis, while having little impact on ILC2 proliferation or cytokine production. These data reveal a subset of miRNAs that are regulated upon ILC2 activation and establish a specific role for miR-155 in regulating ILC2 survival following activation

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4+ T cells potentiates type 2 immunity and promotes parasitic helminth expulsion

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity

MicroRNA-155 Protects Group 2 Innate Lymphoid Cells From Apoptosis to Promote Type-2 Immunity

Group-2 innate lymphoid cells (ILC2) play critical roles in the initiation and maintenance of type-2 immune responses, predominantly through their production of the type-2 cytokines IL-5, IL-9, and IL-13. ILC2 are essential for the efficient elimination of helminth parasites, but also contribute to the detrimental type-2 immune responses that underlie diseases such as asthma and allergy. While several transcription factors have been identified that regulate the development and function of ILC2, less is known about the post-transcriptional mechanisms that regulate these processes. We identified micro-RNAs (miRNAs) that are co-ordinately regulated in ILC2 from mice exposed to two different stimuli, namely IL-33 “alarmin” administration or Nippostrongylus brasiliensis parasitic worm infection. miR-155 is upregulated in ILC2 in response to both stimuli and miR-155−/− mice had impaired IL-33-driven ILC2 responses. Using mixed bone marrow chimeras, we demonstrate that this deficit is intrinsic to ILC2 and that miR-155 protects ILC2 from apoptosis, while having little impact on ILC2 proliferation or cytokine production. These data reveal a subset of miRNAs that are regulated upon ILC2 activation and establish a specific role for miR-155 in regulating ILC2 survival following activation

Development of a bioartificial kidney (BAK) as a renal transport model

DMPK (Drug Metabolism and Pharmacokinetics) studies are essential for drug development but 90% of potential compounds are rejected in clinical trials, thus increasing the cost and time required for any drug to enter the market. Current in vitro models utilise 2D cell cultures that do not accurately represent the in vivo environment resulting in altered cell functions. Therefore, new models are needed that incorporate mechanical and structural conditions in a cell culture in order to mimic the in vivo environment. The aim of this project is to develop a hollow fibre based BAK (Bioartificial Artificial Kidney) device as a 3D cell culture model, similar to the proximal tubule physiology and study the active transport of renal transporter specific compounds and substrates to accurately predict renal clearance.Firstly, polymer biomaterials were produced and characterised in terms of cell adhesion, viability and function. PVDF (Polyvinylidene fluoride) polymers have shown be excellent biomaterials for cell adhesion without the use of any coatings while cell viability was not affected. Furthermore, HEK-OCT2 and MDCK-Mdr1a cells attached on the PVDF flat membranes were able to uptake their specific substrates in similar ratios as cells attached on TCP (Tissue culture plastic). However, PVDF proved to be an unsuitable material for HF (Hollow Fibre) production by using a conventional wet spinning process. PVDF HFs were not homogeneous with large pores making them unsuitable for the BAK device. As an alternative, commercially polypropylene based HFs (P1LX) were chosen instead.Secondly, fluorescence-based assays were developed to assess the functionality of the three main drug transporters involved in the renal transport of pharmaceutical compounds (P-gp, BCRP and OCT2). Primary RPTECs and cell lines were utilised to compare cell type variability while fluorescent substrates were used as an alternative to standard compounds. Fluorescent substrates alternatives Rho123, MTX-FITC and ASP+ were identified as suitable for studying the functions of P-gp, BCRP and OCT2 renal transporters, respectively.Finally, custom made BAK devices that could house a single HF were designed and produced that would allow for media perfusion both in the HF lumen and ECS (Extracapillary space). The renal similar basement membrane matrix coating Geltrex was used to enhance cell attachment on the HF membrane and flow rate of 3 μL/min was applied to expose cells to a physiological shear stress. MDCK cells were able to form a tight barrier under flow conditions after 7 days and were able to maintain it for at least 3 days. Human cells (HEK and RPTECs) however were not able to reach confluency within the HF and thus only uptake transport was studied.Cells grown in a 3D environment within the BAK device had different gene expression profiles compared to 2D grown cells. Most notably, the upregulation of the microvilli marker CD-133 and the downregulation of the renal transporters P-gp and BCRP while active transport of fluorescent substrates was significantly reduced. OCT2 expression and function on the other hand remained unchanged. These results demonstrate the differences between the function of cells grown in 2D static and 3D dynamic environments.This is a progress towards the development of a new renal model that incorporates a physiological environment to aid the improvement and efficiency of DMPK studies.</div

Supplementary Information Files for Development of a hollow fibre-based renal module for active transport studies

Supplementary Information Files for Development of a hollow fibre-based renal module for active transport studiesUnderstanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, diferences between assays and the body are common, indicating the importance of in vitro–in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fbre (Plasmaphan P1LX, 3M) that serves as a porous scafold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant fow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2 . MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fuorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown signifcant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fvefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efciency compared to Transwell cell cultures while efux of the P-gp-specifc substrates Hoechst and Rhodamine 123 was decreased. These results further support the efect of the microenvironment and fuidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies.<br

Development of a hollow fibre-based renal module for active transport studies

Understanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, diferences between assays and the body are common, indicating the importance of in vitro–in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fbre (Plasmaphan P1LX, 3M) that serves as a porous scafold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant fow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2 . MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fuorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown signifcant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fvefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efciency compared to Transwell cell cultures while efux of the P-gp-specifc substrates Hoechst and Rhodamine 123 was decreased. These results further support the efect of the microenvironment and fuidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies

MicroRNA-155 Protects Group 2 Innate Lymphoid Cells From Apoptosis to Promote Type-2 Immunity.

Group-2 innate lymphoid cells (ILC2) play critical roles in the initiation and maintenance of type-2 immune responses, predominantly through their production of the type-2 cytokines IL-5, IL-9, and IL-13. ILC2 are essential for the efficient elimination of helminth parasites, but also contribute to the detrimental type-2 immune responses that underlie diseases such as asthma and allergy. While several transcription factors have been identified that regulate the development and function of ILC2, less is known about the post-transcriptional mechanisms that regulate these processes. We identified micro-RNAs (miRNAs) that are co-ordinately regulated in ILC2 from mice exposed to two different stimuli, namely IL-33 ?alarmin? administration or Nippostrongylus brasiliensis parasitic worm infection. miR-155 is upregulated in ILC2 in response to both stimuli and miR-155?/? mice had impaired IL-33-driven ILC2 responses. Using mixed bone marrow chimeras, we demonstrate that this deficit is intrinsic to ILC2 and that miR-155 protects ILC2 from apoptosis, while having little impact on ILC2 proliferation or cytokine production. These data reveal a subset of miRNAs that are regulated upon ILC2 activation and establish a specific role for miR-155 in regulating ILC2 survival following activation

Bcl11b is essential for group 2 innate lymphoid cell development

Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s

Bcl11b is essential for group 2 innate lymphoid cell development

© 2015 Walker et al. Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b -/- fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt + ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s.Link_to_subscribed_fulltex