The intrinsic role of SOCS1 in intestinal epithelial cells

The SOCS1 protein negatively regulate cytokine signaling. To define its role in intestinal epithelial cells (IEC), our laboratory created a mouse model of Socs1 gene deletion in IEC lineages of the small and large intestines. Analysis of this Socs1ΔIEC mouse model suggests that lethal ulcerative colitis might be driven by Paneth and goblet cell defects, like those seen in inflammatory bowel disease patients and mouse models with defective genes regulating autophagy and endoplasmic reticulum (ER) stress response. Ensuing dysbiosis and bacteria invasion would then trigger immune responses for bacteria killing, whereas erosion of the colon epithelium in colitis Socs1ΔIEC mice seems driven by STAT1-dependent sensitization of Socs1- null IECs to apoptosis induced by immune cells-derived IFNγ in response to bacterial invasion. To define SOCS1 IEC-intrinsic role in the epithelium integrity and renewal, and for confirming that SOCS1 deficiency sensitizes IECs to IFNγ-driven cell death, crypt-derived colonoid populations from the WT, Ifng-/- and Socs1-/- x Ifng-/- (DKO) mouse models were produced. Morphological analysis, gene and/or protein expression profiling of markers of IEC populationages (Lg5, E-cadherin, Muc2 and Tff3), autophagy (P62), ER stress response (spliced Xbp1) and apoptosis (cleaved caspase 3 protein), as well as EdU incorporation proliferation assays were then performed. Our results indicate that the loss of Ifng and Socs1 is not affecting colonoid growth and renewal capacity of stem cells, goblet cell differentiation, or IEC proliferation. However, we detected Ifng expression in WT colonoids, which could explain the E-cadherin upregulation, P62 protein reduced levels, and enhanced Xpb1 splicing in the Ifng-/- colonoids. Likewise, DKO colonoids also displayed elevated E-cadherin protein expression, but Xbp1 splicing was further increased and P62 protein slightly restored when compared to Ifng-/- colonoids. These results indicate that loss of SOCS1 in IECs might promote deficiency in ER stress response and enhanced autophagy. Furthermore, the loss of IFNg or SOCS1 seems to increase STAT1 protein levels in IECs at steady state and upon IFNg treatment, but only simultaneous deletion of Ifng and Socs1 genes was found to boost IFNg-induced STAT1 phosphorylation. Importantly morphological assessment combined with measure of cleaved caspase 3 protein levels by WB and immunofluorescent microscopy strongly suggest that the loss of SOCS1 makes IECs more prone to death by a non-apoptotic mechanism at steady state and to apoptosis upon IFNg stimulation. Although our results are preliminary and need to be validated in independent colonoids s, they support that goblet cell dysfunction in Socs1ΔIEC mice could be linked to defects in autophagy and ER stress response, and that loss of SOCS1 sensitizes IECs to IFNγ-induced cell death in a STAT1-dependent manner.The SOCS1 protein negatively regulate cytokine signaling. To define its role in intestinal epithelial cells (IEC), our laboratory created a mouse model of Socs1 gene deletion in IEC lineages of the small and large intestines. Analysis of this Socs1ΔIEC mouse model suggests that lethal ulcerative colitis might be driven by Paneth and goblet cell defects, like those seen in inflammatory bowel disease patients and mouse models with defective genes regulating autophagy and endoplasmic reticulum (ER) stress response. Ensuing dysbiosis and bacteria invasion would then trigger immune responses for bacteria killing, whereas erosion of the colon epithelium in colitis Socs1ΔIEC mice seems driven by STAT1-dependent sensitization of Socs1- null IECs to apoptosis induced by immune cells-derived IFNγ in response to bacterial invasion. To define SOCS1 IEC-intrinsic role in the epithelium integrity and renewal, and for confirming that SOCS1 deficiency sensitizes IECs to IFNγ-driven cell death, crypt-derived colonoid populations from the WT, Ifng-/- and Socs1-/- x Ifng-/- (DKO) mouse models were produced. Morphological analysis, gene and/or protein expression profiling of markers of IEC populationages (Lg5, E-cadherin, Muc2 and Tff3), autophagy (P62), ER stress response (spliced Xbp1) and apoptosis (cleaved caspase 3 protein), as well as EdU incorporation proliferation assays were then performed. Our results indicate that the loss of Ifng and Socs1 is not affecting colonoid growth and renewal capacity of stem cells, goblet cell differentiation, or IEC proliferation. However, we detected Ifng expression in WT colonoids, which could explain the E-cadherin upregulation, P62 protein reduced levels, and enhanced Xpb1 splicing in the Ifng-/- colonoids. Likewise, DKO colonoids also displayed elevated E-cadherin protein expression, but Xbp1 splicing was further increased and P62 protein slightly restored when compared to Ifng-/- colonoids. These results indicate that loss of SOCS1 in IECs might promote deficiency in ER stress response and enhanced autophagy. Furthermore, the loss of IFNg or SOCS1 seems to increase STAT1 protein levels in IECs at steady state and upon IFNg treatment, but only simultaneous deletion of Ifng and Socs1 genes was found to boost IFNg-induced STAT1 phosphorylation. Importantly morphological assessment combined with measure of cleaved caspase 3 protein levels by WB and immunofluorescent microscopy strongly suggest that the loss of SOCS1 makes IECs more prone to death by a non-apoptotic mechanism at steady state and to apoptosis upon IFNg stimulation. Although our results are preliminary and need to be validated in independent colonoids s, they support that goblet cell dysfunction in Socs1ΔIEC mice could be linked to defects in autophagy and ER stress response, and that loss of SOCS1 sensitizes IECs to IFNγ-induced cell death in a STAT1-dependent manner.

Name thesis:degree_name

M. Sc.

Level thesis:degree_level

Maîtrise

Discipline thesis:degree_discipline

Biologie cellulaire

Grantor dc:publisher

Université de Sherbrooke

Year dc:date.issued

2021

Author dc:creator

• Emami, Seyed Alireza

Advisor dc:contributor.advisor

• Saucier, Caroline

• SOCS1
• Inflammatory bowel disease
• IFNγ
• Colonoid
• Intestinal epithelial cells
• Maladie inflammatoire de l'intestin
• Colonoïde
• Cellules épithéliales intestinales

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/11143/18680

OAI identifier oai:identifier

oai:usherbrooke.scholaris.ca:11143/18680