Periodic Reporting for period 1 - PI3K-in-tolerance (PI3K delta role in dendritic cell antigen processing and presentation to control gut tolerance)
Reporting period: 2019-11-01 to 2021-10-31
Effective mammalian host defence against pathogenic microorganisms involve activation of dendritic cells (DCs) with potent priming ability to orchestrate adaptive T cell responses. Paradoxically within the intestine, the mammalian host encounters trillions of microbes and their components that alternatively stimulate DCs to promote immune tolerance to commensal bacteria. Aberrations in microbial surveillance or tolerance result in intestinal inflammation and inflammatory bowel disease (IBD) that affects the industrialised world. The mechanisms involved in colitis development and maintenance involve host and microbiota interplay, yet more studies regarding the molecular mechanisms of host gut immunity are required to uncover effective therapeutic strategies.
Phosphatidylinositol-3-OH kinases (PI3Ks) control pattern recognition receptor (PRR) signaling and coordinate phagosome dynamics. The class I PI3K p110 delta (p110δ) isoform is predominantly expressed in the hematopoietic compartment and inborn errors of the mammalian p110δ PI3K leads to defects in antimicrobial immunity and colitis, yet little known how p110δ safeguards host immunity. In this project, we have utilised molecular immunology, biochemistry, and mouse genetics tools and uncovered akey role of p110δ PI3K in DCs that orchestrate priming and activation of antigen specific T cells, summarised as (1) the presentation of phagosomal antigens within the MHCI and MHCII that upon microbial recognition by PRRs (2) DC-intrinsic p110δ role in imprinting tolerogenic regulatory T cells in the gut and (3) characterised the molecular components under p110δ, coordinating antigen processing and presentation process in DC phagosomes to orchestrate peripheral T cell tolerance to intestinal microorganisms and immune homeostasis. Our study provides a strong rationale to therapeutically target PI3K signalling in DCs to parse out cellular and molecular interactions within the mammalian intestine.
Phosphatidylinositol-3-OH kinases (PI3Ks) control pattern recognition receptor (PRR) signaling and coordinate phagosome dynamics. The class I PI3K p110 delta (p110δ) isoform is predominantly expressed in the hematopoietic compartment and inborn errors of the mammalian p110δ PI3K leads to defects in antimicrobial immunity and colitis, yet little known how p110δ safeguards host immunity. In this project, we have utilised molecular immunology, biochemistry, and mouse genetics tools and uncovered akey role of p110δ PI3K in DCs that orchestrate priming and activation of antigen specific T cells, summarised as (1) the presentation of phagosomal antigens within the MHCI and MHCII that upon microbial recognition by PRRs (2) DC-intrinsic p110δ role in imprinting tolerogenic regulatory T cells in the gut and (3) characterised the molecular components under p110δ, coordinating antigen processing and presentation process in DC phagosomes to orchestrate peripheral T cell tolerance to intestinal microorganisms and immune homeostasis. Our study provides a strong rationale to therapeutically target PI3K signalling in DCs to parse out cellular and molecular interactions within the mammalian intestine.
PI3K isoform p110δ role was explored in ubiquitous kinase deleted or conditional PI3K-gene-targeted mice in models of gut inflammation in vivo and PRR-associated events in bone marrow-derived dendritic cells (DCs) in vitro.
The in vivo inactivation of p110δ rendered dysbiosis and a hyper-inflammatory state revealed by 1) DC hyperactivation in the colon lamina propria (cLP) and mesenteric lymph node (mLN), 2) an exaggerated mucosal IFN-γ and IL-17 cytokine production in CD8 T cell in cLP and mLN, and 3) significantly reduced in Treg response (Foxp3 and IL10 producing) T CD4 cells in cLP and mLN. Indeed, we could demonstrate that all the referred results were similarly reproduced in DC-restricted deletion of p110δ kinase.
At molecular level, PRR-mediated activation of DCs defective in p110δ activity shown defect in Akt phosphorylation in parallel with increased stress MAPK signalling and NF-KB activation. These events culminate in increased release of inflammatory mediators and hyperactivation of caspase-1 and inflammasome activation. Besides, DCs defective in p110δ activity shown significative reduction in the production of intra-phagosomal reactive oxygen species (ROS) creating an state of increased acidification inside the vesicle. This acidic state observed in p110δ defective DCs leads to increased antigen processing and degradation impacting negatively in antigen presentation. Still in the mechanisms orchestrated by p110δ, we demonstrated, for the first time, the interaction between p110δ and RAC2 associating the lack of the referred interaction with the defect in PI3K signalling, increased inflammatory mediators and increased inflammasome activation under PRR activation. In addition, we demonstrate that p110δ and RAC2 interaction is important for ROS production vesicle alkalinization that leads to preserve antigen for presentation.
The results of the proposed research have led to the identification of a new role and mechanism of action of a lipid kinase p110δ PI3K isoform in 1. DC-intrinsic NOD2-dependent activation of PI3K signalling pathway, 2. DC-intrinsic anti-inflammatory role in PRR signalling in vitro and in vivo, 3. New PRR-driven regulatory pathways that induce gut tolerance through DC anti-inflammatory properties keeping gut homeostasis, 4. New mechanisms involved in PRR signalling triggered by p110δ PI3K required for proper antigen presentation and gut tolerance. All aspects studied here could be explored to generate anti-/pro-inflammatory DCs in vitro and in vivo.
The results were presented in local events in the Biochemical Pharmacology Department meetings and William Harvey Research Institute seminars and externally at The Francis Crick Institute.
The in vivo inactivation of p110δ rendered dysbiosis and a hyper-inflammatory state revealed by 1) DC hyperactivation in the colon lamina propria (cLP) and mesenteric lymph node (mLN), 2) an exaggerated mucosal IFN-γ and IL-17 cytokine production in CD8 T cell in cLP and mLN, and 3) significantly reduced in Treg response (Foxp3 and IL10 producing) T CD4 cells in cLP and mLN. Indeed, we could demonstrate that all the referred results were similarly reproduced in DC-restricted deletion of p110δ kinase.
At molecular level, PRR-mediated activation of DCs defective in p110δ activity shown defect in Akt phosphorylation in parallel with increased stress MAPK signalling and NF-KB activation. These events culminate in increased release of inflammatory mediators and hyperactivation of caspase-1 and inflammasome activation. Besides, DCs defective in p110δ activity shown significative reduction in the production of intra-phagosomal reactive oxygen species (ROS) creating an state of increased acidification inside the vesicle. This acidic state observed in p110δ defective DCs leads to increased antigen processing and degradation impacting negatively in antigen presentation. Still in the mechanisms orchestrated by p110δ, we demonstrated, for the first time, the interaction between p110δ and RAC2 associating the lack of the referred interaction with the defect in PI3K signalling, increased inflammatory mediators and increased inflammasome activation under PRR activation. In addition, we demonstrate that p110δ and RAC2 interaction is important for ROS production vesicle alkalinization that leads to preserve antigen for presentation.
The results of the proposed research have led to the identification of a new role and mechanism of action of a lipid kinase p110δ PI3K isoform in 1. DC-intrinsic NOD2-dependent activation of PI3K signalling pathway, 2. DC-intrinsic anti-inflammatory role in PRR signalling in vitro and in vivo, 3. New PRR-driven regulatory pathways that induce gut tolerance through DC anti-inflammatory properties keeping gut homeostasis, 4. New mechanisms involved in PRR signalling triggered by p110δ PI3K required for proper antigen presentation and gut tolerance. All aspects studied here could be explored to generate anti-/pro-inflammatory DCs in vitro and in vivo.
The results were presented in local events in the Biochemical Pharmacology Department meetings and William Harvey Research Institute seminars and externally at The Francis Crick Institute.
PI3Ks are evolutionarily conserved enzymes involved in immune cell signalling and phagocytosis processes. We expect that our study will have generated new and important knowledge and assisted in better understanding of the immunological and cell signalling events involved in DC-mediated immune resistance to microbes and also how tolerance can be achieved to the commensal microbes in mucosal environment such as the intestine.
The data generated from the project have important ramifications for the human health and therapies in which PI3K-mediated regulation of immune signals could be reciprocally mapped and defined leading to 1) discovery of new therapeutic strategies in cancer and inflammation and 2) understanding adverse effects of drugs involved in intestinal homeostasis and tolerance by academia and pharmaceutical industry.
Our results could be beneficial for academics, clinicians and pharmaceutical industry for therapeutic harnessing of the p110δ PI3K-driven antigen presentation in DCs will be important in immunotherapy, in which temporal inhibition of p110δ activity in specific immune cell populations, particularly in regulatory T cells, would be beneficial in cancer and chronic viral infections, by boosting an effective immune response.
Our work demonstrate that p110δ PI3K is a key molecule in innate and adaptive immune cell populations and that inborn errors of p110δ or chronic chemical inactivation can result in immune deficiencies that can lead to gut inflammation and thus requires spatially and temporally manipulated to avoid adverse effects on the host immunity. This could be explored in terms of the potential to develop personalized therapeutic strategies that could lead to potential savings for the health system and affect the quality of progress beyond the ‘state-of-art'.
It is important to highlight that the project has been successfully carried out and completed throughout the Covid19 pandemic, a significant achievement for the Fellow's future career path and socio-economic and societal impact. The Researcher has developed local, national and international networks and benefited from technical and scientific skills training through access to outstanding equipment and facilities in William Harvey Research Institute, exposure to the academic and discuss problems encountered during regular group (weekly) and institutional meetings (semi-annually). Unfortunately, the limitations of participation in the face-to-face scientific or social events and public engagement was not allowed during the fellowship and had to be carried online via the internet platforms. The results of this study is likely to be submitted and published in a high-impact peer-reviewed journals.
The data generated from the project have important ramifications for the human health and therapies in which PI3K-mediated regulation of immune signals could be reciprocally mapped and defined leading to 1) discovery of new therapeutic strategies in cancer and inflammation and 2) understanding adverse effects of drugs involved in intestinal homeostasis and tolerance by academia and pharmaceutical industry.
Our results could be beneficial for academics, clinicians and pharmaceutical industry for therapeutic harnessing of the p110δ PI3K-driven antigen presentation in DCs will be important in immunotherapy, in which temporal inhibition of p110δ activity in specific immune cell populations, particularly in regulatory T cells, would be beneficial in cancer and chronic viral infections, by boosting an effective immune response.
Our work demonstrate that p110δ PI3K is a key molecule in innate and adaptive immune cell populations and that inborn errors of p110δ or chronic chemical inactivation can result in immune deficiencies that can lead to gut inflammation and thus requires spatially and temporally manipulated to avoid adverse effects on the host immunity. This could be explored in terms of the potential to develop personalized therapeutic strategies that could lead to potential savings for the health system and affect the quality of progress beyond the ‘state-of-art'.
It is important to highlight that the project has been successfully carried out and completed throughout the Covid19 pandemic, a significant achievement for the Fellow's future career path and socio-economic and societal impact. The Researcher has developed local, national and international networks and benefited from technical and scientific skills training through access to outstanding equipment and facilities in William Harvey Research Institute, exposure to the academic and discuss problems encountered during regular group (weekly) and institutional meetings (semi-annually). Unfortunately, the limitations of participation in the face-to-face scientific or social events and public engagement was not allowed during the fellowship and had to be carried online via the internet platforms. The results of this study is likely to be submitted and published in a high-impact peer-reviewed journals.