Periodic Reporting for period 4 - Plat-IL-1 (Pathophysiology of platelet-derived Interleukin 1)
Período documentado: 2021-09-01 hasta 2022-08-31
Our innate immune system is the first line of defense against pathogens. It comprises our natural barriers (skin and mucosa), soluble antimicrobial peptides, pore-forming proteins, signaling molecules, and cells such as phagocytes (monocytes, macrophages, neutrophils, mast cells, and natural-killer cells).
However, recent research has identified immune functions and significant contributions of other non-immune cells (i.e. red blood cells and platelets) to an effective immune response. Likewise, there are emerging connections between the coagulation, immune, and complement systems.
In the ERC-funded project "PLAT-IL-1" our team explored multiple ways platelets interact with cells of the innate immune system and how their interactions influence immune response, host defense, and autoimmunity.
Blood innate immune cells, such as monocytes and neutrophils, are largely outnumbered by platelets. These cells travel together through the vasculature and are in constant contact. Similarly, macrophages and other tissue-resident immune cells often interact with platelets during inflammation, wound, or tissue infections. Nevertheless, most literature on macrophages, neutrophils, or monocytes studied them in isolation.
Among the signaling molecules that orchestrate innate immune mechanisms are the cytokines of the Interleukin-1 family. The pro-inflammatory cytokines IL-1b and IL-1a are among the first cytokines produced in the inflammatory tissue site. These molecules operate as an alarm system, inducing robust immune activation and coordinating the recruitment of inflammatory cells to the site. As nearly all cells express the IL-1 receptor (IL-1R), IL-1 cytokines can influence innate and adaptive immune responses and exert broad effects on the body. Indeed, numerous severe autoinflammatory diseases are caused by dysregulation in IL-1 signaling. The production of IL-1 cytokines is regulated by the inflammasomes, an intracellular signaling platform that enables the maturation and secretion of IL-1 cytokines.
Our PLAT-IL-1 team was particularly interested in finding the first responders: the initial inflammasome-activated cells and primary sources of IL-1 during early inflammatory events. Platelets have been proposed to assemble inflammasomes, produce IL-1 cytokines, and are seen in the inflammatory tissue site before other innate immune cells arrive. We, therefore, speculated that they could be the first responders and significant sources of IL-1 within the tissue or in the vasculature.
However, recent research has identified immune functions and significant contributions of other non-immune cells (i.e. red blood cells and platelets) to an effective immune response. Likewise, there are emerging connections between the coagulation, immune, and complement systems.
In the ERC-funded project "PLAT-IL-1" our team explored multiple ways platelets interact with cells of the innate immune system and how their interactions influence immune response, host defense, and autoimmunity.
Blood innate immune cells, such as monocytes and neutrophils, are largely outnumbered by platelets. These cells travel together through the vasculature and are in constant contact. Similarly, macrophages and other tissue-resident immune cells often interact with platelets during inflammation, wound, or tissue infections. Nevertheless, most literature on macrophages, neutrophils, or monocytes studied them in isolation.
Among the signaling molecules that orchestrate innate immune mechanisms are the cytokines of the Interleukin-1 family. The pro-inflammatory cytokines IL-1b and IL-1a are among the first cytokines produced in the inflammatory tissue site. These molecules operate as an alarm system, inducing robust immune activation and coordinating the recruitment of inflammatory cells to the site. As nearly all cells express the IL-1 receptor (IL-1R), IL-1 cytokines can influence innate and adaptive immune responses and exert broad effects on the body. Indeed, numerous severe autoinflammatory diseases are caused by dysregulation in IL-1 signaling. The production of IL-1 cytokines is regulated by the inflammasomes, an intracellular signaling platform that enables the maturation and secretion of IL-1 cytokines.
Our PLAT-IL-1 team was particularly interested in finding the first responders: the initial inflammasome-activated cells and primary sources of IL-1 during early inflammatory events. Platelets have been proposed to assemble inflammasomes, produce IL-1 cytokines, and are seen in the inflammatory tissue site before other innate immune cells arrive. We, therefore, speculated that they could be the first responders and significant sources of IL-1 within the tissue or in the vasculature.
During the PLAT-IL-1 project, we discovered that human and mouse platelets are likely not cellular sources of IL-1 cytokines. Using a series of complementary techniques and experiments in human cells and mouse models, we demonstrated that platelets do not express inflammasome proteins and are unable to produce IL-1 or many other pro-inflammatory cytokines that drive innate immune responses.
However, while studying the interactions of platelets with human innate immune cells, we found that, despite not expressing inflammasomes themselves, platelets are essential for the inflammasome function in other immune cells, such as macrophages, neutrophils, and monocytes. Co-culture with platelets boosted the inflammasome activation and production of IL-1α, IL-1β, and IL-18 from human macrophages and neutrophils and were crucial for the optimal production of IL-1 cytokines by human monocytes. Platelets influenced the inflammasome activation of these cells by enhancing the transcription of NLRP3 and pro-IL-1β. In line with their importance in vitro, platelet depletion in mice attenuated LPS-induced IL-1β responses in vivo, and blood platelet counts correlated positively with plasma levels of IL-1β in human cohorts. Moreover, we found an enriched platelet gene signature in the whole blood transcriptomics of pediatric patients with IL-1-driven auto-inflammatory diseases. Thus, in this first part of the project, we discovered a pivotal role for platelets in the development of IL-1-driven autoinflammatory diseases (Rolfes et al., 2020).
Another discovery during the PLAT-IL-1 action began after we noticed that primary monocytes, isolated from human blood using commercially available immuno-magnetic isolation kits, contain platelets as contaminants. We then noticed that the immune function of monocytes was impaired when contaminant platelets were removed. Removal of platelets caused monocyte immunoparalysis, characterized by a impaired in their ability to produce cytokines in response to inflammatory triggers. The anergic state of platelet-depleted monocytes was not permanent and was reversed by their supplementation with autologous platelets. Notably, cytokine functions in monocytes from patients with immune thrombocytopenia (ITP), an autoimmune condition characterized by low blood platelet counts, were naturally impaired. Strikingly, the supplementation with healthy platelets reverted immunoparalysis in ITP monocytes, revealing the possibility of using platelet therapy for these patients. Mechanistically, we discovered that platelets are cellular sources of transcription factors that they supply to monocytes, and that the platelet-derived transcription factor MAPK14, was necessary for the full cytokine function of human monocytes. These findings are of fundamental clinical relevance, as platelet transfusion, a worldwide therapy used to counteract thrombocytopenia, is highly inflammatory and known to cause numerous adverse and fatal effects. This study was published as a pre-print (Hawwari et al., 2022) and is currently undergoing peer review.
The experimental setup and animal models used in the PLAT-IL-1 action allowed us to investigate the inflammasomes' activation in vivo and raised the possibility of testing novel biologicals able to interfere with its activity. Together with Dr. Florian Schmidt, a cell biologist with expertise in innate immunity and virology, we developed camelid-derived nanobodies targeting ASC, a central component of the inflammasomes. ASC nanobodies were highly efficient in disaggregating pre-formed inflammasomes and ameliorating arthritis in mice (Bertheloot et al., 2022). This approach represents a novel biologic-based treatment for inflammasome-initiated inflammatory diseases. The study was published and highlighted in the June cover of EMBO Molecular Medicine in 2022.
Overall, the PLAT-IL-1 project was fundamental to establishing my group as a reference in platelet immunology in Bonn and the international community. It also increased the visibility of our research and consolidated me as an independent scientist in a prestigious German university.
REFERENCES:
Bertheloot, D., et al. 2022. EMBO Mol Med e15415.
Hawwari, I., et al. 2022. bioRxiv 2022.2008.2010.503291.
Rolfes, V., et al. 2020. Cell Rep 31:107615.
However, while studying the interactions of platelets with human innate immune cells, we found that, despite not expressing inflammasomes themselves, platelets are essential for the inflammasome function in other immune cells, such as macrophages, neutrophils, and monocytes. Co-culture with platelets boosted the inflammasome activation and production of IL-1α, IL-1β, and IL-18 from human macrophages and neutrophils and were crucial for the optimal production of IL-1 cytokines by human monocytes. Platelets influenced the inflammasome activation of these cells by enhancing the transcription of NLRP3 and pro-IL-1β. In line with their importance in vitro, platelet depletion in mice attenuated LPS-induced IL-1β responses in vivo, and blood platelet counts correlated positively with plasma levels of IL-1β in human cohorts. Moreover, we found an enriched platelet gene signature in the whole blood transcriptomics of pediatric patients with IL-1-driven auto-inflammatory diseases. Thus, in this first part of the project, we discovered a pivotal role for platelets in the development of IL-1-driven autoinflammatory diseases (Rolfes et al., 2020).
Another discovery during the PLAT-IL-1 action began after we noticed that primary monocytes, isolated from human blood using commercially available immuno-magnetic isolation kits, contain platelets as contaminants. We then noticed that the immune function of monocytes was impaired when contaminant platelets were removed. Removal of platelets caused monocyte immunoparalysis, characterized by a impaired in their ability to produce cytokines in response to inflammatory triggers. The anergic state of platelet-depleted monocytes was not permanent and was reversed by their supplementation with autologous platelets. Notably, cytokine functions in monocytes from patients with immune thrombocytopenia (ITP), an autoimmune condition characterized by low blood platelet counts, were naturally impaired. Strikingly, the supplementation with healthy platelets reverted immunoparalysis in ITP monocytes, revealing the possibility of using platelet therapy for these patients. Mechanistically, we discovered that platelets are cellular sources of transcription factors that they supply to monocytes, and that the platelet-derived transcription factor MAPK14, was necessary for the full cytokine function of human monocytes. These findings are of fundamental clinical relevance, as platelet transfusion, a worldwide therapy used to counteract thrombocytopenia, is highly inflammatory and known to cause numerous adverse and fatal effects. This study was published as a pre-print (Hawwari et al., 2022) and is currently undergoing peer review.
The experimental setup and animal models used in the PLAT-IL-1 action allowed us to investigate the inflammasomes' activation in vivo and raised the possibility of testing novel biologicals able to interfere with its activity. Together with Dr. Florian Schmidt, a cell biologist with expertise in innate immunity and virology, we developed camelid-derived nanobodies targeting ASC, a central component of the inflammasomes. ASC nanobodies were highly efficient in disaggregating pre-formed inflammasomes and ameliorating arthritis in mice (Bertheloot et al., 2022). This approach represents a novel biologic-based treatment for inflammasome-initiated inflammatory diseases. The study was published and highlighted in the June cover of EMBO Molecular Medicine in 2022.
Overall, the PLAT-IL-1 project was fundamental to establishing my group as a reference in platelet immunology in Bonn and the international community. It also increased the visibility of our research and consolidated me as an independent scientist in a prestigious German university.
REFERENCES:
Bertheloot, D., et al. 2022. EMBO Mol Med e15415.
Hawwari, I., et al. 2022. bioRxiv 2022.2008.2010.503291.
Rolfes, V., et al. 2020. Cell Rep 31:107615.
Through the PLAT-IL-1 Action, my lab has generated valuable research tools, which will be instrumental in driving new discoveries in the next 2-3 years. These include transcriptomic and proteomic datasets, new platelet and inflammasome reporter models, and novel therapeutics against inflammasomes that can be further exploited for clinical use. We will make these resources available to other researchers at the University. These tools will also make us a highly sought-out group by international collaborators and contribute to the growing international recognition of our team.