Periodic Reporting for period 4 - IMMUNE CELL SWARMS (Innate Immune Cell Swarms: Integrating and Adapting Single Cell and Population Dynamics in Inflamed and Infected Tissues)
Okres sprawozdawczy: 2021-08-01 do 2022-01-31
The inflammatory response protects the body against infection and injury but can itself become dysregulated with deleterious consequences to the organism. As the key cell type for eliminating bacteria and fungi, neutrophils accumulate as one of the first inflammatory cells at local sites of tissue injury and infection. While studies in living organisms have identified many factors that guide neutrophil migration, we hardly understand how these cells integrate the plethora of signals arising in inflammatory environments and coordinate their dynamic behavior with other tissue-resident immune cells. Recent intravital microscopy studies have discovered an unexpected phenomenon during this response: Swarm formation of neutrophils. It is now clear that intercellular communication among neutrophils amplifies their recruitment in a feed-forward manner. By secreting attractants that act through cell surface–expressed G protein–coupled receptors (GPCRs) on neighboring cells, neutrophils use this form of intercellular communication and coordinate their hunt for pathogens as a swarm. However, the stop signals for neutrophil swarming have not been defined yet. It is completely unknown how the swarming response is terminated to avoid unlimited neutrophil accumulation and prevent excessive inflammation. The failure to shutdown these pro-inflammatory circuits is considered critical in non-healing wounds and at the onset of chronic inflammation, which in turn may contribute to degenerative diseases such as cancer, diabetes and autoimmune diseases. Hence, there is an urgent need for more detailed information to understand the molecular control of neutrophil swarm dynamics and their contribution to the delicate balance between host protection and tissue destruction in inflammatory and infectious diseases.
In this project we investigated the mechanisms that stop neutrophil swarming in mammalian tissues. The stop signals may be derived from cells of the surrounding inflammatory environment or from neutrophils themselves. We hypothesized that the attractants released by neutrophils may become highly concentrated at sites where these cells cluster in larger numbers. It is well established that high chemoattractant concentrations can attenuate cellular responses by a process termed GPCR desensitization. We hypothesized a self-limiting mechanism for swarming: The local accumulation of the same neutrophil-expressed attractants that amplify swarming during early stages would cause desensitization of their respective GPCRs at later stages of neutrophil clustering. This led us to investigate the role of GPCR desensitization in neutrophil tissue navigation and host defense.
Our results describe a cell-intrinsic stop mechanism for the self-organization of neutrophil collectives in infected tissues, which is based on sensing the local accumulation of the same cell-secreted attractants that amplify swarming during early stages. GPCR desensitization acts as a negative feedback control mechanism to stop neutrophil migration in swarm aggregates. A protein called G-protein receptor coupled kinase 2 (GRK2) critically controls GPCR desensitization during neutrophil swarming. This navigation mechanism allows neutrophils to self-limit their dynamics within forming swarms and ensures optimal elimination of bacteria. However, this response is not a general mechanism used by all immune cells, as we find other roles of GRK proteins in other immune cell subsets.
In this project we investigated the mechanisms that stop neutrophil swarming in mammalian tissues. The stop signals may be derived from cells of the surrounding inflammatory environment or from neutrophils themselves. We hypothesized that the attractants released by neutrophils may become highly concentrated at sites where these cells cluster in larger numbers. It is well established that high chemoattractant concentrations can attenuate cellular responses by a process termed GPCR desensitization. We hypothesized a self-limiting mechanism for swarming: The local accumulation of the same neutrophil-expressed attractants that amplify swarming during early stages would cause desensitization of their respective GPCRs at later stages of neutrophil clustering. This led us to investigate the role of GPCR desensitization in neutrophil tissue navigation and host defense.
Our results describe a cell-intrinsic stop mechanism for the self-organization of neutrophil collectives in infected tissues, which is based on sensing the local accumulation of the same cell-secreted attractants that amplify swarming during early stages. GPCR desensitization acts as a negative feedback control mechanism to stop neutrophil migration in swarm aggregates. A protein called G-protein receptor coupled kinase 2 (GRK2) critically controls GPCR desensitization during neutrophil swarming. This navigation mechanism allows neutrophils to self-limit their dynamics within forming swarms and ensures optimal elimination of bacteria. However, this response is not a general mechanism used by all immune cells, as we find other roles of GRK proteins in other immune cell subsets.
Neutrophils circulate in our bodies and hunt in infected tissues to ingest, kill, and digest harmful pathogens. To become such effective killers in the very complex situation of an inflamed tissue, they work together as a collective. They release chemical signals to attract other cells to form clusters of cells and attack as a swarm. By studying isolated mouse neutrophils in the culture dish, we identified that the G-protein coupled receptor kinase 2 (GRK2) regulates a process termed G-protein coupled receptor (GPCR) desensitization and thus the responsiveness of neutrophils to swarm-secreted chemoattractants. Intravital imaging of injured skin and infected lymph nodes of mice showed that GRK2 and GPCR desensitization play critical roles at sites where swarming neutrophils accumulate and self-generate local fields of high swarm attractant concentration. Desensitization-resistant neutrophils moved faster and explored larger areas of lymph node tissue infected with the bacterium Pseudomonas aeruginosa. Such behavior suggested more effective bacterial sampling throughout the infected organ. Surprisingly, mice with GRK2-deficient neutrophils showed impaired rather than improved bacterial clearance. This finding could not be explained by altered antibacterial effector functions. In vitro assays for the detailed analysis of swarming behavior and bacterial growth revealed that GPCR desensitization to swarm attractants is required to induce neutrophil arrest for optimal bacterial phagocytosis and containment in swarm clusters. In summary, we identified a navigation strategy, which allows neutrophils to self-limit their swarming dynamics, thus ensuring optimal elimination of bacteria. This work has been published (Kienle et al., Science 2021) and presented on several international conferences. We have also set this work into the context of current knowledge and work of other in two review articles (Glaser et al., Current Opinion in Cell Biology 2021; Mihlan et al., Frontiers in Cell and Developmental Biology, appearance in April/May 2022). Moreover, we compared the GPCR-based navigation strategies of several immune cell types, including neutrophils, in another review article (Lämmermann & Kastenmüller, Immunological Reviews 2019).
Moreover, we also explored the functional role of GRK-controlled GPCR desensitization in immune cells other than neutrophils. We considered this process as a general mechanism that might influence the dynamics of other immune cells in a similar manner to neutrophils. However, we made the unexpected finding that GRK-deficient dendritic cells, T cells and B cells have completely different phenotypes than GRK-deficient neutrophils, and we further delineated distinct functional roles of GRKs in specific immune cell subsets. A research manuscript is currently prepared for the presentation of these findings. Lastly, in our efforts to develop an integrated view of how different immune cell populations influence neutrophil swarming, we made the unexpected observation that mast cells, a tissue-resident myeloid immune cell type, can influence swarming neutrophils. We could identify the signals that mast cells release to initiate the formation of neutrophil swarms. These unexpected findings shed a new light on the role of neutrophils and mast cells during allergic reactions. A research manuscript is currently prepared for the presentation of these findings.
Moreover, we also explored the functional role of GRK-controlled GPCR desensitization in immune cells other than neutrophils. We considered this process as a general mechanism that might influence the dynamics of other immune cells in a similar manner to neutrophils. However, we made the unexpected finding that GRK-deficient dendritic cells, T cells and B cells have completely different phenotypes than GRK-deficient neutrophils, and we further delineated distinct functional roles of GRKs in specific immune cell subsets. A research manuscript is currently prepared for the presentation of these findings. Lastly, in our efforts to develop an integrated view of how different immune cell populations influence neutrophil swarming, we made the unexpected observation that mast cells, a tissue-resident myeloid immune cell type, can influence swarming neutrophils. We could identify the signals that mast cells release to initiate the formation of neutrophil swarms. These unexpected findings shed a new light on the role of neutrophils and mast cells during allergic reactions. A research manuscript is currently prepared for the presentation of these findings.
We discovered desensitization to a self-produced activation signal as a principle of biological self-organization. These results pave the way for a better understanding of neutrophil biology, which is essential for immune host defense against bacteria and could inform therapeutic approaches in the future. Moreover, the swarming behavior and underlying mechanisms could also inform other categories of collective behavior and self-organization in cells and insects.