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Periodic Report Summary - LEMPIT (Leukocyte microdomains and platelet interactions in thrombo-inflammatory injury)

Inflammation is a process that aims to restore the normal function of organisms when this is altered by infection or trauma. Paradoxically, inflammation is also the number one killer in western societies. This occurs because the cellular and molecular mechanisms that protect the organism are also responsible for unwanted damage to tissues.

In my laboratory, we study neutrophils, a major cellular component of the inflammatory response. Neutrophils are responsible for the initial responses to infection or injury, such that alterations in their number or function results in high susceptibility to infection. Neutrophils achieve their anti-microbial functions by rapidly migrating to injured areas and locally releasing peptides and enzymes or by producing toxic substances such as highly reactive oxygen species. Under certain conditions, however, neutrophils can activate at wrong times or places and damage healthy tissue. A dramatic example of this inflammatory damage occurs when neutrophil activating agents, such as antibodies or endotoxin, appear in blood. In these cases, neutrophils adhere to the vessel-lining endothelium and release their toxic cargo which results in vascular damage and leads to tissue injury and even death. The interests of my laboratory are to both understand how neutrophil activation originates and to uncover the physiological control mechanisms that regulate this activation. To achieve these goals, we use intravital microscopy in various mouse mutants and inflammatory scenarios.

Previous work from our group and others uncovered an early process of neutrophil activation that we have postulated is important for neutrophil function. Leukocytes that accumulate in inflamed areas use glyco-conjugate ligands to bind endothelail selectins (P- and E-selectins) present on activated vascular cells. Through interactions mediated by these adhesive molecules cells undergo a rolling-like motion which allows them to sample other activating and adhesive cues on the endothelial cells, such as chemokines and integrin ligands. Resulting from these secondary activating signals, leukocytes stop rolling and adhere to the vessel wall. Also as a consequence of these signals they lose their round morphology and even distribution of receptors and cytoplasmic proteins, a process known as polarisation. Polarised neutrophils are characterised by the formation of a region of active actin reorganisation where integrins are enriched or leading edge, and a rounded structure that protrudes from the endothelial contact and is enriched in highly glycosylated receptors or trailing edge. We discovered that neutrophils that accumulate in inflamed areas are polarised and that they 'capture' circulating erythrocytes and platelets with receptors located in the leading or trailing edges. Interactions through the leading edge were mediated by the integrin Mac-1 (aMb2) and promoted vascular injury. For example, in the context of sickle cell anemia, sickled erythrocytes interact with the leading edge of adherent neutrophils and occlude small venules, which result in ischemia of the irrigated tissues and even death. In models of pulmonary injury in which anti-leukocyte antibodies are used to trigger inflammation, the interaction with circulating platelets activates neutrophils and the production of reactive oxygen species, resulting in vascular damage and tissue dysfunction.

Our current work is employing murine models of localised vascular inflammation to analyse the behaviour of neutrophils in live tissues. We use intravital microscopy at resolutions that allow discrimination of the leading and trailing edges in adherent neutrophils and the capture of circulating platelets. We are using a knock-in mouse where subcellular regions can be identified by the presence of the DOCK2-GFP chimeric protein, and will allow assessing the formation of the leading and trailing domains in leukocytes in real-time. A second goal of this project is to identify the domains and receptors that mediate leukocyte-platelet interactions, so that we can build a 'molecular interaction map' that will allow us to study the contribution of each microdomain (leading or trailing domains) to neutrophil function. We have begun the characterisation of mice deficient in several receptors that we hypothesised might be important in each domain. For this purpose, we have obtained mice deficient in the integrin Mac-1, the selectins P and E, and in selectin ligands PSGL-1 and ESL-1. This array of mouse models will help us build the proposed molecular map. Our results indicate that the integrin Mac-1 is essential for the interactions mediated by the leading edge, whereas P-selectin and PSGL-1 are important for the interactions mediated by the trailing edge. Using mice deficient in the different receptors associated to the leading- or trailing domains we have assessed alterations in the in vivo behaviour of neutrophils. Mutants in Mac-1 display a marked reduction in heterotypic interactions and impaired ability to crawl on the endothelium, while absence in PSGL-1 and P-selectin, which are important for interactions with the trailing edge, display the opposite behaviour.

A third aim of our studies is to study the contribution of each leukocyte domain to inflammatory disease. A particularly useful model for these studies is transfusion-related acute lung injury (TRALI), in which injection of an anti-MHC-II antibody mimics the human syndrome and provokes lung injury. Because this model requires a particular genetic background in mice (Balb/c strain) to render them susceptible to TRALI, we have devoted the past two years to generate all the mutant mice required for these studies in this genetic background. We have now transferred the mutations in Mac-1, endothelial selectins, PSGL-1 and ESL-1 into the Balb/c background for 10 generations, so that we are in a perfect position to investigate the contribution of each subcellular domain to the induction of injury in this inflammatory model.

Through the studies that we are conducting, we expect to define the subcellular domains and molecular mediators that regulate the behaviour of neutrophils in the inflamed vasculature. The ultimate goal of this work is to design strategies to prevent thrombo-inflammatory injury, the number one killer in western societies.

The overall objective of the BIONANOTOOLS project is the understanding of the principles that underlie protein structure, stability and function by protein design. This research not only contributes to our understanding of the fundamental physics of protein folding, but also provides guidelines for the design of novel functional proteins with new and useful activities.

In particular, we focus on a type of proteins called tetratricopeptide repeats (TPR). They present a simple modular structure, where a small structural unit is repeated in tandem. Overall TPR domains are a very robust system to study protein structure, folding, and function, and to use them as building blocks for protein engineering to generate new functional nano-molecules.

The main objectives of the BIONANOTOOLS project are:

(1) characterisation of thermodynamic stability of scaffold proteins;
(2) design of novel stable scaffold;
(3) design of protein libraries for selection of novel functional domains;
(4) application of designed modules in nanobiotechnology.

Towards these objectives, during the first two years of BIONANOTOOLS project, we have made the following progresses:

We performed studies on protein stability and folding of design proteins to gain a better understanding on how the protein sequence determines the structure and thermodynamic stability of the proteins. We realised biophysical thermodynamic studies on newly designed repeat proteins. In this work, we showed that the modular structure of repeat proteins translates into modular stability. Additionally, we showed how by simple design principles protein stability can be increased, in a predictable fashion, in which the properties of individual units can be manipulated to generate a collective and predictable effect on the ensemble.

We have also successfully designed repeat protein modules that bind novel target peptides and demonstrated that these novel functional modules exhibit the desired binding activity in vitro and are also active in cells. We have generated new libraries that we are currently screening using high-throughput technologies for novel small molecule-binding domains.

Additionally, we are using our designed recognition modules for their immobilisation onto ordered surfaces synthesised of block-copolymers for their applications as biosensors.

Finally, we started to use the intrinsic self-assembly properties of redesigned stable repeat proteins to assemble nanostructured macroscopic films made purely of proteins.

The main results achieved so far are described below:

(1) The generation by rational design of extremely stable protein frameworks. These proteins with improved physical and biological properties will be useful in biotechnological applications, such as generation of novel biomaterials and will present enhanced efficiency as therapeutics.
(2) The design and selection of novel binding modules that have many potential applications, such as functional substitutes for antibodies, and represent new tools for cellular and molecular biology.
(3) The combination of those designed modules with block-copolymer technologies to generate biofunctional patterned surfaces.
(4) The preliminary generation of ordered materials by self-assembly of designed protein modules.

At the end of the project, we plan to have a profound understanding of our protein modules in both thermodynamics and functionality. Therefore, we plan to successfully generate an array of proteins with defined structure, stability and functionality. Those proteins will be extremely useful bio-tools that will be applied to monitor and investigate biological processes in vivo, as biosensors for diagnosis to detect markers of different disease.

Additionally, the final results of the characterisation of stability and self-assembly properties of these proteins will provide us a better understanding of the protein modules for their use as building blocks in biomaterials design. The ability to rationally design and direct biopolymer self-assembly, and endow it with reactive groups, will allow for functional materials with tailored morphology and mechanical properties. These kinds of materials show promise for a wide range of applications: from nanotechnology to biomedicine.

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