Microtubules as an integrator of the physical and chemical properties of the microenvironment.

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Leukocyte recruitment: from blood to the site of infection

The recruitment of blood leukocytes to the site of infection in tissues involves several steps of migration through the blood vessel endothelium and into the interstitial space. Neutrophils—the most abundant blood leukocytes—are essential for pathogen clearance, and their recruitment to the site of infection is the most studied (Weninger et al., 2014).

Transendothelial migration

The transendothelial migration of neutrophils in inflamed tissues involves a well-established multi-step adhesion cascade. Selectin expression on activated endothelial cells allows neutrophils to slow down and roll along the walls of inflamed blood vessels. Integrin engagement and chemokine sensing leads to firm adhesion of neutrophils, followed by their transmigration through the endothelial cell monolayer (Vestweber, 2015). After transmigration, the neutrophils crawl into the subendothelial space confined by the dense basement membrane and pericytes surrounding the blood vessels. The neutrophils extravasate into the tissue through low-density regions of extracellular proteins in the basement membrane. Chemokines secreted by the perivascular immune cells guide the neutrophil extravasation (Vestweber, 2015).
Blood leukocyte transmigration is a limiting step that requires specific signaling events triggered by inflammation. Thus, leukocyte infiltration is limited to the site of infection (Pober and Sessa, 2007).

Interstitial migration

Once in the interstitial space, leukocytes navigate in a three-dimensional environment composed of a network of cross-linked extracellular matrix fibers (Lämmermann and Germain, 2014). To reach the site of infection, they integrate chemoattractant signals from tissue-resident immune cells, damaged cells, or pathogens. The neutrophil population exhibits coordinated chemotactic migration, leading to the formation of a dense cell cluster that contains the injury (Kienle and Lämmermann, 2016; Ng et al., 2011). This swarming behavior relies on the secretion of chemokines that act as a paracrine signal among the neutrophils. This process of self-amplification of a local inflammatory signal facilitates neutrophil recruitment throughout the tissue at a long distance from the site of injury (200–300 µm) and in a short time (~20 min) (Lämmermann et al., 2013).
Leukocyte migration into the interstitial space does not require high-affinity integrin signaling. Thus, immune cell recruitment is mainly orchestrated by chemokines secreted during the inflammation process and is independent of the composition and structure of the extracellular matrix. This migration mechanism, which will be described in detail later in this thesis, is a critical step in the immune response that requires leukocytes to be able to rapidly reach any type of tissues  upon detection of inflammatory signals (Lämmermann et al., 2008). Integrins transduce signals of arrest rather than migration during the immune response. They induce leukocyte adhesion on the inflamed blood vessels (Vestweber, 2015) and are necessary for neutrophil accumulation at the site of injury (Lämmermann et al., 2013).
At the site of infection, innate immune cells release molecules and enzymes that directly eliminate the pathogen or prevent its spread (Alberts et al., 2002a). The molecules are specific to the pathogen family. Phagocytic cells, mainly neutrophils and macrophages, engulf pathogens and remove cellular debris. This process is essential for the resolution of inflammation at a later stage of the immune response (Westman et al., 2020). While innate immune cells control the spread of pathogens, they release a second set of cytokines that orchestrate the activation of an adaptive immune response (Iwasaki and Medzhitov, 2015).

Dendritic cell maturation

Antigen transport from the injury site to the lymph nodes is essential for the initiation of an adaptive immune response. Microbial detection triggers DC maturation, a multi-step process that leads to mature DCs (mDCs) capable of activating T lymphocytes. This process involves antigen uptake and presentation on MHC, integration of the inflammatory context to activate appropriate adaptive immune cells, and acquisition of the ability to migrate to the lymph node (Cella et al., 1997a).

Antigen uptake and processing

Before microbial detection, immature DCs (iDCs) scan the tissues for harmful particles. Tissue surveillance is ensured by projecting long cell protrusions in random directions or by active cell migration. In vitro studies suggest that the migration patterns of iDCs follow an intermittent random walk model. They alternate between rapid migration phases and arrest phases when sampling their surroundings; this behavior optimizes the pathogen search in the environment (Chabaud et al., 2015).
Direct pathogen detection through PRRs or inflammatory chemokine sensing transiently increases the ability of DCs to internalize extracellular components by endocytosis, phagocytosis, or macropinocytosis. Subsequently, internalized pathogenic particles are degraded into antigenic peptides and loaded onto MHC II for presentation to CD4+ TLs and onto MHC I for presentation to  CD8+ TLs (West et al., 2004).
MHC II binds to antigens degraded in the lysosomal pathway. In the absence of antigens, the invariant chain (Ii) stabilizes MHC II. After pathogen detection, the lysosomal protease cathepsin-S cleaves Ii and allows peptide loading onto MHC II. The complex is then transported to the plasma membrane to present the antigen to T lymphocytes (Neefjes et al., 2011).
MHC I binds to antigens degraded by cytosolic and nuclear proteasomes. Cytosolic peptides are transported to the endoplasmic reticulum (ER) where they are loaded onto MHC I molecules (Neefjes et al., 2011). Extracellular antigen presentation on MHC I requires a process called cross-presentation. There are two main pathways. In the vacuolar route, MHC I vesicles encounter the lysosomal pathway, and antigens degraded in the lysosomes are loaded onto MHC I. The second pathway is cytosolic: internalized particles escape from endosomes and are degraded by the cytosolic proteasome before being loaded onto MHC I in the ER (Alloatti et al., 2016).
Antigen processing is tightly regulated to avoid the presentation of self-antigens in an inflammatory context. Maturation of DCs increases MHC expression and translocation to the cell surface (Cella et al., 1997b) and modulates the lysosomal activity to process antigens properly (Trombetta et al., 2003). Increasing lysosomal acidification promotes antigen processing on MHC II, whereas maintaining a high pH promotes cross-presentation on MHC I (Samie and Cresswell, 2015). Cytokines and PRRs such as TLRs are known to signal from the endolysosomal compartments. PRRs directly modulate antigen presentation by co-trafficking with pathogenic endocytic particles (Watts et al., 2010) and locally regulate lysosomal activity to promote pathogen particle degradation into antigens (Roche and Furuta, 2015). Thus, he endolysosomal compartment plays a role as a signaling platform that integrates the inflammatory context to control antigen presentation.

Cytokine and co-stimulatory molecules

Antigens are not the only type of information acquired by DCs at the site of infection; they also integrate PAMPs and DAMPs present in the microenvironment and cytokines secreted by other immune cells. These factors provide information on the pathogenicity and location of the microbe as well as the level of tissue damage. This inflammatory context shapes the cytokine profile secreted by the mDCs and the nature of the co-stimulatory molecules expressed on their surface (Iwasaki and Medzhitov, 2015).

Mechanisms of cell retraction

In addition to the pushing force that drives cell protrusions, the actomyosin cytoskeleton also generates pulling forces associated with cell retraction (Cramer, 2013).
MyoII contributes significantly to cell retraction. This molecular motor generates a contractile force by sliding actin bundles at the cell cortex. The coupling between the membrane and the cortex causes the cells to retract (Vicente-Manzanares et al., 2009). Ultimately, the compressive stress exerted by MyoII on F-actin might cause their disassembly (Vogel et al., 2013). This, in turn, induces the compaction of the cortical actin and reinforces the retraction of the cells (Wilson et al., 2010).
However, several studies show that cells can retract without MyoII activity (Lämmermann et al., 2008; Wessels et al., 1988). They revealed that actin disassembly, mainly driven by ADF/cofilin (Mseka and Cramer, 2011) and compaction of the actin network by crosslinkers, produce sufficient tensile force to cause cell retraction (Sun etal., 2010).

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Generation of an actin flow

The mechanisms of cell movement described above highlight the central role of actin turnover in this process. Actin assembly at the protrusion sites and simultaneous disassembly at the retraction sites result in actin flow. Mechanically, the plasma membrane opposes resistance to F-actin assembly; thus, when a filament polymerizes against it, it is pushed back while the membrane protrudes (Cramer, 1997). MyoII activity is not required for this flow but further enhances the retrograde movement of the cortex (Renkawitz et al., 2009; Wilson et al., 2010). It can be observed
along the entire cell length (Renkawitz et al., 2009) or limited to the protruding area (Svitkina et al., 1997; Theriot and Mitchison, 1991). This actin flow generates the driving force that leads to cell migration (Pollard and Borisy, 2003). However, its transduction into traction force on the substrate and polarization across the cell are necessary to induce a net displacement (Vicente-Manzanares et al., 2005). I will describe this mechanism in the following parts.

Table of contents :

I. Principles and organization of the immune system
A. Two types of immune response
1. Innate immune response
2. Adaptive immune response
B. Steady-state organization of the immune system
C. Innate immune cells
D. Adaptive immune cells
1. T lymphocytes
2. B lymphocytes
E. Antigen-presenting cells
1. Macrophages
2. Dendritic cells
II. Local immune response: activation of the innate system
A. Inflammation
B. Leukocyte recruitment: from blood to the site of infection
1. Transendothelial migration
2. Interstitial migration
C. Dendritic cell maturation
1. Antigen uptake and processing
2. Cytokine and co-stimulatory molecules
3. Migration to the lymph nodes
III. Systemic immune response: activation of the adaptive system by dendritic cells
A. Antigen presentation to CD4+ T cells
B. Differentiation of CD4+ T cells and activation of the specific immune cell effectors
I. Intracellular forces for cell deformation
A. Acto-Myosin Cytoskeleton
1. Actin filament
2. Actin network organization
a) Branched network – Arp2/3
b) Non-Branched Network – Formins
3. Myosin
B. Different types of cell protrusions
1. Actin-rich protrusion
2. « Bleb » protrusion
C. Mechanisms of cell retraction
D. Generation of an actin flow
II. Force transmission to the substrate
A. Adhesion-dependent interaction
B. Anchorage-independent
1. Actin flow friction
2. Hydrostatic pressure and ‘Chimneying.’
III. Maintenance of front-rear polarization
A. Definition of front and rear domains
1. Rho family of small GTPases paradigm
2. Cortical polarization
B. Front-to-rear coupling during migration
1. Membrane tension
2. Sustained actin flow
3. Long-range cytoskeleton structures: microtubule network
a) Organization of the microtubule network
b) The role of microtubules in cell polarization
I. Physical and chemical properties of the interstitial space: challenges for cell migration
A. Chemical properties
1. The extracellular matrix
2. Chemokines
B. Physical properties
C. Challenges for DCs interstitial migration
1. In vitro models of the interstitial migration
2. Main characteristics of mDCs migration in 3D confined-environment and limiting factors
II. Maturation optimizes DCs migration machinery for rapid and directed migration
A. Remodelling of the actomyosin cytoskeleton
B. Coordination of antigen capture and rapid migration
III. DCs strategies to overcome physical obstacles
A. Cell branching coordination in a porous environment
1. Rac and Cdc42 control front protrusions in mDCs
2. Microtubule network coordinate protrusion and retraction
B. mDCs migration in confined environments
1. The mechanical properties of the nucleus will limit cell migration
2. Selection of the path of least resistance
a) Hydraulic resistance
b) Pore size selection
3. Mechanism of Nuclear deformation
a) MyoII-based contractility
b) Arp2/3-dependent actin network
c) Different requirements for nuclear deformation
d) Specific mechanisms for mDCs entry into the lymphatic vessels
I. Bone marrow-derived dendritic cells as a cell model
A. Culture protocol
B. Characteristics of bone marrow-derived dendritic cells
II. Microfabricated devices to study mDCs migration under different degrees of confinement. 
A. Manufacturing protocol
B. MDCs migration in microchannels of different dimensions
1. Devices for live-cell imaging
2. Microscopic set-up and image analysis
3. Device developed for immunofluorescence staining of cells
C. Mature DCs migration in microchannels of varying dimensions
1. Design of the microfabricated devices
2. Microscopic set-up and image analysis
D. Arrays of pillars combining cell constraints and branching
III. From microfabricated devices to ex-vivo tissue explants
A. Mature DC migration assay in mouse ear explant
B. A simple method to image tissue collagen structures without second harmonic generation
I. Myosin II activity is selectively needed for migration in highly confined microenvironments in mature dendritic cells
II. Confinement triggers ROCK-dependent actomyosin reorganization associated with mature dendritic cell speed in confined microchannels.
A. Characterization of actomyosin organization at different degrees of confinement
B. Dynamic cytoskeleton remodeling in microchannels of varying dimensions
C. ROCK activity controls actin remodeling during confined migration
III. Impact of confinement on the intracellular organization of mature dendritic cells.
A. Confined migration decreases actin fraction at the nucleus and triggers rupture of the perinuclear lamina
B. Confinement triggers actin accumulation around the lysosomes and Golgi apparatus clustered around the microtubule organizing center.
1. Actin and microtubule interplay during confined migration
2. Golgi apparatus and lysosomes form a compact structure clustered on the MTOC surrounded by cortical actin
3. Confinement-induced fast actin accumulation around the lysosomal cluster
4. Confinement-induced lysosomal actin network is dependent on formin activity.
5. Microtubule depolymerization affects the confinement-induced actin network at the center of the cell
6. Fragmentation of the Golgi apparatus does not affect confinement-induced actin remodeling
I. Summary of the results
II. Mechanisms of cell plasticity
A. Does dendritic cell intra-cellular reorganization favor cell migration in confined areas?
B. Cytoskeleton reorganization and plasticity of the mode of migration
C. Potential mechano-responses and confinement sensors in mature dendritic cells
III. Microtubules as an integrator of the physical and chemical properties of the microenvironment.
IV. Impact of confine migration on mDCs immune functions
A. Dense cortical actin and antigen transport.
B. Actin and microtubules interplay with antigen processing in lysosomes.
C. Confined migration impacts the nuclear shell.
I. Publications list
II. Synthèse en français
A. Préambule
B. Contexte
C. Méthodes
D. Résultats
1. Réorganisation du cytosquelette d’actine induit par le confinement des DCs
2. La structure d’actine à l’arrière de la cellule est dépendante de l’activité de la kinase ROCK et permet le maintien de la vitesse des DCs dans les espaces restrein
3. La dépolymérisation des microtubules affecte l’actine induite par le confinement au centre de la cellule
E. Conclusions et perspectives


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