Interendothelial Slits Research Articles

Biomechanics of phagocytosis of red blood cells by macrophages in the human spleen

The clearance of senescent and altered red blood cells (RBCs) in the red pulp of the human spleen involves sequential processes of prefiltration, filtration, and postfiltration. While prior work has elucidated the mechanisms underlying the first two processes, biomechanical processes driving the postfiltration phagocytosis of RBCs retained at interendothelial slits (IES) are still poorly understood. We present here a unique computational model of macrophages to study the role of cell biomechanics in modulating the kinetics of phagocytosis of aged and diseased RBCs retained in the spleen. After validating the macrophage model using in vitro phagocytosis experiments, we employ it to probe the mechanisms underlying the kinetics of phagocytosis of mechanically altered RBCs, such as heated RBCs and abnormal RBCs in hereditary spherocytosis (HS) and sickle cell disease (SCD). Our simulations show pronounced deformation of the flexible and healthy RBCs in contrast to minimal shape changes in altered RBCs. Simulations also show that less deformable RBCs are engulfed faster and at lower adhesive strength than flexible RBCs, consistent with our experimental measurements. This efficient sensing and engulfment by macrophages of stiff RBCs retained at IES are expected to temper splenic congestion, a common pathogenic process in malaria, HS, and SCD. Altogether, our combined computational and in vitro experimental studies suggest that mechanical alterations of retained RBCs may suffice to enhance their phagocytosis, thereby adapting the kinetics of their elimination to the kinetics of their mechanical retention, an equilibrium essential for adequately cleaning the splenic filter to preserve its function.

Red blood cell passage through deformable interendothelial slits in the spleen: Insights into splenic filtration and hemodynamics

The spleen constantly clears altered red blood cells (RBCs) from the circulation, tuning the balance between RBC formation (erythropoiesis) and removal. The retention and elimination of RBCs occur predominantly in the open circulation of the spleen, where RBCs must cross submicron-wide inter-endothelial slits (IES). Several experimental and computational studies have illustrated the role of IES in filtrating the biomechanically and morphologically altered RBCs based on a rigid wall assumption. However, these studies also reported that when the size of IES is close to the lower end of clinically observed sizes (less than 0.5 μm), an unphysiologically large pressure difference across the IES is required to drive the passage of normal RBCs, sparking debates on the feasibility of the rigid wall assumption. In this work, We propose two deformable IES models, namely the passive model and the active model, aiming to explore the impact of the deformability of IES on the filtration function of the spleen. In the passive model, we implement the worm-like string model to depict the IES’s deformation as it interacts with blood plasma and allows RBC to traverse. In contrast, the active model involved regulating the IES deformation based on the local pressure surrounding the slit. To demonstrate the validity of the deformable model, we simulate the filtration of RBCs with varied size and stiffness by IES under three scenarios: (1) a single RBC traversing a single slit; (2) a suspension of RBCs traversing an array of slits, mimicking in vitro spleen-on-a-chip experiments; (3) RBC suspension passing through the 3D spleen filtration unit known as’the splenon’. Our simulation results of RBC passing through a single slit show that the deformable IES model offers more accurate predictions of the critical cell surface area to volume ratio that dictate the removal of aged RBCs from circulation compared to prior rigid-wall models. Our biophysical models of the spleen-on-a-chip indicate a hierarchy of filtration function stringency: rigid model > passive model > active model, providing a possible explanation of the filtration function of IES. We also illustrate that the biophysical model of ‘the splenon’ enables us to replicate the ex vivo experiments involving spleen filtration of malaria-infected RBCs. Taken together, our simulation findings indicate that the deformable IES model could serve as a mesoscopic representation of spleen filtration function closer to physiological reality, addressing questions beyond the scope of current experimental and computational models and enhancing our understanding of the fundamental flow dynamics and mechanical clearance processes within in the human spleen.

A combined computational and experimental investigation of the filtration function of splenic macrophages in sickle cell disease.

Being the largest lymphatic organ in the body, the spleen also constantly controls the quality of red blood cells (RBCs) in circulation through its two major filtration components, namely interendothelial slits (IES) and red pulp macrophages. In contrast to the extensive studies in understanding the filtration function of IES, fewer works investigate how the splenic macrophages retain the aged and diseased RBCs, i.e., RBCs in sickle cell disease (SCD). Herein, we perform a computational study informed by companion experiments to quantify the dynamics of RBCs captured and retained by the macrophages. We first calibrate the parameters in the computational model based on microfluidic experimental measurements for sickle RBCs under normoxia and hypoxia, as those parameters are not available in the literature. Next, we quantify the impact of key factors expected to dictate the RBC retention by the macrophages in the spleen, namely, blood flow conditions, RBC aggregation, hematocrit, RBC morphology, and oxygen levels. Our simulation results show that hypoxic conditions could enhance the adhesion between the sickle RBCs and macrophages. This, in turn, increases the retention of RBCs by as much as four-fold, which could be a possible cause of RBC congestion in the spleen of patients with SCD. Our study on the impact of RBC aggregation illustrates a 'clustering effect', where multiple RBCs in one aggregate can make contact and adhere to the macrophages, leading to a higher retention rate than that resulting from RBC-macrophage pair interactions. Our simulations of sickle RBCs flowing past macrophages for a range of blood flow velocities indicate that the increased blood velocity could quickly attenuate the function of the red pulp macrophages on detaining aged or diseased RBCs, thereby providing a possible rationale for the slow blood flow in the open circulation of the spleen. Furthermore, we quantify the impact of RBC morphology on their tendency to be retained by the macrophages. We find that the sickle and granular-shaped RBCs are more likely to be filtered by macrophages in the spleen. This finding is consistent with the observation of low percentages of these two forms of sickle RBCs in the blood smear of SCD patients. Taken together, our experimental and simulation results aid in our quantitative understanding of the function of splenic macrophages in retaining the diseased RBCs and provide an opportunity to combine such knowledge with the current knowledge of the interaction between IES and traversing RBCs to apprehend the complete filtration function of the spleen in SCD.

Mechanisms of Red Blood Cell Splenic Filtration Revealed By Multiscale Modeling and in-Vitro Microfluidic Experiments

Interendothelial slits in the spleen fulfill the major physiological function of continuously filtering red blood cells (RBCs) from the bloodstream to remove abnormal and aged cells. To date, the process of passage of 8 µm RBCs through 0.3-µm wide slits remains enigmatic. Should the slits increase their caliber during RBC passage as sometimes proposed in the literature? Here, we elucidated the mechanisms that govern the passage dynamics or retention of RBCs in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with slits of defined physiological dimensions, including submicron width. We observed that healthy RBCs pass through 0.28-µm wide rigid slits at body temperature. To achieve this tour de force, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, they must be able to locally unfold their spectrin cytoskeleton inside the slits. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through slits scales with in-slit pressure drop and slit width to the -1 and -3 power, respectively. This transit dynamics is similar to that of a Newtonian fluid in a 2D Poiseuille flow, thus showing that it is controlled by the RBC cytoplasmic viscosity. Altogether, our results clearly show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in-vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Transit Time Theory for a Droplet Passing through a Slit in Pressure-Driven Low Reynolds Number Flows.

Soft objects squeezing through small apertures are crucial for many in vivo and in vitro processes. Red blood cell transit time through splenic inter-endothelial slits (IESs) plays a crucial role in blood filtration and disease progression, while droplet velocity through constrictions in microfluidic devices is important for effective manipulation and separation processes. As these transit phenomena are not well understood, we sought to establish analytical and numerical solutions of viscous droplet transit through a rectangular slit. This study extends from our former theory of a circular pore because a rectangular slit is more realistic in many physiological and engineering applications. Here, we derived the ordinary differential equations (ODEs) of a droplet passing through a slit by combining planar Poiseuille flow, the Young-Laplace equations, and modifying them to consider the lubrication layer between the droplet and the slit wall. Compared to the pore case, we used the Roscoe solution instead of the Sampson one to account for the flow entering and exiting a rectangular slit. When the surface tension and lubrication layer were negligible, we derived the closed-form solutions of transit time. When the surface tension and lubrication layer were finite, the ODEs were solved numerically to study the impact of various parameters on the transit time. With our solutions, we identified the impact of prescribed pressure drop, slit dimensions, and droplet parameters such as surface tension, viscosity, and volume on transit time. In addition, we also considered the effect of pressure drop and surface tension near critical values. For this study, critical surface tension for a given pressure drop describes the threshold droplet surface tension that prevents transit, and critical pressure for a given surface tension describes the threshold pressure drop that prevents transit. Our solutions demonstrate that there is a linear relationship between pressure and the reciprocal of the transit time (referred to as inverse transit time), as well as a linear relationship between viscosity and transit time. Additionally, when the droplet size increases with respect to the slit dimensions, there is a corresponding increase in transit time. Most notably, we emphasize the initial antagonistic effect of surface tension which resists droplet passage but at the same time decreases the lubrication layer, thus facilitating passage. Our results provide quantitative calculations for understanding cells passing through slit-like constrictions and designing droplet microfluidic experiments.

Physical mechanisms of red blood cell splenic filtration.

The splenic interendothelial slits fulfill the essential function of continuously filtering red blood cells (RBCs) from the bloodstream to eliminate abnormal and aged cells. To date, the process by which 8 [Formula: see text]m RBCs pass through 0.3 [Formula: see text]m-wide slits remains enigmatic. Does the slit caliber increase during RBC passage as sometimes suggested? Here, we elucidated the mechanisms that govern the RBC retention or passage dynamics in slits by combining multiscale modeling, live imaging, and microfluidic experiments on an original device with submicron-wide physiologically calibrated slits. We observed that healthy RBCs pass through 0.28 [Formula: see text]m-wide rigid slits at 37 °C. To achieve this feat, they must meet two requirements. Geometrically, their surface area-to-volume ratio must be compatible with a shape in two tether-connected equal spheres. Mechanically, the cells with a low surface area-to-volume ratio (28% of RBCs in a 0.4 [Formula: see text]m-wide slit) must locally unfold their spectrin cytoskeleton inside the slit. In contrast, activation of the mechanosensitive PIEZO1 channel is not required. The RBC transit time through the slits follows a [Formula: see text]1 and [Formula: see text]3 power law with in-slit pressure drop and slip width, respectively. This law is similar to that of a Newtonian fluid in a two-dimensional Poiseuille flow, showing that the dynamics of RBCs is controlled by their cytoplasmic viscosity. Altogether, our results show that filtration through submicron-wide slits is possible without further slit opening. Furthermore, our approach addresses the critical need for in vitro evaluation of splenic clearance of diseased or engineered RBCs for transfusion and drug delivery.

Computational investigation of flow dynamics and mechanical retention of age-associated red blood cells in the spleen

The shape and deformability of red blood cells (RBCs) change with age, increasing their likelihood of becoming trapped in the splenic interendothelial slits (IESs) and being removed from the circulation. Existing evidence suggests that cell size and viscoelasticity vary with age, which impedes understanding of the mechanical retention of aged RBCs in the spleen. We present a computational study of the flow dynamics, shape changes, and mechanical retention of age-associated RBCs in the splenic IES. The results show that increasing RBC membrane viscosity slows the flow process early in aging, while increased sphericity due to cell size reduction eventually leads to RBC retention.

Microfluidic study of retention and elimination of abnormal red blood cells by human spleen with implications for sickle cell disease

The spleen clears altered red blood cells (RBCs) from circulation, contributing to the balance between RBC formation (erythropoiesis) and removal. The splenic RBC retention and elimination occur predominantly in open circulation where RBCs flow through macrophages and inter-endothelial slits (IESs). The mechanisms underlying and interconnecting these processes significantly impact clinical outcomes. In sickle cell disease (SCD), blockage of intrasplenic sickled RBCs is observed in infants splenectomized due to acute splenic sequestration crisis (ASSC). This life-threatening RBC pooling and organ swelling event is plausibly triggered or enhanced by intra-tissular hypoxia. We present an oxygen-mediated spleen-on-a-chip platform for in vitro investigations of the homeostatic balance in the spleen. To demonstrate and validate the benefits of this general microfluidic platform, we focus on SCD and study the effects of hypoxia on splenic RBC retention and elimination. We observe that RBC retention by IESs and RBC-macrophage adhesion are faster in blood samples from SCD patients than those from healthy subjects. This difference is markedly exacerbated under hypoxia. Moreover, the sickled RBCs under hypoxia show distinctly different phagocytosis processes from those non-sickled RBCs under hypoxia or normoxia. We find that reoxygenation significantly alleviates RBC retention at IESs, and leads to rapid unsickling and fragmentation of the ingested sickled RBCs inside macrophages. These results provide unique mechanistic insights into how the spleen maintains its homeostatic balance between splenic RBC retention and elimination, and shed light on how disruptions in this balance could lead to anemia, splenomegaly, and ASSC in SCD and possible clinical manifestations in other hematologic diseases.

Cyclic Mechanical Stresses Alter Erythrocyte Membrane Composition and Microstructure and Trigger Macrophage Phagocytosis.

Red blood cells (RBCs) are cleared from the circulation when they become damaged or display aging signals targeted by macrophages. This process occurs mainly in the spleen, where blood flows through submicrometric constrictions called inter‐endothelial slits (IES), subjecting RBCs to large‐amplitude deformations. In this work, RBCs are circulated through microfluidic devices containing microchannels that replicate the IES. The cyclic mechanical stresses experienced by the cells affect their biophysical properties and molecular composition, accelerating cell aging. Specifically, RBCs quickly transition to a more spherical, less deformable phenotype that hinders microchannel passage, causing hemolysis. This transition is associated with the release of membrane vesicles, which self‐extinguishes as the spacing between membrane‐cytoskeleton linkers becomes tighter. Proteomics analysis of the mechanically aged RBCs reveals significant losses of essential proteins involved in antioxidant protection, gas transport, and cell metabolism. Finally, it is shown that these changes make mechanically aged RBCs more susceptible to macrophage phagocytosis. These results provide a comprehensive model explaining how physical stress induces RBC clearance in the spleen. The data also suggest new biomarkers of early "hemodamage" and inflammation preceding hemolysis in RBCs subjected to mechanical stress.

Pitting of malaria parasites in microfluidic devices mimicking spleen interendothelial slits

The spleen is a hematopoietic organ that participates in cellular and humoral immunity. It also serves as a quality control mechanism for removing senescent and/or poorly deformable red blood cells (RBCs) from circulation. Pitting is a specialized process by which the spleen extracts particles, including malaria parasites, from within circulating RBCs during their passage through the interendothelial slits (IES) in the splenic cords. To study this physiological function in vitro, we have developed two microfluidic devices modeling the IES, according to the hypothesis that at a certain range of mechanical stress on the RBC, regulated through both slit size and blood flow, would force it undergo the pitting process without affecting the cell integrity. To prove its functionality in replicating pitting of malaria parasites, we have performed a characterization of P. falciparum-infected RBCs (P.f.-RBCs) after their passage through the devices, determining hemolysis and the proportion of once-infected RBCs (O-iRBCs), defined by the presence of a parasite antigen and absence of DAPI staining of parasite DNA using a flow cytometry-based approach. The passage of P.f.-RBCs through the devices at the physiological flow rate did not affect cell integrity and resulted in an increase of the frequency of O-iRBCs. Both microfluidic device models were capable to replicate the pitting of P.f.-RBCs ex vivo by means of mechanical constraints without cellular involvement, shedding new insights on the role of the spleen in the pathophysiology of malaria.

How the spleen reshapes and retains young and old red blood cells: A computational investigation.

The spleen, the largest secondary lymphoid organ in humans, not only fulfils a broad range of immune functions, but also plays an important role in red blood cell’s (RBC) life cycle. Although much progress has been made to elucidate the critical biological processes involved in the maturation of young RBCs (reticulocytes) as well as removal of senescent RBCs in the spleen, the underlying mechanisms driving these processes are still obscure. Herein, we perform a computational study to simulate the passage of RBCs through interendothelial slits (IES) in the spleen at different stages of their lifespan and investigate the role of the spleen in facilitating the maturation of reticulocytes and in clearing the senescent RBCs. Our simulations reveal that at the beginning of the RBC life cycle, intracellular non-deformable particles in reticulocytes can be biomechanically expelled from the cell upon passage through IES, an insightful explanation of why this peculiar “pitting” process is spleen-specific. Our results also show that immature RBCs shed surface area by releasing vesicles after crossing IES and progressively acquire the biconcave shape of mature RBCs. These findings likely explain why RBCs from splenectomized patients are significantly larger than those from nonsplenectomized subjects. Finally, we show that at the end of their life span, senescent RBCs are not only retained by IES due to reduced deformability but also become susceptible to mechanical lysis under shear stress. This finding supports the recent hypothesis that transformation into a hemolyzed ghost is a prerequisite for phagocytosis of senescent RBCs. Altogether, our computational investigation illustrates critical biological processes in the spleen that cannot be observed in vivo or in vitro and offer insights into the role of the spleen in the RBC physiology.

Erythrocyte flow through the interendothelial slits of the splenic venous sinus.

The flow patterns of red blood cells through the spleen are intimately linked to clearance of senescent RBCs, with clearance principally occurring within the open flow through the red pulp and slits of the venous sinus system that exists in humans, rats, and dogs. Passage through interendothelial slits (IESs) of the sinus has been shown by MacDonald et al. (Microvasc Res 33:118-134, 1987) to be mediated by the caliber, i.e., slit opening width, of these slits. IES caliber within a given slit of a given sinus section has been shown to operate in an asynchronous manner. Here, we describe a model and simulation results that demonstrate how the supporting forces exerted on the sinus by the reticular meshwork of the red pulp, combined with asymmetrical contractility of stress fibers within the endothelial cells comprising the sinus, describe this vital and intriguing behavior. These results shed light on the function of the sinus slits in species such as humans, rats, and dogs that possess sinusoidal sinuses. Instead of assuming a passive mechanical filtering mechanism of the IESs, our proposed model provides a mechanically consistent explanation for the dynamically modulated IES opening/filtering mechanism observed in vivo. The overall perspective provided is also consistent with the view that IES passage serves as a self-protective mechanism in RBC vesiculation and inclusion removal.

Quantitative prediction of flow dynamics and mechanical retention of surface-altered red blood cells through a splenic slit

Normal red blood cells (RBCs) have remarkable properties of deformability, which enable them to squeeze through tiny splenic inter-endothelial slits (IESs) without any damage. Decreased surface-area-to-volume (SA/V) ratio through the loss of membrane surface is a key determinant of splenic entrapment of surface-altered RBCs due to cell aging or disease. Here, we investigate the flow dynamics and mechanical retention of the surface-altered RBCs with different extents of surface area loss, using a multiscale RBC (MS-RBC) model implemented in dissipative particle dynamics (DPD). We show that the DPD-based MS-RBC simulations can accurately reproduce the ex vivo experimentally measured rate of RBC mechanical retention when we take into account the distribution of RBC surface area (i.e., the size difference within the RBC population). We also examine the cumulative effect of the cell surface area loss on the traversal dynamics of the surface-altered RBCs, where we found that the final values of cell surface area (or the SA/V ratio) play a key role in determining the RBC traversal dynamics, regardless of the loss pathway of cell surface area. Taken together, these simulation results have implications for understanding the sensitivity of the splenic IESs to retain and clear the surface-altered RBCs with increased surface area loss, providing an insight into the fundamental flow dynamics and mechanical clearance of the surface-altered RBCs by the human spleen.

Reticulocyte and red blood cell deformation triggers specific phosphorylation events.

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC's response to mechanical stimuli and for the study of reticulocyte maturation.

Response modes of erythrocytes in high-frequency oscillatory shear flows

Due to its capability of duplicating the deformation scenario of erythrocytes (red blood cells), in in vivo time scales, passing through interendothelial slits in the spleen, the understanding of the dynamic response of erythrocytes in oscillatory shear flows is of critical importance to the development of an effective in vitro methodology to study the mechanics, metabolism, and aging procedure in vivo [R. Asaro et al., “Erythrocyte aging, protection via vesiculation: An analysis methodology via oscillatory flow,” Front. Physiol. 9, 1607 (2018)]. Accordingly, we conducted a systematic computational investigation of the dynamics of erythrocytes in high-frequency oscillatory shear flows by using a fluid-cell interaction model based on the Stokes-flow framework and a multiscale structural depiction of the cell. Within the range of parameters we consider, we identify five different response modes (wheeling, tilted wheeling, tank treading mode 1, tank treading mode 2, and irregular). The occurrence and stability of these response modes depend on the frequency of the flow, the peak capillary number, the viscosity ratio, the initial orientation of the cell, and the stress-free state of the protein skeleton. Through long-term simulations [O(102) periods], mode switching events have been discovered, during which the cell transfers from one mode to another, often via an intermediate transient mode. The deformation of the skeleton and the contact stress between the skeleton and the lipid bilayer are computed since these are of direct importance to describing vital cell phenomena such as vesiculation by which the cell protects itself from premature elimination.

Boundary integral simulations of a red blood cell squeezing through a submicron slit under prescribed inlet and outlet pressures

We developed a boundary integral formulation to simulate a red blood cell (RBC) squeezing through a submicron slit under prescribed inlet and outlet pressures. The main application of this computational study is to investigate splenic filtrations of RBCs and the corresponding in vitro mimicking microfluidic devices, during which RBCs regularly pass through inter-endothelial slits with a width less than 1.0 µm. The diseased and old RBCs are damaged or destroyed in this mechanical filtration process. We first derived the boundary integral equations of a RBC immersed in a confined domain with prescribed inlet and outlet pressures. We applied a unified self-adaptive quadrature to accurately evaluate singular and nearly singular integrals, which are especially important in this fluid-structure interaction problem with strong lubrication. A multiscale model is applied to calculate forces from the RBC membrane, and it is coupled to boundary integral equations to simulate the fluid-structure interaction. After multi-step verifications and validations against analytical and experimental results, we systematically investigated the effects of pressure drop, volume-to-surface-area ratio, internal viscosity, and membrane stiffness on RBC deformation and internal stress. We found that spectrins of RBCs could be stretched by more than 2.5 times under high hydrodynamic pressure and that the bilayer tension could be more than 500 pN/μm, which might be large enough to open mechanosensitive channels but too small to rupture the bilayer. On the other hand, we found that the bilayer-cytoskeletal dissociation stress is too low to induce bilayer vesiculation.

Splenic Mechanisms of Thrombocytopenia

The spleen is the largest secondary lymphoid organ in the body and contains up to 25 percent of the body's lymphocyte populations. It is not only responsible for initiating immune responses against a multitude of infectious antigens within its white pulp, it also has the exquisite ability to filter the blood and remove, for example, senescent erythrocytes and platelets. This natural process is carried out within the red pulp of the spleen which is composed monocyte-rich connective tissue cords of Billroth intertwined with sinus cavities lined by parallel-oriented endothelial cells that have interendothelial slits which allow for the mechanical sorting of "old" cells. This occurs because of the inability of the senescent cells to properly migrate through the endothelial fenestrae into the venous circulation allowing them to be identified by cells of the reticuloendothelial system (RES) and quickly destroyed by phagocytosis. This process also allows for the efficient recycling of iron from destroyed erythrocyte hemoglobin molecules. There are a wide variety of clinical conditions that can significantly alter the ability of the RES to destroy blood cells including hereditary blood cell defects, inflammation, cancer and abnormal immune responses. This lecture will focus on the central role that the spleen plays in not only generating immune responses against platelets but also in primarily causing the destruction of both senescent and antibody-opsonized platelets leading to thrombocytopenia. It will discuss the soluble and cellular mechanisms of splenic sequestration, destruction and the ability of the spleen to modulate anti-platelet immunity. Mechanisms involving complement activation, Fc Receptor-mediated phagocytosis, antibody dependent cellular cytotoxicity and platelet self-destruction will be addressed. It will compare the spleen's platelet destructive capabilities with other organs, particularly the liver and will detail how immune responses generated in the white pulp can modulate platelet destructive processes in the red pulp. Disclosures Semple: Amgen: Consultancy, Honoraria, Speakers Bureau; Rigel: Consultancy, Honoraria; UCB: Consultancy, Honoraria.

Multiscale Modeling of Red Blood Cells Squeezing through Submicron Slits

A multiscale model is applied to study the dynamics of healthy red blood cells (RBCs), RBCs in hereditary spherocytosis, and sickle cell disease squeezing through submicron slits. This study is motivated by the mechanical filtration of RBCs by inter-endothelial slits in the spleen. First, the model is validated by comparing the simulation results with experiments. Secondly, the deformation of the cytoskeleton in healthy RBCs is investigated. Thirdly, the mechanisms of damage in hereditary spherocytosis are investigated. Finally, the effects of cytoplasm and membrane viscosities, especially in sickle cell disease, are examined. The simulations results provided guidance for future experiments to explore the dynamics of RBCs under extreme deformation.

A Microengineered Matrix to Decouple the Biophysical and Biochemical Mechanisms of Blood Cell Interactions with Thrombi and Vascular Wall Matrices

Biochemical aspects of cellular process are well characterized, but more recently, it has been shown that cells dynamically sense and respond to biophysical cues such as substrate stiffness and geometrical constraints; physical cues even direct cell differentiation and stem cell lineage (Discher et al, Science, 2005). In hematology, we know that platelets are shear activated and attenuate force based on substrate stiffness, and that endothelial cells align with flow and are activated by shear stress. Blood cells pass through, and interact with, biological matrices such as fibrin clots and the vascular wall, but the physical and biochemical aspects of these interactions are indistinguishable from one another in vivo. As such, there is a gap in knowledge as to how blood cells respond to matrices as they transit through them.To decouple the physical and biochemical interactions of blood cells and biological matrices, we sought to recreate the physical geometry of a fibrin network in a controlled, non-biological, in vitro microfluidic system. To this end, we designed a two-part microfluidic device comprised of an array of micron sized pillars (~1 µm diameter, 3 µm height, and 2 µm gap between pillars) overlaid with a microfluidic channel (Fig 1). The dimensions of the pillars are on the order of the diameter of fibrin fibers and the mesh size of a fibrin gel (Okada et al, J. Biol. Chem, 1985), while channels of various dimensions can be bonded over the pillar array to represent various biological scenarios. Standard microfluidic processes cannot produce pillars with the feature sizes reported herein, so electron beam lithography was used to create the mold from which the elastomeric pillars are made.The biophysical interaction of platelets flowing through fibrin mesh (absent biological factors) was recreated by a pillar array oriented perpendicular to the direction of flow in a 6 µm tall channel. When washed platelets are perfused through the system, they adhere to the pillars, aggregate, and form an occlusive mass that extends to the edges of the array (Fig 2A). Platelet adhesion initiates exclusively at the pillars and aggregation propagates to the extents of the channel area perpendicular to flow, resulting in channel occlusion and flow cessation. These findings show that in the absence of platelet agonists and biological ligands, platelets are activated by the shear environment afforded by the presence of fibrin fibers. Thus, in addition to the biochemical players in clot formation, the geometry of the fibrin mesh plays a role in platelet adhesion, and clot propagation. As expected, passive adsorption of fibrinogen and collagen to the pillar surfaces enhances platelet aggregation, as evidenced by a decrease in time to channel occlusion from 10 min to 2 min and 6 min, respectively. With thus we see the synergistic effect of biophysical and biochemical factors in clot propagation. This novel microfluidic system both separates biophysical and biochemical aspects of clot formation and allows researchers to specify the precise location and extent of clot formation in vitro.Platelets are not the only blood cells to interact with and react to physical barriers. Red blood cell (RBC) deformation has been historically studied in single cell assays, SEM studies of fixed clots, and more recently after RBCs have passed through a filtration system comprised of either beads or long slits (Deplaine et al, Blood, 2010); however, real time visualization of RBC deformation in geometries representative of biological matrices has remained elusive. The deformation (and possible fragmentation) that RBCs undergo when passing through the physical challenges of a fibrin matrix or the interendothelial slits of the spleen can be visualized in our system: an array of pillars overlaid by a 3 µm channel. Our findings visually suggest that red blood cells are able to deform through the matrix with little effect on their membranes, and that exposure to high shear gradients alone does not cause cell fragmentation (Figure 2B). The ready deformation and transit of healthy RBCs in our system confirms recent computational studies of RBC filtration by the spleen (Pivkin et al, PNAS, 2016). Further studies will give insight into the deformation and transit of sickled cells and malaria infected RBCs in physical matrices. Overall, our microfluidic studies give novel insight into the biophysical aspect of blood cell interactions with biological matrices. [Display omitted] [Display omitted] DisclosuresLam:Sanguina, LLC: Equity Ownership.

Modeling Transmigration of Malaria Infected Red Blood Cells through Inter-Endothelial Slits in Human Spleens using Dissipative Particle Dynamics

We simulate the transmigration of malaria-infected red blood cells (RBCs) through the inter-endothelial slits in the human spleen by using Dissipative Particle Dynamics based two-component RBC model. We modeled the spectrin-actin network and the lipid bilayer separately and considered the real number of the structural proteins in the model. The mechanical properties of the bilayer-cytoskeletal interactions, such as stiffness and friction, are calibrated by comparing with membrane fluctuations and tank-treading experiments. First, we further validated our numerical model by comparing the predicted retention rates of healthy and pathological cells in an ‘artificial spleen’ consisting of micro beads with the experimental measurements. To explore the possibility of the bilayer-cytoskeletal detachment during this transmigration process, which is strongly related to RBC aging and hereditary spherocytosis, we predicted the maximum interaction force between the spectrin-actin cytoskeleton and the lipid bilayer and compared the value with the previously predicted bilayer-cytoskeletal bond strength. Furthermore, we systematically studied the effects of cell rigidity, cell shape and inter-endothelial slit dimensions on the critical pressure gradient for RBCs to pass through the spleen. We found that the cell shape plays a much more important role than the cell rigidity in the transmigration process, which may guide the future experiments and the drug design for eradicating malaria and treating anemia such as hereditary spherocytosis.