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Open Access Publications from the University of California

Scholarly Works (29 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2015)

Atherosclerosis is an underlying cause of two of the leading causes of death in the world, heart attacks and strokes. It is a chronic inflammatory disease characterized by plaque build-up in large and medium arteries. During disease progression, monocytes in the blood invade the arterial wall, where they can differentiate into macrophages and dendritic cells. Together, these cells phagocytose lipids, clear apoptotic cell debris, present antigen to T cells, and produce pro- and anti-inflammatory cytokines. Both monocyte recruitment to the plaque and macrophage function within the plaque require cell motion. The purpose of this work is to study monocyte and macrophage movement in the context of atherosclerosis.

First, the mechanism of how the atypical chemokine receptor Duffy Antigen / Receptor for Chemokines (DARC) influences the progression of atherosclerosis is investigated. DARC alters the localization of chemokines and it was thought that through this it could influence cell recruitment to the arteria wall. It was confirmed that the absence of DARC does promote plaque formation, but a molecular mechanism of action was not discovered.

Next, to study myeloid cell motion more directly, I developed a system for imaging fluorescent leukocytes in atherosclerotic arteries in vivo. The major challenge in intravital imaging of large arteries is the motion artifacts due to the expansion of the arterial wall with the heartbeat. This new system utilizes cardiac triggering and novel image post-processing algorithms to remove these motion artifacts and allow for cell motion quantification. Apoe-/- Cx3cr1GFP/+ Cd11cYFP mice were generated to visualize labeled monocytes and macrophages. Monocytes were observed patrolling the endothelium of atherosclerotic arteries and the motion characteristics were quantified. Macrophages were seen actively probing their local environment as well as migrating through the plaque. Finally, these macrophages were extensively phenotyped by cell surface markers to link observed motion to known subsets and function.

Supplemental videos include a demonstration of the improvement in movie quality with cardiac triggering and image post-processing, examples movies of monocytes patrolling atherosclerotic arteries and macrophages migrating within the plaque and an example 3D reconstruction of atherosclerotic plaque acquired in vivo. Supplemental SNP analysis of the mice used in this work is also provided.

  • 10 supplemental videos
  • 1 supplemental file
Cover page: Intravital Live cell Triggered Imaging System Reveals Monocyte Patrolling and Macrophage Migration in Atherosclerosis
  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2016)

This project focuses on the proof-of-concept development of a separation construct to isolate antigen-specific CD4 T cells using a buoyant lipid microparticle (microbubble) reagent attaching major histocompatibility complex class II (MHCII) molecules. These cells are implicated in the progression of cardiovascular disease. The project relied critically on preexisting reagents produced by collaborators: microbubbles and biotinylated recombinant MHCII monomers.

Current cell separation methods rely primarily on either magnetic separation or flow-cytometry based sorting. The method presented here provides the basis for a new avenue of investigation using a principally different separation scheme based on buoyancy. It is intended to be extensible to the separation of antigen-specific T cells in other contexts and was originally intended to be a removable separation reagent (unsuccessful). The ultimate goal is the development of a separation system which may be used to further clinical work or research relying on cell isolation.

Here, the buoyancy-based method is first used to perform bulk B and CD4 T cell separations. These are common lab procedures for which many commercial kits are available, which made them an ideal testbed for initial study. Flow cytometric analysis and confocal microscopy were performed to confirm successful cell enrichment.

The separation system was then further developed to separate antigen-specific CD4 T cells reactive to the endogenous murine peptide P6 (TGAYSNASSTESASY), relevant in the study of atherosclerosis. Flow cytometric and microscopic confirmation of successful separation was performed in this section as well.

Several devices for centrifugation were designed and constructed in the course of this project. These attempted to create a pendant droplet between the buoyant and non-buoyant fractions in a single column of liquid formed by centrifugation, allowing for these layers to be completely sequestered from one another. Though these devices were unsuccessful, they merit further study. Another device, a centrifuge adapter, was created as an all-purpose experimental aid to hold and centrifuge all combinations of reagent tubes used in the cell separation protocol. Computer graphics animations and diagrams of various facets of the project were also made, including molecular-scale models of separation constructs derived from Protein Data Bank (PDB) structural data.

    Cover page: Buoyancy-based separation of antigen-specific T cells
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2007)

    Background

    We present a multilevel, agent based, in silico model that represents the dynamics of rolling, activation, and adhesion of individual leukocytes in vitro. Object-oriented software components were designed, verified, plugged together, and then operated in ways that represent the molecular and cellular mechanisms believed responsible for leukocyte rolling and adhesion. The result is an in silico analogue of an experimental in vitro system. The experimentally measured, phenotypic attributes of the analogue were compared and contrasted to those of leukocytes in vitro from three different experimental conditions.

    Results

    The individual in silico dynamics of "rolling" on simulated P-selectin, and separately on simulated VCAM-1, were an acceptable match to individual in vitro distance-time and velocity-time measurements. The analogues are also able to represent the transition from rolling to adhesion on P-selectin and VCAM-1 in the presence of GRO-alpha chemokine. The individual in silico and in vitro behavioral similarities translated successfully to population level measures. These behavioral similarities were enabled in part by subdividing the functionality of the analogue's surface into 600 independent, "cell"-controlled, equally capable modules of comparable functionality.

    Conclusion

    The overlap in phenotypic attributes of our analogue with those of leukocytes in vitro confirm the considerable potential of our model for studying the key events that determine the behavioral outcome of individual leukocytes during rolling, activation, and adhesion. Our results provide an important foundation and framework for future in silico research into plausible causal links between well-documented, subcellular molecular level events and the variety of systemic phenotypic attributes that distinguish normal leukocyte adhesion from abnormal disease-associated adhesion.
      Cover page: Dynamics of in silico leukocyte rolling, activation, and adhesion
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2023)
      Abstract: Neutrophils are the most abundant leukocytes in human blood. Neutrophil malfunction may cause various immunological diseases. Beta2 integrins are critical for neutrophil recruitment. Kindlin-3 and talin-1 are important regulators for beta2 integrin activation. We have confirmed this using conformation-specific antibodies KIM127 and mAb24 that recognize activation epitopes in beta2 integrin. Besides activation, clustering is also critical for beta2 integrin avidity and adhesion of neutrophils. Whether kindlin-3 and talin-1 are involved in beta2 integrin clustering remains unclear. Here we used super-resolution stochastic optical reconstruction microscopy (STORM) to study their roles in beta2 integrin clustering on human neutrophil-like HL60 cells. We find that lymphocyte function-associated antigen 1 (LFA-1) and macrophage-1 antigen (Mac-1) are significantly clustered in wild-type cells after IL-8 or fMLP stimulation. Using CRISPR-Cas9-mediated kindlin-3 and talin-1 knockout HL60 cells, we found that kindlin-3 but not talin-1 are responsible for beta2 integrin clustering. Our study provides new insights into the regulation of beta2 integrin clustering and leukocyte recruitment. Supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454), and a startup fund from UConn Health.
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2015)
        Neutrophil recruitment in most tissues is limited to postcapillary venules, where E- and P-selectins are inducibly expressed by venular endothelial cells. These molecules support neutrophil rolling via binding of PSGL-1 and other ligands on neutrophils. Selectins extend ≤ 38 nm above the endothelial plasma membrane, and PSGL-1 extends to 50 nm above the neutrophil plasma membrane. However, endothelial cells are covered with an ESL composed of glycosaminoglycans that is ≥ 500 nm thick and has measurable resistance against compression. The neutrophil surface is also covered with a surface layer. These surface layers would be expected to completely shield adhesion molecules; thus, neutrophils should not be able to roll and adhere. However, in the cremaster muscle and in many other models investigated using intravital microscopy, neutrophils clearly roll, and their rolling is easily and quickly induced. This conundrum was thought to be resolved by the observation that the induction of selectins is accompanied by ESL shedding; however, ESL shedding only partially reduces the ESL thickness (to 200 nm) and thus is insufficient to expose adhesion molecules. In addition to its antiadhesive functions, the ESL also presents neutrophil arrest-inducing chemokines. ESL heparan sulfate can also bind L-selectin expressed by the neutrophils, which contributes to rolling and arrest. We conclude that ESL has both proadhesive and antiadhesive functions. However, most previous studies considered either only the proadhesive or only the antiadhesive effects of the ESL. An integrated model for the role of the ESL in neutrophil rolling, arrest, and transmigration is needed.
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2018)
          Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development.
            Cover page: Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development
            • Article
            • Peer Reviewed
            UC San Diego Previously Published Works (2023)
            Direct contact between cells expressing either ephrin ligands or Eph receptor tyrosine kinase produces diverse developmental responses. Transmembrane ephrinB ligands play active roles in transducing bi-directional signals downstream of EphB/ephrinB interaction. However, it has not been well understood how ephrinB relays transcellular signals to neighboring cells and what intracellular effectors are involved. Here, we report that kindlin2 can mediate bi-directional ephrinB signaling through binding to a highly conserved NIYY motif in the ephrinB2 cytoplasmic tail. We show this interaction is important for EphB/ephrinB-mediated integrin activation in mammalian cells and for blood vessel morphogenesis during zebrafish development. A mixed two-cell population study revealed that kindlin2 (in ephrinB2-expressing cells) modulates transcellular EphB4 activation by promoting ephrinB2 clustering. This mechanism is also operative for EphB2/ephrinB1, suggesting that kindlin2-mediated regulation is conserved for EphB/ephrinB signaling pathways. Together, these findings show that kindlin2 enables EphB4/ephrinB2 bi-directional signal transmission.
              Cover page: Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2020)
              β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E+ H+ ) β2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.
                Cover page: Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2016)
                Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E(+)) and acquire a high-affinity conformation with an 'open' headpiece (H(+)). The canonical switchblade model of integrin activation proposes that the E(+) conformation precedes H(+), and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E(-)H(+) conformation. E(-)H(+) β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation.
                  Cover page: Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2018)
                  Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure.
                    Cover page: Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages
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