Kevin G. Phillips

IL-23–Mediated Psoriasis-Like Epidermal Hyperplasia Is Dependent on IL-17A

IL-23 and Th17 cells producing IL-17A and IL-22 are found in excess in skin affected by psoriasis. Previous studies showed that IL-22, but not IL-17A, mediates psoriasis-like epidermal hyperplasia following recombinant murine (rm)IL-23 injections into skin. To further investigate the role of IL-17A, ears of mice were injected with rmIL-23. Investigators blinded to treatment conditions and mouse genotypes measured ear swelling, epidermal thickness, and cytokine expression. In wild-type (WT) mice, rmIL-23 induced ear swelling (p < 0.001, all p values versus saline), epidermal hyperplasia by histology (p < 0.001) and confocal microscopy (p < 0.004), and expression of both IL-17A and IL-22. As expected, rmIL-23 injections into IL-22−/− mice resulted in relatively little ear swelling (p < 0.09) and epidermal hyperplasia (p < 0.51 by histology and p < 0.75 by confocal microscopy). Notably, rmIL-23 injections into IL-17A−/− mice produced little ear swelling (p < 0.001, versus IL-23–injected WT mice) and epidermal hyperplasia (p < 0.001 by histology and p < 0.005 by confocal microscopy), even though IL-22 was readily induced in these mice. Furthermore, systemic delivery of blocking Abs directed against either IL-22 or IL-17A completely inhibited IL-23–induced epidermal hyperplasia in WT mice. These results demonstrate that IL-17A, like IL-22, is a downstream mediator for IL-23–induced changes in murine skin and that both of these Th17 cytokines are necessary to produce IL-23–mediated skin pathology. IL-17A may represent an attractive therapeutic target in individuals with psoriasis by blocking downstream effects of IL-23.

Optical Quantification of Cellular Mass, Volume, and Density of Circulating Tumor Cells Identified in an Ovarian Cancer Patient

Clinical studies have demonstrated that circulating tumor cells (CTCs) are present in the blood of cancer patients with known metastatic disease across the major types of epithelial malignancies. Recent studies have shown that the concentration of CTCs in the blood is prognostic of overall survival in breast, prostate, colorectal, and non-small cell lung cancer. This study characterizes CTCs identified using the high-definition (HD)-CTC assay in an ovarian cancer patient with stage IIIC disease. We characterized the physical properties of 31 HD-CTCs and 50 normal leukocytes from a single blood draw taken just prior to the initial debulking surgery. We utilized a non-interferometric quantitative phase microscopy technique using brightfield imagery to measure cellular dry mass. Next we used a quantitative differential interference contrast microscopy technique to measure cellular volume. These techniques were combined to determine cellular dry mass density. We found that HD-CTCs were more massive than leukocytes: 33.6 ± 3.2 pg (HD-CTC) compared to 18.7 ± 0.6 pg (leukocytes), p < 0.001; had greater volumes: 518.3 ± 24.5 fL (HD-CTC) compared to 230.9 ± 78.5 fL (leukocyte), p < 0.001; and possessed a decreased dry mass density with respect to leukocytes: 0.065 ± 0.006 pg/fL (HD-CTC) compared to 0.085 ± 0.004 pg/fL (leukocyte), p < 0.006. Quantification of HD-CTC dry mass content and volume provide key insights into the fluid dynamics of cancer, and may provide the rationale for strategies to isolate, monitor or target CTCs based on their physical properties. The parameters reported here can also be incorporated into blood cell flow models to better understand metastasis.

Activated factor XI inhibits chemotaxis of polymorphonuclear leukocytes

PMN leukocytes are the most abundant leukocytes in the circulation and play an important role in host defense. PMN leukocyte recruitment and inflammatory responses at sites of infection are critical components in innate immunity. Although inflammation and coagulation are known to have bidirectional relationships, little is known about the interaction between PMN leukocytes and coagulation factors. Coagulation FXI participates in the intrinsic coagulation pathway upon its activation, contributing to hemostasis and thrombosis. We have shown previously that FXI‐deficient mice have an increased survival and less leukocyte accumulation into the peritoneum in severe polymicrobial peritonitis. This result suggests a role for FXI in leukocyte trafficking and/or function. In this study, we characterized the functional consequences of FXIa binding to PMN leukocytes. FXIa reduced PMN leukocyte chemotaxis triggered by the chemokine, IL‐8, or the bacterial‐derived peptide, fMLP, perhaps as a result of the loss of directed migration. In summary, our data suggest that FXIa modulates the inflammatory response of PMN leukocytes by altering migration. These studies highlight the interplay between inflammation and coagulation and suggest that FXIa may play a role in innate immunity.

ASSESSMENT OF OPTICAL CLEARING AGENTS USING REFLECTANCE-MODE CONFOCAL SCANNING LASER MICROSCOPY

The mechanism of action of clearing agents to improve optical imaging of mouse skin during reflectance-mode confocal microscopy was tested. The dermal side of excised dorsal mouse skin was exposed for one hour to saline, glycerin, or 80% DMSO, then the clearing agent was removed and the dermis placed against a glass cover slip through which a confocal microscope measured reflectance at 488 nm wavelength. An untreated control was also measured. The axial attenuation of reflectance signal, R(zf) versus increasing depth of focus zf behaved as R = ρexp(-μzf2G), where ρ is tissue reflectivity and μ is attenuation [cm-1]. The factor 2G accounts for the in/out path of photons, and the numerical aperture of the lens. The ρ, μ data were mapped to values of scattering coefficient (μs [cm-1]) and anisotropy of scattering (g). Images showed that glycerin significantly increased the g of dermis from about 0.7 to about 0.99, with little change in the μs of dermis at about 300 cm-1. DMSO and saline had only slight and inconsistent effects on g and μs.

Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation

The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.

p21-Activated Kinase (PAK) Regulates Cytoskeletal Reorganization and Directional Migration in Human Neutrophils

Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

Quantification of cellular volume and sub-cellular density fluctuations: comparison of normal peripheral blood cells and circulating tumor cells identified in a breast cancer patient.

Cancer metastasis, the leading cause of cancer-related deaths, is facilitated in part by the hematogenous transport of circulating tumor cells (CTCs) through the vasculature. Clinical studies have demonstrated that CTCs circulate in the blood of patients with metastatic disease across the major types of carcinomas, and that the number of CTCs in peripheral blood is correlated with overall survival in metastatic breast, colorectal, and prostate cancer. While the potential to monitor metastasis through CTC enumeration exists, the basic physical features of CTCs remain ill defined and moreover, the corresponding clinical utility of these physical parameters is unknown. To elucidate the basic physical features of CTCs we present a label-free imaging technique utilizing differential interference contrast (DIC) microscopy to measure cell volume and to quantify sub-cellular mass-density variations as well as the size of subcellular constituents from mass-density spatial correlations. DIC measurements were carried out on CTCs identified in a breast cancer patient using the high-definition (HD) CTC detection assay. We compared the biophysical features of HD-CTC to normal blood cell subpopulations including leukocytes, platelets (PLT), and red blood cells (RBCs). HD-CTCs were found to possess larger volumes, decreased mass-density fluctuations, and shorter-range spatial density correlations in comparison to leukocytes. Our results suggest that HD-CTCs exhibit biophysical signatures that might be used to potentially aid in their detection and to monitor responses to treatment in a label-free fashion. The biophysical parameters reported here can be incorporated into computational models of CTC-vascular interactions and in vitro flow models to better understand metastasis.

The thrombotic potential of circulating tumor microemboli: computational modeling of circulating tumor cell-induced coagulation.

Thrombotic events can herald the diagnosis of cancer, preceding any cancer-related clinical symptoms. Patients with cancer are at a 4- to 7-fold increased risk of suffering from venous thromboembolism (VTE), with ∼7,000 patients with lung cancer presenting from VTEs. However, the physical biology underlying cancer-associated VTE remains poorly understood. Several lines of evidence suggest that the shedding of tissue factor (TF)-positive circulating tumor cells (CTCs) and microparticles from primary tumors may serve as a trigger for cancer-associated thrombosis. To investigate the potential direct and indirect roles of CTCs in VTE, we characterized thrombin generation by CTCs in an interactive numerical model coupling blood flow with advection-diffusion kinetics. Geometric measurements of CTCs isolated from the peripheral blood of a lung cancer patient prior to undergoing lobectomy formed the basis of the simulations. Singlet, doublet, and aggregate circulating tumor microemboli (CTM) were investigated in the model. Our numerical model demonstrated that CTM could potentiate occlusive events that drastically reduce blood flow and serve as a platform for the promotion of thrombin generation in flowing blood. These results provide a characterization of CTM dynamics in the vasculature and demonstrate an integrative framework combining clinical, biophysical, and mathematical approaches to enhance our understanding of CTCs and their potential direct and indirect roles in VTE formation.

A physical sciences network characterization of circulating tumor cell aggregate transport

Circulating tumor cells (CTC) have been implicated in the hematogenous spread of cancer. To investigate the fluid phase of cancer from a physical sciences perspective, the multi-institutional Physical Sciences-Oncology Center (PS-OC) Network performed multidisciplinary biophysical studies of single CTC and CTC aggregates from a patient with breast cancer. CTCs, ranging from single cells to aggregates comprised of 2-5 cells, were isolated using the high-definition CTC assay and biophysically profiled using quantitative phase microscopy. Single CTCs and aggregates were then modeled in an in vitro system comprised of multiple breast cancer cell lines and microfluidic devices used to model E-selectin mediated rolling in the vasculature. Using a numerical model coupling elastic collisions between red blood cells and CTCs, the dependence of CTC vascular margination on single CTCs and CTC aggregate morphology and stiffness was interrogated. These results provide a multifaceted characterization of single CTC and CTC aggregate dynamics in the vasculature and illustrate a framework to integrate clinical, biophysical, and mathematical approaches to enhance our understanding of the fluid phase of cancer.

Dermal reflectivity determined by optical coherence tomography is an indicator of epidermal hyperplasia and dermal edema within inflamed skin.

Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of inflammation in psoriasis remain unclear. We undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to noninvasively document cutaneous alterations in mouse skin treated topically with Imiquimod (IMQ), an established model of a psoriasis-like disease. Quantitative appraisal of dermal architectural changes was achieved through a two parameter fit of OCT axial scans in the dermis of the form A(x, y, z) = ρ(x, y)exp [-μ(x, y)z]. Ensemble averaging over 2000 axial scans per mouse in each treatment arm revealed no significant changes in the average dermal attenuation rate, , however the average local dermal reflectivity , decreased significantly following 1, 3, and 6 days of IMQ treatment (p < 0.001) in comparison to vehicle-treated control mice. In contrast, epidermal and dermal thickness changes were only significant when comparing controls and 6-day IMQ treated mice. This suggests that dermal alterations, attributed to collagen fiber bundle enlargement, occur prior to epidermal thickness changes due to hyperplasia and dermal thickness changes due to edema. Dermal reflectivity positively correlated with epidermal hyperplasia (r(epi)(2) = 0.78) and dermal edema (r(derm)(2) = 0.86). Our results suggest that dermal reflectivity as measured by OCT can be utilized to quantify a psoriasis-like disease in mice, and thus has the potential to aid in the quantitative assessment of psoriasis in humans.