Nanorobotic investigation identifies novel visual, structural and functional correlates of autoimmune pathology in a blistering skin disease model

Copyright © 2014 Seiffert-Sinha et al. There remain major gaps in our knowledge regarding the detailed mechanisms by which autoantibodies mediate damage at the tissue level. We have undertaken novel strategies at the interface of engineering and clinical medicine to integrate nanoscale visual and structural data using nanorobotic atomic force microscopy with cell functional analyses to reveal previously unattainable details of autoimmune processes in real-time. Pemphigus vulgaris is a life-threatening autoimmune blistering skin condition in which there is disruption of desmosomal cell-cell adhesion structures that are associated with the presence of antibodies directed against specific epithelial proteins including Desmoglein (Dsg) 3. We demonstrate that pathogenic (blister-forming) anti-Dsg3 antibodies, distinct from non-pathogenic (non-blister forming) anti-Dsg3 antibodies, alter the structural and functional properties of keratinocytes in two sequential steps - an initial loss of cell adhesion and a later induction of apoptosis-related signaling pathways, but not full apoptotic cell death. We propose a ''2-Hit'' model for autoimmune disruption associated with skin-specific pathogenic autoantibodies. These data provide unprecedented details of autoimmune processes at the tissue level and offer a novel conceptual framework for understanding the action of selfreactive antibodies.published_or_final_versio

Anti-Thyroid Peroxidase Reactivity Is Heightened in Pemphigus Vulgaris and Is Driven by Human Leukocyte Antigen Status and the Absence of Desmoglein Reactivity

Pemphigus vulgaris (PV) belongs to an autoimmune disease cluster that includes autoimmune thyroid disease (AITD), suggesting common mechanisms driving autoimmune susceptibility. Our group has shown that PV patients exhibit significant reactivity to AITD-related anti-thyroid peroxidase (anti-TPO), and anti-TPO antibodies affect signaling pathways in keratinocytes similar to anti-desmoglein (Dsg) 3 antibodies. To further assess the relevance of anti-TPO reactivity in PV, we analyzed anti-TPO levels in 280 PV and 167 healthy control serum samples across a comprehensive set of variable and static parameters of disease activity and etiopathogenesis. PV patients have significantly higher activity rates (A.R.s) for anti-TPO than healthy controls, but levels do not differ between phases of clinical activity and remission. Patients that carry both the PV-associated human leukocyte antigen (HLA) alleles DRB1*0402 and DQB1*0503, or DQB1*0503 alone show a low prevalence of anti-TPO (A.R. 9.5 and 4.8%, respectively), while patients that lack expression of these alleles or carry DRB1*0402 alone have a much higher prevalence of anti-TPO (A.R. 23.1 and 15.8%, respectively), suggesting that the absence of DQB1*0503 may predispose patients to the development of anti-TPO antibodies. Similarly, anti-Dsg1−/3− patients have a higher anti-TPO A.R. (26.9%) than anti-Dsg1−/3+ (18.8%), anti-Dsg1+/3− (14.3%), and anti-Dsg1+/3+ (3.9%) patients. Our data suggest that anti-TPO reactivity in PV is driven by genetic markers that may be in linkage disequilibrium with the established PV-susceptibility alleles and that this association drives the selection of a combination of anti-Dsg and anti-TPO antibodies, with anti-TPO filling the gap in active patients that do not carry the established PV-associated autoantibodies and/or are lacking the established PV-HLA-susceptibility alleles

Modulation of Mechanical Stress Mitigates Anti-Dsg3 Antibody-Induced Dissociation of Cell–Cell Adhesion

It is becoming increasingly clear that mechanical stress in adhesive junctions plays a significant role in dictating the fate of cell–cell attachment under physiological conditions. Targeted disruption of cell–cell junctions leads to multiple pathological conditions, among them the life-threatening autoimmune blistering disease pemphigus vulgaris (PV). The dissociation of cell–cell junctions by autoantibodies is the hallmark of PV, however, the detailed mechanisms that result in tissue destruction remain unclear. Thus far, research and therapy in PV have focused primarily on immune mechanisms upstream of autoantibody binding, while the biophysical aspects of the cell– cell dissociation process leading to acantholysis are less well studied. In work aimed at illuminating the cellular consequences of autoantibody attachment, it is reported that externally applied mechanical stress mitigates antibody-induced monolayer fragmentation and inhibits p38 MAPK phosphorylation activated by anti-Dsg3 antibody. Further, it is demonstrated that mechanical stress applied externally to cell monolayers enhances cell contractility via RhoA activation and promotes the strengthening of cortical actin, which ultimately mitigates antibody-induced cell–cell dissociation. The study elevates understanding of the mechanism of acantholysis in PV and shifts the paradigm of PV disease development from a focus solely on immune pathways to highlight the key role of physical transformations at the target cell

Inheritance-Specific Dysregulation of Th1- and Th17-Associated Cytokines in Alopecia Areata

Autoimmune diseases tend to cluster in families, suggesting genetic predisposition to autoimmunity associated with familial background. We have previously reported similarities in gene expression patterns and PTPN22 polymorphisms between alopecia areata (AA) patients and their healthy relatives, but not unrelated healthy controls. However, the spectrum of disease promoting (or preventing) pathways that may be activated in blood relatives of AA patients remains to be defined. Here, we investigated the extent to which cytokines associated with the Th1 and Th17 pathway are differentially expressed in the blood of patients with AA and its clinical subtypes in comparison to both healthy relatives as well as unrelated healthy controls. A comprehensive set of Th1- and Th17-related cytokines were evaluated by ELISA. We found a significant elevation of the Th17 inducer IL-23, the Th17 product IL-17A, the Th1 hallmark cytokine IFNγ, and TNFα, a Th1 cytokine with relevance to the Th17 pathway in AA patients, regardless of disease subtype, compared to healthy individuals. On further examination, we found that healthy family members grouped together with patients in terms of elevated Th1- and Th17-pathway cytokines in an inheritance-specific manner, distinct from unrelated controls. The elevation of Th17-associated cytokines in healthy controls related to AA patients indicates that Th1 and Th17 dysregulation in AA may be genetically based. Of note, one unrelated control displayed elevated levels of IL-17A and IL-23 similar to those detected in patients. One year after initial blood draw, areas of beard hair loss consistent with the diagnosis of AA were reported by this individual, indicating that the elevation in Th17-related cytokines may have predictive value

Bionanomanipulation using atomic force microscopy

In recent years, atomic force microscopy (AFM) has become an increasingly versatile technique for biomedical applications [1] [3]. AFM offers several advantages for biomedical investigation. It requires little sample preparation; thereby, native biomolecules can be directly studied; it provides threedimensional (3-D) images of surface topography and quantitative measures of biological specimens in a physiologically stable environment; and it can be performed on viable samples. © 2010 IEEE.Link_to_subscribed_fulltex

Development of cell fixture for in-situ imaging and manipulation of membrane protein structure

The development of atomic force microscopy (AFM) opens an entirely new way to investigate the dynamic changes of cellular structures, such as cell junctions and protein molecules. Since these structures are in nanometer scale, it cannot be observed and studied by conventional optical microscopy. Besides, AFM can also be used for precise manipulation of these nano-sized molecules such that the interaction and structure-function can be studied at the single molecular level. Because of cell surface inhomogeneities and movements in living conditions, it presents a major challenge to perform the in-situ imaging and manipulation of these nanoscale biostructures. Here, we present the design and development of a biocompatible porous polymer micro-mesh for immobilizing live cells and protein molecules. Based on the novel AFM nanomanipulation and imaging system with the use of the cell fixture, the cell junctions and individual protein molecules in physiological conditions with unparalleled resolution can be easily observed and studied. © 2009 IEEE NANO Organizers.Link_to_subscribed_fulltex

Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy (AFM)

We present the use of atomic force microscopy (AFM) to visualize and quantify the dynamics of epithelial cell junction interactions under physiological and pathophysiological conditions at the nanoscale. Desmosomal junctions are critical cellular adhesion components within epithelial tissues and blistering skin diseases such as Pemphigus are the result in the disruption of these components. However, these structures are complex and mechanically inhomogeneous, making them difficult to study. The mechanisms of autoantibody mediated keratinocyte disassembly remain largely unknown. Here, we have used AFM technology to image and measure the mechanical properties of living skin epithelial cells in culture. We demonstrate that force measurement data can distinguish cells cultured with and without autoantibody treatment. Our demonstration of the use of AFM for in situ imaging and elasticity measurements at the local, or tissue level opens potential new avenues for the investigation of disease mechanisms and monitoring of therapeutic strategies in blistering skin diseases. © 2011 IEEE.Link_to_subscribed_fulltex

Micro fixture enabled in-situ imaging and manipulation of cell membrane protein

This paper presents a new and reliable approach for in-situ imaging and manipulation of membrane protein molecules. The major problematic issue in medicine is the lack of fundamental understanding of the molecular mechanisms that cause major life-threatening and incurable diseases. The development of atomic force microscopy (AFM) opens an entirely new way to address these medical questions. A biocompatible porous polymer micro-mesh is designed for immobilizing living cells and protein molecules during AFM scanning. We demonstrate its usefulness by imaging cancerous B-cells as well as cell-junction molecules associated with living epithelial cells at high resolution. This technology can not only be used for imaging, but also for nanomanipulation. Current results suggest that this AFM nanomanipulation system and the novel cell fixture apparatus can be used to study the dynamic changes and function of cellular structures with unparalleled resolution both under physiological and pathophysiological conditions. ©2010 IEEE.Link_to_subscribed_fulltex

Analysis of keratinocytes stiffness after desmosome disruption using Atomic Force Microscopy based nanomanipulation

Desmosomes are cell-cell junctions that provide mechanical strength for cells and maintain integrity of tissues in animal skins and hearts. Disruption of the desmosome like in some autoimmune diseases will therefore change the mechanical property of the anchoring cells and causing blistering. In the paper, we used Atomic Force Microscopy (AFM) to visualize the keratinocytes intercellular structure. The AFM based nanomanipulation system is employed to perform the nanoindentation experiment for measuring keratinocytes nanomechanical property under both physiological and pathophysiological conditions. Our demonstration of the use of AFM for in situ imaging and nanomanipulation for quantifying stiffness in nanoscale can facilitate the investigation of diseases related to blistering and development of therapeutic strategies accordingly. © 2009 IEEE NANO Organizers.Link_to_subscribed_fulltex