Systemic remodeling of the redox regulatory network due to RNAi perturbations of glutaredoxin 1, thioredoxin 1, and glucose-6-phosphate dehydrogenase

<p>Abstract</p> <p>Background</p> <p>Cellular clearance of reactive oxygen species is dependent on a network of tightly coupled redox enzymes; this network rapidly adapts to oxidative conditions such as aging, viral entry, or inflammation. Current widespread use of shRNA as a means to perturb specific redox couples may be misinterpreted if the targeted effects are not monitored in the context of potential global remodeling of the redox enzyme network.</p> <p>Results</p> <p>Stable cell lines containing shRNA targets for glutaredoxin 1, thioredoxin 1, or glucose-6-phosphate dehydrogenase were generated in order to examine the changes in expression associated with altering cytosolic redox couples. A qRT PCR array revealed systemic off-target effects of altered antioxidant capacity and reactive oxygen species formation. Empty lentiviral particles generated numerous enzyme expression changes in comparison to uninfected cells, indicating an alteration in antioxidant capacity irrespective of a shRNA target. Of the three redox couples perturbed, glutaredoxin 1, attenuation produced the most numerous off-target effects with 10/28 genes assayed showing statistically significant changes. A multivariate analysis extracted strong co-variance between glutaredoxin 1 and peroxiredoxin 2 which was subsequently experimentally verified. Computational modeling of the peroxide clearance dynamics associated with the remodeling of the redox network indicated that the compromised antioxidant capacity compared across the knockdown cell lines was unequally affected by the changes in expression of off-target proteins.</p> <p>Conclusions</p> <p>Our results suggest that targeted reduction of redox enzyme expression leads to widespread changes in off-target protein expression, changes that are well-insulated between sub-cellular compartments, but compensatory in both the production of and protection against intracellular reactive oxygen species. Our observations suggest that the use of lentivirus can in itself have off-target effects on dynamic responses to oxidative stress due to the changes in species concentrations.</p

Investigating the functional consequences of heterogeneity within immune cell populations

A well-functioning immune system is the result of the combined efforts of a multitude of different cell types, performing separate but coordinated tasks. Diversity within these historically categorized “cell types” has functional consequences, and deeper characterization of cell heterogeneity within immune cell populations is crucial for a better understanding of immune functionality. The main objectives of this research were to investigate heterogeneity in immune cell populations by 1) leveraging emerging microfluidics techniques for transcriptomic and phenotypic analysis, 2) developing systems-level computational models, and 3) interrogating cells using dynamic immunological stimuli. Through parallelized microfluidics-based gene expression assays on primary neutrophils and T cells, we collected information on a wide range of gene targets, including not traditionally assayed surface markers, key cytokine receptors, intracellular signaling components and metabolic enzymes. Through the comparison of data pre-processing and analysis methods, we identified novel subgroups within the immune cell populations, as defined by single cell gene expression. We observed that the prevalence of these subgroups differs between individual donors. Noting the wide range of expression values associated with the IL-2 receptor, we subsequently investigated the functional consequences of diversity in cytokine receptors on an individual cell basis. We coupled time-lapse fluorescent microscopy and tightly controlled delivery of fluctuating extracellular IL-2 cytokine concentrations in a microfluidic cell trap developed for the capture and immobilization of non-adherent cells. A computational model of IL-2 signaling established experimental timescales of interest for delivery of oscillatory stimulus. The combined computational modeling and microfluidics technologies allowed us to investigate features of T cell responses to IL-2 under physiologically relevant conditions. The computational model established the importance of specific receptor subunit expression on the dynamic responses to extracellular IL-2 ligand. The question of how to correctly process and analyze single cell data is as of yet unanswered, despite the fact that such analysis is crucial to correct interpretation of the biological information that such data can reveal. In this work, detailed single cell analyses at the transcriptomic and phenotypic levels are described for enhancing our understanding of population behaviors.Ph.D

Oscillatory IL-2 stimulus reveals pertinent signaling timescales of T cell responsiveness.

Cell response to extracellular ligand is affected not only by ligand availability, but also by pre-existing cell-to-cell variability that enables a range of responses within a cell population. We developed a computational model that incorporates cell heterogeneity in order to investigate Jurkat T cell response to time dependent extracellular IL-2 stimulation. Our model predicted preferred timing of IL-2 oscillatory input for maximizing downstream intracellular STAT5 nuclear translocation. The modeled cytokine exposure was replicated experimentally through the use of a microfluidic platform that enabled the parallelized capture of dynamic single cell response to precisely delivered pulses of IL-2 stimulus. The in vitro results demonstrate that single cell response profiles vary with pulsatile IL-2 input at pre-equilibrium levels. These observations confirmed our model predictions that Jurkat cells have a preferred range of extracellular IL-2 fluctuations, in which downstream response is rapidly initiated. Further investigation into this filtering behavior could increase our understanding of how pre-existing cellular states within immune cell populations enable a systems response within a preferred range of ligand fluctuations, and whether the observed cytokine range corresponds to in vivo conditions

Increased level of soluble HLA class I antigens in systemic lupus erythematosus: correlation with anti-DNA antibodies and leukopenia

The concentration of soluble HLA class I (sHLA-I) was measured by ELISA in serum samples from 30 well-characterised SLE patients at high and low disease activity states and from 100 healthy controls. HLA-A allotypes in the patients were analysed by a PCR-based typing technique. A higher level of sHLA-I was found in SLE patient sera both at high and low disease activity than in controls (P< 0.001). The sHLA-I level was further increased during active disease (P< 0.01). Concentrations of sHLA-I correlated with anti-dsDNA antibodies at high disease activity, but not with disease activity as analysed by a modified SLEDAI. Numbers of leukocytes and lymphocytes, as well as levels of C1q and C3 correlated inversely with sHLA-I concentration. In five serial samples from ten patients the sHLA-I level co-varied with disease activity. Presence of HLA allotype A9 was associated with higher sHLA-I levels in both patients (P< 0.001) and controls (P< 0.001). We conclude that the increased sHLA-I concentration in SLE patients was related to several laboratory parameters reflecting disease activity suggesting that sHLA-I molecules are connected with the disease process. Increased sHLA-I level due to HLA-A allotype was not a disease susceptibility factor for SLE

Mesenchymal stromal cells for bone trauma, defects, and disease: Considerations for manufacturing, clinical translation, and effective treatments

Bone is a complex tissue capable of natural repair to injury, however, the healing process is often impaired by the untoward effects of trauma, defects, and disease. Thus, therapeutic modalities, including the use of cells involved in the body's natural healing processes, are investigated to promote or complement natural bone repair. Herein, several modalities and innovative approaches for using mesenchymal stromal cells (MSCs) to treat bone trauma, defects, and diseases are discussed. Given the evidence that supports the promising potential of MSCs, we highlight important considerations for advancing the clinical use of MSCs including the standardization of procedures from the harvest to delivery to patients and realized solutions to manufacturing. A better understanding of the current approaches implemented to address the challenges of using therapeutic MSCs will help improve study designs and, ultimately, achieve effective outcomes for restoring bone health

Cell-based versus corticosteroid injections for knee pain in osteoarthritis: a randomized phase 3 trial.

Various types of cellular injection have become a popular and costly treatment option for patients with knee osteoarthritis despite a paucity of literature establishing relative efficacy to each other or corticosteroid injections. Here we aimed to identify the safety and efficacy of cell injections from autologous bone marrow aspirate concentrate, autologous adipose stromal vascular fraction and allogeneic human umbilical cord tissue-derived mesenchymal stromal cells, in comparison to corticosteroid injection (CSI). The study was a phase 2/3, four-arm parallel, multicenter, single-blind, randomized, controlled clinical trial with 480 patients with a diagnosis of knee osteoarthritis (Kellgren-Lawrence II-IV). Participants were randomized to the three different arms with a 3:1 distribution. Arm 1: autologous bone marrow aspirate concentrate (n = 120), CSI (n = 40); arm 2: umbilical cord tissue-derived mesenchymal stromal cells (n = 120), CSI (n = 40); arm 3: stromal vascular fraction (n = 120), CSI (n = 40). The co-primary endpoints were the visual analog scale pain score and Knee injury and Osteoarthritis Outcome Score pain score at 12 months versus baseline. Analyses of our primary endpoints, with 440 patients, revealed that at 1 year post injection, none of the three orthobiologic injections was superior to another, or to the CSI control. In addition, none of the four groups showed a significant change in magnetic resonance imaging osteoarthritis score compared to baseline. No procedure-related serious adverse events were reported during the study period. In summary, this study shows that at 1 year post injection, there was no superior orthobiologic as compared to CSI for knee osteoarthritis. ClinicalTrials.gov Identifier: NCT03818737