Differential regulation of innate immune cytokine production through pharmacological activation of Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) in burn patient immune cells and monocytes

Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0–48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells

Whole Blood, Fixed Ratio, or Goal-Directed Blood Component Therapy for the Initial Resuscitation of Severely Hemorrhaging Trauma Patients: A Narrative Review

This narrative review explores the pathophysiology, geographic variation, and historical developments underlying the selection of fixed ratio versus whole blood resuscitation for hemorrhaging trauma patients. We also detail a physiologically driven and goal-directed alternative to fixed ratio and whole blood, whereby viscoelastic testing guides the administration of blood components and factor concentrates to the severely bleeding trauma patient. The major studies of each resuscitation method are highlighted, and upcoming comparative trials are detailed

Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease

Video analysis of cRBC sickling.

<p>(<b>A</b>) Video at 300X magnification of sodium metabisulfite (MBS)-induced sickling of cRBCs from four separate mock (Control), and two IHK-β-globin, or four IHK-β^T87Q-globin nucleofections. Sickling was analyzed at 256 frame intervals with a cell scoring positive for a rapid morphologic change from the previous frame; and each cell treated as a separate event for a total of 568 cells in the control (black dotted line), 310 cells in the IHK-β-globin treated trials (light grey dashed line), and 609 cells in the IHK-β^T87Q-globin treated trials (dark grey solid line). Data shown as mean percent sickled, * <i>p</i> < 0.01, Gehan-Breslow-Wilcoxon test. (<b>B</b>) Representative frames from one IHK-β^T87Q-globin treated field and one control field at 4, 8, and 12 min following MBS induction. (<b>C</b>) Spiculated cRBC examples from MBS-induced controls.</p

Imaging cytometry of cRBC sickling.

<p>(<b>A</b>) Images from ImageStream of an MBS-induced SS control trial ordered by sickle score and corresponding to histogram data. (<b>B</b>) Histogram and gating of an β^S/β^S CD34⁺ (SS) control trial with and without MBS induction and a β^wt/β^wt CD34⁺ (AA) with and without MBS induction. Gates were set for ^~ 10% spillover in AA trials. (<b>C</b>) Percent of mature cRBCs scoring positive for sickled shape distortion from β^S/β^S cRBCs from seven mock nucleofected trials (SS Control), six IHK-β^wt-globin, six IHK-β^T87Q-globin, and four β^wt/β^wt mock nucleofected groups (AA control). Data shown as mean ± SD, * <i>p</i> < 0.05, students t test. AA controls were statistically significant from all SS trials, <i>p</i> < 0.05, students t test.</p

Imaging cytometry scoring of sickling.

<p>(<b>A</b>) Examples of cells selected by hand as examples of ‘sickle’ or ‘non-sickle’ for training sets. (<b>B</b>) Object masks (as defined by IDEAS software) and representations of perimeter and object axis metrics used by the software to quantify cell morphology. (<b>C</b>) Examples of single cells from an β^S/β^S trial with and without induced sickling and used for statistical comparison (<b>D</b>) The 10 highest Fisher’s discriminant (FD) of means values as calculated by IDEAS for software-defined measures. Bold indicates measure used in combined analysis. Data shown from separate MBS-induced SS controls and a matched uninduced SS control.</p

Erythroid culture and differentiation.

<p>(<b>A</b>) Fields from an IHK-β^T87Q-globin treated β^S/β^S CD34+ differentiation at day 0 (post-nucleofection), day 8, day 12 (on feeder layer), day 17 (on feeder layer), and day 21 (washed and replated without a feeder layer prior to analysis). All images were captured at 300X magnification, scale bar = 20 μm. (<b>B</b>) Flow cytometry of the same trial as panel A at day 21 using antibodies specific for CD235a, CD45, CD34, and DNA. Both panels depict the same singlet-gated population. (<b>C</b>) Approximately 3x10⁷ non-adherent cRBCs at day 21 suspended in PBS at an approximate hematocrit of 0.1% (left) and as a pellet (right). (<b>D</b>) pKT2/meIF-SB100X//CAGGS-dsRed nucleofected cells at day 4 in brightfield (left panel), and dsRed epifluorescence (right panel). Images at 300X magnification, scale bars = 20 μm. (<b>E</b>) Cell yields relative to non-nucleofected cells from day 0 to day 4 and from day 4 to day 21. * indicates <i>p</i> < 0.05 day 0 to day 4, students t-test; ‡ indicates <i>p</i> < 0.05 day 4 to day 21, students t-test, error bars shown as mean ± SD.</p