Andrea Szilagyi

Murine hematopoietic stem cells and progenitors express adrenergic receptors

Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments monocytopoiesis and alters the beta-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of alpha1-, alpha2- and beta(2)-AR in enriched populations of ER-MP209(+) and ER-MP12(+) myeloid progenitors, CD117(+) and CD34(+) multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development.

Inhibition of multidrug-resistant Acinetobacter baumannii by nonviral expression of hCAP-18 in a bioengineered human skin tissue.

When skin is compromised, a cascade of signals initiates the rapid repair of the epidermis to prevent fluid loss and provide defense against invading microbes. During this response, keratinocytes produce host defense peptides (HDPs) that have antimicrobial activity against a diverse set of pathogens. Using nonviral vectors we have genetically modified the novel, nontumorigenic, pathogen-free human keratinocyte progenitor cell line (NIKS) to express the human cathelicidin HDP in a tissue-specific manner. NIKS skin tissue that expresses elevated levels of cathelicidin possesses key histological features of normal epidermis and displays enhanced antimicrobial activity against bacteria in vitro. Moreover, in an in vivo infected burn wound model, this tissue results in a two log reduction in a clinical isolate of multidrug-resistant Acinetobacter baumannii. Taken together, these results suggest that this genetically engineered human tissue could be applied to burns and ulcers to counteract bacterial contamination and prevent infection.

Perturbed bone marrow monocyte development following burn injury and sepsis promote hyporesponsive monocytes.

The mechanism of monocyte deactivation in critically injured burn patients remains unresolved. Two functionally distinct F4/80+Gr-1+ and F4/80+Gr-1− monocyte subsets have been characterized based on their homing to inflammatory or noninflammatory tissues, respectively. We hypothesized that the posttraumatic milieu in the bone marrow (BM) blunts the production of “inflammatory” monocytes. C57Blk/J male mice were divided into sham (S), burn (B), and burn sepsis (BS) groups. B and BS received a 15% dorsal scald burn and BS was inoculated with 15K colony forming units Pseudomonas aeruginosa at the burn site. Animals were killed and blood and femoral BM were collected 48, 72, and 96 hours after injury. ER-MP20+ monocyte progenitors were isolated from BM and differentiated into macrophage (MØ) or dendritic cells (DCs) and characterized by the cell surface expression of F4/80 and CD11c, respectively. In both cell types, TLR-4 agonist induced cytokine levels were determined. Results showed a 2-fold increase in the F4/80+Gr-1+ subset at 48 hours in BS that started to decline at 72 hours and remained low at 96 hours. ER-MP20+ progenitors isolated at 48 hours exhibited robust MØ differentiation potential but a significant decline in the percentage of the F4/80+Gr-1+ subset (P < .05 vs S) with a concomitant decrease in tumor necrosis factor α production. DC development from ER-MP20+ progenitors and LPS-stimulated tumor necrosis factor α production were impaired. Therefore, BM progenitor derived MØ will replace the transient hyper-responsive circulating monocytes later during the course of the septic insult. Hypo-reactivity of the developing monocytes and DC in the BM and their subsequent egress to the periphery provide a plausible explanation for the immunosuppression that ensues a critical burn injury and sepsis.

Propranolol restores the tumor necrosis factor-alpha response of circulating inflammatory monocytes and granulocytes after burn injury and sepsis.

Beta-adrenergic blockade ameliorates the hypermetabolism and catabolism in severe burn injury. Despite the salutary effects of beta-adrenergic blockade, the immunologic responses that accompany beta-blockade are not known. We have shown that burn sepsis is associated with increased sympathetic activation leading to altered monocytopoiesis and cytokine release in macrophages (MØ). Recent evidence suggests that murine MØ expressing F4/80+Gr1+ are the inflammatory phenotype. Here, we report that propranolol given after burn sepsis modulates the number and function of myeloid cells in circulation. B6D2F1 male mice were divided into sham (S), burn (B), and burn sepsis (BS) groups. Dorsal hair was shaved from S, B, and BS; B and BS received 15% scald burn; BS was inoculated with Pseudomonas Aeruginosa (PA 14, 4000–5000 colony-forming units) at the burn site. Mice from each group were then subjected to two different treatment regimens. One set received subcutaneous injections of propranolol (5 mg/kg body weight) at 24 and 48 hours after the injury while the control groups received saline. Blood was collected by cardiac puncture at 72 hours. The distribution of total F4/80+ monocyte population was determined by flow cytometry. Inflammatory monocyte subset was gated on Gr1+ expression in the F4/80+ fraction. Lipopolysaccharide-stimulated intracellular tumor necrosis factor (TNF)-&agr; (ic-TNF) was also measured as an indicator of inflammatory response. The total F4/80+ monocyte fraction was significantly increased in BS (45 ± 0.8%) vs S and B (10 ± 0.8%; 9.5 ± 0.6%). Propranolol treatment for 2 days reduced the number of circulating monocytes by 60% in BS. The mean fluorescent intensity (MFI) of ic-TNF produced per cell (F4/80+Gr1+ MØ) was significantly decreased in B and BS (S: 3043 ± 213, B: 1638 ± 343, BS: 1463 ± 67). Of importance, propranolol treatment partially restored the MFI of ic-TNF (2177 ± 114) and increased the percentage of inflammatory monocyte subset (F4/80+Gr1+) in BS by 70% compared with saline treatment. In contrast, beta-blockade after BS increased the percentage of granulocytes in circulation (28.4 ± 3.6% in BS propranolol vs 15.4 ± 0.3% in BS saline; P < .05) and augmented their TNF production (MFI = 903 ± 102 in BS propranolol vs 644 ± 5 in BS saline; P < .05). Propranolol reverses burn sepsis-induced monocytosis and simultaneously increases the number of granulocytes and enhances the inflammatory potential of the granulocytes and inflammatory monocyte subsets in circulation suggesting that monitoring MØ subsets and granulocytes in blood is a reliable biomarker to predict the efficacy of beta-blockade.

Nonviral human beta defensin-3 expression in a bioengineered human skin tissue: a therapeutic alternative for infected wounds.

The innate immune system differentially regulates the expression of host defense peptides to combat infection during wound healing. We enhanced the expression of a host defense peptide, human beta defensin‐3 (hBD‐3), in keratinocytes to generate a three‐dimensional biologic dressing to improve healing of infected wounds. The NIKS human keratinocyte cell line was stably transfected ex vivo with a construct containing an epidermis‐specific promoter driving hBD‐3 (NIKShBD‐3) using nonviral methods. Levels of hBD‐3 mRNA and protein in three‐dimensional skin tissue produced from NIKShBD‐3 were determined using quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Tissue architecture was characterized by hematoxylin and eosin staining and by indirect immunofluorescence using proliferation and keratinocyte differentiation markers. Antimicrobial activity was assessed using an in vitro bacterial growth assay and in vivo using a murine burn infection model. Three‐dimensional full thickness skin tissues containing epidermal NIKShBD‐3 or control NIKS possessed histologic features of interfollicular epidermis and exhibited normal tissue growth and differentiation. NIKShBD‐3 tissue contained approximately fivefold more hBD‐3 protein than tissue containing unmodified control NIKS. In vitro studies showed that NIKShBD‐3 tissue produced a significant reduction in the growth of Staphylococcus aureus multiple peptide resistance factor (mprF) compared with control tissue. In an in vivo infected murine burn model, NIKShBD‐3 tissue resulted in a 90% reduction in bacterial growth. These results demonstrate that sustained delivery of hBD‐3 by a bioengineered skin tissue results in a therapeutically relevant reduction in growth of a S. aureus strain in an animal model of infected third‐degree burn wounds.

High MafB expression following burn augments monocyte commitment and inhibits DC differentiation in hemopoietic progenitors

We have previously shown that perturbed bone marrow progenitor development promotes hyporesponsive monocytes following experimental burn sepsis. Clinical and experimental sepsis is associated with monocyte deactivation and depletion of mDCs. Decrease in circulating DCs is reported in burn patients who develop sepsis. In our 15% TBSA scald burn model, we demonstrate a significant reduction in the circulating MHC‐II+ population and mDCs (Gr1negCD11b+CD11c+) with a corresponding decrease in bone marrow MHC‐II+ cells and mDCs for up to 14 days following burn. We explored the underlying mechanism(s) that regulate bone marrow development of monocytes and DCs following burn injury. We found a robust bone marrow response with a significant increase in multipotential HSCs (LSK) and bipotential GMPs following burn injury. GMPs from burn mice exhibit a significant reduction in GATA‐1, which is essential for DC development, but express high levels of MafB and M‐CSFRs, both associated with monocyte production. GMPs obtained from burn mice differentiated 1.7 times more into Mφ and 1.6‐fold less into DCs compared with sham. Monocytes and DCs expressed 50% less MHC‐II in burn versus sham. Increased monocyte commitment in burn GMPs was a result of high MafB and M‐CSFR expressions. Transient silencing of MafB (siRNA) in GMP‐derived monocytes from burn mice partially restored DC differentiation deficits and increased GATA‐1 expression. We provide evidence that high MafB following burn plays an inhibitory role in monocyte‐derived DC differentiation by regulating M‐CSFR and GATA‐1 expressions.

A novel antibacterial gene transfer treatment for multidrug-resistant Acinetobacter baumannii-induced burn sepsis.

Sepsis caused by multidrug-resistant bacterial infections in critically injured patients has become a major clinical problem. Recently, Acinetobacter baumannii (AB) wound infections, especially in our critically injured soldiers fighting in Iraq and Afghanistan, is posing a major clinical problem and an economic burden. ConjuGon, Inc., has developed a novel antibacterial therapeutic technology using bacterial conjugation. The donor cells are attenuated Escherichia coli carrying a conjugative plasmid. The expression of bactericidal genes cloned on the plasmid is tightly repressed in the donor cells but becomes de-repressed once mobilized into a pathogen and disrupts protein synthesis. Here, we tested the efficacy of this novel conjugation technology to control and eradicate a drug-resistant clinical isolate of AB wound infection both in vitro and in a murine burn sepsis model. C57Blk/6J mice were divided into burn (B) and burn sepsis (BS) groups. All animals received a 12% TBSA dorsal scald full-thickness burn. The BS group was inoculated with multidrug-resistant AB (1 × 105 colony-forming units [CFU]) at the burn wound site. BS animals were either untreated or treated with increasing concentrations (103–1010 CFU) of attenuated donor E. coli encoding bactericidal proteins. The survival rate was monitored for 10 days. The ability of donor cells to significantly diminish AB levels in the burn wound 24 hours after injury was determined by quantitative cultures. Donor cells were highly effective in killing AB in vitro. In the burn sepsis model, 90% B group animals survived, and 40% to 50% BS animals survived with no treatment in 5 to 6 days. Treatment with donor cells at 1010 to 106 provided significant survival advantage (P < .05). Quantitative cultures of burn wounds revealed that AB numbers increased from 3 × 104 CFU to 7.8 ± 4.4 × 109 CFU in 24 hours in the untreated group. Single treatment with donor cells (1010 CFU) significantly reduced AB in the burn wound to less than the levels seeded into the wound (1.23 ± 0.5 × 104 CFU; P < .05). Taken together, these results indicate that this novel technology is an efficient method to control drug-resistant AB burn wound infections and prevent their systemic spread.

β-Adrenergic stimulation increases macrophage CD14 expression and E. coli phagocytosis through PKA signaling mechanisms

CD14 is a glycoprotein that binds bacterial LPS in MØ. It is an essential component of the phagocytic system and is increased in septic shock. Critical injury and sepsis result in elevated endogenous CA levels. CAs have a significant impact on MØ inflammatory functions. We tested the hypothesis that β‐adrenergic stimulation regulates CD14 expression and bacterial phagocytosis in BMØ. Murine BMØ stimulated with isoproterenol (>8 h) induced a dose‐dependent increase in cell surface CD14 expression. Specific PKA inhibitor (H‐89) and gene‐silencing (siRNA) studies demonstrated the role of cAMP‐dependent PKA in mediating this response. In addition, we observed a correlation between an isoproterenol‐mediated increase in CD14 expression and live Escherichia coli uptake in BMØ. Further, the essential role of CD14 in an isoproterenol‐mediated increase in E. coli uptake was highlighted from experiments using CD14−/− mice. Moreover, the dose response of isoproterenol stimulation to CD14 expression and E. coli phagocytosis overlapped with similar EC50. Additionally, isoproterenol‐mediated E. coli phagocytosis was prevented by H‐89, suggesting that β‐adrenergic stimulus in BMØ increases CD14 expression and live E. coli phagocytosis through a common signaling pathway. Our studies indicate the potential impact of β‐adrenergic agents on important innate immune functions.

Burn injury dampens erythroid cell production through reprioritizing bone marrow hematopoietic response.

BACKGROUND Anemia in burn patients is due to surgical blood loss and anemia of critical illness. Because the commitment paradigm of common bone marrow progenitors dictates the production of erythroid, myeloid, and lymphoid cells, we hypothesized that skewed bone marrow lineage commitment decreases red cell production and causes anemia after a burn injury. METHODS After anesthesia, B(6)D(2)F(1) mice received a 15% total body surface area dorsal scald burn. The sham group did not receive scald burn. Femoral bone marrow was harvested on 2, 5, 7, 14, and 21 postburn days (PBD). Total bone marrow cells were labeled with specific antibodies to erythroid (CD71/Ter119), myeloid (CD11b), and lymphoid (CD19) lineages and analyzed by flow cytometry. To test whether erythropoietin (EPO) could increase red blood cell production, EPO was administered to sham and burn animals and their reticulocyte response was measured on PBD 2 and PBD 7. RESULTS Burn injury reduced the erythroid cells of the bone marrow from 35% in sham to 17% by PBD 5 and remained at similar level until PBD 21. Myeloid cells, however, increased from 42% in sham to 60% on PBD 5 and 77% on PBD 21. Burn injury reduced reticulocyte counts on PBD 2 and PBD 7 indicating that the erythroid compartment is severely depleted. This depleted compartment, however, responded to EPO but was not sufficient to change red cell production. CONCLUSION Burn injury skews the bone marrow hematopoietic commitment away from erythroid and toward myeloid cells. Shrinkage of the erythroid compartment contributes to resistance to EPO and the anemia of critical illness.

Dendritic cell depletion in burn patients is regulated by MafB expression.

Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23–68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.