John G. Noel

Effect of Glutamine on Phagocytosis and Bacterial Killing by Normal and Pediatric Burn Patient Neutrophils

Glutamine is essential for the function of lymphocytes and macrophages, where it serves, among other things, as a source of energy. Little information is available concerning the fuel that polymorphonuclear cells use for their metabolic and bactericidal functions. It was the purpose of this study to determine whether glutamine would enhance the in vitro bactericidal function of normal neutrophils and whether the amino acid would restore the observed impaired function in burn patients to or above the normal level. Twelve burn patients with total body surface area burns ranging from 32% to 87% were studied. At various postburn times, neutrophils were isolated and their ability to kill Staphylococcus aureus in the presence and absence of glutamine was determined and compared with that in normal subjects. Glutamine enhanced the bactericidal function of normal neutrophils. In every patient, at all but two postburn times, glutamine caused an improvement in the observed abnormal neutrophil bactericidal function and often restored it to or slightly above the normal level. Glutamine had no effect on the expression of C3b receptors (CR1 or CD35) or on phagocytosis by the cells. This study confirms the beneficial effects of glutamine in at least one arm of the immune system and adds evidence for the possible advantage of including this amino acid in the diets of burn and other trauma patients.

γδ T cells mitigate the organ injury and mortality of sepsis

Sepsis is a difficult condition to treat and is associated with a high mortality rate. Sepsis is known to cause a marked depletion of lymphocytes, although the function of different lymphocyte subsets in the response to sepsis is unclear. γδ T cells are found largely in epithelial‐rich tissues, and previous studies of γδ T cells in models of sepsis have yielded divergent results. In the present study, we examined the function of γδ T cells during sepsis in mice using cecal ligation and puncture (CLP). Mice deficient in γδ T cells had decreased survival times and increased tissue damage after CLP compared with wild‐type mice. Furthermore, bacterial load was increased in γδ T cell‐deficient mice, yet antibiotic treatment did not change mortality. Additionally, we found that recruitment of neutrophils and myeloid suppressor cells to the site of infection was diminished in γδ T cell‐deficient mice. Finally, we found that circulating levels of IFN‐γ were increased, and systemic levels of IL‐10 were decreased in γδ T cell‐deficient mice after CLP compared with wild‐type mice. γδ T cell‐deficient mice also had increased intestinal permeability after CLP compared with wild‐type mice. Neutralization of IFN‐γ abrogated the increase in intestinal permeability in γδ T cell‐deficient mice. The intestines taken from γδ T cell‐deficient mice had decreased myeloperoxidase yet had increased tissue damage as compared with wild‐type mice. Collectively, our data suggest that γδ T cells modulate the response to sepsis and may be a potential therapeutic target.

Thermal injury elevates the inflammatory monocyte subpopulation in multiple compartments.

Recent publications have demonstrated that human resident and inflammatory monocyte (IM) subpopulations have equivalents in rodents. The effect of thermal injury upon these subpopulations has not been studied. Mice were given a scald burn and killed on postburn days (PBDs) 2, 4, and 8. Bone marrow, blood, and spleen white cells were isolated, and the percentage of resident monocytes (CD11b+ LY6C+), IMs (CD11b+ LY6C++), and monocyte progenitors (macrophage-colony-forming unit [M-CFU]) were determined. The ability of each monocyte population to make TNF-α was determined by intracellular cytokine staining. Finally, the ability of sorted fractions from PBD 8 spleen to inhibit lymphocyte proliferation was performed. We noted that there was an increase in M-CFU in the blood and spleen at PBD 8, but the marrow only had a nonsignificant increase in M-CFU. All compartments showed a significant increase in the number of IMs by PBD 8, but no significant changes in resident monocytes were seen. In all compartments, IMs were a major source of TNF-α. The postburn increase in IMs and monocyte progenitors in the spleen was accompanied by an increase in the monocyte chemokine monocyte chemoattractant protein 1 and constitutively high levels of the progenitor chemokine stromal-derived factor 1α. After burn injury, mice deficient in the receptor for soluble TNF-α had equal levels of splenic M-CFU and monocytes, as did wild-type mice, suggesting that this cytokine is not essential for this effect. We conclude that in this model, IMs are a significant source of in vivo TNF-α.

Effect of thermal injury on splenic myelopoiesis.

Thermal injury increases the number of macrophage progenitors in the bone marrow but leads to a decrease in the number of granulocyte progenitors. In the spleen, thermal injury increases the numbers of myeloid progenitors, but the lineage commitment of these cells is unknown. In this study mice were given a scald burn, and the number of splenic myeloid progenitors as well as their progeny was determined. BrdU uptake was used to monitor the de novo production of splenocytes for 8 days after the burn. Burn injury increased the numbers of splenic granulocyte-macrophage (GM), granulocyte (G), and macrophage (M) progenitors at postburn day 8 by 12-, 11-, and 18-fold, respectively. Scald injury increased the number of mature PMN (CD11b+ GR1bright) in the spleen and increased the number of white pulp monocyte/macrophages. Increased numbers of BrdU-positive PMN and monocyte/macrophages were seen after injury. Burn macrophages produced increased levels of the anti-inflammatory hematopoietic cytokine G-CSF. Our work clearly shows that the increased myelopoiesis observed postinjury leads to the production of mature myeloid cells. However, the effects of thermal injury on progenitors in the spleen and marrow are not equivalent.

G-CSF Drives a Posttraumatic Immune Program That Protects the Host from Infection

Traumatic injury is generally considered to have a suppressive effect on the immune system, resulting in increased susceptibility to infection. Paradoxically, we found that thermal injury to the skin induced a robust time-dependent protection of mice from a lethal Klebsiella pneumoniae pulmonary challenge. The protective response was neutrophil dependent and temporally associated with a systemic increase in neutrophils resulting from a reprioritization of hematopoiesis toward myeloid lineages. A prominent and specific activation of STAT3 in the bone marrow preceded the myeloid shift in that compartment, in association with durable increases in STAT3 activating serum cytokines G-CSF and IL-6. Neutralization of the postburn increase in serum G-CSF largely blocked STAT3 activation in marrow cells, reversing the hematopoietic changes and systemic neutrophilia. Daily administration of rG-CSF was sufficient to recapitulate the changes induced by injury including hematopoietic reprioritization and protection from pulmonary challenge with K. pneumoniae. Analysis of posttraumatic gene expression patterns in humans reveals that they are also consistent with a role for G-CSF as a switch that activates innate immune responses and suppresses adaptive immune responses. Our findings suggest that the G-CSF STAT3 axis constitutes a key protective mechanism induced by injury to reduce the risk for posttraumatic infection.

Keratinocyte growth factor supports pulmonary innate immune defense through maintenance of alveolar antimicrobial protein levels and macrophage function.

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of toxic and infectious insults. In prior studies we found that recombinant human KGF accelerates clearance of bacteria from the murine lung by augmenting the function of alveolar macrophages (AM). In this study we tested the hypothesis that endogenous KGF plays a role in the maintenance of innate pulmonary defense against gram-negative bacterial infections. KGF-deficient mice exhibited delayed clearance of Escherichia coli from the lungs, attenuated phagocytosis by AM, and decreased antimicrobial activity in bronchoalveolar lavage (BAL) fluid, due in part to reductions in levels of surfactant protein A, surfactant protein D, and lysozyme. These immune deficits were accompanied by lower alveolar type II epithelial cell counts and reduced alveolar type II epithelial cell expression of collectin and lysozyme genes on a per cell basis. No significant between-group differences were detected in selected inflammatory cytokines or BAL inflammatory cell populations at baseline or after bacterial challenge in the wild-type and KGF-deficient mice. A single intranasal dose of recombinant human KGF reversed defects in bacterial clearance, AM function, and BAL fluid antimicrobial activity. We conclude that KGF supports alveolar innate immune defense through maintenance of alveolar antimicrobial protein levels and functions of AM. Together these data demonstrate a role for endogenous KGF in maintenance of normal pulmonary innate immune function.

Mitogenic stimulation accelerates influenza-induced mortality by increasing susceptibility of alveolar type II cells to infection

Significance Influenza is a recurring global health threat that preferentially targets vulnerable groups such as the very young, the pregnant, the elderly, and the infirm. The spread of influenza A virus (IAV) from the epithelium of the conducting airway to the alveolar epithelium is a pivotal event in the pathogenesis of primary viral pneumonia. Host susceptibility to IAV pneumonia is often attributed to altered immunity, and cell autonomous vulnerability states of the alveolar epithelium, such as proliferative tone, are rarely considered. Here we demonstrate that mitogenic stimulation of alveolar epithelial type II cells renders them susceptible to IAV infection in an mTOR-dependent manner. Development of pneumonia is the most lethal consequence of influenza, increasing mortality more than 50-fold compared with uncomplicated infection. The spread of viral infection from conducting airways to the alveolar epithelium is therefore a pivotal event in influenza pathogenesis. We found that mitogenic stimulation with keratinocyte growth factor (KGF) markedly accelerated mortality after infectious challenge with influenza A virus (IAV). Coadministration of KGF with IAV markedly accelerated the spread of viral infection from the airways to alveoli compared with challenge with IAV alone, based on spatial and temporal analyses of viral nucleoprotein staining of lung tissue sections and dissociated lung cells. To better define the temporal relationship between KGF administration and susceptibility to IAV infection in vivo, we administered KGF 120, 48, 24, and 0 h before intrapulmonary IAV challenge and assessed the percentages of proliferating and IAV-infected, alveolar type II (AECII) cells in dispersed lung cell populations. Peak AECII infectivity coincided with the timing of KGF administration that also induced peak AECII proliferation. AECII from mice that were given intrapulmonary KGF before isolation and then infected with IAV ex vivo exhibited the same temporal pattern of proliferation and infectious susceptibility. KGF-induced increases in mortality, AECII proliferation, and enhanced IAV susceptibility were all reversed by pretreatment of the animals with the mTOR inhibitor rapamycin before mitogenic stimulation. Taken together, these data suggest mTOR signaling-dependent, mitogenic conditioning of AECII is a determinant of host susceptibility to infection with IAV.

Thermal injury of the skin induces G-CSF-dependent attenuation of EPO-mediated STAT signaling and erythroid differentiation arrest in mice

Inflammation-mediated impairment of erythropoiesis plays a central role in the development of the anemia of critical illness (ACI). ACI develops despite elevation of endogenous erythropoietin (EPO), does not respond to exogenous erythropoietin (EPO) supplementation, and contributes significantly to transfusion requirements in burned patients. We have reported previously that the reduction of red blood cell mass in the bone marrow of a burn-injured ACI mouse model is granulocyte colony-stimulating factor (G-CSF) dependent. Given that elevated G-CSF levels also have been associated with lower hemoglobin levels and increased transfusion requirements in trauma victims, we postulated that G-CSF mediates postburn EPO resistance. In ACI mice, we found that bone marrow erythroid differentiation, viability, and proliferation are impaired after thermal injury of the skin. These changes in the marrow were associated with attenuated phosphorylation of known EPO-responsive signaling nodes, signal transducer and activator of transcription 5 (STAT5) Y694 and STAT3 S727, in bone marrow erythroid cells and developed despite highly elevated levels of endogenous EPO. Severely blunted STAT5 Y694 phosphorylation in bone marrow erythroid cells after exogenous EPO supplementation confirmed that EPO signaling was impaired in ACI mice. Importantly, parenteral administration of anti-G-CSF largely rescued postburn bone marrow erythroid differentiation arrest and EPO signaling in erythroid cells. Together, these data provide strong evidence for a role for G-CSF in the development of ACI after burn injury through suppression of EPO signaling in bone marrow erythroid cells.