Cory Deburghgraeve

Advanced Age Impairs Macrophage Polarization

Aging affects many aspects of the cellular function of macrophages. Macrophages play a critical role in innate immunity, acting as sentinels to fight pathogens, promoting wound healing, and orchestrating the development of the specific acquired immune response. However, little is known about how age influences the ability of macrophage to change phenotypes in response to environmental factors. This study examined the age-associated defects on macrophage polarization toward a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Adherent splenocytes enriched for macrophages were cultured with or without lipopolysaccharide (LPS), a combination of interferon (IFN)-γ and tumor necrosis factor (TNF)-α or interleukin (IL)-4. A panel of M1 markers, inducible nitric oxide synthase (iNOS), IL-6, IL-1β, and TNF-α, and M2 markers, including arginase-1 (Arg1), Ym1, and Found In Inflammatory Zone 1 (FIZZ1), were analyzed. IL-6 mRNA in cells from aged mice was decreased by 78% and 58% compared with young after stimulation with LPS or IFN-γ and TNF-α (P<0.05), respectively. Also, there was a marked reduction in the induced levels of iNOS, IL-1β, and TNF-α in cells from aged mice relative to young controls. Similarly, IL-4 exposure resulted in a reduction of M2 markers in adherent splenocytes from aged mice compared with younger animals. This was consistent with a 28% decrease in splenic F4/80(+)IL-4R(+) cells in aged mice relative to controls, although IL-4R expression on these cells did not vary between age groups. In contrast, levels of M1 and most M2 markers, save for FIZZ1, in bone marrow-derived macrophages were similar between the age groups, irrespective of stimuli. These data imply that impaired macrophage polarization in the elderly may dysregulate the development of the host response, making them more susceptible to infectious diseases and that the aging microenvironment may be a key modulator of these macrophage-elicited responses.

Decreased Pulmonary Inflammation Following Ethanol and Burn Injury in Mice Deficient in TLR4 but not TLR2 Signaling

BACKGROUND Clinical and laboratory evidence suggests that alcohol consumption prior to burn injury leads to dysregulated immune function and subsequent higher rates of morbidity and mortality. Our laboratory previously observed higher levels of pro-inflammatory cytokines and leukocyte infiltration in the lungs of mice following ethanol and burn injury. To understand the mechanism of the increased inflammatory response, we looked at different signaling initiators of inflammation including toll-like receptors 2 and 4 (TLR2 and 4) pathways. METHODS Wild-type, TLR2, and TLR4 knockout mice were treated with vehicle or a single binge dose of ethanol (1.11 g/kg) and subsequently given a sham or burn injury. Twenty-four hours postinjury, systemic and pulmonary levels of pro-inflammatory cytokines were quantified, and differences in neutrophil infiltration were determined by histological examination. RESULTS Higher numbers of neutrophils were observed in the lungs of wild-type mice following the combined insult of ethanol and burn injury relative to either injury alone. This increase in leukocyte accumulation was absent in the TLR4 knockout mice. Circulating levels of IL-6 and tumor necrosis factor-α were also elevated in wild-type mice but not in TLR4 knockout mice. Consistent with these findings, pulmonary levels of KC and IL-6 were increased in wild-type mice following burn and ethanol compared to burn injury alone as well as to their TLR4 knockout counterparts. In contrast, TLR2 knockout mice displayed similar levels, to wild-type mice, of neutrophil infiltration as well as IL-6 and KC in the lung. CONCLUSIONS These data suggest that TLR4 signaling is a crucial contributory component in the exuberant inflammation after ethanol and burn injury. However, TLR2 does not appear to play a vital role in the aberrant pulmonary inflammation.

Development of a combined radiation and burn injury model.

Combined radiation and burn injuries are likely to occur after nuclear events, such as a meltdown accident at a nuclear energy plant or a nuclear attack. Little is known about the mechanisms by which combined injuries result in higher mortality than by either insult alone, and few animal models exist for combined radiation and burn injury. Herein, the authors developed a murine model of radiation and scald burn injury. Mice were given a single dose of 0, 2, 4, 5, 6, or 9 Gray (Gy) alone, followed by a 15% TBSA scald burn. All mice receiving ≤4 Gy of radiation with burn survived combined injury. Higher doses of radiation (5, 6, and 9 Gy) followed by scald injury had a dose-dependent increase in mortality (34, 67, and 100%, respectively). Five Gy was determined to be the ideal dose to use in conjunction with burn injury for this model. There was a decrease in circulating white blood cells in burn, irradiated, and combined injury (5 Gy and burn) mice by 48 hours postinjury compared with sham (49.7, 11.6, and 57.3%, respectively). Circulating interleukin-6 and tumor necrosis factor-&agr; were increased in combined injury at 48 hours postinjury compared with all other treatment groups. Prolonged overproduction of proinflammatory cytokines could contribute to subsequent organ damage. Decreased leukocytes might exacerbate immune impairment and susceptibility to infections. Future studies will determine whether there are long lasting consequences of this early proinflammatory response and extended decrease in leukocytes.

Age-related differences in the neutrophil response to pulmonary pseudomonas infection

BACKGROUND Pseudomonas aeruginosa pneumonia is more common and more lethal in the elderly. The immunologic underpinnings of this increased incidence and mortality have not been evaluated, however are assumed to be a complication of age-associated immune dysfunction. METHODS Young (10-12week old) and aged (18-20month old) BALB/c mice were subjected to intratracheal infection of P. aeruginosa. Animals were sacrificed 24h after inoculation. The lungs were collected for analysis of lung pathology, chemokine levels, neutrophil counts, and myeloperoxidase activity. RESULTS Pulmonary levels of the neutrophil chemokine KC are significantly higher in aged mice relative to young following P. aeruginosa infection. Despite this, neutrophil counts are higher in young mice compared to aged mice after infection. Furthermore, the neutrophils are predominantly found in the air space of young infected mice. This correlated with increased myeloperoxidase activity from bronchoalveolar lavage specimens of young mice relative to aged mice after infection. CONCLUSIONS Neutrophil migration into the lungs is impaired in aged mice 24h after intratracheal infection despite elevated chemokine levels, suggesting that immunosenescence is impairing neutrophil migration.

Prolonged chemokine expression and excessive neutrophil infiltration in the lungs of burn-injured mice exposed to ethanol and pulmonary infection.

Pulmonary infections are a major cause of mortality in the critically ill burn patient. Alcohol consumption before burn increases the risk of pulmonary infection. Previously, we have shown an elevated mortality and lung pathology in mice given ethanol before burn and intratracheal infection relative to controls. Here we examine the cellular composition at 24 and 48 h in the circulation and the alveoli of infected mice given alcohol and burn. At 24 h after injury, blood neutrophils obtained from mice exposed to ethanol before burn and infection were 2-fold above those of the experimental controls (P < 0.05). By 48 h, the number of circulating neutrophils decreased and was comparable to levels found in untreated animals. Moreover, at 24 h, bronchoalveolar lavage cells obtained from all treatment groups had similar frequencies and contained 80% neutrophils regardless of treatment. In contrast, the following day, neutrophils were elevated 2-fold only in the alveoli of infected burn animals and 5-fold when ethanol preceded the injury (P < 0.05). These data were confirmed by immunofluorescence microscopy using a neutrophil-specific marker (P < 0.05). Levels of neutrophil chemoattractants, KC and macrophage inflammatory protein 2, and the cytokine, IL-1&bgr;, were 2-fold greater in the lungs of infected mice given burn, regardless of ethanol exposure, relative to infected sham injured animals (P < 0.05). Like the number of neutrophils, by the second day after injury, KC and macrophage inflammatory protein 2 remained 5-fold higher in the animals given ethanol, burn, and infection, when compared with other groups (P < 0.05). A similar pattern was seen for pulmonary levels of IL-1&bgr; (P < 0.05). Additionally, a reduction in neutrophil apoptosis was observed at the 24-h time point in infected mice exposed to ethanol and burn (P < 0.05). Targeting proinflammatory mediators in mice exposed to ethanol before burn and infection may help alleviate prolonged neutrophil accumulation in the lungs.

Combined Radiation and Burn Injury Results in Exaggerated Early Pulmonary Inflammation

Events such as a nuclear meltdown accident or nuclear attack have potential for severe radiation injuries. Radiation injury frequently occurs in combination with other forms of trauma, most often burns. Thus far, combined injury studies have focused mainly on skin wound healing and damage to the gut. Since both radiation exposure and remote burn have pulmonary consequences, we examined the early effects of combined injury on the lung. C57BL/6 male mice were irradiated with 5 Gy of total body irradiation followed by a 15% total body surface area scald burn. Lungs from surviving animals were examined for evidence of inflammation and pneumonitis. At 48 h post-injury, pathology of the lungs from combined injury mice showed greater inflammation compared to all other treatment groups, with marked red blood cell and leukocyte congestion of the pulmonary vasculature. There was excessive leukocyte accumulation, primarily neutrophils, in the vasculature and interstitium, with occasional cells in the alveolar space. At 24 and 48 h post-injury, myeloperoxidase levels in lungs of combined injury mice were elevated compared to all other treatment groups (P < 0.01), confirming histological evidence of neutrophil accumulation. Pulmonary levels of the neutrophil chemoattractant KC (CXCL1) were 3 times above that of either injury alone (P < 0.05). Further, monocyte chemotactic protein-1 (MCP-1, CCL2) was increased two- and threefold compared to burn injury or radiation injury, respectively (P < 0.05). Together, these data suggest that combined radiation and burn injury augments early pulmonary congestion and inflammation. Currently, countermeasures for this unique type of injury are extremely limited. Further research is needed to elucidate the mechanisms behind the synergistic effects of combined injury in order to develop appropriate treatments.

Challenges in the diagnosis of 2009 H1N1 in a lung transplant patient and the long-term implications for prevention and treatment: a case report.

Although respiratory viral infections have been associated with acute rejection and bronchiolitis obliterans syndrome, the long-term impact of the novel pandemic influenza A (2009 H1N1) virus on lung transplant patients has not been defined. We describe the diagnostic challenges and long-term consequences of 2009 H1N1 infection in a lung transplant patient, discuss the potential implications for prevention and treatment, and conclude that even timely antiviral therapy may be insufficient to prevent long-term morbidity.