Ann M. Kelly

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-γ–primed eosinophils to release mitochondrial DNA in a reactive oxygen species–dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner—in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Murine Cytomegalovirus Interference with Antigen Presentation Has Little Effect on the Size or the Effector Memory Phenotype of the CD8 T Cell Response

As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV’s three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8lowCD62L−CD11c+NKG2A+) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Δm4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Δm4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.

Differential Role for CD80 and CD86 in the Regulation of the Innate Immune Response in Murine Polymicrobial Sepsis

Background Inflammation in the early stages of sepsis is governed by the innate immune response. Costimulatory molecules are a receptor/ligand class of molecules capable of regulation of inflammation in innate immunity via macrophage/neutrophil contact. We recently described that CD80/86 ligation is required for maximal macrophage activation and CD80/86−/− mice display reduced mortality and inflammatory cytokine production after cecal ligation and puncture (CLP). However, these data also demonstrate differential regulation of CD80 and CD86 expression in sepsis, suggesting a divergent role for these receptors. Therefore, the goal of this study was to determine the individual contribution of CD80/86 family members in regulating inflammation in sepsis. Methodology/Principal Findings CD80−/− mice had improved survival after CLP when compared to WT or CD86−/− mice. This was associated with preferential attenuation of inflammatory cytokine production in CD80−/− mice. Results were confirmed with pharmacologic blockade, with anti-CD80 mAb rescuing mice when administered before or after CLP. In vitro, activation of macrophages with neutrophil lipid rafts caused selective disassociation of IRAK-M, a negative regulator of NF-κB signaling from CD80; providing a mechanism for preferential regulation of cytokine production by CD80. Finally, in humans, upregulation of CD80 and loss of constitutive CD86 expression on monocytes was associated with higher severity of illness and inflammation confirming the findings in our mouse model. Conclusions In conclusion, our data describe a differential role for CD80 and CD86 in regulation of inflammation in the innate immune response to sepsis. Future therapeutic strategies for blockade of the CD80/86 system in sepsis should focus on direct inhibition of CD80.

Mouse eosinophils possess potent antibacterial properties in vivo.

ABSTRACT Eosinophils are best known as the predominant cellular infiltrate associated with asthma and parasitic infections. Recently, numerous studies have documented the presence of Toll-like receptors (TLRs) on the surfaces of eosinophils, suggesting that these leukocytes may participate in the recognition and killing of viruses and bacteria. However, the significance of this role in the innate immune response to bacterial infection is largely unknown. Here we report a novel role for eosinophils as antibacterial defenders in the host response. Isolated mouse eosinophils possessed antipseudomonal properties in vitro. In vivo, interleukin-5 transgenic mice, which have profound eosinophilia, demonstrated improved clearance of Pseudomonas aeruginosa introduced into the peritoneal cavity. The findings of improved bacterial clearance following adoptive transfer of eosinophils, and impaired bacterial clearance in mice with a congenital eosinophil deficiency, established that this effect was eosinophil specific. The data presented also demonstrate that eosinophils mediate this antibacterial effect in part through the release of cationic secondary granule proteins. Specifically, isolated eosinophil granules had antibacterial properties in vitro, and administration of eosinophil granule extracts significantly improved bacterial clearance in vivo. These data suggest a potent yet underappreciated antibacterial role for eosinophils in vivo, specifically for eosinophil granules. Moreover, the data suggest that the administration of eosinophil-derived products may represent a viable adjuvant therapy for septic or bacteremic patients in the intensive care unit.

Interleukin 5 Is Protective during Sepsis in an Eosinophil-Independent Manner

RATIONALE The immune response in sepsis is characterized by overt immune dysfunction. Studies indicate immunostimulation represents a viable therapy for patients. One study suggests a potentially protective role for interleukin 5 (IL-5) in sepsis; however, the loss of eosinophils in this disease presents a paradox. OBJECTIVES To assess the protective and eosinophil-independent effects of IL-5 in sepsis. METHODS We assessed the effects of IL-5 administration on survival, bacterial burden, and cytokine production after polymicrobial sepsis. In addition, we examined the effects on macrophage phagocytosis and survival using fluorescence microscopy and flow cytometry. MEASUREMENTS AND MAIN RESULTS Loss of IL-5 increased mortality and tissue damage in the lung, IL-6 and IL-10 production, and bacterial burden during sepsis. Therapeutic administration of IL-5 improved mortality in sepsis. Interestingly, IL-5 administration resulted in neutrophil recruitment in vivo. IL-5 overexpression in the absence of eosinophils resulted in decreased mortality from sepsis and increased circulating neutrophils and monocytes, suggesting their importance in the protective effects of IL-5. Furthermore, novel data demonstrate IL-5 receptor expression on neutrophils and monocytes in sepsis. IL-5 augmented cytokine secretion, activation, phagocytosis, and survival of macrophages. Importantly, macrophage depletion before the onset of sepsis eliminated IL-5-mediated protection. The protective effects of IL-5 were confirmed in humans, where IL-5 levels were elevated in patients with sepsis. Moreover, neutrophils and monocytes from patients expressed the IL-5 receptor. CONCLUSIONS Taken together, these data support a novel role for IL-5 on noneosinophilic myeloid populations, and suggest treatment with IL-5 may be a viable therapy for sepsis.

OX40 Ligand Regulates Inflammation and Mortality in the Innate Immune Response to Sepsis

The initial phase of sepsis is characterized by massive inflammatory cytokine production that contributes to multisystem organ failure and death. Costimulatory molecules are a class of receptors capable of regulating cytokine production in adaptive immunity. Recent studies described their presence on neutrophils and monocytes, suggesting a potential role in the regulation of cytokine production in innate immunity. The purpose of this study was to determine the role for OX40–OX40 ligand (OX40L) interaction in the innate immune response to polymicrobial sepsis. Humans with sepsis demonstrated upregulation of OX40L on monocytes and neutrophils, with mortality and intensive care unit stay correlating with expression levels. In an animal model of polymicrobial sepsis, a direct role for OX40L in regulating inflammation was indicated by improved survival, decreased cytokine production, and a decrease in remote organ damage in OX40L−/− mice. The finding of similar results with an OX40L Ab suggests a potential therapeutic role for OX40L blockade in sepsis. The inability of anti-OX40L to provide significant protection in macrophage-depleted mice establishes macrophages as an indispensable cell type within the OX40/OX40L axis that helps to mediate the clinical signs of disease in sepsis. Conversely, the protective effect of anti-OX40L Ab in RAG1−/− mice further confirms a T cell-independent role for OX40L stimulation in sepsis. In conclusion, our data provide an in vivo role for the OX40/OX40L system in the innate immune response during polymicrobial sepsis and suggests a potential beneficial role for therapeutic blockade of OX40L in this devastating disorder.