Caroline M. Percopo

Functionally Competent Eosinophils Differentiated Ex Vivo in High Purity from Normal Mouse Bone Marrow

We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to 4 days of culture with recombinant mouse FLT3-L and recombinant mouse stem cell factor followed by recombinant mouse IL-5 alone thereafter, the resulting bone marrow-derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5R α-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common β-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-γ, IL-4, MIP-1α, and IL-6. The rodent pathogen pneumonia virus of mice replicates in bmEos and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells.

Lactobacillus-Mediated Priming of the Respiratory Mucosa Protects against Lethal Pneumovirus Infection

The inflammatory response to respiratory virus infection can be complex and refractory to standard therapy. Lactobacilli, when targeted to the respiratory epithelium, are highly effective at suppressing virus-induced inflammation and protecting against lethal disease. Specifically, wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus plantarum or Lactobacillus reuteri were completely protected against lethal infection with the virulent rodent pathogen, pneumonia virus of mice; significant protection (60% survival) persisted for at least 13 wk. Protection was not unique to Lactobacillus species, and it was also observed in response to priming with nonpathogenic Gram-positive Listeria innocua. Priming with live lactobacilli resulted in diminished granulocyte recruitment, diminished expression of multiple proinflammatory cytokines (CXCL10, CXCL1, CCL2, and TNF), and reduced virus recovery, although we have demonstrated clearly that absolute virus titer does not predict clinical outcome. Lactobacillus priming also resulted in prolonged survival and protection against the lethal sequelae of pneumonia virus of mice infection in MyD88 gene-deleted (MyD88−/−) mice, suggesting that the protective mechanisms may be TLR-independent. Most intriguing, virus recovery and cytokine expression patterns in Lactobacillus-primed MyD88−/− mice were indistinguishable from those observed in control-primed MyD88−/− counterparts. In summary, we have identified and characterized an effective Lactobacillus-mediated innate immune shield, which may ultimately serve as critical and long-term protection against infection in the absence of specific antiviral vaccines.

Mouse and Human Eosinophils Degranulate in Response to Platelet-Activating Factor (PAF) and LysoPAF via a PAF-Receptor–Independent Mechanism: Evidence for a Novel Receptor

Platelet-activating factor (PAF [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine]) is a phospholipid mediator released from activated macrophages, mast cells, and basophils that promotes pathophysiologic inflammation. Eosinophil responses to PAF are complex and incompletely elucidated. We show in this article that PAF and its 2-deacetylated metabolite (lysoPAF) promote degranulation (release of eosinophil peroxidase) via a mechanism that is independent of the characterized PAFR. Specifically, we demonstrate that receptor antagonists CV-3988 and WEB-2086 and pertussis toxin have no impact on PAF- or lysoPAF-mediated degranulation. Furthermore, cultured mouse eosinophils from PAFR−/− bone marrow progenitors degranulate in response to PAF and lysoPAF in a manner indistinguishable from their wild-type counterparts. In addition to PAF and lysoPAF, human eosinophils degranulate in response to lysophosphatidylcholine, but not phosphatidylcholine, lysophosphatidylethanolamine, or phosphatidylethanolamine, demonstrating selective responses to phospholipids with a choline head-group and minimal substitution at the sn-2 hydroxyl. Human eosinophils release preformed cytokines in response to PAF, but not lysoPAF, also via a PAFR-independent mechanism. Mouse eosinophils do not release cytokines in response to PAF or lysoPAF, but they are capable of doing so in response to IL-6. Overall, our work provides the first direct evidence for a role for PAF in activating and inducing degranulation of mouse eosinophils, a crucial feature for the interpretation of mouse models of PAF-mediated asthma and anaphylaxis. Likewise, we document and define PAF and lysoPAF-mediated activities that are not dependent on signaling via PAFR, suggesting the existence of other unexplored molecular signaling pathways mediating responses from PAF, lysoPAF, and closely related phospholipid mediators.

Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6

Eosinophils are recruited to the lung in response to infection with pneumovirus pathogens and have been associated with both the pathophysiologic sequelae of infection and, more recently, with accelerated virus clearance. Here, we demonstrate that the pneumovirus pathogens, respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM), can infect human and mouse eosinophils, respectively, and that virus infection of eosinophils elicits the release of disease-related proinflammatory mediators from eosinophils. RSV replication in human eosinophils results in the release of infectious virions and in the release of the proinflammatory mediator, interleukin-6 (IL-6). PVM replication in cultured bone marrow eosinophils (bmEos) likewise results in release of infectious virions and the proinflammatory mediators IL-6, IP-10, CCL2, and CCL3. In contrast to the findings reported in lung tissue of RSV-challenged mice, PVM replication is accelerated in MyD88 gene-deleted bmEos, whereas release of cytokines is diminished. Interestingly, exogenous IL-6 suppresses virus replication in MyD88 gene-deleted bmEos, suggesting a role for a MyD88-dependent cytokine-mediated feedback circuit in modulating this response. Taken together, our findings suggest that eosinophils are targets of virus infection and may have varied and complex contributions to the pathogenesis and resolution of pneumovirus disease.

Interferon-gamma coordinates CCL3-mediated neutrophil recruitment in vivo

BackgroundWe have shown previously that acute infection with the respiratory pathogen, pneumonia virus of mice (PVM), results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice.ResultsIn this work, we demonstrate that CCL3-mediated neutrophil recruitment is coordinated by interferon-gamma (IFNγ). Neutrophil recruitment in response to PVM infection was diminished five-fold in IFNγ receptor gene-deleted mice, although neutrophils from IFNγR -/- mice expressed transcripts for the CCL3 receptor, CCR1 and responded functionally to CCL3 ex vivo. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNγ. Interestingly, although supplemental IFNγ restored neutrophil recruitment and resulted in a sustained weight loss among CCL3-overexpressing IFNγ -/- mice, CCL3-mediated neutrophil recruitment alone did not result in the pulmonary edema or respiratory failure characteristic of severe viral infection, suggesting that CCL3 and IFN-γ together are sufficient to promote neutrophil recruitment but not pathologic activation.ConclusionOur findings reveal a heretofore unrecognized hierarchical interaction between the IFNγ and CCL3, which demonstrate that IFNγ is crucial for CCL3-mediated neutrophil recruitment in vivo.

Anti-la antibody diminishes ocular inflammation in experimental autoimmune uveitis

An experimental model of inflammatory eye disease, experimental autoimmmune uveitis (EAU), was established by injecting rats in the footpad with S-antigen in complete Freunds adjuvant. This model system was used to evaluate the role of major histocompatibility complex (MHC) class II antigens (la) in the pathogenesis of this T cell mediated disease. One day prior to S-antigen priming, rats were injected with either anti-la antibodies or with mouse ascites. Clinical and histopathological analysis of eyes from rats treated with anti-la antibody showed less ocular inflammation as well as a delay in onset of EAU when compared to controls (p=0.01). Furthermore, immunocytochemical evaluation demonstrated that tissue obtained from animals receiving anti-la therapy also expressed less la antigen, as well as a diminution in the number of infiltrating macrophages and lymphocytes. These data show that anti-la treatment significantly modifies the course of EAU in the rat. In addition, this study suggests that MHC cla...

Eosinophils from Lineage-Ablated ΔdblGATA Bone Marrow Progenitors: The dblGATA Enhancer in the Promoter of GATA-1 Is Not Essential for Differentiation Ex Vivo

A critical role for eosinophils in remodeling of allergic airways was observed in vivo upon disruption of the dblGATA enhancer that regulates expression of GATA-1, which resulted in an eosinophil-deficient phenotype in the ΔdblGATA mouse. We demonstrate here that bone marrow progenitors isolated from ΔdblGATA mice can differentiate into mature eosinophils when subjected to cytokine stimulation ex vivo. Cultured ΔdblGATA eosinophils contain cytoplasmic granules with immunoreactive major basic protein and they express surface Siglec F and transcripts encoding major basic protein, eosinophil peroxidase, and GATA-1, -2, and -3 to an extent indistinguishable from cultured wild-type eosinophils. Fibroblast coculture and bone marrow cross-transplant experiments indicate that the in vivo eosinophil deficit is an intrinsic progenitor defect, and remains unaffected by interactions with stromal cells. Interestingly, and in contrast to those from the wild type, a majority of the GATA-1 transcripts from cultured ΔdblGATA progenitors express a variant GATA-1 transcript that includes a first exon (1EB), located ∼3700 bp downstream to the previously described first exon found in hemopoietic cells (1EA) and ∼42 bp upstream to another variant first exon, 1EC. These data suggest that cultured progenitors are able to circumvent the effects of the ΔdblGATA ablation by using a second, more proximal, promoter and use this mechanism to generate quantities of GATA-1 that will support eosinophil growth and differentiation.

Activated mouse eosinophils protect against lethal respiratory virus infection

Eosinophils are recruited to the airways as a prominent feature of the asthmatic inflammatory response where they are broadly perceived as promoting pathophysiology. Respiratory virus infections exacerbate established asthma; however, the role of eosinophils and the nature of their interactions with respiratory viruses remain uncertain. To explore these questions, we established acute infection with the rodent pneumovirus, pneumonia virus of mice (PVM), in 3 distinct mouse models of Th2 cytokine-driven asthmatic inflammation. We found that eosinophils recruited to the airways of otherwise naïve mice in response to Aspergillus fumigatus, but not ovalbumin sensitization and challenge, are activated by and degranulate specifically in response to PVM infection. Furthermore, we demonstrate that activated eosinophils from both Aspergillus antigen and cytokine-driven asthma models are profoundly antiviral and promote survival in response to an otherwise lethal PVM infection. Thus, although activated eosinophils within a Th2-polarized inflammatory response may have pathophysiologic features, they are also efficient and effective mediators of antiviral host defense.

IL-33 promotes eosinophilia in vivo and antagonizes IL-5-dependent eosinophil hematopoiesis ex vivo.

IL-33 is an IL-1 family cytokine that elicits IL-5-dependent eosinophilia in vivo. We show here that IL-33 promotes minimal eosinophil hematopoiesis via direct interactions with mouse bone marrow progenitors ex vivo and that it antagonizes eosinophil hematopoiesis promoted by IL-5 on SCF and Flt3L primed bone marrow progenitor cells in culture. SCF and Flt3L primed progenitors respond to IL-33 by acquiring an adherent, macrophage-like phenotype, and by releasing macrophage-associated cytokines into the culture medium. IL-33-mediated antagonism of IL-5 was reproduced in part by the addition of GM-CSF and was inhibited by the actions of neutralizing anti-GM-CSF antibody. These findings suggest that the direct actions of IL-33 on bone marrow progenitors primed with SCF and Flt3L are antagonistic to the actions of IL-5 and are mediated in part by GM-CSF.

Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection.

We showed previously that wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus species were fully (100%) protected against the lethal sequelae of infection with the virulent pathogen, pneumonia virus of mice (PVM), a response that is associated with diminished expression of proinflammatory cytokines and diminished virus recovery. We show here that 40% of the mice primed with live Lactobacillus survived when PVM challenge was delayed for 5months. This robust and sustained resistance to PVM infection resulting from prior interaction with an otherwise unrelated microbe is a profound example of heterologous immunity. We undertook the present study in order to understand the nature and unique features of this response. We found that intranasal inoculation with L. reuteri elicited rapid, transient neutrophil recruitment in association with proinflammatory mediators (CXCL1, CCL3, CCL2, CXCL10, TNF-alpha and IL-17A) but not Th1 cytokines. IFNγ does not contribute to survival promoted by Lactobacillus-priming. Live L. reuteri detected in lung tissue underwent rapid clearance, and was undetectable at 24h after inoculation. In contrast, L. reuteri peptidoglycan (PGN) and L. reuteri genomic DNA (gDNA) were detected at 24 and 48h after inoculation, respectively. In contrast to live bacteria, intranasal inoculation with isolated L. reuteri gDNA elicited no neutrophil recruitment, had minimal impact on virus recovery and virus-associated production of CCL3, and provided no protection against the negative sequelae of virus infection. Isolated PGN elicited neutrophil recruitment and proinflammatory cytokines but did not promote sustained survival in response to subsequent PVM infection. Overall, further evaluation of the responses leading to Lactobacillus-mediated heterologous immunity may provide insight into novel antiviral preventive modalities.