Eosinophil-associated ribonuclease 11 is a macrophage chemoattractant.

RNase A is the prototype of an extensive family of divergent proteins whose members share a unique disulfide-bonded tertiary structure, conserved catalytic motifs, and the ability to hydrolyze polymeric RNA. Several members of this family maintain independent roles as ribonucleases and modulators of innate immunity. Here we characterize mouse eosinophil-associated RNase (Ear) 11, a divergent member of the eosinophil ribonuclease cluster, and the only known RNase A ribonuclease expressed specifically in response to Th2 cytokine stimulation. Mouse Ear 11 is differentially expressed in somatic tissues at baseline (brain ≪ liver < lung < spleen); systemic stimulation with IL-33 results in 10-5000-fold increased expression in lung and spleen, respectively. Ear 11 is also expressed in response to protective priming of the respiratory mucosa with Lactobacillus plantarum; transcripts are detected both locally in lung as well as systemically in bone marrow and spleen. Mouse Ear 11 is enzymatically active, although substantially less so than mEar 1 and mEar 2; the relative catalytic efficiency (kcat/Km) of mEar 11 is diminished ∼1000-1500-fold. However, in contrast to RNase 2/EDN and mEar 2, which have been characterized as selective chemoattractants for CD11c(+) dendritic cells, mEar 11 has prominent chemoattractant activity for F4/80(+)CD11c(-) tissue macrophages. Chemoattractant activity is not dependent on full enzymatic activity, and requires no interaction with the pattern recognition receptor, Toll-like receptor 2 (TLR2). Taken together, this work characterizes a divergent RNase A ribonuclease with a unique expression pattern and function, and highlights the versatility of this family in promoting innate immunity.

B cells are not essential for Lactobacillus-mediated protection against lethal pneumovirus infection.

We have shown previously that priming of respiratory mucosa with live Lactobacillus species promotes robust and prolonged survival from an otherwise lethal infection with pneumonia virus of mice, a property known as heterologous immunity. Lactobacillus priming results in a moderate reduction in virus recovery and a dramatic reduction in virus-induced proinflammatory cytokine production; the precise mechanisms underlying these findings remain to be elucidated. Because B cells have been shown to promote heterologous immunity against respiratory virus pathogens under similar conditions, in this study we explore the role of B cells in Lactobacillus-mediated protection against acute pneumovirus infection. We found that Lactobacillus-primed mice feature elevated levels of airway Igs IgG, IgA, and IgM and lung tissues with dense, B cell (B220(+))-enriched peribronchial and perivascular infiltrates with germinal centers consistent with descriptions of BALT. No B cells were detected in lung tissue of Lactobacillus-primed B cell deficient µMT mice or Jh mice, and Lactobacillus-primed µMT mice had no characteristic infiltrates or airway Igs. Nonetheless, we observed diminished virus recovery and profound suppression of virus-induced proinflammatory cytokines CCL2, IFN-γ, and CXCL10 in both wild-type and Lactobacillus-primed µMT mice. Furthermore, Lactobacillus plantarum-primed, B cell-deficient µMT and Jh mice were fully protected from an otherwise lethal pneumonia virus of mice infection, as were their respective wild-types. We conclude that B cells are dispensable for Lactobacillus-mediated heterologous immunity and were not crucial for promoting survival in response to an otherwise lethal pneumovirus infection.

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice.

Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ(-/-) mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNß, IFNλ2/3 and IFN-stimulated 2-5' oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ(-/-) mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include over-production of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ(-/-) mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.

Protocols for identifying, enumerating, and assessing mouse eosinophils.

Eosinophils are prominent in allergic diseases, and their effector functions are studied in numerous gene-deleted and transgenic mouse models. However, mouse eosinophils and human eosinophils are not structurally or functionally equivalent, and assays designed to evaluate the properties of human eosinophils may or may not be reliable or effective in experiments targeting their murine counterparts. In this chapter, we emphasize methods focused on detection, isolation, and functional assessment of eosinophils from mouse tissue and present a protocol that promotes the growth and differentiation of eosinophils from unselected mouse bone marrow progenitors. Overall, these protocols provide a scaffold on which the relative contributions of mouse eosinophils can be evaluated.

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.

The Pneumonia Virus of Mice (PVM) model of acute respiratory infection.

Pneumonia Virus of Mice (PVM) is related to the human and bovine respiratory syncytial virus (RSV) pathogens, and has been used to study respiratory virus replication and the ensuing inflammatory response as a component of a natural host­pathogen relationship. As such, PVM infection in mice reproduces many of the clinical and pathologic features of the more severe forms of RSV infection in human infants. Here we review some of the most recent findings on the basic biology of PVM infection and its use as a model of disease, most notably for explorations of virus infection and allergic airways disease, for vaccine evaluation, and for the development of immunomodulatory strategies for acute respiratory virus infection.

Antigen profiles for the quantitative assessment of eosinophils in mouse tissues by flow cytometry.

Much of our current understanding of eosinophil-associated pathologies has developed from the use of mouse models. While mouse eosinophils can be readily detected by flow cytometric methods, most studies do not document the efficiency of this process compared to direct counting of stained cells. Our intent was to address this knowledge gap by identifying one or more eosinophil-specific antigen profiles that yielded flow cytometric data that was statistically consistent with direct counts. We found that anti-CD193 (CCR3) and anti-CD125 (IL-5Rα) antibodies were effective at detecting eosinophils in bone marrow of interleukin-5 transgenic mice, but these antibodies under-reported the percent positive cells. In contrast, anti-Siglec F alone or in combination with anti-CD45 can be used for the quantitative detection of eosinophils in mouse bone marrow and spleen. The antigen profile CD45(+)SiglecF(+)CD11c(-) was effective at detecting eosinophils in the lung as well as bone marrow and spleen, and the results obtained correlated with direct morphometric counts under all conditions evaluated (r(2)=0.98-0.99). To the best of our knowledge, this is the first systematic analysis presenting definitive correlations between percent eosinophils detected by cell surface markers and direct counting of stained cells in multiple tissues and at varying degrees of eosinophilia.

Defective eosinophil hematopoiesis ex vivo in inbred Rocky Mountain White (IRW) mice.

We examine the proliferation and differentiation of bone marrow (BM) progenitors from inbred Rocky Mountain White (IRW) mice, a strain used primarily for retrovirus infection studies. In contrast to findings with BALB/c and C57BL/6 strains, IRW BM cells cannot proliferate or generate pure eosinophil cultures ex vivo in response to a defined cytokine regimen. Analysis of IRW BM at baseline was unremarkable, including 0.08 ± 0.03% Lin(-)Sca-1(+)c-kit(+) (LSK) hematopoietic stem cells and 5.2 ± 0.3% eosinophils; the percentage of eosinophil progenitors (EoPs; Lin(-)Sca-1(-)c-kit(+)CD34(+)IL-5Rα(+)) was similar in all three mouse strains. Transcripts encoding GM-CSFRα and the IL-3/IL-5/GM-CSF common ß chain were detected at equivalent levels in IRW and BALB/c BM, whereas expression of transcripts encoding IL-5Rα, IL-3Rα, and GATA-2 was diminished in IRW BM compared with BALB/c. Expression of membrane-bound IL-5Rα and intracellular STAT5 proteins was also diminished in IRW BM cells. Diminished expression of transcripts encoding IL-5Rα and GATA-2 and immunoreactive STAT5 in IRW BM persisted after 4 days in culture, along with diminished expression of GATA-1. Western blot revealed that cells from IRW BM overexpress nonsignaling soluble IL-5Rα protein. Interestingly, OVA sensitization and challenge resulted in BM and airway eosinophilia in IRW mice; however, the responses were significantly blunted. These results suggest that IRW mice have diminished capacity to generate eosinophils in culture and in vivo, likely as a result of diminished signaling via IL-5Rα.