Peroxidasin and eosinophil peroxidase, but not myeloperoxidase, contribute to renal fibrosis in the murine unilateral ureteral obstruction model.

Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.

Impact of eosinophil-peroxidase (EPX) deficiency on eosinophil structure and function in mouse airways.

Eosinophil peroxidase (EPX) is a major constituent of the large cytoplasmic granules of both human and mouse eosinophilic leukocytes. Human EPX deficiency is a rare, autosomal-recessive disorder limited to the eosinophil lineage. Our intent was to explore the impact of EPX gene deletion on eosinophil content, structure, and function. In response to repetitive intranasal challenge with a filtrate of the allergen, Alternaria alternata, we found significantly fewer eosinophils peripherally and in the respiratory tracts of EPX-/- mice compared to wild-type controls; furthermore, both the major population (Gr1-/lo ) and the smaller population of Gr1hi eosinophils from EPX-/- mice displayed lower median fluorescence intensities (MFIs) for Siglec F. Quantitative evaluation of transmission electron micrographs of lung eosinophils confirmed the relative reduction in granule outer matrix volume in cells from the EPX-/- mice, a finding analogous to that observed in human EPX deficiency. Despite the reduced size of the granule matrix, the cytokine content of eosinophils isolated from allergen-challenged EPX-/- and wild-type mice were largely comparable to one another, although the EPX-/- eosinophils contained reduced concentrations of IL-3. Other distinguishing features of lung eosinophils from allergen-challenged EPX-/- mice included a reduced fraction of surface TLR4+ cells and reduced MFI for NOD1. Interestingly, the EPX gene deletion had no impact on eosinophil-mediated clearance of gram-negative Haemophilus influenzae from the airways. As such, although no clinical findings have been associated with human EPX deficiency, our findings suggest that further evaluation for alterations in eosinophil structure and function may be warranted.

Frontline Science: Eosinophil-deficient MBP-1 and EPX double-knockout mice link pulmonary remodeling and airway dysfunction with type 2 inflammation.

Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1-/-/EPX-/- mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1-/-/EPX-/- mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1-/-/EPX-/- mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1-/-/EPX-/- mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.

SiglecF+Gr1hi eosinophils are a distinct subpopulation within the lungs of allergen-challenged mice.

Although eosinophils as a group are readily identified by their unique morphology and staining properties, flow cytometry provides an important means for identification of subgroups based on differential expression of distinct surface Ags. Here, we characterize an eosinophil subpopulation defined by high levels of expression of the neutrophil Ag Gr1 (CD45+CD11c-SiglecF+Gr1hi). SiglecF+Gr1hi eosinophils, distinct from the canonical SiglecF+Gr1- eosinophil population, were detected in allergen-challenged wild-type and granule protein-deficient (EPX-/- and MBP-1-/-) mice, but not in the eosinophil-deficient ΔdblGATA strain. In contrast to Gr1+ neutrophils, which express both cross-reacting Ags Ly6C and Ly6G, SiglecF+Gr1hi eosinophils from allergen-challenged lung tissue are uniquely Ly6G+ Although indistinguishable from the more-numerous SiglecF+Gr1- eosinophils under light microscopy, FACS-isolated populations revealed prominent differences in cytokine contents. The lymphocyte-targeting cytokines CXCL13 and IL-27 were identified only in the SiglecF+Gr1hi eosinophil population (at 3.9 and 4.8 pg/106 cells, respectively), as was the prominent proinflammatory mediator IL-13 (72 pg/106 cells). Interestingly, bone marrow-derived (SiglecF+), cultured eosinophils include a more substantial Gr1+ subpopulation (∼50%); Gr1+ bmEos includes primarily a single Ly6C+ and a smaller, double-positive (Ly6C+Ly6G+) population. Taken together, our findings characterize a distinct SiglecF+Gr1hi eosinophil subset in lungs of allergen-challenged, wild-type and granule protein-deficient mice. SiglecF+Gr1hi eosinophils from wild-type mice maintain a distinct subset of cytokines, including those active on B and T lymphocytes. These cytokines may facilitate eosinophil-mediated immunomodulatory responses in the allergen-challenged lung as well as in other distinct microenvironments.

High vascular endothelial growth factor levels in NZW mice do not correlate with collagen deposition in allergic asthma.

BACKGROUND: Eosinophils contribute to the early features of allergic lung inflammation through the generation and release of a plethora of mediators. Eosinophil peroxidase (EPO) is one of the eosinophil granule proteins involved in the early response, but its participation in airway remodeling is not established. The present study addressed this question comparing an EPO-deficient mouse strain (NZW) with BALB/c and C57Bl/c strains. METHODS: Mice were immunized with ovalbumin/alum, challenged twice with ovalbumin aerosol, and lung responses were measured at day 22 or 28. Collagen, mucus and eosinophils were determined in lung sections stained with picrosirius, periodic acid-Schiff or hematoxylin-eosin; transforming growth factor-beta and vascular endothelial growth factor were determined by ELISA, lipid bodies by enumeration in osmium-stained eosinophils, and airway reactivity to methacholine in isolated lung preparations. RESULTS: NZW mice showed significantly less collagen around bronchi and blood vessels, less mucus and less eosinophils around bronchi. Eosinophil lipid body formation and airway hyperreactivity were comparable among strains. Levels of transforming growth factor-beta were also comparable; however, the NZW mice showed much higher levels of vascular endothelial growth factor, even under basal conditions. CONCLUSIONS: In allergic lung inflammation, the combination of EPO deficiency and overexpression of VEGF found in NZW mice is associated with less collagen deposition, less mucus and reduced tissue eosinophilia. Eosinophil activation and airway hyperreactivity in NZW mice were similar to the other strains.

Lack of eosinophil peroxidase or major basic protein impairs defense against murine filarial infection.

Eosinophils are a hallmark of allergic diseases and helminth infection, yet direct evidence for killing of helminth parasites by their toxic granule products exists only in vitro. We investigated the in vivo roles of the eosinophil granule proteins eosinophil peroxidase (EPO) and major basic protein 1 (MBP) during infection with the rodent filaria Litomosoides sigmodontis. Mice deficient for either EPO or MBP on the 129/SvJ background developed significantly higher worm burdens than wild-type mice. Furthermore, the data indicate that EPO or MBP is involved in modulating the immune response leading to altered cytokine production during infection. Thus, in the absence of MBP, mice showed increased interleukin-10 (IL-10) production after stimulation of macrophages from the thoracic cavity where the worms reside. In addition to elevated IL-10 levels, EPO(-/-) mice displayed strongly increased amounts of the Th2 cytokine IL-5 by CD4 T cells as well as a significantly higher eosinophilia. Interestingly, a reduced ability to produce IL-4 in the knockout strains could even be seen in noninfected mice, arguing for different innate propensities to react with a Th2 response in the absence of either EPO or MBP. In conclusion, both of the eosinophil granule products MBP and EPO are part of the defense mechanism against filarial parasites. These data suggest a hitherto unknown interaction between eosinophil granule proteins, defense against filarial nematodes, and cytokine responses of macrophages and CD4 T cells.