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

Eosinophil-deficient MBP-1 and EPX double-knockout mice show how eosinophils are necessary for increased IL-4/IL-13 expression linked with acute and chronic pulmonary inflammation.

Abstract

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.

Introduction

The unresolved and potentially underappreciated roles of eosinophils in allergic respiratory disease have been driving forces toward the creation of disease-management approaches for patients that efficaciously target these cells (reviewed in [1, 2]). This unresolved character of eosinophil-mediated events is likely a consequence of the wider array of eosinophil activities recently identified in several studies. Specifically, in addition to tissue-damaging activities (reviewed in [3]), eosinophils appear to be important regulatory cells capable of modulating pulmonary immune responses and remodeling/repair events in the lung [4–7]. In particular, studies using the available congenitally eosinophil-deficient strains of mice, PHIL [8] and ΔdblGATA [9], and the inducible eosinophil-deficient strain, iPHIL [10], have each suggested that pulmonary eosinophils appear to mediate a variety of immune responses and/or events, including T effector cell recruitment [11, 12], the polarization of immune responses by suppression of Th1/Th17 pathways [13], and the expression of Th2-associated cytokines and chemokines [14, 15]. Despite these data, issues of concern have surrounded eosinophil-deficient strains of mice and may have led to differences in reported data (see e.g., [16–18]), obscuring the definition of eosinophil effector functions and their role in health and disease.

Our studies of eosinophil granule proteins and their roles as eosinophil-derived mediators of inflammation have led to the creation of knockout mice deficient in each of the abundant granule proteins, MBP-1 [19] and EPX [20]. Surprisingly, the singular loss of each of those granule proteins had no demonstrable effects on the pulmonary changes linked with acute allergen sensitization/airway challenge. Our subsequent attempts to breed those knockout mice led to the discovery that the eosinophilopoietic capacity of mice was absolutely dependent on the expression of both of those abundant granule protein genes. That is, double-knockout mice deficient in both MBP-1 and EPX (i.e., MBP-1^−/−/EPX^−/− mice) are eosinophil deficient with no observable effects on any of the other leukocyte lineages [21]. We further showed in that study that the dependency of eosinophilopoiesis on granule biogenesis was mechanistically the result of targeting the survival of eosinophil marrow progenitors. Moreover, recent studies by other groups support our linkage of eosinophil production with the expression of granule constituents [22, 23]. Thus, the availability of MBP-1^−/−/EPX^−/− mice offers an important alternative model system with which to define eosinophil-mediated events. In particular, the novel, multigene targeting of eosinophils in this strain that results in the specific loss of eosinophils is accomplished without confounding issues, such as the expression of cytotoxins [17] or altering the expression of transcription factors in multiple leukocyte lineages [16]. We capitalized on the availability of those double-knockout mice to reexamine and further define the role of pulmonary eosinophils in both an acute setting after allergen provocation and a chronic type 2–driven setting resulting from the ectopic expression of the eosinophil agonist cytokine IL-5 and chemokine eotaxin-2 (I5/hE2 mice [24, 25]). These data confirmed that the presence of pulmonary eosinophils was necessary for the induction of airway type 2 cytokine expression (i.e., IL-4 and IL-13) in the lungs of mice that, in turn, appeared to be responsible for the inflammatory changes and lung dysfunction that occurred after acute allergen challenge of eosinophil-sufficient WT mice. Moreover, our data with the I5/hE2 chronic type 2 inflammatory model showed that the selective targeting of eosinophils occurring in MBP-1^−/−/EPX^−/− mice reduced pulmonary fibrosis and all but eliminated the goblet cell metaplasia occurring in that model. Collectively, these data support the hypothesis that eosinophils are important regulatory cells that modulate the immune responses and remodeling/repair events in the lung and highlight underappreciated activities contributing to the pulmonary pathologies linked with allergic inflammatory settings.

MATERIALS AND METHODS

Mice

WT, ∆dblGATA [26], PHIL [8], MBP-1^−/− [19], EPX^−/− [20], and MBP-1^−/−/EPX^−/− [21] (each on a C57BL/6J background >20 generations) were used for assessment of baseline eosinophil numbers in bone marrow and peripheral blood. The effects of pulmonary allergic inflammation were studied in 8–16-wk-old WT and MBP-1^−/−/EPX^−/− mice. Double-transgenic I5/hE2 mice [24], as well as I5/hE2 mice crossed with MBP-1^−/−/EPX^−/− mice (8–12 wk old) were used as part of studies evaluating the role(s) of eosinophils in the pathologies that develop in a chronic, severe, type 2 inflammatory setting. Mice in this study were housed in ventilated microisolator cages in the specific pathogen-free animal facility at the Mayo Clinic Arizona (Scottsdale, AZ, USA). All experimental manipulations and studies involving animals were performed in accordance with the U.S. National Institutes of Health (Bethesda, MD, USA) and Mayo Foundation (Rochester, MN, USA) institutional guidelines.

Induction of allergic airway inflammation

Mice were sensitized and challenged with chicken OVA, as previously described [24]. Mice were assessed for pulmonary/airway cellular infiltrates, eosinophil degranulation, histopathologies, and lung function on d 28 (i.e., 2 d after the last OVA challenge) in this protocol.

Histopathologic evaluations

Formalin-fixed, paraffin-embedded lungs were sectioned and stained with H&E for general morphology and global assessments of inflammation (i.e., qualitative assessments of cellular infiltrates and gross morphologic changes). Masson’s Trichrome was used to visualize collagen deposition. In some cases, the extent of the collagen deposition among the groups of mice examined was quantified with Picrosirius red–stained slides imaged in polarized light [25]. Briefly, under polarized light, the submucosal, interstitial area surrounding all central airways within the entire pulmonary parenchyma represented in coronal lung sections from n = 7–11 mice/group was evaluated at a magnification of ×200 (i.e., 6–21 central airways/section, evaluating all airways ≤0.7 mm in diameter). The extent and intensity of Picrosirius red staining was quantified as the sum total (Σ) of pixel values (Pixel number × Average pixel intensity within the region surrounding the basement membrane of a given central airway), normalized to the total length of central airway basement membrane evaluated.

PAS was used to visualize and quantify airway epithelial cell mucin accumulation. Specifically, mucin quantifications inside airway epithelial cells were performed according to a previously published algorithm [27] and expressed as a Mucus index. All evaluations were performed in duplicate as independent, observer-blinded assessments. Image analyses were performed with ImagePro Plus software (Media Cybernetics, Rockville, MD, USA) and ImageJ (U.S. National Institutes of health, https://imagej.nih.gov/ij/).

Lung function assessments

Assessments of baseline airway resistance and airway hyperresponsiveness to a nonspecific agonist were performed with a forced ventilation technique (FlexiVent, SCIREQ, Montreal, QC, Canada) as previously described [8]. Briefly, mice were treated with pentobarbital (90 µg/g of body weight; Nembutal sodium solution; Oak Pharmaceuticals, Lake Forest, IL, USA) and pancuronium bromide (0.5 µg/g of body weight; Sigma-Aldrich, St. Louis, MO, USA), intubated, and after baseline airflow assessments, subjected to increasing doses of aerosolized methacholine (Sigma-Aldrich). A single-frequency “SnapShot-150” maneuver was used to obtain values of airway dynamic resistance that fit a single-compartment model. The peak responses were used to create the methacholine dose–response curves presented.

Assessment of blood, tissue, and airway eosinophilia

Bone marrow brush smears, peripheral blood films, and cytospins were prepared as previously described [28]. Hematologic evaluations were performed with slides stained with a Romanowsky dye set (Diff-Quik; Siemens, Munich, Germany) to visualize eosinophils based on dye staining and nuclear morphology. Eosinophils were also visualized and quantified by immunohistochemistry with a rat anti-mouse eosinophil-associated ribonuclease mAb (MT3-25.1.1) that recognizes mouse eosinophil-associated ribonucleases Ear-1, -2, -6/7, and -5/11 [21]. Sections were counterstained with methyl green to highlight tissue architecture. Flow cytometric assessments of eosinophils were performed on a BD LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with the FlowJo software package (Tree Star, Ashland, OR, USA) with cells stained with CCR3-FITC (R&D Systems, Minneapolis, MN, USA) and Siglec-F-PE (Becton Dickinson, Franklin Lakes, NJ, USA), after a pregating strategy of live granulocytes (Molecular Probes Live/Dead Fixable Aqua-staining; Thermo Fisher Scientific, Waltham, MA, USA) and FSC and SSC scatter patterns. Total cellularity and the number of eosinophils present were determined with manual cell counts using a hemocytometer (Thermo Fisher Scientific) followed by FACS assessments of the percentage of recovered leukocytes.

Cytokine measurements

IL-4, IL-13, IFN-γ, and IL-17 were measured in BAL fluid by sandwich ELISA (R&D Systems, Minneapolis, MN, USA), per the manufacturer’s instructions.

Statistical analysis

Data were analyzed and plotted using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). ANOVA and Tukey’s method were used for multiple group comparisons. The differences among means were considered significant at P < 0.05. The results were plotted as means ± sem.

RESULTS

Baseline eosinophil levels in MBP-1^−/−/EPX^−/− mice were comparable to congenitally eosinophil-deficient mice

We have previously demonstrated that the concurrent loss of MBP-1 and EPX disrupts eosinophilopoiesis through a mechanism promoting the turnover of lineage-committed progenitors [21]. The presence of eosinophils at homeostatic baseline in both the bone marrow and peripheral circulation of MBP-1^−/−/EPX^−/− mice was put into context by concurrently performing similar assessments in WT and congenitally eosinophil-deficient PHIL [8] and ΔdblGATA [9] mice. Romanowsky-stained slide preparations revealed that eosinophils and their progenitors were abundantly present and readily visible in the bone marrow of WT mice (Fig. 1A). However, the concurrent loss of MBP-1 and EPX in double-knockout mice represented an obstacle to that staining and the unambiguous identification of eosinophils. That is, the unambiguous identification is difficult to accomplish because the abundant granule proteins are the bulk of the cationic characteristics of the granules, which is responsible for the eosin staining of those granulocytes with Romanowsky-based dye sets. That same loss of granule proteins also prevented using either of the gold standard granule protein Abs available to detect tissue-infiltrating eosinophils, anti-MBP-1 [8]m and anti-EPX [29]. Thus, stained marrow preparations appeared to show that, similar to congenitally eosinophil-deficient strains of mice (i.e., PHIL and ΔdblGATA), MBP-1^−/−/EPX^−/− mice were also deficient in eosinophil-lineage–committed cells. Our solution to these logistical issues was the use of yet another granule protein-specific mAb we created, which recognizes the collective group of eosinophil-associated ribonucleases (i.e., Ears [30]). Those data confirmed our classic hematologic assessments in a more direct fashion by immunohistochemical staining of femoral bone marrow sections; eosinophil-lineage–committed cells represented 8–12% of the hematopoietic compartment cells [28]. The bone marrow from the two congenitally eosinophil-deficient strains (i.e., PHIL and ΔdblGATA) demonstrated that they were generally devoid of eosinophils and their progenitors (Fig. 1A). Interestingly, the eosinophil-deficiency, mediated by the loss of MBP-1 and EPX gene expression (i.e., MBP-1^−/−/EPX^−/−), resulted in a similar, but less absolute, ablation of eosinophils from the marrow compartment relative to the established eosinophil-deficient strains of mice (Fig. 1A). However, the severe character of the targeting of eosinophils and their progenitors in all 3 mouse models (i.e., PHIL [>98%], ΔdblGATA [>98%], and MBP-1^−/−/EPX^−/− [>95%]) resulted in the virtual loss of peripheral, circulating eosinophils. That is, each strain displayed an ablation of peripheral blood at the baseline, with no observable effect on the total white blood cell counts relative to that of the WT mice (Fig. 1B).

Figure 1. The concurrent loss of MBP-1 and EPX expression significantly reduces the number of eosinophil-lineage–committed marrow progenitors and nearly eliminates terminally differentiated, peripheral blood eosinophils.

(A) The upper panels are brush smear preparations of bone marrow leukocytes from mice of each genotype to determine the presence or absence of eosinophil-lineage–committed progenitors/cell (arrows). Scale bar of the main photomicrographs in each panel = 50 µm. Scale bar of the inset photomicrographs in each panel = 20 µm. The lower panels are photomicrographs of femur sections from mice of each genotype examined after immunohistochemical staining (dark staining cells) using a rat mAb (MT3-25.1.1) that recognized the eosinophil-associated granule ribonucleases Ear-1, -2, -6/7, and -5/11. The negative control was rat normal serum IgG. Scale bar = 50 µm. (B) Cell counts and hematologic cell differentials of peripheral blood films showed that MBP-1^−/−/EPX^−/− mice have an eosinophil deficiency (without effects on the composition of the other prominent leukocytes, which was equivalent to the deficiency observed in the established, engineered strains of mice congenitally deficient in eosinophils (i.e., PHIL [8] and ∆dblGATA [9] mice). The data presented are expressed as means ± sem; n = 6–9 mice/genotype.

MBP-1^−/−/EPX^−/− mice failed to mount the pulmonary tissue eosinophil infiltrate and airway eosinophilia characteristics of acute allergen sensitization/challenge in WT mice

MBP-1^−/−/EPX^−/− mice were subjected to an established OVA acute sensitization/airway challenge protocol [24] to determine whether the immune responses linked with an allergen sensitization/challenge provided additional hematopoietic pressure that would overcome the blockade of eosinophilopoiesis observed in MBP-1^−/−/EPX^−/− mice. That determination was initially addressed by immunohistochemical staining of lung sections with our anti-Ear antibody after the OVA challenge of sensitized mice. Those data showed that the hematopoietic pressures mediated by an allergen challenge (saline-sensitized mice were used as negative controls) were unable to overcome the disruption of eosinophilopoiesis in MBP-1^−/−/EPX^−/− mice. MBP-1^−/−/EPX^−/− mice did not develop a robust, peribronchial and perivascular inflammatory cell infiltrate after OVA sensitization/airway challenge (Fig. 2A). Immunohistochemistry using our anti-Ear mAb unambiguously demonstrated the loss of lung tissue–infiltrating eosinophils. That loss of eosinophils extended to the airways in which flow cytometric assessments of BAL of MBP-1^−/−/EPX^−/− mice displayed a significant loss of airway cellularity (Fig. 2B), which again resulted from a ≥97% decrease in FSC^med/SSC^hi/CCR3⁺/Siglec-F⁺ [21] BAL eosinophils relative to the airways of WT mice after OVA challenge (Fig. 2C).

Figure 2. MBP-1^−/−/EPX^−/− mice fail to develop pulmonary eosinophilia in response to an acute OVA airway challenge.

(A) Midcoronal lung sections from OVA-sensitized/challenged WT and MBP-1^−/−/EPX^−/− mice (control animals of each genotype were challenged with saline alone) were stained to determine the eosinophil dependency of the induced lung pathologies occurring in the model: 1) the upper panels are H&E-stained lung sections. Scale bar = 100 µm, and 2) The lower panels are photomicrographs of lung sections from mice of each genotype examined after immunohistochemical staining (dark staining cells) using a rat anti-Ear mAb (MT3-25.1.1). The negative control was rat normal serum IgG. Scale bar = 100 µm. Flow cytometric assessments of total BAL cellularity (B) and eosinophil levels (FSC^med/SSC^hi/CCR3⁺/Siglec-F⁺ cells) (C). The data presented are expressed as means ± sem (n = 4–8.). *P < 0.05.

Loss of lung eosinophils in MBP-1^−/−/EPX^−/− mice is linked to a reduction of allergen-induced histopathologies and a concomitant reduction of pulmonary type 2 immune responses

H&E-stained lung sections showed that the absence of eosinophils in MBP-1^−/−/EPX^−/− mice led to significant decreases in the gross pathologic changes associated with allergic respiratory inflammation, including induced airway epithelial hypertrophy and expansion of resident pulmonary inflammatory cell infiltrates (Fig. 2A). The loss of induced inflammatory changes in MBP-1^−/−/EPX^−/− mice also extended to the development of GM/MA. PAS staining of lung sections showed significantly lower levels of allergen-induced goblet cell metaplasia in MBP-1^−/−/EPX^−/− mice relative to the OVA-challenged, WT mice (Fig. 3A). In particular, a quantitative assessment of GM/MA in these mice showed that the loss of eosinophils in MBP-1^−/−/EPX^−/− mice resulted in an ∼60% decrease in the GM/MA relative to OVA-sensitized/airway-challenged, WT animals (Fig. 3A). The loss of GM/MA in MBP-1^−/−/EPX^−/− mice was also accompanied by significant reductions in the allergen-induced type 2 cytokines most often linked with goblet metaplasia: IL-4 (Fig. 3B; 6.0 ± 1.0 pg/ml vs. 1.6 ± 0.3 pg/ml, respectively) and IL-13 (Fig. 3C; 104.4 ± 15.7 pg/ml vs. 39.4 ± 9.4 pg/ml, respectively). Cytokines associated with Th1 (e.g., IFN-γ) and Th17 (e.g., IL-17) were undetectable.

Figure 3. The loss of eosinophils in MBP-1^−/−/EPX^−/− mice significantly reduces OVA-induced goblet cell metaplasia/epithelial cell mucin accumulation (GM/MA) and the concomitant OVA-induced airway expression of IL-4 and IL-13.

(A) The GM/MA occurring in OVA-sensitized/aerosol-challenged MBP-1^−/−/EPX^−/− mice was assessed relative to WT mice (WT mice challenged with saline alone were used as a negative control group). Midcoronal lung sections were subjected to PAS staining (dark-staining central airway cells. Quantitative mucin content indices of each cohort of mice are displayed as means ± sem (12–16 animals/group). Scale bar = 100 µm. *P < 0.05. The eosinophil deficiency in MBP-1^−/−/EPX^−/− mice was also linked with concomitant changes in the induced expression of type 2 cytokines in the airways of OVA-sensitized/aerosol-challenged mice with significant decreases in both IL-4 (B) and IL-13 (C) BAL levels. Dashed lines represent the limits of detection associated with each ELISA (IL-4, 2 pg/ml; IL-13, 1.5 pg/ml). Assessments of IFN-γ and IL-17 remained at undetectable levels in both WT and MBP-1^−/−/EPX^−/− mice. The data are expressed as means ± sem (n = 3–5 mice). *P < 0.05.

Induced AHR in OVA-challenged mice is abolished in eosinophil-deficient MBP-1^−/−/EPX^−/− mice

Invasive measurements of lung function were performed to define any potential contribution of eosinophils to the methacholine-induced AHR that occurs after OVA airway challenge. Those data showed that, similar to what was observed with PHIL [8] and ΔdblGATA [9] mice, the loss of eosinophils in MBP-1^−/−/EPX^−/− mice abolished the allergen-induced AHR that occurred in eosinophil-sufficient, WT mice after OVA challenge (Fig. 4).

Figure 4. The eosinophil deficiency associated with MBP-1^−/−/EPX^−/− mice was associated with a significantly reduced allergen-induced methacholine response after allergen challenge.

Aerosolized methacholine-induced airway resistance was assessed with an invasive ventilator-based technique (FlexiVent; SCIREQ). C57BL/6J WT mice challenged with saline alone served as negative controls for these studies. In contrast to WT OVA-sensitized/airway-challenged mice, which displayed typical dose-dependent patterns of increased sensitivity/reactivity in response to methacholine provocation, those changes were abolished in OVA-treated MBP-1^−/−/EPX^−/− mice. Those reductions in airway resistance were significant enough to reduce the dose-dependent responses of MBP-1^−/−/EPX^−/− mice to levels comparable to WT or MBP-1^−/−/EPX^−/− mice challenged with saline alone. The data are expressed as means ± sem (n = 3–6 mice). *P < 0.05.

Targeted deletion of EPX and MBP-1 is a sufficient eosinophilopoietic blockade preventing the induced pulmonary eosinophilia associated with constitutive IL-5 and eotaxin-2 overexpression in transgenic mice

The link between eosinophils, pulmonary inflammation, and type 2 cytokine expression was further defined by crossing MBP-1^−/−/EPX^−/− mice with a double-transgenic model of chronic type 2 pulmonary inflammation. Those mice (i.e., I5/hE2 [24, 25]) constitutively expressed IL-5 from all mature T cells and concurrently overexpressed eotaxin-2 from lung Clara (club) cells. In this model, the IL-5–mediated eosinophilopoietic pressure on the marrow of I5/hE2 mice was both significant and chronic [31]. Moreover, the robust, constitutive overexpression of eotaxin-2 from lung Clara (club) cells elicited and maintained a prominent pulmonary eosinophilia. Collectively, the ectopic overexpression of these eosinophil agonists promoted a significant lung phenotype, characterized by histopathologies, pulmonary dysfunction, and the induced expression of the type 2 cytokines IL-4 and IL-13 [25]. Moreover, earlier studies showed that all those pathologic manifestations were eosinophil dependent [24, 25]. Histologic assessments of lung sections from compound transgenic-gene knockout mice deficient in MBP-1 and EPX (i.e., I5/hE2/MBP-1^−/−/EPX^−/− mice) showed that even the constitutive overexpression of IL-5 and eotaxin-2 was unable to overcome the disruption of eosinophilopoiesis caused by the loss of MBP-1 and EPX (Fig. 5A). Specifically, H&E-stained sections of I5/hE2/MBP-1^−/−/EPX^−/− mice revealed the loss of massive proinflammatory infiltrates characteristic of the parental I5/hE2 model. The targeted effect on eosinophils in these mice was again shown by immunohistochemistry with our anti-Ear mAb, which demonstrated a profound loss of eosinophils in I5/hE2/MBP-1^−/−/EPX^−/− relative to that of the I5/hE2 mice (Fig. 5A). That targeting of tissue-infiltrating eosinophils also resulted in effects on the induced airway cellularity of I5/hE2/MBP-1^−/−/EPX^−/− mice, which displayed a significant decrease in total BAL cells (Fig. 5B) that accompany a 99% decrease in the induced airway eosinophilia (Fig. 5C).

Figure 5. The collapse of eosinophilopoiesis promoted by the concurrent loss of MBP-1 and EPX is a sufficient blockade to prevent the accumulation of pulmonary eosinophilia, even in the context of transgenic mice (I5/hE2) constitutively expressing eosinophil-agonist cytokines and chemokines.

(A) Midcoronal lung sections from the parental I5/hE2 transgenic model of chronic type 2 pulmonary inflammation were compared to sections recovered from compound transgenic-gene knockout mice deficient for MBP-1 and EPX (i.e., I5/hE2/MBP-1^−/−/EPX^−/−) with C57BL/6J mice serving as negative controls. The eosinophil dependency of the induced lung pathologies occurring in compound-transgenic mice relative to the parental I5/hE2 model was determined by staining sections via multiple venues: 1) the upper panels were stained with H&E for gross morphologic assessments; scale bar = 100 µm; and 2) the lower panels had immunohistochemical staining (dark red staining cells) with our rat anti-Ear mAb (MT3-25.1.1) to demonstrate the dramatic character of the eosinophil reduction occurring in I5/hE2/MBP-1^−/−/EPX^−/− mice relative to the parental I5/hE2 model; scale bar = 100 µm. Flow cytometric assessments of total BAL cellularity (B) and eosinophil levels (FSC^med/SSC^hi/CCR3⁺/Siglec-F⁺ cells) (C). The data presented are expressed as means ± sem (n = 4–7). *P < 0.05.

Eosinophil deficiency linked with I5/hE2/MBP-1^−/−/EPX^−/− mice is sufficient to abolish pulmonary pathologies and lung remodeling events that accompany induced, type 2 cytokine expression occurring in the lungs of the parental I5/hE2 model

H&E-stained lung sections (Fig. 5A) showed that, similar to targeting eosinophils in I5/hE2 mice through crosses with either of the congenital eosinophil-deficient strains of mice (i.e., I5/hE2/PHIL [24] or I5/hE2/ΔdblGATA [32]), the loss of eosinophils prompted by MBP-1 and EPX deficiency (i.e., I5/hE2/MBP-1^−/−/EPX^−/− mice) abolished the massive proinflammatory cell aggregates occurring in the parental I5/hE2 model. Moreover, those data showed that the MBP-1/EPX–dependent loss of eosinophils also attenuated the induced airway-remodeling events that occur in I5/hE2 mice, such as the hypertrophy of the central airway epithelium. The targeted effects on GM/MA in I5/hE2/MBP-1^−/−/EPX^−/− mice were also significant and indistinguishable from those we previously observed in eosinophil-deficient I5/hE2/PHIL mice [24]. PAS-stained lung sections from I5/hE2/MBP-1^−/−/EPX^−/− mice showed a near-complete loss of GM/MA that, when quantified, represented a nearly 90% (mucus index of 18.4 vs. 2.2, respectively) decrease relative to the parental I5/hE2 model (Fig. 6A). The loss of GM/MA in I5/hE2/MBP-1^−/−/EPX^−/− mice was accompanied by significant reductions in the induced, ectopic pulmonary expression of type 2 cytokines linked with goblet metaplasia that occurs in I5/hE2 animals. Specifically, the loss of eosinophils in I5/hE2/MBP-1^−/−/EPX^−/− mice, similar to the targeting of eosinophils in allergen-challenged MBP-1^−/−/EPX^−/− mice (see Fig. 3), resulted in significant decreases in the airway levels of both IL-4 (Fig. 6B; 212.2 ± 10.3 pg/ml vs. 16.0 ± 5.3 pg/ml, respectively) and IL-13 (Fig. 6C; 97.9 ± 8.1 pg/ml vs. 6.6 ± 2.1 pg/ml, respectively). Assessments of cytokines characteristic of Th1 (e.g., IFN-γ) and Th17 (e.g., IL-17) immune responses were undetectable in both I5/hE2/MBP-1^−/−/EPX^−/− mice and the parental I5/hE2 model.

Figure 6. The loss of eosinophils in compound I5/hE2 transgenic-gene knockout mice deficient in MBP-1 and EPX nearly abolishes OVA-induced GM/MA and the ectopic airway expression of IL-4 and IL-13 occurring in the parental I5/hE2 model.

(A) The GM/MA occurring in I5/hE2/MBP-1^−/−/EPX^−/− mice was assessed with PAS staining (dark staining central airway cells) relative to the parental I5/hE2 model (C57BL/6J mice were used as a negative control group). Mucin content indices are displayed as means ± sem (4–7 animals/group). All evaluations were performed as independent, observer-blinded assessments. Scale bar = 100 µm. *P < 0.05. The eosinophil deficiency induced by the loss of MBP-1 and EPX mice was also linked with concomitant changes in the induced expression of type 2 cytokines occurring in the parental I5/hE2 model with the near loss of BAL IL-4 (B) and IL-13 (C) levels. Dashed lines represent the limits of detection associated with each ELISA (IL-4, 2 pg/ml; IL-13, 1.5 pg/ml). Assessments of IFN-γ and IL-17 remained at undetectable levels in both I5/hE2/MBP-1^−/−/EPX^−/− mice and the parental I5/hE2 model. The data are expressed as means ± sem (n = 4–6 mice). *P < 0.05.

Assessments of the induced fibrosis (collagen-deposition) occurring in response to the loss of eosinophils in I5/hE2/MBP-1^−/−/EPX^−/− mice also paralleled previous observations of congenitally deficient I5/hE2/PHIL mice [25]. Namely, qualitative assessments of collagen deposition using MT-stained lung sections suggested that the loss of eosinophils in I5/hE2/MBP-1^−/−/EPX^−/− mice was accompanied by a decrease in pulmonary fibrosis relative to the parental I5/hE2 chronic inflammatory model (Fig. 7A). This suggestion was confirmed quantitatively by the evaluation of Picrosirius red–stained sections under polarized light (Fig. 7B), which revealed a significant decrease in airway collagen deposition in I5/hE2/MBP-1^−/−/EPX^−/− vs. I5/hE2 mice (213.6 ± 29.8 vs. 143.7 ± 14.3, respectively; P < 0.05).

Figure 7. The pulmonary fibrosis (i.e., airway collagen deposition) occurring in I5/hE2 mice was significantly reduced in eosinophil-deficient, compound-transgenic I5/hE2 mice deficient in MBP-1 and EPX.

(A) Representative airways from midcoronal lung sections stained with MT from C57BL/6J WT mice (negative controls) and age-matched (12 wk postpartum) I5/hE2 mice (positive controls) are shown in comparison to airways in similar sections from eosinophil-deficient I5/hE2/MBP-1^−/−/EPX^−/− mice. Qualitative assessments of collagen deposition (blue extracellular matrix staining) showed that the loss of eosinophils in I5/hE2/MBP-1^−/−/EPX^−/− mice appeared to accompany a decrease in the induced airway fibrosis occurring in the parental I5/hE2 model. Picrosirius red–stained slides from each group allowed a quantifiable, visual assessment of the decreased collagen deposition (red extracellular staining) occurring in eosinophil-deficient I5/hE2/MBP-1^−/−/EPX^−/− mice. Scale bars = 100 µm. B, bronchiole; PV, pulmonary vessel. (B) Quantitative assessments of Picrosirius red–stained lung sections demonstrated that the pulmonary fibrosis occurring in the parental I5/hE2 model was significantly reduced in I5/hE2 mice deficient in MBP-1 and EPX (I5/hE2/MBP-1^−/−/EPX^−/−). *P < 0.05.

Loss of eosinophils in I5/hE2/MBP-1^−/−/EPX^−/− mice abolishes spontaneous AHR occurring in the parental I5/hE2 model

Force-ventilator assessments of lung function demonstrated that, although allergen-naïve I5/hE2 mice displayed a unique methacholine-induced AHR, the loss of eosinophils occurring in I5/hE2/MBP-1^−/−/EPX^−/− mice abolished that AHR (Fig. 8). The ablation was virtually complete, reducing the methacholine dose-dependent curve to levels observed in allergen-naïve, WT control mice as well as eosinophil-deficient I5/hE2/PHIL mice [24]. Interestingly, I5/hE2/MBP-1^−/−/EPX^−/− mice also displayed a significant improvement in baseline airway resistance relative to the parental I5/hE2 model (0.70 ± 0.03 vs. 0.97 ± 0.08 cm H₂O × s/ml, respectively; P < 0.05), suggesting a role for eosinophil-mediated remodeling activities leading to fixed airway obstruction in chronic settings.

Figure 8. The dose-dependent airway sensitivity to methacholine provocation occurring in the chronic I5/hE2 inflammatory model was lost in eosinophil-deficient I5/hE2/MBP-1^−/−/EPX^−/− mice.

Aerosolized methacholine-induced airway resistance was assessed with an invasive ventilator-based technique (FlexiVent; SCIREQ). Unlike C57BL/6J WT controls, I5/hE2 mice displayed enhanced dose-dependent responses to aerosolized methacholine. However, the absence of an induced eosinophilia linked with the loss of MBP-1 and EPX (i.e., I5/hE2/MBP-1^−/−/EPX^−/− mice) eliminated all evidence of AHR, returning airway resistance as a function of methacholine dose to levels observed in C57BL/6J WT controls. Baseline airway resistance (cm H₂O × s/ml) of each experimental cohort (means ± sem): C57BL/6J WT, 0.87 ± 0.06; I5/hE2, 0.97 ± 0.08; I5/hE2/MBP-1^−/−/EPX^−/−, 0.07 ± 0.0.03. The data are expressed as means ± sem (n = 8–9 mice). *P < 0.05.

DISCUSSION

The first generations of congenitally eosinophil-deficient mice [8, 9] have been remarkable tools with which investigators have finally been able to begin the process of defining specific, eosinophil-mediated activities of functional significance. These mice have allowed “Koch postulates-like” studies to establish a causative relationship between eosinophils and a given disease setting, particularly their role(s) in asthma and other allergic diseases. Despite the value of these mice, some concerns have arisen that require caution when using these early models. For example, PHIL [8] mice have achieved their eosinophil-less character through the expression of diphtheria toxin. Although this strain of mice has been shown to uniquely target eosinophils with no demonstrable effects on other hematopoietic cell lineages, some have argued that the presence of diphtheria toxin may have cell-killing consequences beyond eosinophils that are not predictable on first principles [17]. ΔdblGATA mice achieved their eosinophil-less character by targeting an autoregulatory loop controlling the expression of a key transcription factor (i.e., GATA1). These mice have also been shown to specifically target eosinophil lineage–committed cells; however, the data in the initial article describing this strain have shown that other hematopoietic cells are affected, including erythroblasts and mast cells [26], and subsequent studies demonstrated effects on the number and activities of basophils [16]. The availability of eosinophil-deficient MBP-1^−/−/EPX^−/− mice [33] now offers a logistical solution to the short-comings of previous congenitally eosinophil-deficient models. That is, unlike PHIL, eosinophil deficiency is not achieved through the expression of a cytocidal protein and thus avoids potential confounding secondary effects mediated by the toxin. Moreover, the unique specificity of the eosinophil ablation in MBP-1^−/−/EPX^−/− mice is achieved by targeting 2 independent genes; both of which must be concurrently expressed in the same cell. In this case, although EPX expression is eosinophil specific [8], MBP-1 appears also to be expressed in basophils [34] and mast cells [35]. However, the only cells expressing both MBP and EPX are eosinophil-lineage–committed leukocytes. As a result, the eosinophil ablation in MBP-1^−/−/EPX^−/− mice arguably displays the greatest degree of eosinophil specificity of all the congenitally eosinophil-deficient strains of mice, with no direct and/or indirect effects on any other cell type [21]. In addition, similar to the earlier strains of eosinophil deficiency, the data here showed that MBP-1^−/−/EPX^−/− mice maintained their eosinophil-less character despite eosinophilopoietic pressures mediated by allergen sensitization/airway challenge or ectopic IL-5 overexpression, allowing their broad use in models of disease settings.

The studies presented here, using both acute allergen challenge with OVA and a chronic constitutive cytokine/chemokine transgenic model, each confirm the observations and now growing importance of the abilities of eosinophils to modulate the pulmonary immune microenvironment. Specifically, early studies using PHIL [8] and iPHIL [10] as well as ΔdblGATA [15] mice each showed that the loss of pulmonary IL-4 and IL-13 expression was a singularly prominent effect of losing eosinophils during an acute airway-challenge protocol. This underappreciated activity was further explored using eosinophil-adoptive transfer studies in the context of an allergen challenge of PHIL mice [13, 14] and through crosses of PHIL [24] and ΔdblGATA [32] with the I5/hE2 transgenic model of chronic type 2 pulmonary inflammation. Our studies of MBP-1^−/−/EPX^−/− mice confirm this very important observation, suggesting that, in addition to other known leukocytes that express IL-4 and/or IL-13 in the lung (e.g., ILC2s [36] and CD4⁺ T cells [37]), eosinophils directly or indirectly (through effects on “third-party” cells) contribute to the expression of pulmonary Th2 cytokines. Furthermore, that contribution appears to be important because the loss of eosinophils in OVA treatment of MBP-1^−/−/EPX^−/− mice and the cross of MBP-1^−/−/EPX^−/− with I5/hE2 mice both result in significant loss of the induced IL-4/-13 pulmonary expression occurring in these settings and, in turn, the loss of lung pathologies linked with the expression of these type 2 cytokines (e.g., AHR, GM/MA, and increased collagen deposition/fibrosis).

The use of both allergen challenge and genetic crosses of MBP-1^−/−/EPX^−/− mice with the I5/hE2 model was logistically important because it allowed us to define these events with more precision (i.e., mechanistically). For example, as was shown in many earlier studies ([38], reviewed in [39]) allergen challenge of WT mice induces an airway eosinophilia but without significant levels of eosinophil degranulation. However, although allergen-induced airway IL-13 expression and an accompanying AHR occurs in WT mice, the loss of eosinophils alone during allergen challenge of MBP-1^−/−/EPX^−/− mice significantly reduced both of those endpoint metrics. In contrast, I5/hE2 mice display airway IL-13 expression and AHR at levels comparable to OVA-sensitized/airway-challenged WT mice but in the presence of extensive eosinophil degranulation, suggesting that additional potential activities mediated by the release of granule proteins do not appear to contribute to, or further exacerbate, the induced IL-13 expression or AHR. The acute allergen challenge vs. I5/hE2 model data presented here, nevertheless, do support our hypothesis that the release of EPX (i.e., degranulation) is potentially a significant and underappreciated contributor to GM/MA [24]. Specifically, although OVA challenge of MBP-1^−/−/EPX^−/− mice (no airway eosinophilia) demonstrated a 60% decrease in GM/MA compared with OVA-treated, WT mice (airway eosinophilia with no evidence of significant degranulation), the difference in GM/MA between I5/hE2 (airway eosinophilia and extensive degranulation) and I5/hE2/MBP-1^−/−/EPX^−/− (no airway eosinophilia) mice was far greater, reaching a decrease of nearly 90%. Given that IL-13 levels in OVA-treated WT and I5/hE2 mice were virtually identical, this suggests that the release of granule proteins in the lungs of I5/hE2 mice enhances the GM/MA linked with IL-13 expression. Indeed, our recent studies demonstrating that the peroxidase activities of EPX in the lung elicit a posttranslational protein modification, known as carbamylation, provides a mechanism of the enhanced GM/M observed in I5/hE2 relative to I5/hE2/MBP-1^−/−/EPX^−/− mice [40]. Specifically, these new studies have shown that carbamylated proteins have direct agonist activities on airway epithelial cells, leading to goblet cell metaplasia and increased mucin gene expression. This conclusion is important because it may represent a significant eosinophil-mediated activity that enhances this IL-13–dependent event and thus may contribute to disease pathologies in settings in which degranulation occurs (e.g., in the airways of patients with allergic asthma).

In summary, the induced loss of eosinophils that occurs in the absence of MBP-1 and EPX has again demonstrated the significance of eosinophil-mediated immune regulatory activities in the lung as part of both acute and chronic respiratory inflammation. Moreover, the definitive specificity of the lineage ablation and the magnitude of eosinophil ablation occurring in MBP-1^−/−/EPX^−/− mice provide a unique model with which to explore the potential importance of eosinophils and eosinophil-mediated activities in health and disease.

AUTHORSHIP

N.A.L. had full access to all of the data reported in this study and had final responsibility for the decision to submit this report for publication. S.I.O., A.D.D., E.A.J., H.H.S., C.G.I., J.J.L., and N.A.L. designed the research study; S.I.O., A.D.D., E.A.J., W.E.L., W.L., C.A.P., K.R.Z., and D.C. performed the research presented; S.I.O., A.D.D., E.A.J., W.L., H.H.S., C.G.I., J.J.L., and N.A.L. analyzed the data; S.I.O., J.J.L., and N.A.L. wrote the initial draft of the manuscript, and S.I.O., E.A.J., A.D.D., H.H.S., C.G.I., J.J.L., and N.A.L. provided critical assessments during the revision process leading to the final submitted manuscript.

Glossary

ΔdblGATA

eosinophil-deficient gene knockout mice modulating expression of the transcription factor GATA-1 through the deletion of a double GATA binding site in the promoter of the GATA1 gene

AHR

airway hyperresponsiveness

BAL

bronchoalveolar lavage

EPX

eosinophil peroxidase

EPX^−/−

eosinophil peroxidase gene knockout mice

FSC

forward light scatter

GM/MA

goblet cell metaplasia/epithelial cell mucin accumulation

I5/hE2

double-transgenic strain constitutively overexpressing IL-5 from T cells and eotaxin-2 from Clara (club) cells

I5/hE2/MBP-1^−/−/EPX^−/−

IL-5/hEotaxin-2 transgenic mice deficient in MBP-1 and EPX

MBP-1

major basic protein-1

MBP-1^−/−

major basic protein-1 gene knockout mice

MBP-1^−/−/EPX^−/−

double-knockout mice deficient in MBP-1 and EPX

MT

Masson’s trichrome

OVA

ovalbumin

PAS

periodic acid–Schiff

PHIL

eosinophil-deficient transgenic mice expressing diphtheria toxin via cis-regulatory sequences from the EPX locus

SSC

side scatter of light

WT

wild-type

DISCLOSURES

The authors declare no conflicts of interest.