An essential role for the Th2-type response in limiting tissue damage during helminth infection

Kinetics of Nb larvae-induced lung hemorrhaging and inflammation

To examine the association between lung damage and the innate immune response, BALB/c mice were inoculated with 500 Nb infective L3 treated with antibiotics to remove residual bacteria. Larvae enter the lung between 24 and 48 hours after inoculation, reside in the lung for 36–48 hours, and migrate to the small intestine by day 3²⁸. At days 2, 4, and 7 after inoculation, lung tissue and bronchial-alveolar lavage (BAL) fluid were collected. Increased RBCs, reflecting extensive hemorrhaging, were observed at day 2 after inoculation, but by day 4 hemorrhaging was reduced and by day 7 undetectable (Fig. 1a). Histologic quantitation of lung inflammation, as assessed using the acute lung injury (ALI) scoring method^33,34, showed ALI by day 2, which was resolved by day 7. Inflammation, primarily by neutrophils, was also pronounced at day 2, reduced by day 4, and at baseline by day 7 (Fig. 1b). Analysis of BAL also showed increased RBCs by day 2 after inoculation, which were reduced by day 7 (Fig. 1c). The correlation of RBCs in the BAL with overall increases in RBCs in lung tissue samples provided a useful quantitative indicator for assessing lung hemorrhaging by counting BAL RBCs. The BAL neutrophil count was also closely correlated with lung neutrophil inflammation (see Fig. 1b). To assess lung function, airway resistance was measured in conscious, spontaneously breathing mice using a whole-body plethysmography system. Enhanced pause was increased at day two, but returned to near normal levels at days 4 and 7 (Fig. 1d). Thus, shortly after Nb inoculation ALI occurs, associated with hemorrhaging, inflammation, and decreased lung function, and the lung injury is quickly resolved.

Lung tissue Il4 and Il13 mRNA were increased by day 4 after inoculation and remained high at day 7 (Fig. 1e). Unexpectedly, Il17 mRNA was also elevated by day 2, reaching peak levels at day 4 and returning to baseline by day 7. Elevations in Ifng were not detected (data not shown). Elevated BAL IL-4, IL-13, and IL-17 protein were also detected. Gene expression of a panel of factors associated with wound healing showed elevations in Arginase 1 (Arg1), insulin-like growth factor 1 (Igf1), and collagen as early as 2 days, while matrix metalloprotease 13 (Mmp13) elevations were detected by day 7 (Supplementary Fig. 1). Th2-type innate cytokines were also examined with elevations in Tslp and Il33, but not Il25 mRNA, by day 2 after inoculation (Supplementary Fig. 1).

Mice were also inoculated with different doses of Nb and decreases in BAL-RBCs, BAL and lung neutrophils, and ALI were observed at lower doses (Supplementary Fig. 2). However, increases in Il13, Arg1, and Il17 mRNA were generally sustained, indicating that parasites, probably through tissue damage to lung tissue during migration, were contributing directly to lung damage.

IL-17 dependent neutrophil recruitment to the lung contributed to acute hemorrhaging

To examine whether lung infiltration of neutrophils enhanced hemorrhaging, neutrophils were depleted by in vivo administration of Ly6G-specific Ab., which reduced RBCs (Fig. 2a) and ALI (Supplementary Fig. 3a). However, neutrophil depletion had no effect on bleeding time indicating that blood coagulation was not decreased (Supplementary Fig. 4 d), as observed in other model systems³⁵. As expected, neutrophils, identified morphologically, were also depleted in the BAL following Ly6G-specific Ab treatment (Fig. 2a) and in the lung as determined by FACS (Supplementary Fig. 4a).

As IL-17 was rapidly elevated after inoculation and as IL-17 can mediate neutrophil recruitment^13,14, we hypothesized that IL-17 may contribute to lung damage through neutrophil recruitment. Lung hemorrhaging and neutrophil recruitment were markedly decreased in BAL of Il17ra^−/− mice compared to WT mice at day 2 after inoculation (Fig. 2a) and the ALI score was significantly reduced (Supplementary Fig. 3b). As IL-17RA is required for IL-25 (IL-17E) as well as IL-17A signaling³⁶, we also used blocking IL-17A-specific antibody. BAL neutrophils and RBCs were significantly decreased (Fig. 2a), as was ALI (Supplementary Fig. 3c) and lung neutrophils (Supplementary Fig. 4c) in Nb-inoculated mice administered IL-17A-specific antibody.

IL-4Rα signaling enhanced resolution of lung damage and promoted factors mediating wound healing and control of inflammation

To assess whether Nb-induced hemorrhaging and influx of neutrophils to the lung were influenced by the Th2-type response, BALB/c Il4ra^−/− and WT mice were inoculated with Nb. Increased BAL RBCs and neutrophil infiltration were observed at day 4 after inoculation of Il4ra^−/− mice (Fig. 2b). Similarly, blockade of IL-4Rα signaling resulted in increased ALI and enhanced airway resistance (Supplementary Fig. 3 d, f). Nb-inoculated Il4^−/− and Il13ra1^−/− mice exhibited no increase in either hemorrhaging or inflammation (Fig. 2b), suggesting that either IL-4 or IL-13 alone can mediate control of hemorrhaging and inflammation through IL-4Rα signaling. No difference was detected in the larvae intestinal count between Il4ra^−/− mice and WT controls at day 4 after Nb inoculation (Fig. 2c), indicating that larval migration was not affected by IL-4Rα signaling blockade. Il4, Il13, and Il10 gene expression were also markedly reduced in BALB/c Il4ra^−/− mice compared to BALB/c WT controls at day 4 after inoculation, while IL-17 was markedly elevated (Fig. 2d).

The increased IL-17 in Nb-inoculated Il4ra^−/− mice suggested that elevated IL-17 contributed to the increased lung injury. Nb-inoculated Il4ra^−/− mice were administered blocking IL-17A-specific antibody, resulting in markedly reduced BAL hemorrhaging and BAL inflammation (Fig. 2e), and ALI (Supplementary Fig. 3e). These findings indicate that IL-4Rα signaling controls lung injury at least partly through down modulating IL-17 activity.

Rapid resolution of Nb-induced hemorrhaging in WT mice suggested that the Th2-type response may promote factors that mediate wound healing. IGF-1, a member of the insulin family of growth hormones, has been shown to be expressed by a number of cell types including T cells, macrophages, and epithelial cells, and recent in vitro studies suggested that it may contribute to localized wound healing effects ^37–39 Arg1 may also be expressed by macrophages and can promote collagen formation and cellular proliferation⁴⁰. Elevations in Igf1 and Arg1 gene expression were blocked in Il4ra^−/− mice at day 4 after Nb inoculation, consistent with their expression being dependent on the Th2-type response (Fig. 3a). In addition, elevations in Collagen1 and Mmp13, both important in regulation of tissue regeneration, were inhibited in Nb-inoculated Il4ra^−/− mice.

Blocking IGF-1Rspecific Ab treatment increased both BAL RBCs and neutrophils (Fig. 3b) and increased ALI (Supplementary Fig. 5a) on day 4 after inoculation with Nb, suggesting that IGF-1 contributed to control of both hemorrhaging and inflammation. Increases in Il4 and Il13 were sustained in Nb-inoculated mice administered IGF-1R-specific Abs, indicating that IGF-1 does not affect expression of Th2 cytokines, while Il17 levels were not significantly elevated (Fig. 3c). Other wound healing factors were not affected by IGF-1R-specific antibody treatment (Fig. 3c). Taken together, these findings suggest that IGF-1 is promoting repair of lung damage in vivo through mechanisms downstream of Th2 cytokine expression.

To examine whether the late increase in IL-10, relative to the earlier IL-4 and IL-13 elevations, also contributed to resolution of lung damage, Il10^−/− were inoculated with Nb. At both days 5 and 7 after inoculation, time points when IL-10 is elevated, significantly more hemorrhaging, neutrophil infiltration, and ALI was detected in Il10 ^−/− mice compared to WT controls (Fig. 4b and Supplementary Fig. 5b). Both Il4 and Il13 were partially suppressed at these later time points in Il10 deficient mice (Fig. 4c), suggesting that although IL-10 can enhance expression of these Th2 cytokines at days 5 and 7, IL-10-independent increases still occurred. In contrast, Il17, which had returned to baseline at these later time points in Nb-inoculated WT mice, was markedly increased in Nb-inoculated Il10 ^−/− mice. Modest elevations in Ifng also occurred, while Inos expression was not affected. Furthermore, elevations in wound healing factors, including Igf1, were not affected by Il10 deficiency. These data suggested that initial increases in IL-4R signaling triggers IL-10 expression, which plays an important role in resolving lung damage, by controlling lung inflammation. Unlike Il4ra deficiency, however, Il10 deficiency does not markedly affect expression of factors important in wound healing.

Macrophages control acute hemorrhaging and inflammation

Macrophages are potential sources of IL-10, IGF-1, MMP-13, and Arg1, all of which were elevated in the lung during the acute wound healing process. Macrophages in BAL (Supplementary Fig. 6a) and lung tissue (Fig. 5a), although present in untreated groups, were markedly increased by day 4 after Nb inoculation. Histological staining revealed numerous RBCs engulfed by individual macrophages at day 4 after inoculation suggesting an important role in RBC clearance. Macrophages were sorted from lung tissue at days 0, 3, and 7 after inoculation with Nb. Although IL-10 was not elevated, increases in Arg1, Igf1, and Mmp13 were detected (Supplementary Fig. 6b). Further cell sorting analyses revealed that IL-10 was elevated in CD4⁺ T cells (data not shown).

To examine whether macrophages were required for the rapid wound healing effect of the Th2-type response, transgenic mice expressing the human diptheria toxin receptor (DTR) gene under the control of the CD11b regulatory sequence were used to conditionally ablate macrophages in vivo through DT administration^41,42. Nb inoculated, CD11bDTR mice administered DT showed increased lung hemorrhaging and inflammation, while transfer of WT macrophages restored control of both hemorrhaging and inflammation, although some lung damage was still observed in whole lung samples (Fig. 5b–e). BAL and lung eosinophils were also depleted following DT administration, while add-back of WT macrophages restored eosinophil levels (Fig. 5c, Supplementary Fig. 6c) indicating that eosinophil recruitment was blocked by macrophage depletion not by nonspecific effects of DT treatment. Macrophage add-back also largely restored lung macrophages to levels of control groups not administered DT (Supplementary Fig. 6d). Arg1, Mmp13, and Igf1 mRNA were all reduced by DT administration of Nb inoculated CD11bDTR mice; macrophage add back largely restored these levels (Fig. 5e). To examine whether arginase contributed to the resolution of lung damage, the arginase 1 antagonist, BEC, was administered to Nb-inoculated WT mice, using doses previously described to block Arg1 activity in vivo^1,43. BEC administration reduced lung arginase activity (Supplementary Fig. 5d) and resulted in increased BAL RBCs and neutrophils (Fig. 5f) and also ALI (Supplementary Fig. 5c), at day 4 after Nb inoculation. Liposome- encapsulated dichloro-methylene-diphosphonate (clodronate) liposomes were used as an alternative approach to inactivate macrophages. Increases in BAL RBCs, BAL neutrophils and ALI, and decreased expression of factors associated with wound healing, were also observed in Nb-inoculated chlodronate liposome-treated mice (Supplementary Fig. 7).

Mice

Female 5–8 weeks old BALB/c mice were purchased from NCI. Il4ra^−/−, Il4^−/− and Il10^−/− BALB/c mice, and CD11bDTR C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Il13ra1^−/− BALB/c mice, were provided by Dr. Thomas Wynn (NIAID, Bethesda, MD). Il17ra^−/− BALB/c mice were from Amgen (Thousand Oaks, CA). All mice were maintained in a specific pathogen-free, virus Ab-free facility during the experiments. The experiments herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, Department of Health, Education and Welfare (US National Institutes of Health).

Parasite infection, antibody, and inhibitor administration

Nb L3 were incubated with 400U penicillin, 400 μg ml⁻¹ streptomycin plus 400 μg ml⁻¹ Neomycin (GIBCO, Rockville, MD) for 2hrs at room temperature, and then washed with sterile PBS⁵⁶. For neutrophil depletion, Ly6G-specific-antibody (BioXcell, West Lebanon, NH) or isotype control IgG was administered to mice both i.p. (0.5 mg in 0.2 ml) and intratracheally (i.t.) (0.2 mg in 0.05 ml) one day before parasite inoculation, as described previously⁵⁷. For in vivo IL-17A neutralization, 17A-specific antibody (R&D systems, CA) was administered to mice both i.t.(30 μg) and i.v.(30 μg) on days −2, −1, 0, and i.v. 1 day after inoculation. IGF-1R-specific antibody (clone: IMC-A12, generously provided by ImClone Systems, NY) was administered to mice i.p. (1 mg in 0.2 ml) one day before inoculation with Nb. For arginase blockade, mice were provided with drinking water that contained 0.2% S-(2--boronoethyl)-L-cysteine (BEC) (Enzo Life Sciences, Farmingdale, NY) one day before parasite infection. Arginase bioactivity was measured using the Arginase Assay Kit (DARG-200) (BioAssay Systems, Hayward, CA). Clodronate-liposomes and PBS-liposomes were administered to mice both i.t. (0.1 ml) and i.p.(0.1 ml) on days −2, 0, +2, after Nb inoculation to deplete macrophages, as previously described^1,58.

Gene Expression by realtime PCR

RNA was extracted from lung tissue and then reverse transcribed to cDNA, as previously described². Quantitative fluorogenic Real-Time PCR was performed using Taqman^® (Applied Biosystems, Foster City, CA) kits and the Applied Biosystems 7700 sequence detector. All data were normalized to 18s ribosomal values and quantification of differences between treatment groups was calculated according to the manufacturer’s instructions. Gene expression is presented as the fold increase over naïve WT controls.

Bronchial-alveolar lavage (BAL)

BAL was collected from anesthetized mice and cell suspensions were prepared as previously described⁵⁹. RBCs were lysed with ACK lysing buffer (Lonza, MD) and differential counts performed on Giemsa-Wright (CAMCO) stained cytospin slides.

Airway responsiveness

Airway responsiveness (baseline measurement) was measured in mice using a whole-body plethysmography system (BuxcoResearch Systems, Wilmington, NC), as previously described⁵⁹. Readings of breathing parameters were taken for 2 min after nebulization during which Penh values were determined. The chamber-pressure-time wave was continuously measured via a transducer connected to a computer data-acquisition system.

Histology

Lungs were formalin-fixed, embedded in paraffin and 5μm sections stained with hematoxylin and eosin (H&E). Using previously described methods⁶⁰, ALI was scored, based on four parameters: alveolar capillary congestion; hemorrhage; infiltration or aggregation of neutrophils in the airspace or the vessel wall; thickness of the alveolar wall. Each parameter was graded from 0–4 based on the damage level (0, no or little damage; 1, less than 25% damage; 2, 25%–50% damage; 3, 50%–75% damage; 4, more than 75% damage). The degree of ALI was assessed by sum of scores of 4 parameters, and the average sum of each was compared among groups.

Flow cytometry

Lung tissue was minced and incubated with stirring at 37 °C for 30 min in Hank’s balanced salt solution (HBSS) with 1.3 mM EDTA (Invitrogen), followed by treatment at 37 °C for 1 hour with collagenase (1 mg ml⁻¹; 170 18–037, Gibco-BRL) in RPMI with 5% fetal calf serum and with 100 μg ml⁻¹ of DNase for 10 min. Cells were lysed with ACK to remove erythrocytes, and 0.1×10⁶ cells were blocked with Fc Block (BD PharMingen), directly stained with FITC- or PE- Ly6G-specific antibody (1A8), Gr-1 (RB6-8C5), or F4/80 (BD Biosciences), and analyzed by flow cytometry¹.

Adoptive transfer of macrophages into CD11bDTR mice

Cell suspensions were prepared from whole lungs of WT naïve mice as described above. Positive selection of macrophage cells (F4/80⁺) was conducted by magnetic cell sorting (MiltenyiBiotec). 2–5×10⁶ cells (in 100 μl of PBS) were transferred into CD11bDTR recipients through i.v. injection. After 2 days, CD11bDTR recipients and control groups received DT (25 mg kg⁻¹) i.v. and were inoculated with parasites the next day.

Statistical analysis

Data were analyzed using SigmaPlot 11 (Systat Software, San Jose, CA, USA) and are reported as means ± SEM. Differences between multiple groups were assessed by one way ANOVA and individual comparisons were analyzed using Holm-sidak test. Differences of p < 0.05 were considered statistically significant.