An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection

Abstract

Helminths induce potent T helper 2 (TH2)-type immune responses that can mediate worm expulsion, but the role of this response in controlling the acute tissue damage caused by migrating multicellular parasites through vital tissues remains uncertain. We used a helminth infection model in which parasitic nematode larvae migrate transiently through the lung, resulting in hemorrhage and inflammation. We found that IL-17 initially contributed to inflammation and lung damage, whereas subsequent IL-4 receptor (IL-4R) signaling reduced elevations in IL-17 mRNA levels, enhanced the expression of insulin-like growth factor 1 (IGF-1) and IL-10 and stimulated the development of M2 macrophages, all of which contributed to the rapid resolution of tissue damage. These studies indicate an essential role for TH2-type immune responses in mediating acute wound healing during helminth infection.

Figure 1. Kinetics of N. brasiliensis-induced lung damage

Mice (five/treatment group) were inoculated with Nippostrongylus brasiliensis (Nb) and 2, 4, and 7 days later airway responsiveness was determined or bronchial alveolar lavage (BAL) and lung tissue was collected. (a) Representative H&E staining of lung tissue (Scale bar, 50 μm) showing peak hemorrhage on day 2 and influx of leukocytes by day 4; acute lung injury scores (ALI) showing increased lung damage at day 2 which was largely resolved by day 7. (b) FACS analysis of neutrophils (Ly6G⁺) vs. forward scatter (FSC) (c) Bronchial alveolar lavage (BAL) fluid with red color caused by increased RBCs; BAL- red blood cell (RBC) count, and BAL-neutrophil (Neu) count. (d) airway hyperresponsiveness (baseline measurement) indicated as penh (pause enhanced) values. (e) Il4, Il13, and Il17 mRNA and corresponding BAL cytokine protein levels. BAL protein levels were measured by ELISA. Mean and SEM is shown and all results are representative of at least two independent experiments.

Figure 2. IL-4R signaling controls acute lung hemorrhaging and IL-17 dependent neutrophil infiltration

(a) WT mice administered Ly6G-specific antibody or isotype control were inoculated with N. brasiliensis (Nb) and on day 2 BAL was collected and RBCs and neutrophils counted. In separate experiments, Il17ra^−/−, WT controls and WT mice administered IL-17A-specific antibody or isotype control were inoculated with Nb and at day 2 BAL was collected and RBCs and neutrophils were counted. (b) Il4ra^−/−, Il4^−/−, and Il13ra1^−/− mice and corresponding WT controls were inoculated with Nb and on day 4 BAL was collected and RBCs and neutrophils were counted. (c) Worms were counted in intestine at day 4 after Nb inoculation of Il4ra^−/− and WT mice. (d) Lung tissue from WT and Il4ra^−/− mice was collected on day 4 after inoculation with N. brasiliensis (Nb) L3. Gene expression of Il4, Il13, Il10 and Il17 were determined using RTPCR. (e) Il4ra^−/− mice were inoculated with N. brasiliensis and administered IL-17A specific antibody or Isotype control. At day 2 after inoculation BAL RBCs and neutrophils were enumerated. Results are reported as mean and SEM from three to five individual mice per group and are representative of two independent experiments. *p< 0.05, ** p< 0.01.

Figure 3. Blockade of IL-4R-dependent IGF-1 signaling inhibits control of inflammation and hemorrhaging in N. brasiliensis- inoculated mice

(a) Lung tissue from WT and Il4ra^−/− mice was collected on day 4 after inoculation with N. brasiliensis (Nb). Gene expresson of Insulin-like growth factor (Igf1), arginase 1 (Arg1), Collagen1, and matrix metalloproteinase 13 (Mmp13) were determined by RT-PCR (b) Mice were administered IGF-1R antibody prior to inoculation with Nb and at day 4 BAL was collected and RBCs and neutrophils counted. (c) Lung tissue from Nb-inoculated mice administered IGF-1R-specific antibody or isotype control antibody were examined for changes in gene expression at day 4 after inoculation using RT-PCR. In all experiments, results are reported as mean and SEM from three to five individual mice per group and are representative of two independent experiments.* p< 0.05, ** p< 0.01.

Figure 4. IL-10 down regulates lung inflammation and IL-17 elevations in N. brasiliensis-inoculated mice

(a) Il10 gene expression at days 2, 4, and 7 after inoculation with N. brasiliensis (Nb). (b) Il10^−/− mice and corresponding WT controls were inoculated with Nb. At day 5 and day 7 BAL was collected and RBCs and neutrophils were counted. (c) At day 7, gene expression of lung Il4, Il13, Il17, Ifng, Inos, Igf1, Arg1, and Mmp13 was determined by RT-PCR. Data are the mean and SEM of five mice per group and are representative of two independent experiments. * p< 0.05, ** p< 0.01.

Figure 5. Macrophages control both lung hemorrhaging and inflammation during infection with N. brasiliensis

(a) Lung tissues were collected at day 0, 2, 4, and 7 after inoculation with N. brasiliensis (Nb), and flow cytometric analysis showed increases in lung macrophages (F4/80⁺). lung tissue, as detected by H&E staining, showed RBCs engulfed by macrophages especially at day 4 (Scale bar, 5μm). (b e) CD11bDTR mice were inoculated with Nb and administered DT to delete macrophages in vivo. In some groups WT lung-derived macrophages were transferred to DT treated CD11bDTR mice and all mice were examined at day 3 after inoculation. (b) left lobe of lung was collected and photographed (Scale bar, 5mm); (c) BAL was collected and RBCs, neutrophils, and eosinophils were counted at day 3 after inoculation; (d) H&E histological staining (Scale bar, 50μm) and ALI score of lung tissue; (e) Gene expression of lung Arg1, Mmp13, and Igf1 was determined by RT-PCR. (g) WT mice were administrated BEC and inoculated with Nb. On day 4, BAL was collected and RBCs, neutrophils, and macrophages were counted. All images are representative examples of experiments with five mice/group and all data are expressed as the mean and SEM of five mice/group. Experiments were repeated two times with similar results. * p< 0.05, ** p< 0.01.

Figure 6. Model of acute resolution of lung damage mediated by a helminth-induced Th2-type response

Invasion of lung tissue by N. brasiliensis (Nb) larvae and associated tissue damage initially triggers up regulation of IL-17 resulting in pronounced neutrophil infiltration within 2 days after inoculation. By 4 days IL-4 and IL-13 are markedly up regulated and together they inhibit IL-17 expression and inflammation. IL-4 and IL-13 stimulate subsequent expression of IL-10, which further contributes to control of inflammation. Macrophages are also activated to express IGF-1, Arg1, and other wound healing factors that directly mediate tissue regeneration and remodeling and macrophages also likely contribute to clearance of debris and erythrocytes in tissue.