Major basic protein from eosinophils and myeloperoxidase from neutrophils are required for protective immunity to Strongyloides stercoralis in mice

Abstract

Eosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response to Strongyloides stercoralis in mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections with S. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. Infecting PHIL mice, which are eosinophil deficient, with S. stercoralis resulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. Treating PHIL mice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected with S. stercoralis had significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

Fig. 1.

Primary and secondary immunity to S. stercoralis in mice deficient in either MBP or EPO. (A) S. stercoralis larvae were implanted in cell-permeable diffusion chambers in naïve C57BL/6, MBP ^−/−, or EPO^−/− mice for 5 days, and parasite survival was determined. In addition, mice were treated with an anti-CCR3 MAb to determine the effect of eosinophil depletion on parasite survival in C57BL/6, MBP ^−/−, or EPO^−/− mice. *, statistically significant difference between larval recoveries from treated and untreated mice. (B) Number of neutrophils (polymorphonuclear leukocytes [PMN]), macrophages (Mφ), and eosinophils (EOS) found within cell-permeable diffusion chambers implanted in untreated naïve C57BL/6, MBP ^−/−, or EPO^−/− mice or mice treated with an anti-CCR3 MAb. (C) S. stercoralis larvae were implanted in cell-permeable diffusion chambers in naïve and immunized C57BL/6, MBP ^−/−, or EPO^−/− mice for 1 day, and parasite survival was determined. *, statistically significant difference between larval recoveries from control and immunized mice. Data shown represent the means and standard deviations from 8 to 22 mice per group.

Fig. 2.

In vitro and in vivo studies on the role of MBP and EPO on killing of larvae by eosinophils. (A) Larvae in vitro with eosinophils derived from IL-5 TG mice were killed if untreated serum was added but not if serum that was heat inactivated (HI) or that was derived from C3^−/− mice was added to the cultures. *, statistically significant difference between larval survival from culture wells in which untreated serum was added and wells in which no serum was added or heat-inactivated serum or serum that was derived from C3^−/− mice was added. (B) Larvae were placed in vitro in wells containing eosinophils derived from IL-5 TG, MBP^−/− × IL-5 TG, and EPO^−/− × IL-5 TG mice in the presence of untreated naïve serum. *, statistically significant difference between larval survival in culture wells in which MBP^−/− × IL-5 TG eosinophils were inserted and survival in wells containing eosinophils from either IL-5 TG or EPO^−/− × IL-5 TG mice. (C) Transfer of eosinophils derived from IL-5 TG, MBP^−/− × IL-5 TG, and EPO^−/− × IL-5 TG mice into cell-impermeable diffusion chambers with larvae and implantation for 1 day in naïve C57BL/6 mice. *, statistically significant difference between parasite survival in the presence of IL-5 TG and EPO^−/− × IL-5 eosinophils and the absence of cells or the presence of eosinophils from MBP^−/− × IL-5 TG mice. Data shown represent the means and standard deviations from 11 to 13 mice per group.

Fig. 3.

Primary and secondary immunity to S. stercoralis in PHIL mice. (A) Parasite survival in naïve or immunized C57BL/6 or PHIL mice after 1, 3, or 5 days. (B) Number of neutrophils (polymorphonuclear leukocytes [PMN]), macrophages (Mφ), and eosinophils (EOS) found within diffusion chambers implanted in naïve or immunized C57BL/6 and PHIL mice. (C) Serial dilutions of parasite-specific IgM response in naïve or immunized C57BL/6 or PHIL mice. OD, optical density; WT, wild type. (D) Production of IL-4 and IL-5 by spleen cells derived from naïve or immunized C57BL/6 or PHIL mice after stimulation with medium, S. stercoralis antigen (Ss Ag), or CD3. Data shown represent the means and standard deviations from 9 to 10 mice per group.

Fig. 4.

Treatment of PHIL mice with MAb to eliminate neutrophils. (A) Number of neutrophils (polymorphonuclear leukocytes [PMN]), macrophages (Mφ), and eosinophils (EOS) found within cell-permeable diffusion chambers implanted for 3 days in untreated naïve C57BL/6 or PHIL mice and mice treated with MAb RB6-8C5 to eliminate neutrophils. (B) Parasite survival in naïve C57BL/6 and PHIL mice treated with a MAb to eliminate neutrophils. *, statistically significant difference between larval survival in mice receiving treatment to eliminate neutrophils and untreated controls. Data shown represent the means and standard deviations from 8 to 10 mice per group.

Fig. 5.

Primary and secondary immunity to S. stercoralis in MPO^−/− mice. Parasite survival in naïve or immunized C57BL/6 or MPO^−/− mice for 1, 3 or 5 days. *, statistically significant difference between larval survival in MPO^−/− mice and C57BL/6 mice. Data shown represent the means and standard deviations from 8 to 10 mice per group.