Early life allergen and air pollutant exposures alter longitudinal blood immune profiles in infant rhesus monkeys

Abstract

Early life is a critical period for the progressive establishment of immunity in response to environmental stimuli; the impact of airborne challenges on this process is not well defined. In a longitudinal fashion, we determined the effect of episodic house dust mite (HDM) aerosol and ozone inhalation, both separately and combined, on peripheral blood immune cell phenotypes and cytokine expression from 4 to 25weeks of age in an infant rhesus monkey model of childhood development. Immune profiles in peripheral blood were compared with lung lavage at 25weeks of age. Independent of exposure, peripheral blood cell counts fluctuated with chronologic age of animals, while IFNγ and IL-4 mRNA levels increased over time in a linear fashion. At 12weeks of age, total WBC, lymphocyte numbers, FoxP3 mRNA and IL-12 mRNA were dramatically reduced relative to earlier time points, but increased to a steady state with age. Exposure effects were observed for monocyte numbers, as well as CCR3, FoxP3, and IL-12 mRNA levels in peripheral blood. Significant differences in cell surface marker and cytokine expression were detected following in vitro HDM or PMA/ionomycin stimulation of PBMC isolated from animals exposed to either HDM or ozone. Lavage revealed a mixed immune phenotype of FoxP3, IFNγ and eosinophilia in association with combined HDM plus ozone exposure, which was not observed in blood. Collectively, our findings show that airborne challenges during postnatal development elicit measureable cell and cytokine changes in peripheral blood over time, but exposure-induced immune profiles are not mirrored in the lung.

Keywords: Cytokine; House dust mite; Infant; Ozone; Peripheral blood.

Figure 1

House dust mite (HDM) and ozone exposure protocol. Infant monkeys received Der p1 and Der p2 antigens via subcutaneous (SQ) injection at 2, 4, 8, and 16 weeks of age. Intranasal (IN) HDM was administered at 3, 5, and 9 weeks of age for HDM aerosol groups only. Beginning at 4 weeks of age, animals were exposed to 11 cycles of HDM aerosol, ozone, or HDM aerosol + ozone as indicated by arrows. Animals were necropsied at 25 weeks of age, 3-4 days following the last HDM aerosol or ozone exposure. Blood collection time points are indicated below the exposure regimen.

Figure 2

Peripheral blood leukocyte numbers as a function of chronologic age and exposure. (A) Total white blood cell (WBC) (B) lymphocytes (C) monocytes, (D) eosinophils and (E) neutrophils in blood samples collected as shown in Figure 1. Each column represents the mean+/-SE values, n=6. Horizontal lines indicate significant age-dependent differences between time points by multiple comparison 2-way ANOVA with Tukey's post-test. Exposure dependent effects at individual time points are listed in Table 2. *p<0.05, **p<0.01, ***P<0.001, ****p<0.0001.

Figure 3

Peripheral blood T and B cell frequency as a function of chronologic age and exposure. (A) Frequency of CD45+CD3+ and (B) CD45+CD19+ cells in whole blood samples collected at 12 and 18 weeks of age as shown in Figure 1. Each column represents the mean+/-SE values, n=6. Horizontal lines indicates significant age-dependent difference between time points by multiple comparison 2-way ANOVA with Tukey's post-test. ***P<0.001.

Figure 4

Peripheral blood cellular marker/cytokine gene expression as a function of chronologic age and exposure. (A) CD3 (B) FoxP3 (C) CCR3 (D) IFNγ (E) IL-4 (F) IL-12 (G) IL-6 (H) IL-13 and (I) IL-17 mRNA measured as copy number normalized to 10⁴ GAPDH housekeeping gene copies in blood samples collected as shown in Figure 1. Each column represents the mean+/-SE values, n=6. Horizontal lines indicate significant differences between week of age time points by multiple comparison 2-way ANOVA with Tukey's post-test. Exposure dependent effects at individual time points are listed in Table 2. *p<0.05, **p<0.01, ***P<0.001, ****p<0.0001.

Figure 5

Intrinsic HDM recall response with exposure. PBMC were collected at necropsy, 3-4 days post final HDM aerosol exposure. Cells were treated in vitro for 24 hours with HDM at 10 or 100ug/ml, or PMA +ionomycin, followed by quantitative RT-PCR analysis. (A) CD3 (B) FoxP3 (C) IL-6 (D) IFNγ and (E) IL-4 mRNA copy number was normalized to GAPDH housekeeping gene copy number. Data are expressed as copy number fold change over unstimulated media control values. Each column represents the mean+/-SE values for 3-6 animals. Exposure* or Treatment* indicates significant overall effects by 2-way ANOVA. Horizontal lines indicate significance by 2-way ANOVA with Tukey's post-test for between exposure group comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6

Comparison of peripheral blood and lung lavage leukocyte populations at 6 months of age: effect of exposure. (A) Peripheral blood total white blood cell (WBC) number, and lymphocyte, eosinophil, monocyte, and neutrophil frequency at 6 months of age in animals exposed to HDM aerosol and/or ozone or filtered air control. (B) Lung lavage total cell number, and lymphocyte, eosinophil, monocyte, macrophage, and neutrophil frequency at 6 months of age in animals exposed to HDM aerosol and/or ozone or filtered air control. Lavage and blood samples were collected at 25 weeks of age (necropsy), 3-4 days following the last HDM and/or ozone exposure. Each point represents an individual animal, with 5-6 animals/group. Mean+/-SE is indicated for each exposure group. Horizontal bars indicate significance by 1-way ANOVA with Tukey's post-test for between exposure group comparisons. *p<0.05, **p<0.01

Figure 7

Comparison of peripheral blood and lung cell/cytokine molecular markers at 6 months of age: effect of exposure. (A) Peripheral blood IL-4, IL-6, IFNγ, CD3, FoxP3, IL-12, IL-13, and CCR3 mRNA expression at 6 months of age in animals exposed to HDM aerosol and/or ozone or filtered air control. (B) Lung lavage cell IL-4, IL-6, IFNγ, CD3, FoxP3, IL-12, IL-13, and CCR3 mRNA expression at 6 months of age in animals exposed to HDM aerosol and/or ozone or filtered air control. Copy number is normalized to GAPDH housekeeping gene copy number. Lavage and blood samples were collected at 25 weeks of age (necropsy), 3-4 days following the last HDM and/or ozone exposure. Each point represents an individual animal, with 5-6 animals/group. Mean+/-SE is indicated for each exposure group. Horizontal bars indicate significance by 1-way ANOVA with Tukey's post-test for between exposure group comparisons. Exposure* indicates significant overall exposure dependent effects by 1-way ANOVA, with no significance by post-test. *p<0.05, **p<0.01.