Postnatal episodic ozone results in persistent attenuation of pulmonary and peripheral blood responses to LPS challenge

Abstract

Early life is a dynamic period of growth for the lung and immune system. We hypothesized that ambient ozone exposure during postnatal development can affect the innate immune response to other environmental challenges in a persistent fashion. To test this hypothesis, we exposed infant rhesus macaque monkeys to a regimen of 11 ozone cycles between 30 days and 6 mo of age; each cycle consisted of ozone for 5 days (0.5 parts per million at 8 h/day) followed by 9 days of filtered air. Animals were subsequently housed in filtered air conditions and challenged with a single dose of inhaled LPS at 1 yr of age. After completion of the ozone exposure regimen at 6 mo of age, total peripheral blood leukocyte and polymorphonuclear leukocyte (PMN) numbers were reduced, whereas eosinophil counts increased. In lavage, total cell numbers at 6 mo were not affected by ozone, however, there was a significant reduction in lymphocytes and increased eosinophils. Following an additional 6 mo of filtered air housing, only monocytes were increased in blood and lavage in previously exposed animals. In response to LPS challenge, animals with a prior history of ozone showed an attenuated peripheral blood and lavage PMN response compared with controls. In vitro stimulation of peripheral blood mononuclear cells with LPS resulted in reduced secretion of IL-6 and IL-8 protein in association with prior ozone exposure. Collectively, our findings suggest that ozone exposure during infancy can result in a persistent effect on both pulmonary and systemic innate immune responses later in life.

Fig. 1.

Experimental timeline for postnatal episodic ozone exposure and LPS challenge. Starting at 30 days of age, infant rhesus monkeys were exposed to 11 cycles of ozone. Each cycle consisted of ozone exposure for 5 days followed by 9 days of filtered air (0.5 parts per million at 8 h/day). Animals were evaluated either at the end of 10 cycles followed by 5 days of ozone (6 mo of age) or 11 cycles followed by 6 mo of filtered air (1 yr of age). LPS challenge took place at 1 yr of age.

Fig. 2.

Effect of postnatal episodic ozone exposure on peripheral blood and lavage leukocyte populations at 6 mo of age. Total cell number (A and C) and frequency of leukocyte phenotype (B and D) were measured in filtered air- and ozone-exposed animals at 6 mo of age. Each column represents the mean ± SE values from 4 to 9 animals. *P < 0.05, **P < 0.01 compared with filtered air values. WBC, peripheral white blood cells; PMN, polymorphonuclear leukocytes; BAL, bronchoalveolar lavage.

Fig. 3.

Effect of postnatal episodic ozone exposure on peripheral blood and lavage leukocyte populations at 1 yr of age. Total cell number (A and C) and frequency of leukocyte phenotype (B and D) were measured in filtered air- and ozone-exposed animals at 1 yr of age. Each column represents the mean ± SE values from 4 animals. *P < 0.05 compared with filtered air values.

Fig. 4.

Effect of postnatal episodic ozone exposure on total WBC (A), PMN frequency (B), and lymphocyte frequency (C) following LPS challenge. WBC samples were collected from filtered air- and ozone-exposed animals at 1 yr of age just before LPS instillation (●), 6 h post-LPS (■), and 22 h post-LPS (▲). Individual values from each of 4 animals are shown. *P < 0.05 for 6- vs. 22-h values.

Fig. 5.

Effect of postnatal episodic ozone exposure on lavage inflammation following LPS challenge. Total cell number (A) and frequency of leukocyte phenotype (B and C) were measured in filtered air- and ozone-exposed animals at 1 yr of age, at 6 and 24 h post-LPS. Each column represents the mean ± SE values from 3 to 4 animals. *P < 0.05 compared with filtered air values.

Fig. 6.

Effect of postnatal episodic ozone exposure on lavage cytokine secretion following LPS challenge. IL-6 (A), TNF-α (B), and IL-1β (C) protein concentration were measured in lavage samples collected from 1-yr-old filtered air- and ozone-exposed animals at 6 h (■) and 24 h (▲) post-LPS. Individual values from each of 4 animals are shown. *P < 0.05 for 6- vs. 24-h values.

Fig. 7.

Effect of postnatal episodic ozone exposure on LPS stimulation of peripheral blood mononuclear cells (PBMC) from 1-yr-old monkeys. PBMC from 1-yr-old filtered air- and ozone-exposed animals were treated with LPS in vitro. Cultures were evaluated at either 6 (A and C) or 24 (B and D) h for cytokine secretion. Concentration of IL-6 (A and B) and IL-8 (C and D) protein were measured in culture supernatant collected at listed time points. Each bar represents the mean ± SE of 2 data points obtained from each of 4 animals per group. *P < 0.05 compared with filtered air values. ND, not detected.