Jessica Scholl1, Dimitrios Nasioudis2, Allison Boester3, Maureen Speleotes3, Amos Grunebaum3, Steven S Witkin2. 1. Division of Maternal-Fetal Medicine, Weill Cornell Medicine, New York, New York. Electronic address: jes9188@med.cornell.edu. 2. Division of Immunology and Infectious Diseases, Weill Cornell Medicine, New York, New York. 3. Division of Maternal-Fetal Medicine, Weill Cornell Medicine, New York, New York.
Abstract
BACKGROUND: Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear. OBJECTIVE: We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women. STUDY DESIGN: We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data. RESULTS: There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women. CONCLUSION: The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage.
BACKGROUND:Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear. OBJECTIVE: We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women. STUDY DESIGN: We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data. RESULTS: There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women. CONCLUSION: The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage.