Hypoxia-Inducible Factor 1 Alpha Is Dispensable for Host Defense of Group B Streptococcus Colonization and Infection

Abstract

Group B Streptococcus (GBS) is a leading cause of neonatal morbidity and mortality, and the primary source of exposure is the maternal vagina. Intrapartum antibiotic prophylaxis for GBS-positive mothers has reduced the incidence of GBS early-onset disease, however, potential long-lasting influence of an antibiotic-altered neonatal microbiota, and the frequent clinical sequelae in survivors of invasive GBS infection, compels alternative treatment options for GBS. Here, we examined the role of transcription factor hypoxia-inducible factor 1 alpha (HIF-1α), widely recognized as a regulator of immune activation during infection, in the host response to GBS. Given the importance of endogenous HIF-1α for innate immune defense, and the potential utility of HIF-1α stabilization in promoting bacterial clearance, we hypothesized that HIF-1α could play an important role in coordinating host responses to GBS in colonization and systemic disease. Counter to our hypothesis, we found that GBS infection did not induce HIF-1α expression in vaginal epithelial cells or murine macrophages, nor did HIF-1α deficiency alter GBS colonization or pathogenesis in vivo. Furthermore, pharmacological enhancement of HIF-1α did not improve control of GBS in pathogenesis and colonization models, while displaying inhibitory effects in vaginal epithelial cytokines and immune cell killing in vitro. Taken together, we conclude that HIF-1α is not a prominent aspect of the host response to GBS colonization or invasive disease, and its pharmacological modulation is unlikely to provide significant benefit against this important neonatal pathogen.

Keywords: Group B streptococcus; Hypoxia-inducible factor 1 alpha; Innate immunity; Macrophage; Vaginal colonization.

Fig. 1

Role of endogenous HIF-1α in GBS colonization of the vaginal tract epithelium. a HIF-1α^fl/fl and HIF-1α^fl/fl K14Cre+ female mice were vaginally administered 2 × 10⁷ CFUs of GBS COH1. Mice were vaginally swabbed daily, and the levels of GBS CFU recovered from swabs are shown. b Normalized bioluminescence of VK2 HRE reporter cells infected with GBS COH1 MOI of 20 for 2 h. AKB-4924 treatment (12 μg/mL) was included as a positive control. Symbols represent biological replicates (a, n = 16–23/group) or the means of 4 independent experimental replicates (b, performed in technical triplicate) with lines indicating medians and interquartile ranges. Dotted line in (a) indicates limit of detection. Data were analyzed by two-way ANOVA with Sidak's multiple comparisons posttest. ****p < 0.0001; **p < 0.01. HRE, hypoxia response element; GBS, group B Streptococcus; HIF-1α, hypoxia-inducible factor 1 alpha; CFU, colony-forming unit.

Fig. 2

Impact of HIF-1α stabilization on vaginal epithelial GBS colonization and cytokine production. a Percent adherence of GBS COH1 to VK2 cells after 30 min of infection, MOI = 0.1 or 1. VK2 cells were pretreated for 5 h with 12 μg/mL AKB-4924 or vehicle treated as a control. Production of IL-8 (b) or IL-1β (c) was measured from VK2 supernatant after 3 h of infection with GBS COH1, MOI = 10. VK2 cells were pretreated for 5 h with 12 μg/mL AKB-4924 or vehicle treated as a control. d WT C57Bl/6 female mice were vaginally administered 2 × 10⁷ CFUs of GBS COH1. Mice were vaginally swabbed daily, and the levels of GBS CFU recovered from swabs are shown. Mice were treated vaginally or i.p. with AKB-4924, or vehicle as a control, as described in Methods. Symbols represent means of 4 independent experimental replicates (a–c, performed in technical triplicate) or biological replicates (d, n = 25–26/group) with lines indicating medians and interquartile ranges. Dotted line in (d) indicates limit of detection. Data were analyzed by two-way ANOVA with Sidak's multiple comparisons posttest (b–d) or Wilcoxon matched-pairs signed rank test (a). *p < 0.05. GBS, group B Streptococcus; HIF-1α, hypoxia-inducible factor 1 alpha; CFU, colony-forming unit; WT, wild type.

Fig. 3

Role of endogenous HIF-1α in GBS phagocytosis, intracellular survival, and host morbidity in a murine model of GBS sepsis. BMDMs or thioglycolate-elicited cells were isolated from HIF-1α^fl/fl and HIF-1α^fl/fl K14Cre+ and infected with GBS COH1 at MOI = 10. GBS uptake and intracellular survival were assessed as described in Methods. Percent nonopsonized GBS uptake (a) and intracellular GBS survival (b) in BMDMs. Percent opsonized GBS uptake (c) and intracellular GBS survival (d) in BMDMs. Percent opsonized GBS uptake (e) and nonopsonized intracellular GBS survival (f) in thioglycolate-elicited cells. g HIF-1α^fl/fl and HIF-1α^fl/fl LysMCre+ mice were infected intraperitoneally with 5 × 10⁷ CFU GBS and survival monitored over 7 days. Symbols and error bars represent means ± standard error (a, c, e) or medians ± interquartile ranges (b, d, f) of 3 or 4 independent experimental replicates. In G, symbols represent death time points on a survival curve (n = 14–21/group) with lines indicating percentage survival between time points. Data were analyzed by Wilcoxon-matched pairs signed rank test (a, c, e), two-way ANOVA with Sidak's multiple comparisons posttest (b, d, f), or log-rank (Mantel-Cox) test (g). **p < 0.01; *p < 0.05. BMDM, bone marrow-derived macrophage; GBS, group B Streptococcus; HIF-1α, hypoxia-inducible factor 1 alpha; CFU, colony-forming unit.

Fig. 4

Impact of HIF-1α stabilization on GBS intracellular survival and host morbidity in a murine model of GBS sepsis. BMDMs were isolated from HIF-1α^fl/fl (a) and HIF-1α^fl/fl LysMCre+ (b), treated with AKB-4924 or a vehicle control, and infected with GBS COH1 at MOI = 10. Intracellular GBS survival was assessed as described in Methods. c WT C57Bl/6 mice were pretreated with AKB-4924, or a vehicle control, and infected intraperitoneally with 1×10⁸ or 2 × 10⁸ CFU GBS COH1 and survival monitored over 7 days. Symbols represent the median of 4 or 5 independent experimental replicates (a, b, performed in technical triplicate), with lines indicating medians and interquartile ranges. In (c) symbols represent death time points on a survival curve (n = 11–15/group) with lines indicating percentage survival between time points. Data were analyzed by two-way ANOVA with Sidak's multiple comparisons posttest (a, b), or log-rank (Mantel-Cox) test (c). *p < 0.05. BMDM, bone marrow-derived macrophage; GBS, group B Streptococcus; HIF-1α, hypoxia-inducible factor 1 alpha; CFU, colony-forming unit; WT, wild type.