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. 2018 Jul;142(1):120-129.e6.
doi: 10.1016/j.jaci.2017.07.042. Epub 2017 Sep 12.

Mast Cell Chymase Decreases the Severity of Group B Streptococcus Infections

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Free PMC article

Mast Cell Chymase Decreases the Severity of Group B Streptococcus Infections

Claire Gendrin et al. J Allergy Clin Immunol. .
Free PMC article

Abstract

Background: Group B Streptococcus (GBS) or Streptococcus agalactiae are β-hemolytic gram-positive bacteria that colonize the lower genital tracts of women and are frequently associated with infections during pregnancy. Innate immune defenses are critical for controlling GBS dissemination and systemic infection. Mast cells are resident sentinel cells that come into contact with pathogens early during colonization and infection.

Objective: We aimed to investigate the contribution of chymase to systemic GBS infection and rates of preterm birth.

Methods: Pharmacologic and genetic approaches using mice deficient in mast cell protease (MCPT) 4, the mouse functional homologue of human chymase, were used.

Results: Our studies show that mast cells release a protease with chymotrypsin-like cleavage specificity in response to GBS. Additionally, increased GBS systemic infection and preterm births were observed in MCPT4-deficient mice versus MCPT4-sufficient mice. Furthermore, we observed that proteolytic cleavage of the host extracellular matrix protein fibronectin by peritoneal cell-derived mast cell lysates diminished GBS adherence. Consistent with this observation, the increase in GBS dissemination and preterm births observed in MCPT4-deficient mice was abolished when GBS was deficient in expression of the fibronectin-binding protein SfbA.

Conclusions: Taken together, our results suggest that the protective effect of MCPT4 against GBS dissemination and preterm labor can be attributed in part to MCPT4-mediated proteolysis of fibronectin. Our studies reveal a novel role of mast cells in defense against bacterial infections.

Keywords: Group B Streptococcus; chymase; fibronectin; mast cells; mouse mast cell protease 4; proteases.

Conflict of interest statement

Disclosure of Conflict of interest: The authors declare that they have no conflict of interests.

Figures

Figure 1
Figure 1. GBS induces the release of a mast cell protease with chymotrypsin-like activity and MCPT4-deficient mice exhibit increased rates of GBS infection associated preterm birth
(A) Approximately 105 PCMCs were treated with 106 CFU of WT GBS (COH1) at a MOI of 10 for 4h. The calcium ionophore A23187 (5 μM) was included as a positive control. Percentage of chymotrypsin-like activity release was assessed by measuring chymotrypsin-like activity in the mast cell supernatants and the cell lysates. Data shown were obtained from four independent experiments (n = 3; **P < 0.01, ***P < 0.001, Students t test; error bars, ±SEM). (B–F) Pregnant, chymase proficient, wild type C57BL6/J mice (Mcpt4+/+) or MCPT4-deficient mice in C57BL6/J background (Mcpt4−/−) were injected in utero with 107 CFU of GBS WT COH1 (n=6/group). Mice were monitored for signs of preterm birth up to 72 h post inoculation. Surgery and GBS inoculation for each pregnant mouse were performed independently. (B) Scheme of pup numbering in utero and injection site between fetuses P1 and P2 is shown. (C) Percent preterm birth in MCPT4-sufficient (Mcpt4+/+) or MCPT4-deficient (Mcpt4−/−) mice. Preterm birth was defined as vaginal bleeding with pup in birth canal or in cage before three days post-infection. * P < 0.05, Barnard’s test. (D) Bacterial burden in representative fetal pups and their corresponding placenta from Mcpt4+/+ and Mcpt4−/− mice infected with GBS WT COH1 (n = 5–6/group; of note, pups that were delivered preterm were excluded from CFU enumeration. CFUs are not significantly different (NS= not significant) between any of the groups (median is shown). (E) Bacterial burden in maternal spleen from pregnant Mcpt4+/+ and Mcpt4−/− mice infected with WT GBS (n = 6/group, median is shown, * P <0.05, Mann Whitney test).
Figure 2
Figure 2. MCPT4-deficient mice exhibit increased GBS dissemination during systemic infection independent of neutrophil recruitment
(A–C) MCPT4-sufficient (Mcpt4+/+) and MCPT4-deficient (Mcpt4−/−) mice were intravenously infected with 5 × 107 CFU WT GBS COH1. At 48 h after infection, bacterial burden was evaluated in blood, spleen and lungs. Data shown are from a representative experiment (out of two independent experiments) performed with seven animals per group. The Mann-Whitney test was used for comparisons between the two groups. Medians are indicated (*P < 0.05; **P < 0.01, ***P<0.005). (D) Percent neutrophils (Ly6G+ CD11b+ cells) in the spleens of uninfected and WT GBS infected Mcpt4+/+ and Mcpt4−/− mice (NS= not significant, Mann Whitney test).
Figure 3
Figure 3. Chymase and MCPT4 do not exhibit direct antibacterial activity and chymase does not diminish neutrophil ROS production in response to GBS
(A) Approximately 1.5 × 104 CFU of WT GBS were incubated with either 1.5 × 105 PCMCs (MOI=0.1) from Mcpt4+/+ or Mcpt4−/− mice or with human recombinant chymase (0.1 μg, 0.5 and 2.5 μg) for 4 h. Surviving bacteria were enumerated at the end of the experiment. Survival index (SI) represents the ratio of the GBS CFU recovered after incubation with either PCMCs or human chymase to the GBS CFU recovered in media alone. Data represent the average of three independent experiments (error bars ± SEM). NS= Not significant, ANOVA. (B) Purified adult human neutrophils were pretreated with 84 μM dihydrorhodamine 123 and were then either left untreated or treated with WT GBS COH1 at MOI 100 in the presence and absence of human recombinant chymase (0.5 ug). PMA (20 nM) was included as a positive control. Oxidation to fluorescent mono-hydrorhodamine 123 (MHR) was monitored at 0 and 60 min after treatment, respectively. Gates reflect percent cell numbers. Data shown are representative of two independent experiments.
Figure 4
Figure 4. Chymase and MCPT4 degrade full length fibronectin and fibronectin fragments derived from fibronectin proteolytic cleavage
(A) PCMCs lysates from Mcpt4+/+ or Mcpt4−/− mice (lysates from 2.5 × 105 cells) were incubated with 5μg of human plasma fibronectin. Fibronectin and its degradation fragments were then visualized by western blot analysis. (B) % Abundance of fibronectin fragments (approximately 160kDa, 140kDa, 125kDa, and 110kDa) relative to the abundance of full length fibronectin. Data represent the average of five independent experiments (error bars ± SEM). A student’s t-test was used for comparison between the two groups. (C) Human recombinant chymase (human chymase, 63.5ng) was incubated with 2 μg of human plasma fibronectin. Fibronectin and its degradation fragments (approximately 160kDa, 125kDa, and 110kDa) were then visualized by western blot analysis. (D) % Abundance of fibronectin fragments (approximately 160kDa, 140kDa, 125kDa, and 110kDa) relative to the abundance of full length fibronectin. Data represent the average of four independent experiments (error bars ± SEM).
Figure 5
Figure 5. MCPT4 mediated-fibronectin degradation via proteolytic cleavage prevents GBS adherence
(A) Scheme describing the assay used to evaluate the role of chymase proteolysis of fibronectin on GBS adherence. Microtiter plates were coated overnight at 4°C with fibronectin or fibronectin fragments (obtained by incubation of 5 μg fibronectin with lysates generated from 2.5 × 105 chymase deficient or proficient PCMCs). Plates were then treated with WT GBS COH1 for 30 min at 37°C. After washing non-adherent bacteria, adherent bacteria were trypsinized and enumerated by plating. Data are shown in panel B. (B) GBS adherence to fibronectin that was pretreated with PCMC lysates generated from Mcpt4+/+ or Mcpt4−/− mice. Data represent the average of three independent experiments (error bars ± SEM). A student’s t-test was used for comparison between the two groups. (***P<0.005).
Figure 6
Figure 6. Chymase diminishes GBS infection by inhibiting GBS interactions with the extracellular matrix
(A–C) Mcpt4+/+ and Mcpt4−/− mice were intravenously infected with 5 × 107 CFU of GBS WT or GBSΔsfbA. At 48h after infection, bacterial burden was evaluated in blood, spleens and lungs. Data shown are from a representative experiment (out of two independent experiments) performed with seven animals per group. The Mann-Whitney test was used for comparisons between the two groups. Medians are indicated (*P < 0.05; **P < 0.01, ***P<0.005, NS= Not significant). (D–E) Pregnant Mcpt4+/+ and Mcpt4−/− mice were injected in utero with 107 CFU of GBS WT COH1 or GBS GBSΔsfbA (n=6/group). Mice were monitored for signs of preterm birth up to 72 h post inoculation. Surgery and GBS inoculation for each pregnant mouse were performed independently. (D) Percent preterm birth in Mcpt4+/+ and Mcpt4−/− mice infected with WT GBS or GBSΔsfbA. Preterm birth was defined as vaginal bleeding with pup in birth canal or in cage before three days post-infection. * P < 0.05, * P < 0.005 Barnard’s test. (E) Bacterial burden in maternal spleen from pregnant Mcpt4+/+ and Mcpt4−/− mice infected with WT GBS or GBSΔsfbA. (Median is shown, * P <0.05, ***P <0.005, NS= not significant, Mann Whitney test).

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