In vivo induction of neutrophil chemotaxis by secretory aspartyl proteinases of Candida albicans

Abstract

Secretory aspartyl proteinases (Saps) of Candida albicans are key virulence traits which cause inflammasome-dependent, aseptic inflammation in a mouse model of vaginitis. In this paper, neutrophil migration in response to Sap2, Sap6 and chemo-attractive products released from Sap-treated vaginal epithelium was measured in vitro, ex vivo and in vivo. Our results show that Sap2 and Sap6 induce neutrophil migration and production of potent chemoattractive chemokines such as IL-8 and MIP-2 by vaginal epithelial cells. Our data suggest that at least part of MIP-2 production depends upon IL-1β activity. The vaginal fluid of Candida-infected mice contained a heat-labile inhibitor of neutrophil candidacidal activity that was absent from the vaginal fluid of Sap-treated mice. Overall, our data provide additional information on the capacity of C. albicans Saps to cause aseptic vaginal inflammation and highlight the potential role of some chemokines released from vaginal epithelial cells in this phenomenon.

Keywords: Candida albicans; aspartyl proteinases; killing activity; neutrophil migration; soluble factors.

Figure 1.

Effect of Saps on neutrophil migration and on MIP-2 production. Human fluorescent neutrophils were incubated for 2 h to the upper compartment of transwell filters containing Medium, IL-8 (100 ng/ml), Sap2, tSap2 or Sap6 (all 0.5 µg/ml) in the presence or absence of Pepstatin A (1 µg/ml) (A, left) or vaginal epithelium, stimulated for 24 h in the presence or absence (Medium) of IL-8 (100 ng/ml), Sap2, tSap2 or Sap6 (all 0.5 µg/ml), pretreated or not with Pepstatin A (1 µg/ml) (A, right), in the lower compartment. The number of migrating neutrophils into the lower compartment was measured by using fluorescence signal. Data are expressed as fluorescence intensity of migrated neutrophils (triplicates samples of 5 different experiments, mean ± SEM). Human MIP-2 was assessed on cultures supernatants of vaginal epithelium stimulated for 24 h in the presence or absence (Medium) of IL-8 (100 ng/ml), C. albicans (CA-6) (1 × 10⁷/ml), LPS (10 µg/ml) plus ATP (5 mM), Sap2 or Sap6 (both 0.5 µg/ml), pretreated or not with Pepstatin A (1 µg/ml), by specific Elisa assay (triplicates samples of 5 different experiments, mean ± SEM) (B). Human MIP-2 was also assessed on cultures supernatants of vaginal epithelium unstimulated (Medium) or stimulated for 24 h with C. albicans (CA-6) (1 × 10⁷/ml), Sap2 or Sap6 (both 0.5µg/ml) in the presence or absence of Anakinra (10 µM) by specific Elisa assay (triplicates samples of 3 different experiments, mean ± SEM) (C). *, p < 0.05 IL-8, CA-6, LPS plus ATP or Saps treated vs Medium treated. #, p < 0.05 Saps + Pepstatin A treated vs Saps treated. # #, p < 0.05 CA-6 or Saps + Anakinra treated vs CA-6 or Saps treated.

Figure 2.

Neutrophil influx and activity during vaginal candidiasis. Human fluorescent neutrophils were incubated for 2 h to the upper compartment of transwell filters containing, in the lower compartment, vaginal washes of mice injected for 24 h with Saline, LPS (50 µg/10 µl/mouse), Sap2 or Sap6 (both 0.5 µg/10 µl/mouse). The number of migrating neutrophils into the lower compartment was measured by using fluorescence signal. Data are expressed as fluorescence intensity of migrated neutrophils (triplicates samples of 3 different experiments, mean ± SEM) (A). Vaginal washes of mice injected as above described were centrifuged, cellular fraction was microscopically analyzed to evaluate neutrophil recruitment (arrow) (representative images of 3 separate experiments with similar results, magnification 10 x, Bar = 50 µm) (B) or the % of double positive CD326⁺/IL-8⁺ cells (triplicates samples of 3 different experiments, mean ± SEM) (C) and supernatants were collected and tested for mouse IL-8 (D) and mouse MIP-2 (E) production by specific Elisa assays (triplicates samples of 3 different experiments, mean ± SEM). Killing activity against C. albicans of human neutrophils mixed with Medium or vaginal washes of mice injected for 24 h with Saline, Sap2 or Sap6 (both 0.5 µg/10 µl/mouse), C. albicans (CA-6) (2 × 10⁷/10µl/mouse) or H. I. vaginal washes of mice injected for 24 h with C. albicans (CA-6) (2 × 10⁷/10µl/mouse) was shown (triplicates samples of 5 different experiments, mean ± SEM) (F). *, p < 0.05 LPS or Saps treated vs Saline treated mice. #, differences between LPS, Sap2 or Sap6 treated animals resulted not significant. †, p < 0.05 CA-6 treated vs Medium treated mice.