The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction (original) (raw)
Fig 6
_Eh_CP5 cysteine protease activity is required to activate α5β1 integrin, ATP release and the NLRP3 inflammasome.
(a) Immunoblot analysis of β1 integrin tyrosine phosphorylation (clone PY20) of anti-α5β1 integrin immunoprecipitates from PMA-differentiated THP-1 macrophages stimulated with Wt Eh or Wt-E64 Eh. (b) Activation status of β1 integrin in THP-1 macrophages stimulated with Wt Eh or Wt-E64 Eh evaluated with anti-β1 integrin mAbs, AG89 and HUTS-4 that recognize the active conformation specific epitope of β1 integrin. (c, d) ATP release from THP-1 macrophages stimulated for 1 min with a 1:30 ratio of Wt Eh or Wt-E64 Eh, or rCP5 (1 μg mL-1), or rCP5 inactivated first in E-64 (100 μM). (e, g, h, i) Immunoblot analysis of IL-1β and caspase-1 activation and IL-1β enzyme-linked immunosorbent assay of PMA-differentiated THP-1 macrophages stimulated for 30 min or indicated time with Wt Eh (e, g, h, i), Wt-E64 Eh (e, g, h), _Eh_CP5- Eh (i) alone or with the addition of rCP5 (1 μg mL-1) (g, h, i), rCP5 RAD (g), rCP5 RGA (g) or rCP5 inactivated in E-64 (100 μM) (h, i). (f) Caspase-1 activation upon contact with Wt or Wt-E64 Eh in which active caspase-1 was stained with YVAD-FLICA, red; mammalian nuclei, blue (Eh nuclei are not stained). Scale bars, 10 μM. Data are representative of two (c, f) or three (a, b, d, e, g-i) separate experiments (error bars SEM). ***P < 0.005.