NLRP3+ macrophages aggravate inflammatory cystitis in diabetes

Abstract

Inflammatory macrophages play a pivotal role in the progression of inflammatory cystitis. Formation of NOD-, LRR- and PYD domains-containing protein 3 (NLRP3) inflammasome triggers the activation of caspase-1/IL-1β signaling cascades to mediate inflammatory response. However, it is not known whether NLRP3 activation in macrophages during cystitis may differ in normal or diabetic setting as well as the importance of it. In this study, we found that NLRP3 levels significantly increased in bladder macrophages in diabetic mice that underwent cystitis. Moreover, bladder macrophages from diabetic mice appeared to have increased their potential of growth, migration and phagocytosis. Furthermore, specific depletion of NLRP3 in macrophages alleviated the severity of cystitis in diabetic mice, but not in non-diabetic mice. Together, our data suggest that NLRP3 depletion in macrophages may be a promising strategy for treating diabetic cystitis.

Keywords: LRR-and PYD domains-containing protein 3 (NLRP3); NOD-; diabetes; inflammation; inflammatory cystitis; macrophages.

Figure 1

Diabetes aggravates CYP-induced cystitis. (A) Schematic of experiment: STZ was used at D0 to develop diabetes in mice. After 6 days when diabetes was confirmed, experimental cystitis was induced by 4 i.p. injection of CYP on D6, 8, 10 and 12. Von Frey test was done at D13, and mice were sacrificed at D16 for end assessments. (B-F) Four groups of mice were used. Group 1: mice received i.p. saline (control for STZ) at D0 and i.p. saline (control for CYP) at D6, 8, 10 and 12 (saline+saline); Group 2: mice received i.p. STZ at D0 and i.p. saline (control for CYP) at D6, 8, 10 and 12 (STZ+saline); Group 3: mice received i.p. saline (control for STZ) at D0 and i.p. CYP at D6, 8, 10 and 12 (saline+CYP); Group 4: mice received i.p. STZ at D0 and i.p. CYP at D6, 8, 10 and 12 (STZ+CYP). (B) Nociceptive score of the mice at Von Frey test at D13. (C) Changes in body weight. (D) Bladder weight at D16. (E) Edema score of the bladder at D16. (F) Vesical vascular permeability in bladder at D16. *p<0.05 (STZ+CYP vs saline+CYP). #p<0.05 (saline+CYP vs saline+saline or STZ+saline).

Figure 2

CYP-induced cystitis does not affect diabetic status in mice. (A) Fasting blood glucose. (B) IPGTT at D16. (C) Beta cell mass at D16. (D) Representative insulin staining in mouse pancreas. *p<0.05 (STZ+CYP vs saline+CYP or STZ+saline vs saline+saline). Scale bars are 100µm.

Figure 3

NLRP3 significantly increases in bladder macrophages in diabetic mice that undergo cystitis. (A, B) Mouse bladders were sorted for bladder macrophages based on positivity for F4/80, and further for M1 (CD163-) and M2 (CD163+) subtypes among all F4/80+ macrophages, shown by representative flow charts (A) and by quantification (B). (C) ELISA for some critical factors for macrophage phenotype and functionality. *p<0.05. ns: no significance.

Figure 4

NLRP3 depletion in macrophages alters genes associated with macrophage phenotype. (A-C) A GEO database GSE183698 was used to analyzed gene profile from mouse macrophages with or without NLRP3 depletion (NLRP3KO, WT). (A) Genes associated with fine determination of macrophage phenotype analyzed by String online tool. (B, C) Gene reads from NLRP3 and some key factors associated with macrophage phenotype and functionality. *p<0.05.

Figure 5

Diabetes increases growth and migration potential of macrophages from inflammatory bladder. (A, B) Bladder macrophages from 4 groups of the mice were analyzed for cell growth in a CCK-8 assay (A) and in assay to determine DNA (B). (C, D) Bladder macrophages from 4 groups of the mice were analyzed for migration, shown by quantification (C) and by representative images (D). *p<0.05. ns: no significance. Scale bars are 100µm.

Figure 6

Diabetes increases phagocytosis of macrophages from inflammatory bladder. (A) A phagocytosis assay that analyzes 30 minutes’ zymosan intake. (B, C) Analysis of the intake of GFP+ bacteria, shown by representative images (B), by the mean cellular GFP density of macrophages (C). *p<0.05. Scale bars are 15µm. ns, no significance.

Figure 7

Specific depletion of NLRP3 in macrophages alleviates the severity of cystitis in diabetic mice. We generated mice with macrophage-depletion of NLRP3, Lys2-Cre; NLRP3(fx/fx) and control NLRP3(fx/fx). (A) Flow cytometry showing isolation of macrophages from the bladders of these mice. (B) ELISA for NLRP3 in macrophages nor non-macrophages from the mouse bladder. (C) STZ was used at D0 to develop diabetes in mice. After 6 days when diabetes was confirmed, experimental cystitis was induced by 4 i.p. injection of CYP on D6, 8, 10 and 12. Von Frey test was done at D13, and mice were sacrificed at D16 for end assessments. Group 1: NLRP3(fx/fx) mice received i.p. saline (control for STZ) at D0 and i.p. CYP at D6, 8, 10 and 12 (saline+CYP/N); Group 2: Lys2-Cre; NLRP3(fx/fx) mice received i.p. saline (control for STZ) at D0 and i.p. CYP at D6, 8, 10 and 12 (saline+CYP/LN); Group 3: NLRP3(fx/fx) mice received i.p. STZ at D0 and i.p. CYP at D6, 8, 10 and 12 (STZ+CYP/N); Group 4: Lys2-Cre; NLRP3(fx/fx) mice received i.p. STZ at D0 and i.p. CYP at D6, 8, 10 and 12 (STZ+CYP/LN). (C) Nociceptive score of the mice at Von Frey test at D13. (D) Changes in body weight. (E) Bladder weight at D16. (F) Edema score of the bladder at D16. (G) Vesical vascular permeability in bladder at D16. *p<0.05. ns: no significance.