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NLRP3 INFLAMMSOME ACTIVATION IN PBMCS CONTROLS THE T CELL RESPONSE IN ADULT PATIENTS WITH CHRONIC IMMUNE THROMBOCYTOPENIA
Author(s): ,
Mingqiang Hua
Affiliations:
Department of Hematology,Qilu Hospital, Shandong University,Jinan,China
,
Xin Li
Affiliations:
Department of Hematology,Qilu Hospital, Shandong University,Jinan,China
,
Ming Hou
Affiliations:
Department of Hematology,Qilu Hospital, Shandong University,Jinan,China
Jun Peng
Affiliations:
Department of Hematology,Qilu Hospital, Shandong University,Jinan,China
(Abstract release date: 05/17/18) EHA Library. Hua M. 06/16/18; 214617; S887
Mingqiang Hua
Mingqiang Hua
Contributions
Abstract

Abstract: S887

Type: Oral Presentation

Presentation during EHA23: On Saturday, June 16, 2018 from 16:15 - 16:30

Location: Room A9

Background
Immune thrombocytopenia (ITP) is an acquired autoimmune disorder, which is characterized by imbalanced adaptive immunity of T cells. NLRP3 inflammasome has recently been reported to be involved in diverse inflammatory or immune diseases. However, NLRP3 inflammasome activation in the pathophysiology of ITP remains unclear.

Aims
To explore the effects of NLRP3 inflammsome activation on the T cells in adult patients with chronic ITP.

Methods
Peripheral blood was obtained from 28 adult patients with chronic ITP. PBMCs were isolated using Ficoll density-gradient centrifugation and the CD4+ cells were purified by positive immuno-magnetic microbeads selection. The PBMCs, CD4+ or CD8+ cells were primed with LPS, then stimulated with ATP and finally cultured in plate-bound anti-human CD3 and soluble anti-human CD28 plus IL-2. After four days, the percentage of CD4+ cells and CD8+ cells, the T helper cell subsets (Th cells) of Th1, Th2, Th17 and T-reg cells, the inhibitory molecules, PD1 and CTLA4, and the CFSE-labeled cells were analyzed by flow cytometry. IFN-γ, IL-4, IL-17 and TGF-β in cultured supernatants were assayed by ELISA. The mRNA expression level of cytokines associated with Th cells (Ifn-γ, Il-6, IL-10, Il-17 ), the key transcription factors of Th cells (T-bet, Gata3, Foxp3, Ror-γt) and the inhibitory molecules (Ctla-4, Btla, Tim3, Lag3, Pd1 and Vista) was determined by Real-Time PCR.

Results
We found that NLRP3 inflammasome activation in PBMCs initiated caspase-1–dependent IL-1β secretion, and thereby weakened the Th1 cell differentiation (unstimulated control 13.3% vs. stimulation 5.3%; p=0.015), which can be restored, at least in part, by caspase-1 inhibitor Z-YVAD-FMK (Figure 1). The production of IFN-γ and the expression of Ifn-γ, were significantly down-regulated after NLRP3 inflammsome activation. However, the percentage of Th2 or Th17 was not significantly different between the stimulated and unstimulated groups. Meanwhile, the percentage of T-reg cells was also decreased after stimulation by LPS plus ATP in PBMCs (unstimulated control 6.67% vs. stimulation 4.39%; p=0.033). More importantly, NLRP3 inflammasome activation significantly suppressed the proliferation but didn’t induce the apoptosis of CD4+ cells and CD8+ cells (Figure 2). Accordingly, the mRNA expression of inhibitory molecules (Ctla-4, Btla, Tim3, Pd1 and Vista) of T cells and the PD1 or CTLA4 (Figure 3) on the membrane surface of CD4+ and CD8+ cells were up-regulated after the activation of NLRP3 inflammasome in PBMCs. Furthermore, the percentage of CD8+ cells significantly decreased after NLRP3 inflammasome activation in PBMCs. However, LPS plus ATP took no effect on purified CD4+ or CD8+ cells in contrast with PBMCs.

Conclusion
NLRP3 inflammsome-mediated innate immune reaction may play an important role in controlling the T cell responses of adaptive immunity by the inhibitory molecules and might prevent disease progression in adult chronic ITP.

Session topic: 33.  Platelets disorders

Keyword(s): T cell response, Immunity, ITP

Abstract: S887

Type: Oral Presentation

Presentation during EHA23: On Saturday, June 16, 2018 from 16:15 - 16:30

Location: Room A9

Background
Immune thrombocytopenia (ITP) is an acquired autoimmune disorder, which is characterized by imbalanced adaptive immunity of T cells. NLRP3 inflammasome has recently been reported to be involved in diverse inflammatory or immune diseases. However, NLRP3 inflammasome activation in the pathophysiology of ITP remains unclear.

Aims
To explore the effects of NLRP3 inflammsome activation on the T cells in adult patients with chronic ITP.

Methods
Peripheral blood was obtained from 28 adult patients with chronic ITP. PBMCs were isolated using Ficoll density-gradient centrifugation and the CD4+ cells were purified by positive immuno-magnetic microbeads selection. The PBMCs, CD4+ or CD8+ cells were primed with LPS, then stimulated with ATP and finally cultured in plate-bound anti-human CD3 and soluble anti-human CD28 plus IL-2. After four days, the percentage of CD4+ cells and CD8+ cells, the T helper cell subsets (Th cells) of Th1, Th2, Th17 and T-reg cells, the inhibitory molecules, PD1 and CTLA4, and the CFSE-labeled cells were analyzed by flow cytometry. IFN-γ, IL-4, IL-17 and TGF-β in cultured supernatants were assayed by ELISA. The mRNA expression level of cytokines associated with Th cells (Ifn-γ, Il-6, IL-10, Il-17 ), the key transcription factors of Th cells (T-bet, Gata3, Foxp3, Ror-γt) and the inhibitory molecules (Ctla-4, Btla, Tim3, Lag3, Pd1 and Vista) was determined by Real-Time PCR.

Results
We found that NLRP3 inflammasome activation in PBMCs initiated caspase-1–dependent IL-1β secretion, and thereby weakened the Th1 cell differentiation (unstimulated control 13.3% vs. stimulation 5.3%; p=0.015), which can be restored, at least in part, by caspase-1 inhibitor Z-YVAD-FMK (Figure 1). The production of IFN-γ and the expression of Ifn-γ, were significantly down-regulated after NLRP3 inflammsome activation. However, the percentage of Th2 or Th17 was not significantly different between the stimulated and unstimulated groups. Meanwhile, the percentage of T-reg cells was also decreased after stimulation by LPS plus ATP in PBMCs (unstimulated control 6.67% vs. stimulation 4.39%; p=0.033). More importantly, NLRP3 inflammasome activation significantly suppressed the proliferation but didn’t induce the apoptosis of CD4+ cells and CD8+ cells (Figure 2). Accordingly, the mRNA expression of inhibitory molecules (Ctla-4, Btla, Tim3, Pd1 and Vista) of T cells and the PD1 or CTLA4 (Figure 3) on the membrane surface of CD4+ and CD8+ cells were up-regulated after the activation of NLRP3 inflammasome in PBMCs. Furthermore, the percentage of CD8+ cells significantly decreased after NLRP3 inflammasome activation in PBMCs. However, LPS plus ATP took no effect on purified CD4+ or CD8+ cells in contrast with PBMCs.

Conclusion
NLRP3 inflammsome-mediated innate immune reaction may play an important role in controlling the T cell responses of adaptive immunity by the inhibitory molecules and might prevent disease progression in adult chronic ITP.

Session topic: 33.  Platelets disorders

Keyword(s): T cell response, Immunity, ITP

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