Mechanism study of IgE high-affinity receptor binding to NPC2 on regulating DNP-BSA-induced mast cell degranulation and histamine release

doi:10.11958/20252449

Abstract

Abstract:

Objective To detect the expression of the high-affinity IgE receptor (FcεRⅠ) and Niemann-Pick disease type C2 protein (NPC2) in mast cells treated with 2,4-dinitrophenyl-conjugated bovine serum albumin (DNP-BSA), and to investigate the regulatory mechanism, by which NPC2 protein binding to FcεRⅠ modulating DNP-BSA-induced mast cell degranulation and histamine release. Methods From June to December 2023, five patients with allergic asthma diagnosed in the Department of Respiratory Medicine at Urumqi Friendship Hospital (asthma group) and five healthy controls (control group) were enrolled. Four- to six-week-old male wild-type C57BL/6 mice (n=5) and NPC2 gene knockout mice (n=3) were selected in this study. For in vitro experiments, bone marrow-derived mast cells (BMMCs) were isolated from two randomly selected wild-type mice. NPC2 knockdown was established via siRNA transfection (siNPC2 group), with corresponding negative control group (NC group) and wild-type (WT group) group. For in vivo experiments, an ovalbumin (OVA)-induced allergic asthma mouse model was established (grouping as above). Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to detect NPC2 and FcεRⅠ messenger RNA (mRNA) expression in peripheral blood from the asthma and control groups. Flow cytometry was used to detect the expression of cluster of differentiation 117 (CD117), FcεRⅠ and lysosome-associated membrane protein 1 (CD107a). Enzyme-linked immunosorbent assay (ELISA) was used to measure levels of inflammatory cytokines, chemokines, colony-stimulating factors and histamine release. Co-immunoprecipitation (Co-IP) was performed to verify the interaction between NPC2 and FcεRⅠ in mast cells. Hematoxylin and eosin (HE) staining was used to examine pathological changes in mouse ear tissue and lung tissue. Results The expression of NPC2 and FcεRⅠ mRNA in peripheral blood was significantly higher in the asthma group compared to the control group (P < 0.05). In vitro flow cytometry confirmed that the purity of the cultured BMMCs reached 93.4%. Compared to the WT group, NPC2 knockdown (siNPC2 group) significantly inhibited DNP-BSA-induced release of inflammatory cytokines [interleukin (IL)-4, IL-5, IL-9, IL-13, tumor necrosis factor-α (TNF-α)], chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.05), and reduced the histamine release rate and CD107a expression level. Co-IP experiments confirmed a direct interaction between NPC2 and FcεRⅠ in mast cells. In vivo experiments showed that NPC2 knockdown/knockout (siNPC2 group) mice exhibited significantly alleviated lung inflammatory injury compared to wild-type (WT group) mice. Conclusion NPC2 promotes mast cell activation and degranulation by binding to FcεRⅠ, and knockout of NPC2 significantly suppresses allergic response.

Key words: asthma, mast cells, cell degranulation, receptors, IgE, Niemann-Pick disease, type C, histamine

CLC Number:

R562.25，R341

Figures/Tables 7

References 27

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基因名称	引物序列（5'→3'）	产物大小/bp
NPC2	上游：CCAGCTGAGCAAAGGACAGT	380
	下游：TCTGCTACAGAGCACCTCCT	380
FcεRⅠ	上游：TAAGGTCTCCTTGAACCCTCCA	315
	下游：CAACCATGGCACCTGAGGAA	315
GAPDH	上游：AGGTCGGTGTGAACGGATTTG	123
	下游：TGTAGACCATGTAGTTGAGGTCA	123

基因名称	引物序列（5'→3'）	产物大小/bp
NPC2	上游：CCAGCTGAGCAAAGGACAGT	380
	下游：TCTGCTACAGAGCACCTCCT	380
FcεRⅠ	上游：TAAGGTCTCCTTGAACCCTCCA	315
	下游：CAACCATGGCACCTGAGGAA	315
GAPDH	上游：AGGTCGGTGTGAACGGATTTG	123
	下游：TGTAGACCATGTAGTTGAGGTCA	123

质量浓度	CCL2/（ng/L）								GM-CSF/（ng/L）								IL-4/（ng/L）
质量浓度	WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L	15.23±1.25			14.98±1.18			0.342		8.45±0.72			8.23±0.68			0.384		12.58±1.08			12.31±1.02			0.314
3 μg/L	45.67±3.82^a			28.43±2.65^a			6.432^＊＊		32.18±2.85^a			18.76±1.92^a			6.764^＊＊		38.75±3.24^a			22.46±2.18^a			7.225^＊＊
5 μg/L	78.92±6.54^ab			42.15±3.87^ab			8.381^＊＊		56.34±4.73^ab			29.82±2.74^ab			8.403^＊＊		65.82±5.47^ab			34.27±3.15^ab			8.657^＊＊
F	264.318^＊＊			147.925^＊＊					238.647^＊＊			175.384^＊＊					220.735^＊＊			152.418^＊＊
DNP-BSA含量		IL-5/（ng/L）								IL-9/（ng/L）								IL-13/（ng/L）
DNP-BSA含量		WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L		10.87±0.94			10.65±0.89			0.294		9.63±0.82			9.42±0.78			0.321		11.24±0.97			10.98±0.91			0.338
3 μg/L		35.42±2.96^a			20.18±1.87^a			7.539^＊＊		28.54±2.38^a			16.87±1.65^a			7.033^＊＊		33.67±2.81^a			19.52±1.83^a			7.308^＊＊
5 μg/L		58.76±4.82^ab			30.45±2.79^ab			8.804^＊＊		47.85±3.92^ab			25.63±2.34^ab			8.430^＊＊		62.38±5.16^ab			32.74±3.02^ab			8.586^＊＊
F		249.382^＊＊			198.647^＊＊					196.528^＊＊			158.739^＊＊					260.417^＊＊			215.683^＊＊
DNP-BSA含量			TNF-α/（ng/L）											组胺/%
DNP-BSA含量			WT组			siNPC2组					t			WT组					siNPC2组			t
0 μg/L			13.76±1.15			13.48±1.12					0.302			5.32±0.43					5.18±0.41			0.408
3 μg/L			42.35±3.58^a			24.87±2.32					7.097^＊＊			28.65±2.54^a					15.73±1.48^a			7.612^＊＊
5 μg/L			71.29±5.94^ab			37.56±3.45^ab					8.504^＊＊			58.50±4.32^ab					21.41±2.43^ab			12.961^＊＊
F			232.816^＊＊			173.952^＊＊								378.425^＊＊					149.287^＊＊

质量浓度	CCL2/（ng/L）								GM-CSF/（ng/L）								IL-4/（ng/L）
质量浓度	WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L	15.23±1.25			14.98±1.18			0.342		8.45±0.72			8.23±0.68			0.384		12.58±1.08			12.31±1.02			0.314
3 μg/L	45.67±3.82^a			28.43±2.65^a			6.432^＊＊		32.18±2.85^a			18.76±1.92^a			6.764^＊＊		38.75±3.24^a			22.46±2.18^a			7.225^＊＊
5 μg/L	78.92±6.54^ab			42.15±3.87^ab			8.381^＊＊		56.34±4.73^ab			29.82±2.74^ab			8.403^＊＊		65.82±5.47^ab			34.27±3.15^ab			8.657^＊＊
F	264.318^＊＊			147.925^＊＊					238.647^＊＊			175.384^＊＊					220.735^＊＊			152.418^＊＊
DNP-BSA含量		IL-5/（ng/L）								IL-9/（ng/L）								IL-13/（ng/L）
DNP-BSA含量		WT组			siNPC2组			t		WT组			siNPC2组			t		WT组			siNPC2组			t
0 μg/L		10.87±0.94			10.65±0.89			0.294		9.63±0.82			9.42±0.78			0.321		11.24±0.97			10.98±0.91			0.338
3 μg/L		35.42±2.96^a			20.18±1.87^a			7.539^＊＊		28.54±2.38^a			16.87±1.65^a			7.033^＊＊		33.67±2.81^a			19.52±1.83^a			7.308^＊＊
5 μg/L		58.76±4.82^ab			30.45±2.79^ab			8.804^＊＊		47.85±3.92^ab			25.63±2.34^ab			8.430^＊＊		62.38±5.16^ab			32.74±3.02^ab			8.586^＊＊
F		249.382^＊＊			198.647^＊＊					196.528^＊＊			158.739^＊＊					260.417^＊＊			215.683^＊＊
DNP-BSA含量			TNF-α/（ng/L）											组胺/%
DNP-BSA含量			WT组			siNPC2组					t			WT组					siNPC2组			t
0 μg/L			13.76±1.15			13.48±1.12					0.302			5.32±0.43					5.18±0.41			0.408
3 μg/L			42.35±3.58^a			24.87±2.32					7.097^＊＊			28.65±2.54^a					15.73±1.48^a			7.612^＊＊
5 μg/L			71.29±5.94^ab			37.56±3.45^ab					8.504^＊＊			58.50±4.32^ab					21.41±2.43^ab			12.961^＊＊
F			232.816^＊＊			173.952^＊＊								378.425^＊＊					149.287^＊＊

质量浓度	WT组	siNPC2组	t
0 μg/L	5.32±0.43	5.18±0.43	0.408
3 μg/L	28.65±2.54^a	15.73±1.48^a	7.612^＊＊
5 μg/L	58.50±4.32^ab	21.41±2.43^ab	12.961^＊＊
F	378.425^＊＊	149.287^＊＊