Fig 1.
Mast cell degranulation by AgNP and DNP across 23 strains of mice.
Bone marrow-derived mast cells (BMMCs) were isolated from 23 inbred and recombinant inbred (BxD) strains of mice. Degranulation was assessed by measuring ß-hexosaminidase release following (A) AgNP treatment at 25 μg/ml for 1 h in un-sensitized BMMCs, or (B) DNP treatment at 100 ng/ml for 0.5 h after BMMC overnight sensitization with IgE anti-DNP. Arrows represent low and high responder strains for each treatment used in RNA-seq (AgNP: green-LP/J, red-C57BL/6J; DNP: red-C57BL/6J, blue-RIIIS/J). Values are expressed as mean ± SEM (n = 3/strain) normalized to strain-specific non-treated control values.
Fig 2.
Association mapping study utilizing 22 strains of mice were performed in conjunction with RNA-sequencing on high and low responder strains. ‘Susceptibility’ and ‘response’ genes were reported.
Fig 3.
Association mapping of AgNP or DNP treatment with mast cell degranulation.
Manhattan plot of chromosomal loci associated with (A) AgNP or (B) DNP treatment across 22 strains of mice. The figure inserts include further information on the peak marker, chromosome, p-values, and associated regions. Loci with p-values ≤ 7×10−5 (red line) and adjusted p-values ≤ 0.05 were considered significant.
Table 1.
Chromosomal loci significantly associated with mast cell degranulation by AgNP or DNP and genes residing in each locus.
Fig 4.
Quantitative comparison of differentially expressed transcripts and the biological responses that are significantly enriched in mast cells by AgNPs.
(A) Venn diagram showing the number of differentially expressed (DE) genes produced in response to DNP treatment (black) and AgNP treatment (grey) with a combined total of 3890 genes. (B) Heat map of the relative expression of 85 DE genes (FDR≤0.05) following AgNP exposure in the low responder (LP/J) and high responder strain (C57BL/6J). Expression patterns for individual genes are in rows. The color represents the direction of difference in expression with red indicating increased expression and green indicated decreased expression after treatment; The intensity of the color represents the magnitude of difference in expression (log2 scale) between AgNP treated cells and non-treated cells. (C) Gene ontology (GO) categories overrepresented in the 34 AgNP-responsive gene set were assessed using Pantherdb.org. GO categories are ranked by number of genes, listing the number of AgNP-responsive genes in each category (white bar) verses the number of genes expected in each category (black bar). Significance was set at p≤0.05 with a fold enrichment cutoff of ≥5. RTK, receptor tyrosine kinase; MAPK, mitogen-activated protein kinase.
Table 2.
Candidate genes identified by RNA-seq that are differentially expressed following AgNP treatment.
Fig 5.
Expression of Txnip following AgNP treatment.
Bone marrow-derived mast cells (BMMCs) were treated with AgNP for 1 h at 25 μg/ml and A) mRNA expression of Txnip was quantified by real-time quantitative polymerase chain reaction (qPCR). Values are expressed as fold change compared to non-treated cells (n = 3/group) normalized to Gapdh. B) Representative immunoblots of TXNIP expression in both high (C57BL/6J) and low (LP/J) responder strains following AgNP treatment (1 h at 25 μg/ml). Values are expressed as mean ± SEM of at least 3 independent experiments. * Indicates significant difference from controlled group (p≤ 0.05). # Indicates significant difference between strains (p≤ 0.05).
Fig 6.
Prostaglandin receptor (EP4) expression and inhibition of AgNP-directed mast cell degranulation.
Bone marrow-derived mast cell (BMMCs) isolated from C57BL/6J mice were evaluated for degranulation by measuring ß-hexosaminidase release following 1 h exposure of AgNP at 25 μg/ml ± pre-treatment with EP4 selective inhibitor (10 μM), prostaglandin (PGE2, 10 μM), or Cox-1 and -2 inhibitors (indomethacin, 5 μM). Values are expressed as mean ± SEM (n = 3). * Indicates significant difference compared to non-treated control and # indicates significant difference compared to AgNP treatment alone (p ≤ 0.05).