Image_3_Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.tif

BackgroundAscaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation.MethodsmoDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry.ResultsCompared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI.ConclusionAl-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.</p

Image_1_Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.tif

BackgroundAscaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation.MethodsmoDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry.ResultsCompared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI.ConclusionAl-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.</p

Image_2_Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.tif

BackgroundAscaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation.MethodsmoDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry.ResultsCompared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI.ConclusionAl-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.</p

Genetic variants associated with the strength of the IgE response in Colombian and Swedish populations

<p>Genetic variants associated with the strength of the IgE response in Colombian and Swedish populations</p

Genetic loci associated with the risk of having high IgE response (≥75^th percentile) to ABA-1 and <i>Ascaris</i> extract in the CGA dataset.

<p>A) Effects of the tagSNP <i>CHIA</i> rs10494133 on the risk of high IgE response to ABA-1 (filled circle) and to the <i>Ascaris</i> extract (filled square) under dominant model. B) Effects of <i>STAT6</i> rs73118440 on the risk of high IgE response to ABA-1. OR: Odds ratio; CI: confidence interval.</p

Descriptive of individuals selected for targeted re-sequencing from the Candidate Genes for Asthma (CGA) cohort

<p>Descriptive of individuals selected for targeted re-sequencing from the Candidate Genes for Asthma (CGA) cohort</p

Genetic loci associated with the risk of having high IgE response to both Ascaris and common allergens.

<p>A) Effects of <i>CHI3L1</i> rs4950928 on the risk of high IgE response to ABA-1 (filled circle) and to the pollen allergen Bet v 1 (white triangle). The association with ABA-1 was detected in the CGA cohort and the association with Bet v 1 in the Swedish Eczema Cohort. <b>B)</b> Effect of <i>ABDH13</i> rs3783118 on the risk of high IgE response (≥75^th percentile) to the extracts of <i>Ascaris</i> and HDM in the CGA dataset. Risk of IgE response to the <i>Ascaris</i> extract (filled square); Risk of IgE response to the <i>D</i>. <i>pteronyssinus</i> extract (filled triangle). OR: Odds ratio; CI: confidence interval.</p

Burden of 70 single nucleotide variants with differential enrichment between high (≥75^th percentile) and low (≤25^th percentile) IgE responders to Ascaris and ABA-1.

<p>Each column corresponds to the pattern of one individual. The color scale indicates the reference genotype (grey) or the presence of single nucleotide variants in heterozygous (blue) or homozygous (red).</p

Haplotype association between genetic variants in <i>CHIA</i> and IgE response to ABA-1 in the CGA cohort (n = 988)

<p>Haplotype association between genetic variants in <i>CHIA</i> and IgE response to ABA-1 in the CGA cohort (n = 988)</p

Distribution of variants in the genes analyzed by targeted resequencing (CGA cohort)

<p>Distribution of variants in the genes analyzed by targeted resequencing (CGA cohort)</p