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Cystatin from the helminth Ascaris lumbricoides (Al-CPI) upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells (moDC)

ABSTRACT: Background: Ascaris lumbricoides cystatin (Al-CPI) prevents development of allergic airway inflammation and 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. Methods: moDC 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 STAR. 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. Results: Compared 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. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of LPS. Regarding the pathway of negative regulation of immune response, we found significant decrease in cell surface expression of CD86, HLA-DR and PD-L1 upon stimulation with 1 μM Al-CPI. Conclusion: Al-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. Several SMADs, members of the TGF-beta and the IL-10 families, including KLF10 were also This is a provisional file, not the final typeset article 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE250463 | GEO | 2024/01/15

REPOSITORIES: GEO

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Project description:Allergic contact dermatitis (ACD) is the most prevalent form of immunotoxicity in humans. Dendritic cells (DC) play a central role in the pathogenesis of ACD, particularly during the sensitization phase to a specific allergen. Upon activation, DCs maturate and migrate to draining lymph nodes which is accompanied by major metabolic and phenotypic alterations that facilitate the presentation of the captured antigen to prime naïve T cells. Nevertheless, knowledge on the molecular effects during maturation of DCs in the context of ACD remain scarce. Here, we present a global proteomic analysis of monocyte-derived dendritic cells (MoDC) treated with NiSO4, the most prominent cause of ACD. Proteomic alterations induced after treatment with NiSO4 were compared to the bacterial trigger lipopolysaccharide (LPS). Both substances possess a similar TLR4 binding capacity, which may help to identify allergy-specific effects compared to bacterial activation. MoDCs treated for 24 h with 2.5 µg/ ml LPS displayed a very strong immunological response, characterized by upregulation of DC activation markers, secretion of proinflammatory cytokines and stimulation of T cell proliferation. Similar immunological responses were observed after treatment with 400 µM NiSO4 but less pronounced. Both triggered TLR4 and TREM1 pathways. However, NiSO4 in addition, also activated hypoxic and apoptotic pathways, which might have overshadowed initial signaling. Moreover, our proteomic data support the importance of Nrf2 as a key player in mediating sensitization since many Nrf2 targets genes were strongly upregulated on protein level selectively after treatment with NiSO4. Strikingly, NiSO4 stimulation induced cellular hypocholesterolemia which was counteracted by the induction of genes and proteins relevant for cholesterol biosynthesis. Our proteomic study allowed for the first time to better characterize some of the fundamental differences between NiSO4 and LPS-triggered activation of MoDCs, providing an important contribution to the molecular understanding of contact allergy.

Project description:Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs. Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested. Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function. Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.

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Cystatin from the helminth Ascaris lumbricoides (Al-CPI) upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells (moDC)

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