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Induction of human tolerogenic dendritic cells by 3'-sialyllactose via TLR4 is explained by LPS contamination.

ABSTRACT: The human milk oligosaccharide 3'-sialyllactose (3'SL) has previously been shown to activate murine dendritic cells (DC) in a Toll-like receptor (TLR) 4-mediated manner ex vivo. In this study we aimed to investigate whether 3'SL has similar immunomodulatory properties on human DC. 3'SL was shown to induce NF-κB activation via human TLR4. However, LPS was detected in the commercially obtained 3'SL from different suppliers. After the removal of LPS from 3'SL, we studied its ability to modify DC differentiation in vitro. In contrast to LPS and 3'SL, LPS-free 3'SL did not induce functional and phenotypical changes on immature DC (iDC). iDC that were differentiated in the presence of LPS or 3'SL showed a semi-mature phenotype (i.e., fewer CD83+CD86+ DC), produced IL-10 and abrogated IL-12p70 and tumor necrosis factor-alpha levels upon stimulation with several TLR ligands. Differentiation into these tolerogenic DC was completely abrogated by LPS removal from 3'SL. In contrast to previous reports in mice, we found that LPS-free 3'SL does not activate NF-κB via human TLR4. In conclusion, removing LPS from (oligo)saccharide preparations is necessary to study their potential immunomodulatory function.

SUBMITTER: Perdijk O

PROVIDER: S-EPMC5993091 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Induction of human tolerogenic dendritic cells by 3'-sialyllactose via TLR4 is explained by LPS contamination.

Perdijk Olaf O van Neerven R J Joost RJJ Meijer Ben B Savelkoul Huub F J HFJ Brugman Sylvia S

Glycobiology 20180301 3

The human milk oligosaccharide 3'-sialyllactose (3'SL) has previously been shown to activate murine dendritic cells (DC) in a Toll-like receptor (TLR) 4-mediated manner ex vivo. In this study we aimed to investigate whether 3'SL has similar immunomodulatory properties on human DC. 3'SL was shown to induce NF-κB activation via human TLR4. However, LPS was detected in the commercially obtained 3'SL from different suppliers. After the removal of LPS from 3'SL, we studied its ability to modify DC di ...[more]

PMID: 29281012

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Project description:BackgroundThe use of tolerogenic DCs is a promising therapeutic strategy for transplantation and autoimmune disorders. Immunomodulatory DCs are primarily generated from monocytes (MDDCs) for in vitro experiments following protocols that fail to fulfil the strict regulatory rules of clinically applicable products. Here, we compared the efficacy of three different tolerance-inducing agents, dexamethasone, rapamycin and vitamin D3, on DC biology using GMP (Good Manufacturing Practice) or clinical grade reagents with the aim of defining their use for human cell therapy.MethodsTolerogenic MDDCs were generated by adding tolerogenic agents prior to the induction of maturation using TNF-α, IL-β and PGE2. We evaluated the effects of each agent on viability, efficiency of differentiation, phenotype, cytokine secretion and stability, the stimulatory capacity of tol-DCs and the T-cell profiles induced.ResultsDifferences relevant to therapeutic applicability were observed with the cellular products that were obtained. VitD3-induced tol-DCs exhibited a slightly reduced viability and yield compared to Dexa-and Rapa-tol-DCs. Phenotypically, while Dexa-and VitD3-tol-DCs were similar to immature DCs, Rapa-tol-DCs were not distinguishable from mature DCs. In addition, only Dexa-and moderately VitD3-tol-DCs exhibited IL-10 production. Interestingly, in all cases, the cytokine secretion profiles of tol-DCs were not modified by a subsequent TLR stimulation with LPS, indicating that all products had stable phenotypes. Functionally, clearly reduced alloantigen T cell proliferation was induced by tol-DCs obtained using any of these agent. Also, total interferon-gamma (IFN-γ) secretion by T cells stimulated with allogeneic tol-DCs was reduced in all three cases, but only T cells co-cultured with Rapa-tol-DCs showed impaired intracellular IFN-γ production. In addition, Rapa-DCs promoted CD4+ CD127 low/negative CD25high and Foxp3+ T cells.ConclusionsOur results demonstrate contrasting influences of different clinical-grade pharmacological agents on human tol-DC generation. This should be taken into account for decisions on the use of a specific agent for the appropriate cellular therapy in the context of a particular disease.

Project description:Tolerogenic dendritic cell (tDC)-based clinical trials for the treatment of autoimmune diseases are now a reality. Clinical trials are currently exploring the effectiveness of tDC to treat autoimmune diseases of type 1 diabetes mellitus, rheumatoid arthritis, multiple sclerosis (MS), and Crohn's disease. This review will address tDC employed in current clinical trials, focusing on cell characteristics, mechanisms of action, and clinical findings. To date, the publicly reported human trials using tDC indicate that regulatory lymphocytes (largely Foxp3+ T-regulatory cell and, in one trial, B-regulatory cells) are, for the most part, increased in frequency in the circulation. Other than this observation, there are significant differences in the major phenotypes of the tDC. These differences may affect the outcome in efficacy of recently launched and impending phase II trials. Recent efforts to establish a catalog listing where tDC converge and diverge in phenotype and functional outcome are an important first step toward understanding core mechanisms of action and critical "musts" for tDC to be therapeutically successful. In our view, the most critical parameter to efficacy is in vivo stability of the tolerogenic activity over phenotype. As such, methods that generate tDC that can induce and stably maintain immune hyporesponsiveness to allo- or disease-specific autoantigens in the presence of powerful pro-inflammatory signals are those that will fare better in primary endpoints in phase II clinical trials (e.g., disease improvement, preservation of autoimmunity-targeted tissue, allograft survival). We propose that pre-treatment phenotypes of tDC in the absence of functional stability are of secondary value especially as such phenotypes can dramatically change following administration, especially under dynamic changes in the inflammatory state of the patient. Furthermore, understanding the outcomes of different methods of cell delivery and sites of delivery on functional outcomes, as well as quality control variability in the functional outcomes resulting from the various approaches of generating tDC for clinical use, will inform more standardized ex vivo generation methods. An understanding of these similarities and differences, with a reference point the large number of naturally occurring tDC populations with different immune profiles described in the literature, could explain some of the expected and unanticipated outcomes of emerging tDC clinical trials.

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Induction of human tolerogenic dendritic cells by 3'-sialyllactose via TLR4 is explained by LPS contamination.

Publications

Induction of human tolerogenic dendritic cells by 3'-sialyllactose via TLR4 is explained by LPS contamination.

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