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DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1.

Paredes-Juarez GA, Sahasrabudhe NM, Tjoelker RS, de Haan BJ, Engelse MA, de Koning EJ, Faas MM, de Vos P - Sci Rep (2015)

Bottom Line: Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules.Another effective strategy was suppressing necroptosis using the inhibitor nec-1.DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1.

View Article: PubMed Central - PubMed

Affiliation: University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Section of Immunoendocrinology, Groningen, 9713 GZ, The Netherlands.

ABSTRACT
In between the period of transplantation and revascularization, pancreatic islets are exposed to low-oxygen and low-nutrient conditions. In the present study we mimicked those conditions in vitro to study the involvement of different cell death processes, release of danger-associated molecular patterns (DAMP), and associated in vitro immune activation. Under low-oxygen and low-nutrient conditions, apoptosis, autophagy and necroptosis occur in human islets. Necroptosis is responsible for DAMP-release such as dsDNA, uric acid, and HMGB1. The sensors of the innate immune system able to recognize these DAMPs are mainly TLR, NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule MyD88, we were able to show that the islet-derived DAMPs signal mainly via TLR. Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules. Another effective strategy was suppressing necroptosis using the inhibitor nec-1. Although the effect on cell-survival was minor, nec-1 was able to reduce the release of HMGB1 and its associated immune activation. Our data demonstrate that in the immediate post-transplant period islets release DAMPs that in vitro enhance responses of innate immune cells. DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1.

No MeSH data available.


Related in: MedlinePlus

TLR dependency of the activation of THP1 cells by islet derived molecules.THP1 cells were stimulated with supernatant of human islets incubated for 1 (a) and 7 (b) days under control and low-nutrition conditions at 20% and 1% of oxygen. NF-κB/AP-1 activation was enhanced under low-nutrients and low oxygen conditions in a TLR fashion, since this activation was virtually absent in THP1 MyD88-deficient cell lines at 1 (c) or 7 (d) days of incubation. Values are presented as median ± IQR (n = 4 separate batches of human islets). A p < 0.05 was consider statistical significant (*p < 0.05; **p < 0.01). LPS (10 μg/ml) and Tri-DAP (10 μg/ml) were used as positive control for THP1 and THP1 MyD88-deficient cell lines respectively.
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f1: TLR dependency of the activation of THP1 cells by islet derived molecules.THP1 cells were stimulated with supernatant of human islets incubated for 1 (a) and 7 (b) days under control and low-nutrition conditions at 20% and 1% of oxygen. NF-κB/AP-1 activation was enhanced under low-nutrients and low oxygen conditions in a TLR fashion, since this activation was virtually absent in THP1 MyD88-deficient cell lines at 1 (c) or 7 (d) days of incubation. Values are presented as median ± IQR (n = 4 separate batches of human islets). A p < 0.05 was consider statistical significant (*p < 0.05; **p < 0.01). LPS (10 μg/ml) and Tri-DAP (10 μg/ml) were used as positive control for THP1 and THP1 MyD88-deficient cell lines respectively.

Mentions: As shown in Fig. 1, even under control conditions immune-activation of THP1-XBlue™-MD2-CD14 was observed at 1 day (Fig. 1a). This activation was more enhanced by low-nutrient conditions than by hypoxia. The effect of low-nutrient conditions was reduced after 7 days of culturing (Fig. 1b) (p < 0.05). The reduction, can be explained by survival of a lower number of islet-cells that contribute to DAMPs production as will be presented in the next section, in which almost 40% of the cells are lost due to the harsh culture circumstances. The activation was TLR-dependent as the activation was profoundly reduced when the same supernatant was co-incubated with THP1 cells expressing the non-functional MyD88 under combined hypoxic and low-nutrient conditions (Fig. 1c,d).


DAMP production by human islets under low oxygen and nutrients in the presence or absence of an immunoisolating-capsule and necrostatin-1.

Paredes-Juarez GA, Sahasrabudhe NM, Tjoelker RS, de Haan BJ, Engelse MA, de Koning EJ, Faas MM, de Vos P - Sci Rep (2015)

TLR dependency of the activation of THP1 cells by islet derived molecules.THP1 cells were stimulated with supernatant of human islets incubated for 1 (a) and 7 (b) days under control and low-nutrition conditions at 20% and 1% of oxygen. NF-κB/AP-1 activation was enhanced under low-nutrients and low oxygen conditions in a TLR fashion, since this activation was virtually absent in THP1 MyD88-deficient cell lines at 1 (c) or 7 (d) days of incubation. Values are presented as median ± IQR (n = 4 separate batches of human islets). A p < 0.05 was consider statistical significant (*p < 0.05; **p < 0.01). LPS (10 μg/ml) and Tri-DAP (10 μg/ml) were used as positive control for THP1 and THP1 MyD88-deficient cell lines respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588515&req=5

f1: TLR dependency of the activation of THP1 cells by islet derived molecules.THP1 cells were stimulated with supernatant of human islets incubated for 1 (a) and 7 (b) days under control and low-nutrition conditions at 20% and 1% of oxygen. NF-κB/AP-1 activation was enhanced under low-nutrients and low oxygen conditions in a TLR fashion, since this activation was virtually absent in THP1 MyD88-deficient cell lines at 1 (c) or 7 (d) days of incubation. Values are presented as median ± IQR (n = 4 separate batches of human islets). A p < 0.05 was consider statistical significant (*p < 0.05; **p < 0.01). LPS (10 μg/ml) and Tri-DAP (10 μg/ml) were used as positive control for THP1 and THP1 MyD88-deficient cell lines respectively.
Mentions: As shown in Fig. 1, even under control conditions immune-activation of THP1-XBlue™-MD2-CD14 was observed at 1 day (Fig. 1a). This activation was more enhanced by low-nutrient conditions than by hypoxia. The effect of low-nutrient conditions was reduced after 7 days of culturing (Fig. 1b) (p < 0.05). The reduction, can be explained by survival of a lower number of islet-cells that contribute to DAMPs production as will be presented in the next section, in which almost 40% of the cells are lost due to the harsh culture circumstances. The activation was TLR-dependent as the activation was profoundly reduced when the same supernatant was co-incubated with THP1 cells expressing the non-functional MyD88 under combined hypoxic and low-nutrient conditions (Fig. 1c,d).

Bottom Line: Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules.Another effective strategy was suppressing necroptosis using the inhibitor nec-1.DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1.

View Article: PubMed Central - PubMed

Affiliation: University of Groningen, University Medical Center Groningen, Department of Pathology and Medical Biology, Section of Immunoendocrinology, Groningen, 9713 GZ, The Netherlands.

ABSTRACT
In between the period of transplantation and revascularization, pancreatic islets are exposed to low-oxygen and low-nutrient conditions. In the present study we mimicked those conditions in vitro to study the involvement of different cell death processes, release of danger-associated molecular patterns (DAMP), and associated in vitro immune activation. Under low-oxygen and low-nutrient conditions, apoptosis, autophagy and necroptosis occur in human islets. Necroptosis is responsible for DAMP-release such as dsDNA, uric acid, and HMGB1. The sensors of the innate immune system able to recognize these DAMPs are mainly TLR, NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule MyD88, we were able to show that the islet-derived DAMPs signal mainly via TLR. Immunoisolation in immunoprotective membranes reduced DAMP release and immune activation via retention of the relative large DAMPs in the capsules. Another effective strategy was suppressing necroptosis using the inhibitor nec-1. Although the effect on cell-survival was minor, nec-1 was able to reduce the release of HMGB1 and its associated immune activation. Our data demonstrate that in the immediate post-transplant period islets release DAMPs that in vitro enhance responses of innate immune cells. DAMP release can be reduced in vitro by immunoisolation or intervention with nec-1.

No MeSH data available.


Related in: MedlinePlus