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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

Autophagy and apoptosis in human pancreatic islets.TR-FRET acceptor in human islets incubated under control conditions in normoxia or under low nutrient conditions and hypoxia (a). Assay windows values are plotted as mean (n = 2 duplicate wells for each concentration of CQ). Percentage of apoptotic islets were calculated as positive islets stained with Annexin V cultured in control normoxic conditions and (b) or hypoxic low nutrients (c). Representative western blot picture for caspase-3 and −9 (d). Values are presented as median ± IQR. A p < 0.05 was considered significant (*p < 0.05).
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f3: Autophagy and apoptosis in human pancreatic islets.TR-FRET acceptor in human islets incubated under control conditions in normoxia or under low nutrient conditions and hypoxia (a). Assay windows values are plotted as mean (n = 2 duplicate wells for each concentration of CQ). Percentage of apoptotic islets were calculated as positive islets stained with Annexin V cultured in control normoxic conditions and (b) or hypoxic low nutrients (c). Representative western blot picture for caspase-3 and −9 (d). Values are presented as median ± IQR. A p < 0.05 was considered significant (*p < 0.05).

Mentions: Islets were subsequently subjected to studies for autophagy and apoptosis (Fig. 3). The autophagy TR-FRET assay serves to quantify autophagosomes in islets27. As shown in Fig. 3a, islets cultured under low-nutrient and hypoxic conditions have almost twice as much autophagic bodies (EC50 = 43.5) when compared to control conditions (EC50 = 23.5). Primary apoptosis was quantified by counting the number of Annexin V positive cells and was found to be statistically significant enhanced by low-nutrient hypoxic conditions (p < 0.05) (Fig. 3c) but not by control normoxic conditions (Fig. 3b). As Annexin V staining identifies apoptotic cells in an early stage, the islets were also subjected to caspase-3 and −9 analysis by western blot, to demonstrate the more irreversible stage of apoptosis28. After incubation under low-nutrients hypoxic and control normoxic conditions, full caspase-3 and −9 was found but not their cleaved form (Fig. 3d). This indicates an active process of cellular death towards necrosis rather than secondary apoptosis2930.


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)

Autophagy and apoptosis in human pancreatic islets.TR-FRET acceptor in human islets incubated under control conditions in normoxia or under low nutrient conditions and hypoxia (a). Assay windows values are plotted as mean (n = 2 duplicate wells for each concentration of CQ). Percentage of apoptotic islets were calculated as positive islets stained with Annexin V cultured in control normoxic conditions and (b) or hypoxic low nutrients (c). Representative western blot picture for caspase-3 and −9 (d). Values are presented as median ± IQR. A p < 0.05 was considered significant (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Autophagy and apoptosis in human pancreatic islets.TR-FRET acceptor in human islets incubated under control conditions in normoxia or under low nutrient conditions and hypoxia (a). Assay windows values are plotted as mean (n = 2 duplicate wells for each concentration of CQ). Percentage of apoptotic islets were calculated as positive islets stained with Annexin V cultured in control normoxic conditions and (b) or hypoxic low nutrients (c). Representative western blot picture for caspase-3 and −9 (d). Values are presented as median ± IQR. A p < 0.05 was considered significant (*p < 0.05).
Mentions: Islets were subsequently subjected to studies for autophagy and apoptosis (Fig. 3). The autophagy TR-FRET assay serves to quantify autophagosomes in islets27. As shown in Fig. 3a, islets cultured under low-nutrient and hypoxic conditions have almost twice as much autophagic bodies (EC50 = 43.5) when compared to control conditions (EC50 = 23.5). Primary apoptosis was quantified by counting the number of Annexin V positive cells and was found to be statistically significant enhanced by low-nutrient hypoxic conditions (p < 0.05) (Fig. 3c) but not by control normoxic conditions (Fig. 3b). As Annexin V staining identifies apoptotic cells in an early stage, the islets were also subjected to caspase-3 and −9 analysis by western blot, to demonstrate the more irreversible stage of apoptosis28. After incubation under low-nutrients hypoxic and control normoxic conditions, full caspase-3 and −9 was found but not their cleaved form (Fig. 3d). This indicates an active process of cellular death towards necrosis rather than secondary apoptosis2930.

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