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Effect of disrupted mitochondria as a source of damage-associated molecular patterns on the production of tumor necrosis factor α by splenocytes from dogs

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  • 1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 2 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 3 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 4 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.
  • | 5 Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, OH 43210.
  • | 6 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210.

Abstract

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells.

SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes.

PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow–differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay.

RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system.

CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.

Abstract

OBJECTIVE To evaluate the effects of damage-associated molecular patterns (DAMPs) derived from disrupted mitochondria on canine splenocytes and other immune cells.

SAMPLES Liver, spleen, and bone marrow samples obtained from 8 cadavers of healthy research Beagles that had been euthanized for other purposes.

PROCEDURES Mitochondria were obtained from canine hepatocytes, and mitochondrial DAMPs (containing approx 75% mitochondrial proteins) were prepared. Mitochondrial DAMPs and the nuclear cytokine high-mobility group box protein 1 were applied to splenocytes, bone marrow–differentiated dendritic cells, and a canine myelomonocytic cell (DH82) line for 6 or 24 hours. Cell culture supernatants from splenocytes, dendritic cells, and DH82 cells were assayed for tumor necrosis factor α with an ELISA. Expression of tumor necrosis factor α mRNA in splenocytes was evaluated with a quantitative real-time PCR assay.

RESULTS In all cell populations evaluated, production of tumor necrosis factor α was consistently increased by mitochondrial DAMPs at 6 hours (as measured by an ELISA). In contrast, high-mobility group box protein 1 did not have any independent proinflammatory effects in this experimental system.

CONCLUSIONS AND CLINICAL RELEVANCE The study revealed an in vitro inflammatory effect of mitochondrial DAMPs (containing approx 75% mitochondrial proteins) in canine cells and validated the use of an in vitro splenocyte model to assess DAMP-induced inflammation in dogs. This experimental system may aid in understanding the contribution of DAMPs to sepsis and the systemic inflammatory response syndrome in humans. Further studies in dogs are needed to validate the biological importance of these findings and to evaluate the in vivo role of mitochondrial DAMPs in triggering and perpetuating systemic inflammatory states.

Contributor Notes

Dr. Friedenberg's present address is Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607.

Dr. Papenfuss and Ms. Strange's present address is WIL Research, 1407 George Rd, Ashland, OH 44805.

Mr. VanGundy's present address is Department of Pharmacy, The Ohio State University Wexner Medical Center, Columbus, OH 43210.

Address correspondence to Dr. Friedenberg (steven_friedenberg@ncsu.edu).