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Updated: Jun 27, 2026
Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages
Published on: November 3, 2014
Targeted delivery of siRNA to cell death proteins in sepsis
Pavan Brahmamdam1, Eizo Watanabe, Jacqueline Unsinger
1Department of Surgery, Washington University in St Louis, School of Medicine, St Louis, Missouri 63110, USA.
Abstract:
Immune suppression is a major cause of morbidity and mortality in the patients with sepsis. Apoptotic loss of immune effector cells such as CD4 T and B cells is a key component in the loss of immune competence in sepsis. Inhibition of lymphocyte apoptosis has led to improved survival in animal models of sepsis. Using quantitative real-time polymerase chain reaction of isolated splenic CD4 T and B cells, we determined that Bim and PUMA, two key cell death proteins, are markedly upregulated during sepsis. Lymphocytes have been notoriously difficult to transfect with small interfering RNA (siRNA). Consequently a novel, cyclodextrin polymer-based, transferrin receptor-targeted, delivery vehicle was used to coadminister siRNA to Bim and PUMA to mice immediately after cecal ligation and puncture. Antiapoptotic siRNA-based therapy markedly decreased lymphocyte apoptosis and prevented the loss of splenic CD4 T and B cells. Flow cytometry confirmed in vivo delivery of siRNA to CD4 T and B cells and also demonstrated decreases in intracellular Bim and PUMA protein. In conclusion, Bim and PUMA are two critical mediators of immune cell death in sepsis. Use of a novel cyclodextrin polymer-based, transferrin receptor-targeted siRNA delivery vehicle enables effective administration of antiapoptotic siRNAs to lymphocytes and reverses the immune cell depletion that is a hallmark of this highly lethal disorder.
Insights
Sepsis causes immune cell death by upregulating Bim and PUMA proteins. A novel delivery system effectively delivered anti-apoptotic small interfering RNA (siRNA) to lymphocytes, reducing cell death and improving survival in sepsis models.
Area of Science:
- Immunology
- Molecular Biology
- Sepsis Pathophysiology
Background:
- Sepsis leads to immune suppression, characterized by lymphocyte apoptosis.
- Loss of CD4 T and B cells contributes to immune incompetence during sepsis.
- Inhibiting lymphocyte apoptosis improves survival in preclinical sepsis models.
Purpose of the Study:
- To investigate the role of Bim and PUMA proteins in sepsis-induced lymphocyte apoptosis.
- To develop and evaluate a novel siRNA delivery system for targeting lymphocytes in sepsis.
Main Methods:
- Quantitative real-time PCR to measure Bim and PUMA expression in splenic CD4 T and B cells.
- Development of a cyclodextrin polymer-based, transferrin receptor-targeted delivery vehicle for siRNA.
- Co-administration of siRNA targeting Bim and PUMA to mice post-cecal ligation and puncture.
- Flow cytometry to assess siRNA delivery, lymphocyte apoptosis, and protein levels in vivo.
Main Results:
- Bim and PUMA were significantly upregulated in splenic CD4 T and B cells during sepsis.
- The novel delivery vehicle successfully delivered siRNA to lymphocytes in vivo.
- siRNA therapy targeting Bim and PUMA markedly reduced lymphocyte apoptosis and prevented cell depletion.
- Intracellular levels of Bim and PUMA proteins were decreased following siRNA administration.
Conclusions:
- Bim and PUMA are critical mediators of immune cell death in sepsis.
- A novel targeted siRNA delivery system effectively targets lymphocytes, reversing sepsis-induced immune cell depletion.
- This approach holds promise for treating immune suppression in sepsis.
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