Sepsis remains a prevalent clinical challenge and the underlying pathophysiology is still poorly understood. To investigate the complex molecular mechanisms of sepsis, various animal models have been developed, the most frequently used being the cecal ligation and puncture (CLP) model in rodents. In this model, sepsis originates from a polymicrobial infectious focus within the abdominal cavity, followed by bacterial translocation into the blood compartment, which then triggers a systemic inflammatory response. A requirement of this model is that it is performed with high consistency to obtain reproducible results. Evidence is now emerging that the accompanying inflammatory response varies with the severity grade of sepsis, which is highly dependent on the extent of cecal ligation. In this protocol, we define standardized procedures for inducing sepsis in mice and rats by applying defined severity grades of sepsis through modulation of the position of cecal ligation. The CLP procedure can be performed in as little as 10 min for each animal by an experienced user, with additional time required for subsequent postoperative care and data collection.

Immunodesign of experimental sepsis by cecal ligation and puncture.

Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4+CD25+ T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4+CD25− T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1β and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2−/− mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.

Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells.

Background:Remarkable progress has been made during the last decade in defining the molecular mechanisms that underlie septic shock. This rapidly expanding field is leading to new therapeutic opportunities in the management of severe sepsis.Aim:To provide the clinician with a timely summary of the m

Molecular biology of inflammation and sepsis: a primer.

Toll-like receptors in immunity and inflammatory diseases: Past, present, and future.

The concept of ‘Compensatory anti-inflammatory response syndrome’ (CARS) was proposed in 1997 by Roger Bone (1941–1997) to qualify the consequences of the counter-regulatory mechanisms initiated to limit the overzealous inflammatory process in patients with infectious (sepsis) or non-infectious systemic inflammatory response syndrome (SIRS). One major consequence of CARS is the modification of the immune status that could favour the enhanced susceptibility of intensive care patients to nosocomial infections. Indeed, most animal ‘two-hit’ models illustrate an enhanced sensitivity to infection after a first insult. However, this observation is highly dependent on the experimental procedure. Numerous functions of circulating leukocytes are altered in sepsis and SIRS patients, as well as in animal models of sepsis or SIRS. However, this is rather a reprogramming of circulating leukocytes, since there is not a global defect of the immune cells functions. Furthermore, within tissues, leukocytes are rather primed or activated than immunosuppressed. Thus, CARS may be considered as an adapted compartmentalized response with the aim to silence some acute proinflammatory genes, and to maintain the possible expression of certain genes involved in the anti-infectious process.

Compensatory anti-inflammatory response syndrome.

Regulatory CD4(+)CD25(+) T cells (Tregs) suppress autoimmune and inflammatory diseases through mechanisms that are only partly understood. Previous studies suggest that Tregs can suppress bacterially triggered intestinal inflammation and respond to LPS through TLRs with enhanced suppressive activity. In this study, we have used murine cecal ligation and puncture as a model of polymicrobial sepsis to explore the effects of adoptive transfer of Tregs on septic outcome. Adoptive transfer of in vitro-stimulated Tregs in both prevention and therapeutic modes significantly improved survival of cecal ligation and puncture mice. Furthermore, the effect was dependent on both the number of Tregs adoptively transferred and the presence of host T cells. Animals that received stimulated Tregs had significantly increased peritoneal mast cells and peritoneal TNF-alpha production. More importantly, adoptive transfer of in vitro-stimulated Tregs significantly improved bacterial clearance, which resulted in improved survival. Our results suggest a novel role for Tregs in sepsis.

Adoptive Transfer of In Vitro-Stimulated CD4+CD25+ Regulatory T Cells Increases Bacterial Clearance and Improves Survival in Polymicrobial Sepsis

Cecal ligation and puncture with total parenteral nutrition: a clinically relevant model of the metabolic, hormonal, and inflammatory dysfunction associated with critical illness.

Chunjin Ding

Papers

Adoptive Transfer of In Vitro-Stimulated CD4+CD25+ Regulatory T Cells Increases Bacterial Clearance and Improves Survival in Polymicrobial Sepsis

Cecal ligation and puncture with total parenteral nutrition: a clinically relevant model of the metabolic, hormonal, and inflammatory dysfunction associated with critical illness.