Chun-Shiang Chung

MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis

Polymicrobial sepsis alters the adaptive immune response and induces T cell suppression and Th2 immune polarization. We identify a GR-1+CD11b+ population whose numbers dramatically increase and remain elevated in the spleen, lymph nodes, and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express interleukin 10 and several other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T cell interferon (IFN) γ production but only modestly suppress antigen-specific and nonspecific CD4+ T cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of Th2 cell–dependent and depression of Th1 cell–dependent antibody production. Signaling through MyD88, but not Toll-like receptor 4, TIR domain–containing adaptor-inducing IFN-β, or the IFN-α/β receptor, is required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T cell suppression and preferential Th2 polarization.

Leukocyte apoptosis and its significance in sepsis and shock

Sepsis and multiple organ failure continue to be significant problems among trauma, burn, and the critically ill patient population. Thus, a number of laboratories have focused on understanding the role of altered apoptotic cell death in contributing to immune and organ dysfunction seen in sepsis and shock. Immune cells that undergo altered apoptotic changes include neutrophils, macrophages, dendritic cells, as well as various lymphocyte populations. Evidence of epithelial as well as endothelial cell apoptotic changes has also been reported. Although mediators such as steroids, tumor necrosis factor, nitric oxide, C5a, and Fas ligand (FasL) appear to contribute to the apoptotic changes, their effects are tissue‐ and cell population‐selective. As inhibiting Fas‐FasL signaling (e.g., gene deficiency, Fas fusion protein, or Fas short interfering RNA administration), caspase inhibition (caspase mimetic peptides), and/or the overexpression of downstream antiapoptotic molecules (e.g., Bcl‐2, Akt) improve survival of septic mice, it not only demonstrates the pathological significance of this process but points to novel targets for the treatment of sepsis.

Increased circulating regulatory T cells (CD4(+)CD25 (+)CD127 (-)) contribute to lymphocyte anergy in septic shock patients.

PurposeSepsis syndrome represents the leading cause of death in intensive care unit. Patients present features consistent with a decline in immune responsiveness potentially contributing to mortality. We investigated whether CD4+CD25+ regulatory T cells (Treg) participate in the induction of lymphocyte anergy after sepsis.MethodObservational study in septic shock patients and experimental study in mice.ResultsWe took advantage of the recently described flow cytometric gating strategy using the measurement of CD25 and CD127 expressions for monitoring Treg (CD4+CD25+CD127−Foxp3+). In patients the increased circulating Treg percentage significantly correlated with a decreased lympho-proliferative response. In a murine model of sepsis mimicking these observations, the ex vivo downregulation of Foxp3 expression using siRNA was associated with a restoration of this response.ConclusionThe relative increase in circulating Treg might play a role in lymphocyte anergy described after septic shock and represent a standardizable surrogate marker of declining proliferative capacity after sepsis.

Regulatory T cell populations in sepsis and trauma

Sepsis syndrome remains the leading cause of mortality in intensive care units. It is now believed that along with the body’s hyperinflammatory response designated to eliminate the underlying pathogen, mechanisms are initiated to control this initial response, which can become deleterious and result in immune dysfunctions and death. A similar state of immune suppression has been described after numerous forms of severe trauma/injury. Although the evidence for immune dysfunctions after sepsis has grown, much remains to be understood about mechanisms underpinning its development and how it acts to increase the morbid state of the critically ill patient. In this context, although the majority of clinical and basic science conducted so far has focused on the roles of myeloid cell populations, the contribution of T lymphocytes and in particular, of regulatory T cells has been somewhat ignored. The studies presented here support the concept that regulatory T lymphocytes (CD4+CD25+ regulatory, γδ, and NK T cells) play a role in the control of immune responses and are affected by injury and sepsis. This may be related to their capacity to interact with components of the innate and adaptive immune responses and to their ability to be activated nonspecifically by bacterial products and/or cytokines and to regulate through direct cell–cell and/or soluble mediators. It is our hope that a better understanding of the mechanism through which those rare lymphocyte subsets exert such a profound effect on the immune response may help in improving our ability not only to diagnose but also to treat the critically ill individual.

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis.

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.

What is the role of interleukin 10 in polymicrobial sepsis: Anti-inflammatory agent or immunosuppressant? ☆ ☆☆

BACKGROUND Controversy exists concerning the role of interleukin 10 (IL-10) in sepsis. When IL-10 is used in models of endotoxemia, it appears to protect (by anti-inflammatory effects), whereas in models of polymicrobial sepsis it seems to be deleterious (by immunosuppression?). However, little direct evidence for such an immunosuppressive role is available for polymicrobial sepsis. Thus the aim of this study was to determine whether IL-10 contributes to lymphocyte immunosuppression in a model of cecal ligation and puncture (CLP) and whether neutralization of IL-10 has any salutary effects on survival after sepsis. METHODS To assess the former, polymicrobial sepsis was induced in male C57BL/6J wild-type (+/+) and C57BL/6J-IL-10 knockout(-/-) mice by CLP. Splenocytes were harvested 24 hours later and stimulated with concanavalin A to assess their proliferative capacity and their ability to release the Th1 lymphokines interleukin 2 and interferon gamma (by enzyme-linked immunosorbent assay, nanograms/millilter). To further verify the immunosuppressive role of IL-10, splenocytes were obtained from male C3H/HeN mice 24 hours after CLP and then stimulated in the presence or absence of anti-IL-10 monoclonal antibody (Mab, 4 micrograms/mL). To assess the in vivo effects of IL-10 neutralization on survival after CLP, C3H/HeN mice (16 per group) were given 250 micrograms of anti-IL-10 Mab (intraperitoneally) either immediately after CLP (before the initiation of the hyperdynamic phase) or 12 hours after CLP (the beginning of the hypodynamic state). Control mice were given nonspecific rat immunoglobulin G. RESULTS These data indicate that IL-10 deficiency (-/-) prevents the depression of the proliferative capacity and Th1 lymphokine production after sepsis. Analysis of the interleukin 2-interferon gamma production patterns and proliferative capacity in lymphocytes treated with anti-IL-10 Mab confirmed the role of IL-10 in suppressing lymphocyte responsiveness in CLP. Interestingly, however, only delayed administration (12 hours after CLP) of anti-IL-10 markedly increased survival of mice (Fishers exact test, P < .05). CONCLUSION The results not only illustrate IL-10s role in septic immune dysfunction but document that anti-IL-10 administration beyond the initial proinflammatory hyperdynamic state of polymicrobial sepsis improves survival of animals subjected to sepsis.

The Contribution of CD4 + CD25 + T-Regulatory-Cells to Immune Suppression in Sepsis

Studies have indicated that there is a development of generalized immune dysfunction after septic insult. However, the mechanisms responsible for these changes remain unclear. Recently, accumulating evidence shows that several lymphocyte subpopulations such as NKT-, CD4+-Th2-T-, CD8+-T-, &ggr;&dgr;-T-, and CD4+CD25+ T regulatory cells are capable of actively contributing to the induction of septic immune suppression. Thus, our aim was to investigate the contribution of CD4+CD25+ cells to the immune dysfunction seen in sepsis. To study this, C57BL/6J, C57BL/6-Il6tm1Kopf (interleukin [IL] 6 −/−), and C57BL/6−Il10tm1Cgn (IL-10 −/−) mice were subjected to cecal ligation and puncture (CLP) or sham operations. Twenty-four hours later, blood was collected, and splenocytes were isolated. Phenotypic expression of CD4/CD25 (by fluorescence-activated cell sorter), cell proliferation (presented as proliferation index = [with anti-CD3]/[without anti-CD3]), and immune suppressive capacity (by in vitro add-back experiments) were assessed. The results indicate a marked elevation in CD4+CD25+ cell levels and their proliferation index after sepsis in background mice. CD4+CD25− cells from sham and CLP mice proliferated equally. However, coculture of CD4+CD25− with CD4+CD25+ cells suppressed their proliferation in both sham and CLP mice. Depletion of CD25+ cells in vivo before CLP markedly restored CD4+CD25− proliferative capacity and Th1 cytokine release while not altering plasma proinflammatory cytokine levels. Subsequently, IL−6 −/− and IL-10 −/− mice were used to elucidate the possible mediator(s) regulating the changes seen after sepsis. Although CD4+CD25+ cells increased after septic insult in both C57BL/6J and IL-6 −/− mice, this was not observed in IL-10 −/− mice. Similarly, in vitro proliferation studies showed that proliferation index increased in CD4+CD25+ cells from septic C57BL/6J and IL6 −/− mice, but it remained the same in IL-10 −/− mice. Surprisingly, depletion of CD25+ cells before inducing sepsis did not alter septic mortality. Together, these findings suggest that although CD4+CD25+ T regulatory cells induced by IL-10 seem to contribute to aspects of sepsis-induced lymphoid immune suppression, the oblation of CD25+ cells does not provide a survival advantage or disadvantage.

In vivo gene silencing (with siRNA) of pulmonary expression of MIP‐2 versus KC results in divergent effects on hemorrhage‐induced, neutrophil‐mediated septic acute lung injury

Lung injury in trauma patients exposed to a secondary infectious/septic challenge contributes to the high morbidity/mortality observed in this population. Associated pathology involves a dys‐regulation of immune function, specifically, sequestration of activated polymorphonuclear neutrophils (PMN) in the lungs. The targeting of PMN is thought to involve the release of chemokines from cells within the local environment, creating a concentration gradient along which PMN migrate to the focus of inflammation. Keratinocyte‐derived chemokine (KC) and macrophage‐inflammatory protein‐2 (MIP‐2) are murine neutrophil chemokines identified as playing significant but potentially divergent roles in the pathogenesis of acute lung injury (ALI). In the current study, we examined the contribution of local pulmonary cells to the production of KC and MIP‐2 and the pathogenesis of ALI. We hypothesized that local silencing of KC or MIP‐2, via the local administration of small interference RNA (siRNA) against KC or MIP‐2, following traumatic shock/hemorrhage (Hem), would suppress signaling for PMN influx to the lung, thereby reducing ALI associated with a secondary septic challenge (cecal ligation and puncture). Assessment of siRNA local gene silencing was done in green fluorescent protein (GFP)‐transgenic, overexpressing mice. A marked suppression of GFP expression was observed in the lung 24 h following intratracheal (i.t.) instillation of GFP siRNA, which was not observed in the liver. To test our hypothesis, siRNA against KC or MIP‐2 (75 ug/C3H/Hen mouse) was instilled (i.t.) 2 h post‐Hem (35 mm Hg for 90 min, 4× LRS Rx.). Twenty‐four hours after, mice were subjected to septic challenge and then killed 24 h later. i.t. MIP‐2 siRNA significantly (P<0.05, ANOVA‐Tukey’s test, n=5–6/group) reduced tissue and plasma interleukin (IL)‐6, tissue MIP‐2 (enzyme‐linked immunosorbent assay), as well as neutrophil influx [myeloperoxidase (MPO) activity]. In contrast, KC siRNA treatment reduced plasma KC, tissue KC, and IL‐6 but produced no significant reduction in plasma IL‐6 or MPO. Neither treatment reduced tissue or plasma levels of tumor necrosis factor α compared with vehicle. These data support not only our hypothesis that local pulmonary chemokine production of MIP‐2, to a greater extent than KC, contributes to the pathogenesis of PMN‐associated ALI following Hem but also the use of siRNA as a potential therapeutic.

Shock and hemorrhage: an overview of animal models.

Shock resulting from life-threatening blood loss (hemorrhage) remains a common complication of traumatic injury. Intensive experimental efforts are needed if we are to understand the pathological effect(s) of hemorrhagic shock, alone or in association with traumatic tissue injury, and to reverse this deleterious process in trauma patients. Here, we overview selected studies that are representative of the different hemorrhagic shock models, considering their advantages and disadvantages from a scientific and clinical perspective. Fixed-pressure versus fixed-volume versus uncontrolled hemorrhage models, with or without tissue injury, will be discussed, as well as small versus large animal models. Most of these models are nonlethal in nature, and allow the researcher to understand the changes that contribute to increased susceptibility to subsequent infection or the development of multiple organ failure. We also consider some of the confounders in these models, including anesthesia, the nature of resuscitation, and the use of anticoagulants. The selection of model must take into consideration not only the need for experimental control but must also adequately reflect the clinical pathobiology of shock if we are to develop better pharmacological interventions.

Pathogenesis of indirect (secondary) acute lung injury

At present, therapeutic interventions to treat acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) remain largely limited to lung-protective strategies, as no real molecular–pathophysiologic-driven therapeutic intervention has yet become available. This is in part the result of the heterogeneous nature of the etiological processes that contribute to the state of ALI/ARDS. This article sets out to understand the development of ALI resulting from indirect pulmonary insults, such as extrapulmonary sepsis and trauma, shock, burn injury or mass transfusion, as opposed to direct pulmonary challenges, such as pneumonia, aspiration or lung contusion. Here, we consider not only the experimental and clinical data concerning the roles of various immune (neutrophil, macrophage, lymphocyte and dendritic) as well as nonimmune (epithelial and endothelial) cells in orchestrating the development of ALI resulting from indirect pulmonary stimuli, but also how these cell populations might be targeted therapeutically.