Alfred Ayala

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.

Effect of gender and sex hormones on immune responses following shock.

ABSTRACT— Several clinical and experimental studies show a gender dimorphism of the immune and organ responsiveness in the susceptibility to and morbidity from shock, trauma, and sepsis. In this respect, cell‐mediated immune responses are depressed in males after trauma‐hemorrhage, whereas they are unchanged or enhanced in females. Sex hormones contribute to this gender‐specific immune response after adverse circulatory conditions. Specifically, studies indicate that androgens are responsible for the immunodepression after trauma‐hemorrhage in males. In contrast, female sex steroids seem to exhibit immunoprotective properties after trauma and severe blood loss, because administration of estrogen prevents the androgen‐induced immunodepression in castrated male mice. Nonetheless, the precise underlying mechanisms for these immunomodulatory effects of sex steroids after shock remain unknown. Although testosterone depletion, testosterone receptor antagonism, or estrogen treatment has been shown to prevent the depression of immune functions after trauma‐hemorrhage, it remains to be established whether differences in the testosterone‐estradiol ratio are responsible for the immune dysfunction. Furthermore, sex hormone receptors have been identified on various immune cells, suggesting direct effects. Thus, the immunomodulatory properties of sex hormones after trauma‐hemorrhage might represent novel therapeutic strategies for the treatment of immunodepression in trauma patients.

Females in proestrus state maintain splenic immune functions and tolerate sepsis better than males.

OBJECTIVES To determine: a) whether the cell-mediated immune response during sepsis differs in females vs. males; and b) whether the survival rate in females is different than in males after a septic insult. DESIGN A prospective, randomized animal study. SETTING University research laboratory. SUBJECTS Male and female proestrus C3H/HeN mice. INTERVENTIONS After anesthesia, male and proestrus female mice underwent cecal ligation puncture to induce sepsis. The mice were killed at 24 hrs after the onset of sepsis. MEASUREMENTS AND MAIN RESULTS Splenocyte proliferation, as well as splenocyte interleukin (IL)-2 and IL-3 release, was determined by bioassay. In additional studies, survival rate after septic challenge was measured over 10 days. Splenocyte proliferative capacity and splenocyte IL-2 and IL-3 release were markedly decreased in male, but not in female, septic mice. Furthermore, the survival rate of septic female proestrus mice was significantly higher than in comparable male mice. CONCLUSIONS These results support the concept that the immune response of females differs from males, and that females are immunologically better positioned to meet the challenge of sepsis.

Evidence favoring the role of the gut as a cytokine-generating organ in rats subjected to hemorrhagic shock

There is increasing evidence of an association between intestinal injury and the development of a septic state and distant organ failure. Since this phenomenon can occur in the absence of detectable systemic bacterial translocation (BT), we tested the hypothesis that shock-induced intestinal injury will result in the gut becoming a cytokine-generating organ by measuring interleukin 6 (IL-6) and tumor necrosis factor (TNF) levels in the portal blood, cardiac blood, and intestinal lymph of rats subjected to sham, 30, 60, or 90 min of hemorrhagic shock (30 mm Hg). These blood and lymph samples, as well as the mesenteric lymph nodes (MLN), spleens, and livers, were cultured for translocating bacteria. Although all the portal and cardiac blood samples were sterile, the portal blood levels of TNF and IL-6 were increased to a greater extent than simultaneously obtained cardiac blood samples in rats subjected to 60 or 90 min of shock (p < .05). The lymph IL-6 levels increased but were similar between the groups. BT was limited to the MLN and occurred in a dose-dependent fashion with 38, 63, and 100% of the animals having culture-positive MLNs after 30, 60, or 90 min of shock, respectively. In conclusion, after hemorrhagic shock, the gut appears to become a cytokine liberating organ even in the absence of detectable bacteria in the portal circulation.

Early kidney TNF-α expression mediates neutrophil infiltration and injury after renal ischemia-reperfusion

The purpose of this study was to determine whether isolated renal ischemia and reperfusion (I/R) induces renal tumor necrosis factor (TNF) mRNA production, TNF protein expression, or TNF bioactivity and, if so, whether local/early TNF production acts as mediator of ischemia-induced, neutrophil-mediated renal injury. After rats were anesthetized, varying periods of renal ischemia, with or without reperfusion, were induced. Kidney mRNA content (RT-PCR), TNF protein expression (ELISA), TNF bioactivity (WEHI-164 cell clone cytotoxicity assay), and neutrophil infiltration [myeloperoxidase (MPO) assay] were determined. In other animals, renal MPO and serum creatinine were assessed after TNF was neutralized [binding protein (TNF-BP)]. Thirty minutes of ischemia induced renal TNF mRNA. TNF protein expression and bioactivity peaked after 1 h ischemia and 2 h reperfusion, whereas neutrophil infiltration peaked at 4 h reperfusion. TNF-BP neutralized TNF bioactivity, reduced neutrophil infiltration, and protected postischemic function. These results constitute the initial demonstration that 1) early renal tissue TNF expression contributes to neutrophil infiltration and injury after I/R and 2) TNF-BP may offer a new adjunctive therapy in renal preservation prior to planned ischemic insults.The purpose of this study was to determine whether isolated renal ischemia and reperfusion (I/R) induces renal tumor necrosis factor (TNF) mRNA production, TNF protein expression, or TNF bioactivity and, if so, whether local/early TNF production acts as mediator of ischemia-induced, neutrophil-mediated renal injury. After rats were anesthetized, varying periods of renal ischemia, with or without reperfusion, were induced. Kidney mRNA content (RT-PCR), TNF protein expression (ELISA), TNF bioactivity (WEHI-164 cell clone cytotoxicity assay), and neutrophil infiltration [myeloperoxidase (MPO) assay] were determined. In other animals, renal MPO and serum creatinine were assessed after TNF was neutralized [binding protein (TNF-BP)]. Thirty minutes of ischemia induced renal TNF mRNA. TNF protein expression and bioactivity peaked after 1 h ischemia and 2 h reperfusion, whereas neutrophil infiltration peaked at 4 h reperfusion. TNF-BP neutralized TNF bioactivity, reduced neutrophil infiltration, and protected postischemic function. These results constitute the initial demonstration that 1) early renal tissue TNF expression contributes to neutrophil infiltration and injury after I/R and 2) TNF-BP may offer a new adjunctive therapy in renal preservation prior to planned ischemic insults.

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.

The complex pattern of cytokines in sepsis. Association between prostaglandins, cachectin, and interleukins.

Although the cytokines tumor necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6) are important mediators of hemodynamic, metabolic, and immunologic alterations in the host during sepsis, it is not known whether there is any association between the release of these cytokines and prostanoids during sepsis. Sepsis induced by cecal ligation and puncture in rats led to a persistent elevation (p ≤ 0.05) of plasma TNF until 10 hours, steadily increasing (p ≤ 0.05) IL-1 plasma levels, and enhanced (p ≤ 0.05) IL-6 plasma levels at all time points compared to the sham group. Prostaglandin E2 plasma levels were elevated (p ≤ 0.05) at 5 hours (153 ± 29 pg/mL; control: 47 ±11 pg/mL) and 10 hours (96 ± 16 pg/mL; control: 21 ± 5 pg/ mL). Prostaglandin E2 production by splenic macrophages (sMø) from septic animals was increased (p ≤ 0.05) at 5 hours (9.1 ±2.2 ng/mL) and 10 hours (5.6 ±1.5 ng/mL) compared to controls (3.3 ± 0.3. ng/mL at 5 hours; 1.3 ± 1.3 ng/mL at 10 hours). Incubation of sMø from septic animals with ibuprofen enhanced (p ≤ 0.05) IL-1 and TNF synthesis, while IL-6 production was reduced (p ≤ 0.05). These results indicate that the alterations in prostanoid release and elevated plasma prostanoids may regulate the release and consequently the circulating levels of cytokines during sepsis.

The compensatory anti-inflammatory response syndrome (CARS) in critically ill patients.

Like the systemic inflammatory response syndrome (SIRS), the compensatory anti-inflammatory response syndrome (CARS) is a complex pattern of immunologic responses to severe infection or injury. The difference is that while SIRS is a proinflammatory response tasked with killing infectious organisms through activation of the immune system, CARS is a global deactivation of the immune system tasked with restoring homeostasis. Much research now suggests that the timing and relative magnitude of this response have a profound impact on patient outcomes.

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.

Hemorrhage induces an increase in serum TNF which is not associated with elevated levels of endotoxin

Although tumor necrosis factor (TNF) and interleukin 6 (IL 6) are purported to be important mediators of inflammatory responses following trauma, it is not known if the serum levels of these cytokines are altered by simple hemorrhage. The objective of this study therefore was to determine whether or not: 1) there is any elevation of TNF or IL 6, and 2) if endotoxin, an important upregulator of these cytokines, is also increased following hemorrhage. To study this, C3H/HeN mice were bled to, and maintained at a mean blood pressure of 35 mmHg for 60 min, and then resuscitated with their own shed blood and adequate fluid. Mice were sacrificed at 30 min into hemorrhage and at 2, 4 or 24 hr post-hemorrhage to obtain serum samples. IL 6 and TNF levels were measured using cytokine dependent cellular assays. Using a quantitative Limulus amebocyte lysate assay, endotoxin levels were determined. TNF levels were significantly elevated at 30 min into hemorrhage, remaining so at 2 hr after resuscitation, but absent by 4 hr. Although there was a trend toward elevated IL 6 levels at 2 hr following hemorrhage, which was sustained up to 24 hr, the values were not significantly different from sham controls. When compared to controls, no marked increase in endotoxin was seen at any time point during or following hemorrhage. These results indicate that hemorrhage, in the absence of significant tissue trauma, causes enhanced TNF release which is not the result of increased endotoxin.