Purpose Immunoparalysis defined by prolonged monocyte human leukocyte antigen DR depression is associated with adverse outcomes in adult severe sepsis and can be reversed with granulocyte macrophage colony-stimulating factor (GM-CSF). We hypothesized that immunoparalysis defined by whole-blood ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha (TNFα) response <200 pg/mL beyond day 3 of multiple organ dysfunction syndrome (MODS) is similarly associated with nosocomial infection in children and can be reversed with GM-CSF. Methods In study period 1, we performed a multicenter cohort trial of transplant and nontransplant multiple organ dysfunction syndrome (MODS) patients (≥2 organ failure). In study period 2, we performed an open-label randomized trial of GM-CSF therapy for nonneutropenic, nontransplant, severe MODS patients (≥3 organ failure) with TNFα response <160 pg/mL. Results Immunoparalysis was observed in 34% of MODS patients (n = 70) and was associated with increased nosocomial infection (relative risk [RR] 3.3, 95% confidence interval [1.8–6.0] p < 0.05) and mortality (RR 5.8 [2.1–16] p < 0.05). TNFα response <200 pg/mL throughout 7 days after positive culture was associated with persistent nosocomial infection, whereas recovery above 200 pg/mL was associated with resolution of infection (p < 0.05). In study period 2, GM-CSF therapy facilitated rapid recovery of TNFα response to >200 pg/mL by 7 days (p < 0.05) and prevented nosocomial infection (no infections in seven patients versus eight infections in seven patients) (p < 0.05). Conclusions Similar to in adults, immunoparalysis is a potentially reversible risk factor for development of nosocomial infection in pediatric MODS. Whole-blood ex vivo TNFα response is a promising biomarker for monitoring this condition.

Macrophage responses to Francisella infection have been characterized previously by subdued proinflammatory responses; however, these studies have generally focused on macrophage cell lines or monocyte-derived macrophages. Therefore, we studied the ability of fresh human blood monocytes to engulf and respond to Francisella by using the live vaccine strain variant and Francisella novicida. Because Francisella organisms have been reported to escape from the phagolysosome into the cytosol, we hypothesized that this escape may trigger the activation of caspase-1. Francisella tularensis variants were readily taken up by fresh human CD14 ؉ monocytes, inducing the release of IL-1␤, as well as IL-8, in a timeand dose-dependent fashion. Importantly, whereas live and dead Escherichia coli, F. novicida, and live vaccine strain, as well as the LPS of E. coli, were able to induce abundant IL-1␤ mRNA synthesis and intracellular pro-IL-1␤ production, only live Francisella induced enhanced IL-1␤ processing and release (51 ؎ 10 vs. 7.1 ؎ 2.1 ng͞ml, for F. novicida vs. E. coli LPS; P ‫؍‬ 0.0032). Cytochalasin D blocked the Francisella internalization and the Francisella-induced monocyte IL-1␤ processing and release but not that induced by the exogenous stimulus E. coli LPS. Also, killing bacteria did not block uptake but significantly diminished the IL-1␤ processing and release that was induced by Francisella. Blocking bacterial escape from the phagosome into the cytosol also decreased IL-1␤ but not IL-8 release. These findings demonstrate that Francisella organisms efficiently induce IL-1␤ processing and release in fresh monocytes by means of a sensing system that requires the uptake of live bacteria capable of phagosome escape.bacteria ͉ caspase-1 ͉ cytokine ͉ phagocytosis M onocytes and macrophages, which are the first-line guards of the innate host-defense system, recognize and phagocytose pathogens. The innate immune system senses the invasion of pathogenic microorganisms through the recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs), of which there are 10 that are known in humans, as well as non-TLRs (1-3). Pathogen recognition induces the release of many proinflammatory and antiinflammatory cytokines, which provide a balanced inflammatory response. At the same time, prototypical intracellular pathogens like Francisella tularensis depend on the intracellular milieu of mononuclear cells for growth and replication (4, 5). In this context, a class of intracellular innate host-defense molecules, which recognize and respond to internalized pathogens, has been identified (3, 6). These molecules [historically called CATERPILLERs (3) and nucleotide-binding oligomerization domain-leucine-rich repeats (NOD-LRRs) (7), and, most recently, NACHT leucine-rich repeats (NLRs) (8)] are homologues of plant-disease-resistance genes and contain leucine-rich repeats, much like TLRs. The human family of NLR consists of 22 (8) or 23 (7) proteins. These NLRs, similar to TLRs, function to regulate pathogen recogniti...

Rationale: Caspase-1 processes interleukin 1␤ (IL-1␤) and IL-18 but may also contribute to apoptosis. In this context, caspase-1 knockout mice have been shown to be protected from endotoxin-induced mortality, whereas IL-1␤ knockout mice are not protected. Objectives: We therefore sought to delineate the mechanisms responsible for the differential responses between caspase-1 and IL-1␤ knockout mice. Methods: Caspase-1 knockout, IL-1␤ knockout, and IL-1␤/IL-18 double knockout mice were compared with wild-type mice for survival after intraperitoneal challenge with live Escherichia coli. Measurements and Main Results: Caspase-1 knockout animals were protected from bacterial challenge, whereas wild-type, IL-1␤ knockout, and IL-1␤/IL-18 double knockout animals were not. Wild-type animals and both IL-1␤ knockout and IL-1␤/IL-18 double knockout mice demonstrated significant splenic B lymphocyte apoptosis, which was absent in the caspase-1 knockout mice. Importantly, IL-1␤/IL-18 double knockout mice were protected from splenic cell apoptosis and sepsis-induced mortality by the caspase inhibitor zVAD-fmk. Furthermore, wild-type but not caspase-1 knockout splenic B lymphocytes induced peritoneal macrophages to assume an inhibitory phenotype. Conclusion: Taken together, these findings suggest that caspase-1 is important in the host response to sepsis at least in part via its ability to regulate sepsis-induced splenic cell apoptosis.Keywords: apoptosis; caspase inhibition; septic shock; spleen More than 500,000 people develop sepsis annually and 175,000 of them die in the United States alone (1). Septic shock activates numerous proinflammatory mediators, which can result in multiple organ injury (2, 3). In addition, executioner cysteineaspartate proteases (caspases) play a key role in the disassembly of cells during septic shock via various proapoptotic stimuli. Pharmacologic blockade of caspase activation improves organ function and survival in animal models of sepsis and ischemia reperfusion injury (4). Interleukin 1␤ (IL-1␤) is one of the major proinflammatory cytokines known to be produced in sepsis (5-8). It is synthesized as an inactive 31-kD precursor that requires a unique cysteine protease, IL-1␤Ϫconverting enzyme (caspase-1), to generate biologically active 17-kD IL-1␤ (9, 10).(Received in original form April 20, 2006; accepted Although caspase-1 plays no part in the spontaneous apoptosis of monocytes and macrophages (11), its activation via intracellular pathogens can induce macrophage apoptosis (12, 13) and its deletion has been linked to survival in animal models of endotoxin shock (14). This protective effect could logically be attributed to caspase-1's role in activating the precursor, pro-IL-1␤. Unexpectedly, however, active IL-1␤ does not regulate survival from endotoxin shock, because IL-1␤ knockout animals are not protected from endotoxin-induced death (15). This difference may hold an important key to understanding the role of caspase-1 in host responses. Importantly, prior caspase-1 knockout experiment...

Rationale: Monocytes are central to the initiation of the inflammatory response in sepsis, with caspase-1 activation playing a key role. Monocyte deactivation during sepsis has been linked to poor outcomes. Objectives: Given the importance of caspase-1 in the immune response, we investigated whether monocytes from patients early in septic shock demonstrate alterations in mRNAs for caspase-1-related molecules. Methods: Patients with septic shock (n 5 26; age .18 years), critically ill intensive care unit patients (n 5 20), and healthy volunteers (n 5 22) were enrolled in a prospective cohort study in a university intensive care unit. Demographic, biological, physiologic, and plasma cytokine measurements were obtained. Monocytes were assayed for ex vivo tumor necrosis factor-a production, and fresh monocyte mRNA was analyzed by quantitative reverse-transcription polymerase chain reaction for Toll-like receptors, NOD-LRR proteins, cytokines, and nuclear factor-kB-related genes. Measurements and Main Results: Relative copy numbers for the inflammasome mRNAs for ASC, caspase-1, NALP1, and Pypaf-7 were significantly lower in patients with septic shock compared with critically ill control subjects. NALP1 mRNA levels were linked to survival in patients with sepsis (P 5 0.0068) and correlated with SAPS II scores (r 5 20.63). Conclusions: These data suggest that monocyte deactivation occurs during the earliest stages of the systemic inflammatory response and that changes in inflammasome mRNA expression are part of this process.

Impairment of the ability to mount an inflammatory response is associated with death from adult critical illness. This phenomenon, characterized by reduced monocyte production of proinflammatory mediators such as tumor necrosis factor ␣ (TNF-␣), is poorly understood in children. We hypothesized that differential expression of inflammation-related genes would be seen in monocytes from children with adverse outcomes from multiple organ dysfunction syndrome (MODS). Ex vivo lipopolysaccharide (LPS)-induced TNF-␣ production and plasma cytokines were prospectively measured biweekly in children with dysfunction of two or more organs. Concomitantly, monocyte expression of 28 pro-and antiinflammatory genes [cytokines, Toll-like receptor (TLR)/nuclear factor B (NF-B) signaling pathway members, inflammasome elements] was measured. Thirty children (22 survivors, eight nonsurvivors) were evaluated. High mRNA levels for interleukin (IL)-10, IL-1 receptor-associated kinase (IRAK-M), and the putative inflammasome inhibitor pyrin were associated with death (p Յ 0.02). Plasma IL-10 levels were higher and ex vivo TNF-␣ production was lower in nonsurvivors (p Ͻ 0.05). Among survivors, high mRNA levels for IL-10, IRAK-M, pyrin, IRAK1, or TLR4 were associated with longer durations of pediatric intensive care unit (PICU) stay and mechanical ventilation (p Յ 0.02). These data suggest that adverse outcomes from pediatric MODS are associated with an antiinflammatory monocyte mRNA phenotype. Future studies are warranted to explore mechanisms of immunodepression in pediatric critical illness. (Pediatr Res 62: 597-603, 2007)