Published ahead of print on February 8, 2008, doi:10.1164/rccm.200703-418OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200703-418OC
Inflammasome mRNA Expression in Human Monocytes during Early Septic Shock1 Division of Pulmonary, Allergy, Critical Care and Sleep Medicine; 2 The Dorothy M. Davis Heart and Lung Research Institute; 3 Nationwide Children's Research Institute; and 4 Department of Statistics, The Ohio State University Medical Center, Columbus, Ohio Correspondence and requests for reprints should be addressed to Mark D. Wewers, M.D., Division of Pulmonary, Critical Care, and Sleep Medicine, 201L DHLRI, 473 West 12th Avenue, Columbus, OH 43210-1252. E-mail: wewers.2{at}osu.edu
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 = 26; age >18 years), critically ill intensive care unit patients (n = 20), and healthy volunteers (n = 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- 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 = 0.0068) and correlated with SAPS II scores (r = –0.63). 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.
Key Words: inflammasome • monocytes • septic shock • messenger RNA • NALP1
Sepsis is a leading cause of death in the United States, accounting for an estimated 215,000 deaths per year (1). Although the precise cause of death in sepsis is unclear, in patients who die there seems to be progression from a generalized inflammatory response, referred to as the systemic inflammatory response syndrome (SIRS) (2), to progressive organ failure, known as multiorgan dysfunction syndrome (3). Of the initial mediators of sepsis, inflammatory cytokines play a key role (4). There is also evidence for increased antiinflammatory cytokine production including IL-10, IL-1RA, and soluble tumor necrosis factor (TNF) receptors I and II (5, 6). After activation of the innate immune response, monocytes play an important role in initiating the SIRS response. Not only do they release inflammatory cytokines, but de novo replication in the bone marrow increases their numbers (7), and with circulation they can migrate to sites of inflammation in tissue (8).
Recent studies of Toll-like receptors (TLRs) (9) and the activation of IL-1β (10), a key inflammatory cytokine, have resulted in new insights into the sepsis response. TLRs enable cells to identify pathogens such as bacteria, fungi, and viruses (11). Activation of TLRs promotes recruitment and activation of adapters and signaling intermediates collectively termed the "signalosome," which results in the translocation of nuclear factor (NF)-
Among these acute reactions, IL-1β secretion, through the activation of caspase-1, is a pivotal event in the inflammatory response (12). The post-translational activation of caspase-1 is tightly regulated by a complex of proteins termed the "inflammasome," the known components of which include caspase-1, apoptosis-associated speck-like protein containing a CARD (ASC), NALP1 (NACHT, leucine-rich repeat and pyrin domain containing 1), and caspase-5 (13). Alternate inflammasome constructions have been suggested to contain pyrin (14, 15), NALP3 (14), and other members of the NOD-LRR (NLR) family (16, 17). ASC facilitates inflammasome assembly, thus triggering caspase-1 activation and IL-1β processing. ASC can also regulate the NF- Animal and human studies have highlighted the importance of the inflammasome pathways in the inflammatory response to sepsis (19). For example, caspase-1 knock-out mice are protected from endotoxin and Escherichia coli–induced sepsis (20, 21), while a naturally occurring polymorphism for human caspase-12, a putative regulator of caspase-1, has been linked to sepsis (22, 23). Thus, caspase-1 activation seems to be a prerequisite for a competent immune response (24). Similarly, pyrin—the protein mutated in familial Mediterranean fever—has been shown to regulate production of mature IL-1β by complexing with pro–caspase-1 and ASC (14). When mutated, as in the case of familial Mediterranean fever, excess inflammation results.
Given the relationship of caspase-1 to sepsis, we sought to determine if there were alterations in the caspase-1 inflammasome in human septic shock. We hypothesized that the molecules involved in IL-1β activation (inflammasome) and NF-
Study Design After institutional review board approval and informed consent was obtained, patients admitted to the medical ICU who met the following criteria (modified from the 2001 Society of Critical Care Medicine/European Society of Intensive Care Medicine/American College of Chest Physicians/American Thoracic Society/Surgical Infections Society consensus statement) for the diagnosis of septic shock (25) were prospectively studied. These included only patients who had confirmed or suspected infection (bacterial, viral, fungal) at the time of screening (suspected infection was defined as the presence of SIRS with hypotension in the absence of obvious causes of the same); who had met three of the following four sepsis criteria within a 24-hour period: (1) fever (core temperature >38.3°C) or hypothermia (core temperature <36°C), (2) heart rate greater than 90 beats per minute or more than 2 SD above the normal value for age (except patients with conditions known to increase heart rate or those receiving treatment that would prevent tachycardia), or (3) tachypnea, defined as a respiratory rate  20 breaths per minute or a PaCO2  32 mm Hg or the use of mechanical ventilation for an acute respiratory process, or (4) leukocytosis (WBC count >12,000/µl) or leukopenia (WBC count <4,000/µl) or normal WBC count with >10% immature forms; and who had cardiovascular dysfunction defined as systolic blood pressure (SBP) 90 mm Hg or mean arterial pressure <60 mm Hg, a decrease of SBP >40 mm Hg from baseline for at least 1 hour despite adequate fluid resuscitation or adequate intravascular volume status, or the use of vasopressors to maintain a SBP 90 mm Hg or a MAP 70 mm Hg. All patients enrolled were required to have had blood sampled within 24 hours of the diagnosis of septic shock. Exclusion criteria primarily excluded patients who were immune compromised and are detailed in the online data supplement (Table E1). Twenty-two healthy volunteers (age, 46 yr; interquartile range [IQR], 41.5–66.2; male/female ratio, 10/12) and 20 critically ill nonseptic ICU patients (age, 53 yr; IQR, 43.2–61.7; male/female ratio, 8/12) were studied for comparison to the sepsis samples. Patients eligible for inclusion in the critically ill nonseptic arm were patients within 24 hours of ICU admission without known or suspected infection based on review of treating physicians' assessment, lack of antibiotic therapy (except for prophylactic therapy), and negative microbiologic and radiographic signs of infection. Admitting diagnoses included gastrointestinal hemorrhage (n = 6), cerebral vascular accident (n = 2), seizure disorder (n = 2), altered mental status/substance abuse (n = 3), and miscellaneous (n = 7). All sepsis and control blood samples were obtained between 7 A.M. and 2 P.M.
Monocyte Purification
Plasma Cytokine Measurements
RNA Isolation
 Ct is the Ct target – Ct reference (average of two housekeeping genes). PCR efficiency was calculated by the equation
Ex Vivo Cytokine Production Assay
Statistical Analysis Specific mRNA expression parameters between all patient groups in the inflammasome, signalosome, and inflammatory cytokine groupings were compared with the Kruskal-Wallis nonparametric test using Bonferroni correction for multiple tests. A P value of 0.002 was accepted as statistically significant. Genes meeting significance were analyzed pairwise between septic shock, critically ill ICU, and healthy control groups using the Wilcoxon rank-sum test, and P < 0.05 was accepted as statistically significant. To evaluate the relationship between mRNA levels and survival, comparisons were made between septic shock survivors and nonsurvivors using nominal logistic regression. Spearman's rank correlations were used to describe associations between continuous variables. All reported P values are based on two-tailed tests. Mortality was defined by survival to hospital discharge unless otherwise noted.
Patients A total of 26 patients with septic shock, 20 critically ill ICU control subjects, and 22 healthy control subjects were enrolled in the study. The majority of the study subjects were male. In the patients with septic shock, the median age was 51.5 years, in-hospital mortality was 57%, and a source of infection was identified in two thirds of patients, similar to previous studies (27). The median times from sepsis onset and septic shock onset to blood sampling were 30 hours (IQR, 15–53) and 10.5 hours (IQR, 8–14), respectively. The median Acute Physiology and Chronic Health Evaluation (APACHE) II score was 34. Critically ill control subjects had a median age of 53 years, an in-hospital mortality of 5%, and a median APACHE II score of 12 (Table 1).
Cytokine Measurements After enrollment, plasma samples taken from patients within 24 hours of onset of septic shock were measured for selected pro- and antiinflammatory cytokines. Plasma cytokine levels were significantly elevated in the patients with septic shock in comparison to critically ill and normal control subjects (Table 2), consistent with the presence of systemic inflammation. When septic survivors were compared with nonsurvivors, plasma IL-6, IL-8, and IL-10 levels were significantly elevated in nonsurvivors (P < 0.0005, P < 0.0012, and P < 0.0096, respectively).
Because monocyte deactivation is considered to be a significant contributor to the sepsis response, we measured whole-blood ex vivo lipopolysaccharide-induced TNF-  production capacity in healthy control subjects and in patients with septic shock within our cohort. The median TNF- release in picograms per milliliter was 649 (IQR, 479–994) for control subjects, 547 (IQR, 204.5–1551) for survivors, and 191 (IQR, 79.3–532.5) for nonsurvivors. Nonsurvivors differed from control subjects (P < 0.008) and trended different from survivors (P = 0.08).
mRNA Expression in Patients with Septic Shock versus Control Groups
Comparison of Inflammasome mRNA Expression between Septic Shock Survivors and Nonsurvivors To determine the relationship between survival and selected mRNA levels, comparisons were made between septic shock survivors and nonsurvivors (Table 4). Of the inflammasome components, only NALP1 was significantly different between hospital survivors and nonsurvivors. NALP1 was higher in patients with septic shock who survived to Day 7, Day 30, and to hospital discharge as compared with nonsurvivors. When NALP1 mRNA copies for the normal subjects, critically ill control, septic shock hospital survivor, and nonsurvivor groups were compared, the nonsurvivor group differed from normal control subjects, critically ill control subjects, and the sepsis survivor groups (Figure 1).
Logistic regression analysis for the sepsis group revealed that NALP1 was an independent determinant of mortality at 7 days (P = 0.0026), in hospital (P = 0.0068), and at 30 days (P = 0.0312). The sensitivity and specificity of NALP1 mRNA to predict survival, using a cut point of 5.0 RCN, were 69% and 92%, respectively, for 7-day survival and 73% and 87%, respectively, for hospital survival. Stated as an odds ratio, for every 5-RCN-unit increase in NALP1, the odds of hospital survival increases 3.6-fold.
Severity of Illness Scores and Survival
As part of the innate host defense, monocytes recognize pathogens with highly conserved sensors, termed pattern recognition receptors. TLRs, which are cell-surface sensors, activate signaling cascades that induce an inflammatory response, whereas intracellular sensors, as exemplified by NLRs, regulate NF-  B and caspase-1 activation (28). Both pattern recognition receptors identify conserved motifs present on many different microorganisms. The TLRs signal the induction of proinflammatory cytokines, including IL-1β, a pivotal cytokine in acute inflammation. Given IL-1β's key role in inflammation, its processing and release is highly regulated. Synthesized in an inactive pro-form, IL-1β requires cleavage by caspase-1 (12). Caspase-1 activity is highly regulated by a complex of proteins termed the inflammasomes (29). The relation of the inflammasome to sepsis may be predicted given the finding that caspase-1–deficient mice are protected when exposed to lethal doses of endotoxin (20) and to lethal peritoneal E. coli infection (21).
The finding that monocyte gene expression for caspase-1, ASC, and NALP1 (major components of the inflammasome) is significantly lower in patients with septic shock than in critically ill control subjects is novel. Indeed, monocyte ex vivo TNF- Whether this monocyte inflammasome down-regulation reflects the normal response to infection in general or is specific to septic shock remains to be determined. Further studies of patients with less severe infections may be informative. Also, because it is likely that the earliest sepsis response is immune activation, studying monocyte activation levels even earlier in the course of the response may be informative and relevant to the role of antiinflammatory therapy in sepsis. Analyzing mRNA patterns over time in patients who resolve their sepsis may provide useful information as to how long patient monocytes remain deactivated.
In relation to monocyte cytokine mRNA, the trend was also one of down-regulation, with TNF- In this context, it is important to point out a caveat of our study. We do not have the mechanism to study monocytes that may have been sequestered intravascularly or into sites of infection or organ injury. Therefore, the monocyte deactivation phenomenon that we have described can only be attributed to cells still in circulation. Future studies addressing tissue monocytes in comparison to circulating cells will be important. Regarding mortality, increased severity of illness scores in our sepsis population were a determinant of hospital mortality, with the SAPS II being most closely linked. Inflammasome components also correlated with disease severity scores, with NALP1 showing the highest correlation, suggesting that regulation of the inflammasome within 24 hours of onset of shock may reflect the severity of illness. Within the sepsis group, of the inflammasome components that were significantly altered, NALP1 alone was an independent determinant of mortality at 7 days, hospital survival, and 30-day mortality and was more specific than SAPS II in determining 7-day survival. In this context, a recent report demonstrated that NALP1 gene polymorphisms in inbred mouse strains correlate with macrophage lethality to anthrax lethal toxin (33). Furthermore, a human NALP1 polymorphism was recently linked to the immunodeficiency state associated with familial vitiligo (34). Thus, the sepsis-induced suppression of NALP1 may induce a transient immunosuppressive phenotype to monocytes. In conclusion, deactivation of monocytes appears within 24 hours of onset of human septic shock and is reflected by a decrease in mRNA levels of inflammasome components and proinflammatory cytokine message. Studies regarding role of the inflammasome in the sepsis response may affect future therapeutic interventions.
The authors gratefully acknowledge the support of the Pulmonary and Critical Care Clinical Trials Office at The Ohio State University.
* These authors contributed equally to this manuscript.  Supported by National Institutes of Health grants HL69899 (R.J.F.), K12HD43372 (M.W.H.), and HL40871 and HL76278 (M.D.W.) and by American Lung Association grant RG52369N (M.C.E.). This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200703-418OC on February 8, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 14, 2007; accepted in final form February 6, 2008
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