Mammalian DNA is an endogenous danger signal that stimulates local synthesis and release of complement factor B.

Complement factor B plays a critical role in ischemic tissue injury and autoimmunity. Factor B is dynamically synthesized and released by cells outside of the liver, but the molecules that trigger local factor B synthesis and release during endogenous tissue injury have not been identified. We determined that factor B is upregulated early after cold ischemia-reperfusion in mice, using a heterotopic heart transplant model. These data suggested upregulation of factor B by damage-associated molecular patterns (DAMPs), but multiple common DAMPs did not induce factor B in RAW264.7 mouse macrophages. However, exogenous DNA induced factor B mRNA and protein expression in RAW cells in vitro, as well as in peritoneal and alveolar macrophages in vivo. To determine the cellular mechanisms involved in DNA-induced factor B upregulation we then investigated the role of multiple known DNA receptors or binding partners. We stimulated peritoneal macrophages from wild-type (WT), toll-like receptor 9 (TLR9)-deficient, receptor for advanced glycation end products (RAGE)⁻/⁻ and myeloid differentiation factor 88 (MyD88)⁻/⁻ mice, or mouse macrophages deficient in high-mobility group box proteins (HMGBs), DNA-dependent activator of interferon-regulatory factors (DAI) or absent in melanoma 2 (AIM2), with DNA in the presence or absence of lipofection reagent. Reverse transcription-polymerase chain reaction, Western blotting and immunocytochemical analysis were employed for analysis. Synthesis of factor B was independent of TLR9, RAGE, DAI and AIM2, but was dependent on HMGBs, MyD88, p38 and NF-κB. Our data therefore show that mammalian DNA is an endogenous molecule that stimulates factor B synthesis and release from macrophages via HMGBs, MyD88, p38 and NF-κB signaling. This activation of the immune system likely contributes to damage following sterile injury such as hemorrhagic shock and ischemia-reperfusion.

Transcriptomic response of murine liver to severe injury and hemorrhagic shock: a dual-platform microarray analysis.

Trauma-hemorrhagic shock (HS/T) is a complex process that elicits numerous molecular pathways. We hypothesized that a dual-platform microarray analysis of the liver, an organ that integrates immunology and metabolism, would reveal key pathways engaged following HS/T. C57BL/6 mice were divided into five groups (n = 4/group), anesthetized, and surgically treated to simulate a time course and trauma severity model: 1) nonmanipulated animals, 2) minor trauma, 3) 1.5 h of hemorrhagic shock and severe trauma (HS/T), 4) 1.5 h HS/T followed by 1 h resuscitation (HS/T+1.0R), 5) 1.5 h HS/T followed by 4.5 h resuscitation (HS/T+4.5R). Liver RNA was hybridized to CodeLink and Affymetrix mouse whole genome microarray chips. Common genes with a cross-platform correlation >0.6 (2,353 genes in total) were clustered using k-means clustering, and clusters were analyzed using Ingenuity Pathways Analysis. Genes involved in the stress response and immunoregulation were upregulated early and remained upregulated throughout the course of the experiment. Genes involved in cell death and inflammatory pathways were upregulated in a linear fashion with elapsed time and in severe injury compared with minor trauma. Three of the six clusters contained genes involved in metabolic function; these were downregulated with elapsed time. Transcripts involved in amino acid metabolism as well as signaling pathways associated with glucocorticoid receptors, IL-6, IL-10, and the acute phase response were elevated in a severity-dependent manner. This is the first study to examine the postinjury response using dual-platform microarray analysis, revealing responses that may enable novel therapies or diagnostics.

Body adipose content is independently associated with a higher risk of organ failure and nosocomial infection in the nonobese patient postinjury.

OBJECTIVE: Obesity defined by a body mass index (BMI) >30 kg/m is associated with increased morbidity and mortality following trauma. Evidence suggests that obesity represents a state of chronic inflammation and that the adipose tissue content may affect the intensity and resolution of inflammatory response. We sought to avoid the confounding effects attributable to obesity and determine the association of BMI and outcomes following injury in nonobese patients. METHODS: Data were obtained from a multicenter prospective cohort study evaluating outcomes in blunt-injured adults with hemorrhagic shock. Only patients with a BMI≥18.5 and<30 were analyzed. Those with isolated traumatic brain injury and cervical cord injury and those who survived<24 hours were excluded. Logistic regression was used to evaluate the effects of BMI on mortality, multiple organ failure (MOF, multiple organs dysfunction score [MODS]>5), and nosocomial infection (NI) after adjusting for differences in demographics, injury severity, early resuscitation needs, shock parameters, and comorbidities. RESULTS: Overall mortality, MOF, and NI rates for the study cohort (n=820) were 13%, 37%, and 46%, respectively. Median Injury Severity Score was 33 (interquartile range, 22-41). Median BMI for the study cohort was 25 (interquartile range, 23-27). As BMI increased, maximum organ dysfunction scores also significantly increased for cardiac, respiratory, and renal systems. Logistic regression revealed no significant association with mortality (odds ratio [OR]=0.95; 95% confidence interval [CI], 0.9-1.0); however, BMI was independently associated with a higher risk of MOF (OR=1.09; 95% CI, 1.02-1.06) and NI (OR=1.07; 95% CI, 1.01-1.13). For every single-point increase in BMI, the risk of MOF and NI increase by 9% and 7%, respectively. CONCLUSION: The risk of MOF and NI increases as BMI increases in the nonobese injured patient. These results suggest that body adipose content may be associated with the magnitude of or extent of the inflammatory response postinjury. Further studies are needed to elucidate the mechanism responsible for this association.

Complement factor 3 deficiency attenuates hemorrhagic shock-related hepatic injury and systemic inflammatory response syndrome.

Although complement activation is known to occur in the setting of severe hemorrhagic shock and tissue trauma (HS/T), the extent to which complement drives the initial inflammatory response and end-organ damage is uncertain. In this study, complement factor 3-deficient (C3(-/-)) mice and wild-type control mice were subjected to 1.5-h hemorrhagic shock, bilateral femur fracture, and soft tissue injury, followed by 4.5-h resuscitation (HS/T). C57BL/6 mice were also given 15 U of cobra venom factor (CVF) or phosphate-buffered saline injected intraperitoneally, followed by HS/T 24 h later. The results showed that HS/T resulted in C3 consumption in wild-type mice and C3 deposition in injured livers. C3(-/-) mice had significantly lower serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and circulating DNA levels, together with much lower circulating interleukin (IL)-6, IL-10, and high-mobility group box 1 (HMGB1) levels. Temporary C3 depletion by CVF preconditioning also led to reduced transaminases and a blunted cytokine release. C3(-/-) mice displayed well-preserved hepatic structure. C3(-/-) mice subjected to HS/T had higher levels of heme oxygenase-1, which has been associated with tissue protection in HS models. Our data indicate that complement activation contributes to inflammatory pathways and liver damage in HS/T. This suggests that targeting complement activation in the setting of severe injury could be useful.

Pivotal advance: The pattern recognition receptor ligands lipopolysaccharide and polyinosine-polycytidylic acid stimulate factor B synthesis by the macrophage through distinct but overlapping mechanisms.

TLRs and complement are critical to the host response in sepsis, trauma, and ischemia/reperfusion. We hypothesize that TLR stimulation leads to synthesis and release of complement components by macrophages, an important source of extrahepatic complement. RAW264.7 macrophages or peritoneal macrophages from WT and TLR4-, TLR3-, TRIF-, or MyD88-deficient mice were cultured under standard conditions. In some experiments, cells were pretreated with inhibitors of MAPKs or a NF-κB inhibitor. Cells were stimulated with TLR ligands at known stimulatory concentrations. Intratracheal and i.p. injections were also performed in mice. RT-PCR, Western blotting, and immunocytochemistry were used for analysis. Using a RT-PCR-based panel, we demonstrate that of 18 complement components tested, factor B of the alternative pathway is the most robustly up-regulated complement component in macrophages in response to LPS. This up-regulation results in release of factor B into the media. Up-regulation of factor B by LPS is dependent on TLR4, TRIF, JNK, and NF-κB. A screen of other TLR ligands demonstrated that stimulation with poly I:C (dsRNA analog) also results in up-regulation of factor B, which is dependent on JNK and NF-κB but independent of TLR3 and TRIF. Up-regulation of factor B is also observed after intratracheal and i.p. injection of LPS or poly I:C in vivo. PRR stimulation profoundly influences production and release of factor B by macrophages. Understanding the mechanisms of PRR-mediated complement production may lead to strategies aimed at preventing tissue damage in diverse settings, including sepsis, trauma, and ischemia/reperfusion.

Staged endourologic and endovascular repair of an infrarenal inflammatory abdominal aortic aneurysm presenting with forniceal rupture.

We present the case of a 79-year-old female who presented with severe left flank pain and a pulsatile abdominal mass. She was diagnosed with left peripelvic urinary extravasation and forniceal rupture secondary to an intact infrarenal inflammatory abdominal aortic aneurysm with extensive periaortic fibrosis. Successful operative repair was performed with staged ureteral and endovascular stenting with subsequent resolution of periaortic inflammation and ureteral obstruction, and shrinkage of the aneurysm sac. Inflammatory abdominal aortic aneurysms (IAAAs) represent 5% to 10% of all abdominal aortic aneurysms. The distinguishing features of inflammatory aneurysms include thickening of aneurysm wall, retroperitoneal fibrosis, and adhesions to adjacent retroperitoneal structures. The most commonly involved adjacent structures are the duodenum, left renal vein, and ureter. Adhesions to the urinary system can cause hydronephrosis or hydroureter and result in obstructive uropathy. An unusual case of IAAA presenting with forniceal rupture is presented, with successful endovascular and endourologic repair.

ClC-5: ontogeny of an alternative chloride channel in respiratory epithelia.

Chloride transport is critical to many functions of the lung. Molecular defects in the best-known chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to impaired function of airway defensins, hydration of airway surface fluid, and mucociliary clearance leading to chronic lung disease, and premature death, but do not cause defects in lung development. We examined the expression of one member of the ClC family of volume- and voltage-regulated channels using the ribonuclease protection assay and Western blot analysis in rats. ClC-5 mRNA and protein are most strongly expressed in the fetal lung, and expression is maintained although downregulated postnatally. In addition, using immunocytochemistry, we find that ClC-5 is predominantly expressed along the luminal surface of the airway epithelium, suggesting that ClC-5 may participate in lung chloride secretion. Identifying candidate genes for critical ion transport functions is essential for understanding normal lung morphogenesis and the pathophysiology of several lung diseases. In addition, the manipulation of non-CFTR chloride channels may provide a viable approach for treating cystic fibrosis lung disease.