Tumor Necrosis Factor α Inhibits Expression of the Iron Regulating Hormone Hepcidin in Murine Models of Innate Colitis

Background: Abnormal expression of the liver peptide hormone hepcidin, a key regulator of iron homeostasis, contributes to the pathogenesis of anemia in conditions such as inflammatory bowel disease (IBD). Since little is known about the mechanisms that control hepcidin expression during states of intestinal inflammation, we sought to shed light on this issue using mouse models. Methodology/Principal Findings: Hepcidin expression was evaluated in two types of intestinal inflammation caused by innate immune activation—dextran sulfate sodium (DSS)-induced colitis in wild-type mice and the spontaneous colitis occurring in T-bet/Rag2-deficient (TRUC) mice. The role of tumor necrosis factor (TNF) $\alpha$ was investigated by in vivo neutralization, and by treatment of a hepatocyte cell line, as well as mice, with the recombinant cytokine. Expression and activation of Smad1, a positive regulator of hepcidin transcription, were assessed during colitis and following administration or neutralization of TNF$\alpha$. Hepcidin expression progressively decreased with time during DSS colitis, correlating with changes in systemic iron distribution. TNF$\alpha$ inhibited hepcidin expression in cultured hepatocytes and non-colitic mice, while TNF$\alpha$ neutralization during DSS colitis increased it. Similar results were obtained in TRUC mice. These effects involved a TNF$\alpha$-dependent decrease in Smad1 protein but not mRNA. Conclusions/Significance: TNF$\alpha$ inhibits hepcidin expression in two distinct types of innate colitis, with down-regulation of Smad1 protein playing an important role in this process. This inhibitory effect of TNF$\alpha$ may be superseded by other factors in the context of T cell-mediated colitis given that in the latter form of intestinal inflammation hepcidin is usually up-regulated

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Commensal Bacteria-Induced Inflammasome Activation in Mouse and Human Macrophages Is Dependent on Potassium Efflux but Does Not Require Phagocytosis or Bacterial Viability

Gut commensal bacteria contribute to the pathogenesis of inflammatory bowel disease, in part by activating the inflammasome and inducing secretion of interleukin-1ß (IL-1ß). Although much has been learned about inflammasome activation by bacterial pathogens, little is known about how commensals carry out this process. Accordingly, we investigated the mechanism of inflammasome activation by representative commensal bacteria, the Gram-positive Bifidobacterium longum subspecies infantis and the Gram-negative Bacteroides fragilis. B. infantis and B. fragilis induced IL-1ß secretion by primary mouse bone marrow-derived macrophages after overnight incubation. IL-1ß secretion also occurred in response to heat-killed bacteria and was only partly reduced when phagocytosis was inhibited with cytochalasin D. Similar results were obtained with a wild-type immortalized mouse macrophage cell line but neither B. infantis nor B. fragilis induced IL-1ß secretion in a mouse macrophage line lacking the nucleotide-binding/leucine-rich repeat pyrin domain containing 3 (NLRP3) inflammasome. IL-1ß secretion in response to B. infantis and B. fragilis was significantly reduced when the wild-type macrophage line was treated with inhibitors of potassium efflux, either increased extracellular potassium concentrations or the channel blocker ruthenium red. Both live and heat-killed B. infantis and B. fragilis also induced IL-1ß secretion by human macrophages (differentiated THP-1 cells or primary monocyte-derived macrophages) after 4 hours of infection, and the secretion was inhibited by raised extracellular potassium and ruthenium red but not by cytochalasin D. Taken together, our findings indicate that the commensal bacteria B. infantis and B. fragilis activate the NLRP3 inflammasome in both mouse and human macrophages by a mechanism that involves potassium efflux and that does not require bacterial viability or phagocytosis

<i>B</i>. <i>infantis</i>-induced IL-1ß secretion by THP-1 macrophages involves caspase 1.

<p><b>A.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> or <i>C</i>. <i>rodentium</i> for 1 hour, washed, then incubated in fresh medium for 4 hours. ZYVAD at a final concentration of 50 or 100 μM was present in the medium, as indicated, throughout the course of the experiment. Supernatants were collected at the end of the 4 hour incubation and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, **<i>p</i> = 0.0002, n = 6 per experimental condition. <b>B.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> for 1 hour, washed, then incubated in fresh medium for the times indicated before preparation of cell lysates. The lysates were analyzed by western blotting with an antibody to caspase 1, with equal loading of lanes being confirmed by blotting with an anti-ß-actin antibody. The p10 processed form of caspase 1 is shown.</p

<i>B</i>. <i>infantis</i>-induced IL-1ß secretion by primary human MDMs.

<p><b>A.</b> Primary human MDMs were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>infantis</i> for 1 hour in the presence or absence 5 μM cytochalasin D (Cyt D) or 50 mM potassium chloride (KCl) as indicated. The cells were washed, then incubated in fresh medium, with potassium chloride added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> Primary human MDMs were infected with heat-killed <i>B</i>. <i>infantis</i> for 1 hour in the presence or absence 5 μM cytochalasin D (Cyt D) or 50 mM potassium chloride (KCl) as indicated. The cells were washed, then incubated in fresh medium, with potassium chloride added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>C.</b> Primary human MDMs were treated with 10 μg/ml of Pam3Cys for 4 hours, after which cellular RNA was analyzed by quantitative RT-PCR to determine IL-1ß mRNA levels (left panel) and supernatants were analyzed by ELISA to determine secreted IL-1ß concentrations (right panel). *<i>p</i> = 0.0002, n = 6 per experimental condition.</p

<i>B</i>. <i>infantis</i> and <i>B</i>. <i>fragilis</i> induce IL-1ß secretion in immortalized mouse macrophages in an NLRP3- and potassium efflux-dependent fashion.

<p><b>A.</b> Immortalized WT mouse macrophages were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> (as well as a 5-fold higher number (5X) in the case of heat-killed <i>B</i>. <i>infantis</i>) for 1 hour in the presence or absence of 5 μM cytochalasin D (CytD) as indicated. The cells were washed and incubated overnight in fresh medium, with cytochalasin D added back as appropriate. IL-1ß concentrations in the supernatants were determined by ELISA. In the condition with 5X heat-killed bacteria, the cells were exposed continuously to the bacteria overnight without the intervening wash step. *<i>p</i> = 0.0001, **<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> WT or NLRP3 KO immortalized mouse macrophages were infected with live <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> for 1 hour. The cells were washed and incubated overnight in fresh medium. IL-1ß concentrations in the supernatants were determined by ELISA. *<i>p</i> < 0.0001, **<i>p</i> = 0.0001, n = 6 per experimental condition. <b>C.</b> Immortalized WT mouse macrophages were infected with live <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> for 1 hour in the presence or absence of 50 mM potassium chloride (KCl) or 2 μM ruthenium red (RR) as indicated. The cells were washed and incubated overnight in fresh medium, with potassium chloride or ruthenium red added back as appropriate. IL-1ß concentrations in the supernatants were determined by ELISA. *<i>p</i> = 0.0002, **<i>p</i> = 0.0011, ***<i>p</i> < 0.0001, n = 6 per experimental condition.</p

<i>B</i>. <i>infantis</i> and <i>B</i>. <i>fragilis</i> induce IL-1ß secretion in mouse BMDMs after overnight incubation.

<p><b>A.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i>, <i>B</i>. <i>fragilis</i> or <i>C</i>. <i>rodentium</i> for 1 hour. The cells were washed and incubated overnight in fresh medium. IL-1ß concentrations in the supernatants were determined by ELISA. *<i>p</i> = 0.0003, **<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i>, <i>B</i>. <i>fragilis</i> or <i>C</i>. <i>rodentium</i> as in <b>A</b> and then incubated overnight (upper panel) or for the times indicated (6 hours or overnight–O/N–lower panel). Cell lysates and supernatants were immunoblotted to detect pro-IL-1ß and mature IL-1ß, respectively, while cell lysates were blotted with an anti-actin antibody to confirm equal loading of lanes. The caspase inhibitor ZYVAD was added to the cells at a concentration of 100 μM where indicated. <b>C.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> for 1 hour. The cells were washed, placed in fresh medium and incubated for a further 4 hours. Total cellular RNA was prepared and used to determine relative IL-1ß mRNA expression by qRT-PCR. *<i>p</i> = 0.0003, **<i>p</i> < 0.0001, n = 6 per experimental condition. <b>D.</b> Mouse BMDMs were infected with <i>B</i>. <i>infantis</i> for 1 hour, washed, then incubated in fresh medium for 4 hours in the presence or absence of 5 mM ATP added exogenously during the last 1 hour. Supernatants were collected at the end of the 4 hour incubation and were analyzed by ELISA to determine IL-1ß concentrations. *<i>p</i> < 0.0001, n = 6 per experimental condition.</p

Bacterial viability and phagocytosis are not required for <i>B</i>. <i>infantis</i>- and <i>B</i>. <i>fragilis</i>-induced IL-1ß secretion by mouse BMDMs.

<p><b>A.</b> Mouse BMDMs were infected with live <i>B</i>. <i>infantis</i> or heat-killed (HK) bacteria, either equivalent in number to the live organisms or a 5-fold excess (5X), for 1 hour. The cells were washed and incubated overnight in fresh medium. IL-1ß concentrations in the supernatants were determined by ELISA. In the condition with 5X heat-killed bacteria, the cells were exposed continuously to the bacteria overnight without the intervening wash. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> Mouse BMDMs were infected with live <i>B</i>. <i>infantis</i> or <i>B</i>. <i>fragilis</i> or an equivalent number of heat-killed (HK) <i>B</i>. <i>fragilis</i> for 1 hour in the presence or absence of 5 μM cytochalasin D (CytD, dissolved in DMSO) as indicated. The cells were washed and incubated overnight in fresh medium, with cytochalasin D added back as appropriate. IL-1ß concentrations in the supernatants were determined by ELISA. n = 6 per experimental condition.</p

<i>B</i>. <i>infantis</i>- and <i>B</i>. <i>fragilis</i>-induced IL-1ß secretion by THP-1 macrophages is dependent on potassium efflux but does not require bacterial viability or phagocytosis.

<p><b>A.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> for 1 hour in the presence or absence of 50 mM potassium chloride (KCl) or 2 μM ruthenium red (RR), as indicated. The cells were washed and incubated in fresh medium, with potassium chloride or ruthenium red added back as appropriate. Supernatants were collected at the end of 4 hours and IL-1ß concentrations determined by ELISA. *<i>p</i> < 0.0001, n = 6 per experimental condition. <b>B.</b> THP-1 macrophages were infected with <i>B</i>. <i>infantis</i> for 1 hour. The cells were washed and incubated in fresh medium, with 5 units/ml of apyrase added as indicated. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA, n = 6 per experimental condition. <b>C.</b> THP-1 macrophages were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>infantis</i> for 1 hour, in the presence of 5 μM cytochalasin D (CytD) where indicated. The cells were washed and incubated in fresh medium, with cytochalasin D added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA, n = 6 per experimental condition. <b>D.</b> THP-1 macrophages were infected with equivalent numbers of live or heat-killed (HK) <i>B</i>. <i>fragilis</i> for 1 hour, in the presence of 5 μM cytochalasin D (CytD) or 50 mM potassium chloride (KCl) where indicated. The cells were washed and incubated in fresh medium, with cytochalasin D, potassium chloride or 5 units/ml of apyrase added back as appropriate. Supernatants were collected after 4 hours and IL-1ß concentrations determined by ELISA. *p < 0.0001, n = 6 per experimental condition.</p