PYHIN protein IFI207 regulates cytokine transcription and IRF7 and contributes to the establishment of K. pneumoniae infection.

PYHIN proteins AIM2 and IFI204 sense pathogen DNA, while other PYHINs have been shown to regulate host gene expression through as-yet unclear mechanisms. We characterize mouse PYHIN IFI207, which we find is not involved in DNA sensing but rather is required for cytokine promoter induction in macrophages. IFI207 co-localizes with both active RNA polymerase II (RNA Pol II) and IRF7 in the nucleus and enhances IRF7-dependent gene promoter induction. Generation of Ifi207-/- mice shows no role for IFI207 in autoimmunity. Rather, IFI207 is required for the establishment of a Klebsiella pneumoniae lung infection and for Klebsiella macrophage phagocytosis. These insights into IFI207 function illustrate that PYHINs can have distinct roles in innate immunity independent of DNA sensing and highlight the need to better characterize the whole mouse locus, one gene at a time.

Caspase-4: A Therapeutic Target for Peptic Ulcer Disease.

Peptic ulcers are caused by the interaction between bacterial and host factors. This study demonstrates enhanced expression of caspase-4 in peptic ulcer patient biopsies, indicating that pyroptosis and noncanonical inflammasome activity may be processes involved in peptic ulcer disease. We show that primary murine macrophages infected with Helicobacter pylori upregulate caspase-11 (the ortholog of human caspase-4), activate caspase-1, and secrete IL-1ß. We demonstrate that misoprostol (a stable PGE1 analogue) decreased IL-1ß secretion and delayed lethality in vivo in a murine peritonitis model. PGE2 was shown to inhibit caspase-11-driven pyroptosis and IL-1ß secretion in macrophages. Overall, we provide evidence for a pathological role of caspase-4/11 in peptic ulcer disease and propose that targeting caspase-4 or inhibiting pyroptosis may have therapeutic potential in the management of peptic ulcers.

Caspase-11 promotes allergic airway inflammation.

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.

Cell Survival and Cytokine Release after Inflammasome Activation Is Regulated by the Toll-IL-1R Protein SARM.

Assembly of inflammasomes after infection or injury leads to the release of interleukin-1ß (IL-1ß) and to pyroptosis. After inflammasome activation, cells either pyroptose or enter a hyperactivated state defined by IL-1ß secretion without cell death, but what controls these different outcomes is unknown. Here, we show that removal of the Toll-IL-1R protein SARM from macrophages uncouples inflammasome-dependent cytokine release and pyroptosis, whereby cells displayed increased IL-1ß production but reduced pyroptosis. Correspondingly, increasing SARM in cells caused less IL-1ß release and more pyroptosis. SARM suppressed IL-1ß by directly restraining the NLRP3 inflammasome and, hence, caspase-1 activation. Consistent with a role for SARM in pyroptosis, Sarm1-/- mice were protected from lipopolysaccharide (LPS)-stimulated sepsis. Pyroptosis-inducing, but not hyperactivating, NLRP3 stimulants caused SARM-dependent mitochondrial depolarization. Thus, SARM-dependent mitochondrial depolarization distinguishes NLRP3 activators that cause pyroptosis from those that do not, and SARM modulation represents a cell-intrinsic mechanism to regulate cell fate after inflammasome activation.

MicroRNA-21 Limits Uptake of Listeria monocytogenes by Macrophages to Reduce the Intracellular Niche and Control Infection.

MiRNAs are important post-transcriptional regulators of gene expression. MiRNA expression is a crucial part of host responses to bacterial infection, however there is limited knowledge of their impact on the outcome of infections. We investigated the influence of miR-21 on macrophage responses during infection with Listeria monocytogenes, which establishes an intracellular niche within macrophages. MiR-21 is induced following infection of bone marrow-derived macrophages (BMDMs) with Listeria. MiR-21-/- macrophages display an increased bacterial burden with Listeria at 30 min and 2 h post-infection. This phenotype was reversed by the addition of synthetic miR-21 mimics to the system. To assess the immune response of wildtype (WT) and miR-21-/- macrophages, BMDMs were treated with bacterial LPS or infected with Listeria. There was no difference in IL-10 and IL-6 between WT and miR-21-/- BMDMs in response to LPS or Listeria. TNF-α was increased in miR-21-/- BMDMs stimulated with LPS or Listeria compared to WT macrophages. We next assessed the production of nitric oxide (NO), a key bactericidal factor in Listeria infection. There was no significant difference in NO production between WT and miR-21-/- cells, indicating that the increased bacterial burden may not be due to impaired killing. As the increased bacterial load was observed early following infection (30 min), we questioned whether this is due to differences in uptake of Listeria by WT and miR-21-/- macrophages. We show that miR-21-deficiency enhances uptake of FITC-dextran and FITC-Escherichia coli bioparticles by macrophages. The previously observed Listeria burden phenotype was ablated by pre-treatment of cells with the actin polymerization inhibitor cytochalasin-D. From analysis of miR-21 targets, we selected the pro-phagocytic regulators myristoylated alanine-rich C-kinase substrate (MARCKS) and Ras homolog gene family, member B (RhoB) for further investigation. MARCKS and RhoB are increased in miR-21-/- BMDMs, correlating with increased uptake of Listeria. Finally, intra-peritoneal infection of mice with Listeria led to increased bacterial burden in livers of miR-21-/- mice compared to WT mice. These findings suggest a possible role for miR-21 in regulation of phagocytosis during infection, potentially by repression of MARCKS and RhoB, thus serving to limit the availability of the intracellular niche of pathogens like L. monocytogenes.

The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1ß transcription and activate the NLRP3 inflammasome.

The NLRP3 inflammasome is a multiprotein complex that regulates the activation of caspase-1 leading to the maturation of the proinflammatory cytokines IL-1ß and IL-18 and promoting pyroptosis. Classically, the NLRP3 inflammasome in murine macrophages is activated by the recognition of pathogen-associated molecular patterns and by many structurally unrelated factors. Understanding the precise mechanism of NLRP3 activation by such a wide array of stimuli remains elusive, but several signaling events, including cytosolic efflux and influx of select ions, have been suggested. Accordingly, several studies have indicated a role of anion channels in NLRP3 inflammasome assembly, but their direct involvement has not been shown. Here, we report that the chloride intracellular channel proteins CLIC1 and CLIC4 participate in the regulation of the NLRP3 inflammasome. Confocal microscopy and cell fractionation experiments revealed that upon LPS stimulation of macrophages, CLIC1 and CLIC4 translocated into the nucleus and cellular membrane. In LPS/ATP-stimulated bone marrow-derived macrophages (BMDMs), CLIC1 or CLIC4 siRNA transfection impaired transcription of IL-1ß, ASC speck formation, and secretion of mature IL-1ß. Collectively, our results demonstrate that CLIC1 and CLIC4 participate both in the priming signal for IL-1ß and in NLRP3 activation.

SARM regulates CCL5 production in macrophages by promoting the recruitment of transcription factors and RNA polymerase II to the Ccl5 promoter.

The four Toll/IL-1R domain-containing adaptor proteins MyD88, MAL, TRIF, and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarily conserved Toll/IL-1R adaptor, sterile α and HEAT/Armadillo motif-containing protein (SARM), has remained more elusive. Recent studies of Sarm(-/-) mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages and discovered a requirement for SARM in CCL5 production, whereas gene induction of TNF, IL-1ß, CCL2, and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-κB and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.

Efficacy of cycling training based on a power field test.

The efficacy of an 8-minute field test to prescribe exercise intensity and assess changes in fitness was evaluated before and after 8 weeks of indoor cycling, and the results were confirmed by laboratory assessment. Changes in maximal steady-state power (MSSP), power at lactate threshold (PT(lact)), maximal power (Pmax), and maximal oxygen uptake (VO2max) were measured on 56 participants (20 women, 36 men; mean +/- SD. 46.5 +/- 10.0 years) who completed 1-hour, biweekly indoor stationary cycling classes on their own road bike outfitted with a Power Tap Pro power meter. The MSSP was defined as the average power during an 8-minute field test, which was administered at the beginning (pre) and end (post) of the training intervention. Individual training ranges were calculated from the pre-MSSP in accordance with Carmichael Training Systems. Laboratory assessments of PT(lact), Pmax, and VO2max were made on 24 of the participants the same weeks MSSP was evaluated. After training, MSSP increased 9.2% (195.4 +/- 56.6 vs. 213.8 +/- 57.2 W; p < 0.05), and PT(lact) increased 12.9% (178.3 +/- 47.1 vs. 201.5 +/- 47.6 W; p < 0.05). The MSSP was approximately 7.5 % higher than PT(lact). Pmax increased approximately 6.7% (315.2 +/- 65.1 to 336.5 +/- 65.9 W), and VO2max increased approximately 6.5% (46.2 +/- 10.7 to 49.1 +/- 10.5 ml x kg(-1) x min(-1)). The MSSP and PT(lact) were highly correlated (r = 0.98) as was MSSP and VO2max (r = 0.90). The results of this research indicated that (a) the field test is a valid measure of fitness and changes in fitness, (b) it provided data for the establishment of training ranges, and (c) a biweekly power-based training program can elicit significant changes in fitness.

MedGem hand-held indirect calorimeter is valid for resting energy expenditure measurement in healthy children.

OBJECTIVE: To assess the validity of a new hand-held indirect calorimeter [MedGem (MG)] in the determination of resting energy expenditure (REE; kilocalories per day) in children. RESEARCH METHODS AND PROCEDURES: One hundred male (n = 54) and female (n = 46) children (10.6 +/- 3.2 years, 43.9 +/- 19.0 kg, 146.1 +/- 18.8 cm, 19.6 +/- 4.9 kg/m(2)) participated. Children arrived at the University of Oklahoma body composition laboratory between 5:30 am and 6:15 am after an overnight fast. On arrival, subjects voided and remained quietly in the supine position for 15 minutes before testing. REE was measured by indirect calorimetry (in random order), with both the MG (sitting upright) and the criterion Delta Trac II (DT) (supine). Data are reported as the mean +/- standard deviation. RESULTS: The mean MG REE (1452 +/- 355 kcal/d) was significantly higher than DT REE (1349 +/- 296 kcal/d, p < 0.001). Bland-Altman analysis revealed a mean bias (MG - DT) of 104 kcal/d, with limits of agreement of -241 to +449 kcal/d. To examine the difference in subject positioning, an independent sample of 38 subjects performed the MG in its normal position (sitting) and holding the MG in a supine position. REE by the MG in the sitting position (1475 +/- 350 kcal/d) was significantly (p < 0.05) higher than the MG in the supine position (1419 +/- 286 kcal/d). DISCUSSION: The mean difference in REE between MG and DT was relatively small (103 kcal/d) but significant; however, a portion of this difference may have been related to differences in subject positioning. These preliminary data indicate that the MG shows promise as a valid tool in the assessment of REE in children.

Variability of measured resting metabolic rate.

BACKGROUND: The necessity of a 12-h fast before resting metabolic rate (RMR) is measured is often a barrier to measuring RMR. OBJECTIVE: We compared RMR measurements obtained in the morning and afternoon and across repeated days to elucidate the magnitude and sources of variability. DESIGN: Healthy men (n = 12) and women (n = 25) aged 21-67 y, with body mass indexes (in kg/m(2)) ranging from 17 to 34 and body fat ranging from 6% to 54%, completed 4 RMR measurements. RMR measurements were made in the morning (after a 12-h fast and 12 h postexercise) and in the afternoon (after a 4-h fast and 12 h postexercise) on 2 separate days with the ventilated-hood technique. Body composition was assessed by dual-energy X-ray absorptiometry. RESULTS: Mean (+/- SE) afternoon RMR was significantly higher than morning RMR on both visit 1 (1593.5 +/- 35.6 compared with 1508.0 +/- 31.5 kcal/d; P = 0.001) and visit 2 (1602 +/- 29.3 compared with 1511.4 +/- 35.9 kcal/d; P = 0.001). The 2 morning measurements (r = 0.93) and the 2 afternoon measurements (r = 0.93) were highly correlated, and no significant differences between measurements were observed. The mean difference between the morning and afternoon measurements was 99.0 +/- 35.8 kcal/d (6%). CONCLUSIONS: Repeated morning and evening measurements of RMR were stable and highly correlated. Day-to-day measurements of RMR were not significantly different. RMR measured in the afternoon after a 4-h fast and exercise was approximately 100 kcal/d higher than RMR measured in the morning.