Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A–C) Representative immunoblots and ELISA for IL-1β in BMDMs or PMs treated with indicated bacteria (106 CFU/ml E. faecalis or 104 CFU/ml E. coli) for 10 hours. (A) Hmox1–/– (–) and Hmox1+/+ (+) BMDMs. (B) Immunoblot quantitation of PMs from LyzM-Cre Hmox1fl/fl and Hmox1fl/fl injected i.p. with E. coli for 2 hours (inset; Hmox1 expression in naive PMs). (C) Retroviral shRNA against HO-1 or latent membrane potential (LMP) control in BMDMs treated as in A. #P < 0.01, LyzM-Cre Hmox1fl/fl versus Hmox1fl/fl; **P < 0.001, *P < 0.05 versus LMP. Results represent average ± SD of 2 independent experiments in triplicate. (D) TNF ELISA in supernatants from BMDMs treated as in A. Results represent mean ± SD of 2 independent experiments in triplicate. (E) Real-time PCR of cDNA from cells treated as in A. Results represent mean fold change versus control ± SD of 2 independent experiments in triplicate. *P < 0.02. (F and G) IL-1β ELISA and immunoblot of BMDM supernatants from Hmox1+/+ and Hmox1–/– treated ± LPS (1 ng/ml) for 5 hours followed by 100 μM, 500 μM, or 1000 μM ATP. Data represent mean ± SD of n = 4–6 from 3 independent experiments. *P < 0.05.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Bacteria, Quantitation Assay, Injection, Expressing, Retroviral, shRNA, Membrane, Control, Real-time Polymerase Chain Reaction

Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A) IL-1β ELISA and immunoblot in BMDMs ± selective HO-1 inhibitors Sn-PP-IX and QC-15 ± CO started 4 hours after bacteria administration (106 CFU/ml) for 6 hours. Note that CO reversed the loss in HO-1 activity found in air treatment. *P < 0.05, air versus air + Sn-PP-IX; &P < 0.01, air versus CO; #P < 0.02, CO + Sn-PP-IX versus Sn-PP-IX. (B) Upper panel: IL-1β in BMDMs from the indicated mice ± E. coli (104 CFU/ml) ± CO as above. Note that CO rescues the IL-1β response in LyzM-Cre Hmox1fl/fl that is absent in air-treated controls (comparing lanes 2 and 5). Lower panel: immunoblot showing disappearance of HO-1 in LyzM-Cre Hmox1fl/fl differentiated over 5 days in response to MCSF (20 ng/ml). Note that as the Lyz promoter becomes active, HO-1 expression decreases. (C) Representative IL-1β and active caspase-1 p20 immunoblots in BMDMs. CO was administered for 6 hours starting 4 hours following E. faecalis infection (106 CFU/ml). (D) TNF ELISA in supernatants from BMDMs infected with E. faecalis as above. All blots represent 2 to 3 independent experiments expressed relative to β-actin as loading control. All ELISA data represent mean ± SD of 2 to 3 independent experiments in triplicate.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Bacteria, Activity Assay, Expressing, Infection, Control

Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A) CFU in BMDM lysates ± E. coli (104) administered for 1 hour followed by exposure to CO or air for 6 hours. #P < 0.05, CO versus air in LyzM-Cre Hmox1fl/fl and Hmox1fl/fl macrophages; *P < 0.05, LyzM-Cre Hmox1fl/fl versus Hmox1fl/fl macrophages. (B) Bacterial counts in BMDM lysates ± E. coli administered for 2 hours followed by addition of penicillin/streptomycin ± CO for 6 hours. Results represent average ± SD of 2 independent experiments in triplicate. *P < 0.001, CO versus air. (C) CFU in supernatants of mouse BMDMs or primary human peripheral blood monocytes (hMo) + E. faecalis (106) for 1 hour prior to CO (white bars) or air (black bars) administration for an additional 4 hours. *P < 0.007; **P < 0.01, CO versus air (C). (D) Growth kinetics of E. faecalis in BMDM supernatants ± CO measured as absorbance at 600 nm. Blue indicates 102, red indicates 106, and violet indicates 1010 CFU/ml. CO, dotted lines; air, solid lines. Data represent mean ± SD of n = 3/group/time point. *P < 0.005; **P < 0.05, versus air at the same bacterial concentration and time point. (E) Growth kinetics of E. faecalis in the medium in the presence or absence of CO. Results represent mean ± SD from 3 independent experiments. Note: similar effects of CO on growth were observed with E. coli.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Concentration Assay

Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A) ATP generation by E. faecalis (106) ± CO for 1 hour measured colorimetrically (inset) or fluorometrically. (B) ATP fluorescence in supernatants from E. coli (104) treated for 6 hours ± CO. *P < 0.05; **P < 0.001. (C and D) Interaction between E. coli–derived 32ATP and immunoprecipitated BMDM P2X7 receptor from WT and P2rx7–/– mice (D) treated with supernatant from CO-exposed 32ATP-producing bacteria. Neg, IgG control; A, ampicillin control. *P < 0.03; #P < 0.05, CO versus air. (E) 32ATP-producing bacteria supernatant exposed to BMDMs where HO-1 was induced by LPS to increase endogenous CO by HO-1 ± QC-15 to block HO-1. *P < 0.01, LPS versus control (C); #P < 0.05, LPS versus LPS + QC. (F) Bacterial counts in BMDM supernatants infected with E. faecalis for 1 hour, then ± ATP (50 μM) for an additional 6 hours. *P < 0.05. (G) ATP in supernatants of WT E. coli MG1665 or ΔatpA E. coli (104) treated 30 minutes with CO or air expressed as fold change to account for proliferation rate differences. Data represent mean ± SD of 3 experiments in triplicate. *P < 0.02, CO + M1655 versus air + M1655. (H) Immunoblot with antibodies against IL-1β lysates of WT or mutant E. coli–treated BMDMs ± CO as above. (I) Bacterial counts expressed as fold change due to differences in proliferative rates. BMDMs were infected for 1 hour, then treated ± CO for an additional 6 hours. **P < 0.01, CO versus WT E. coli (102). *P < 0.05, CO + ΔcyoB versus air + ΔcyoB; &P < 0.04, CO + Δcyd versus air + Δcyd. (J) WT or mutant E. coli growth ± CO. Results represent mean ± SD from 3 independent experiments.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Fluorescence, Derivative Assay, Immunoprecipitation, Bacteria, Control, Blocking Assay, Infection, Western Blot, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A and B). ATP measured by IVIS bioluminescence or fluorescence in mouse peritoneal lavages. Mice (n = 4/group) were infected with E. coli (109) for 1 hour followed by CO for 1 hour. ATP levels were imaged and quantitated 30 minutes after injection of luciferase. *P < 0.05, CO versus air. The stronger signal intensity reflects increased ATP levels evidenced by greater luciferase activity.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Fluorescence, Infection, Injection, Luciferase, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A) Intracellular potassium (K+) levels measured fluorometrically in BMDMs infected with E. coli (104) ± CO. ATP (200 μM) was used as a positive control. Note that both ATP and CO + E. coli decreased intracellular K+ fluorescence, suggesting a K+ efflux. *P < 0.0001, ATP versus WT; #P < 0.001, M1655+CO versus WT; &P < 0.001, M1655 or CO versus WT. White bars, air; black bars, CO. (B) IL-1β ELISA of peritoneal lavage fluid from mice treated ± E. faecalis (106) ± K+ channel inhibition with TEA ± CO. CO was started 1 hour after infection and continued for 1 hour. *P < 0.01 CO versus control (C). All results represent mean ± SD of 2 to 4 independent experiments with n = 3–6/group.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Infection, Positive Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Inhibition, Control

Journal: The Journal of Clinical Investigation

Article Title: Macrophages sense and kill bacteria through carbon monoxide–dependent inflammasome activation

doi: 10.1172/JCI72853

Figure Lengend Snippet: (A) IL-1β in peritoneum from mice infected with E. faecalis (109 CFU) for 1 hour ± CO. Data represent mean ± SD of n = 6/group in triplicate. *P < 0.01; #P < 0.05, CO versus air. (B) Immunoblot of cleaved caspase-1 and IL-1β in PM lysates from mice infected with E. faecalis for 1 hour prior to 1 hour treatment with air or CO. Representative blot from 3 independent experiments. (C) Immunoblots of macrophages lysates from mice treated as in B. (D) ALT in infected mice treated as above. Results represent mean ± SD (n = 4–6/group) from 3 experiments. *P < 0.001, CO versus air. (E) H&E staining 8 hours after E. faecalis ± CO; 6 to 10 images per animal. Arrows indicate lesions. Original magnification, ×20. (F) Bacterial counts in peritoneal lavagates. Inset: IVIS images. Results represent mean ± SD of 6 to 8/group. *P < 0.05; **P < 0.01. (G) Survival in mice injected with lethal E. coli (1010 CFU, i.p.) or CLP ± 4 hours CO initiated 6 hours later. P < 0.02, air + CLP versus CO + CLP; P < 0.02, air + E. coli versus CO + E. coli. (H) Survival of WT and Nalp3–/– mice treated as in G with or without CO (started 1 or 6 hours after bacteria). CO was unable to protect Nalp3–/– mice (P < 0.01). (I) Bacteria counts in peritoneum of E. coli–infected mice. Results represent mean ± SD of 4 to 6/group repeated twice. *P < 0.03, CO versus air; #P < 0.05, Casp1–/– versus Casp1+/+ in both air and CO-treated mice. (J) Survival ± Sn-PP-IX to block HO-1 then ± 4 hours CO beginning 1 hour after bacteria. P < 0.05, 10/group. (K) Survival of indicated mice ± CO as in G. P < 0.03. n = 10–30/group.

Article Snippet: Bacteria isolation and propagation E. coli (DH5α strain, ATCC), E. coli Xen 14 (chemiluminescent EPEC WS2572 –parental strain, Caliper Life Sciences), Salmonella enterica ( S. typhimurium ) (ATCC), Mtb-BCG (provided by William Bishai, Johns Hopkins University, Baltimore, Maryland, USA), and E. faecalis or E. coli (single colony isolated from mouse intestinal flora) were used.

Techniques: Infection, Western Blot, Staining, Injection, Bacteria, Blocking Assay