Carbon monoxide down modulates Toll like receptor 4/MD2 expression on innate immune cells and reduces endotoxic shock susceptibility
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(2) S. A. Riquelme et al. from Gram-negative bacteria, has been reported as one of the major DC stimulators that cause an inflammatory environment in infected tissues. Such an effect takes place as the result of the significantly high secretion of pro-inflammatory cytokines by DC after LPS stimulation, which is higher than the response observed by macrophages after the same stimulus.9–15 Hence, after priming with LPS, DC play a major role in inflammation.9 It has been reported that LPS activates DC and other myeloid cells by binding to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD2) complex, which works as an LPS receptor expressed on the surface of these cells.13,16–20 Binding of LPS to this receptor triggers the secretion of pro-inflammatory cytokines, such as interleukin-12 (IL-12), IL-6 and tumour necrosis factor-a through the activation of, for example, nuclear factor-jB (NF-jB) and mitogen-activated protein kinase (MAPK) signalling pathways.4,10,13,14,20 In innate cells, the TLR4/ MD2 complex has been described as the main upstream activator of cell signalling pathways during a challenge with LPS.21,22 For instance, it was reported that monocytes lacking this receptor (TLR4 knockout cells) fail to secrete inflammatory cytokines in response to endotoxin stimulation.14,18 Furthermore, TLR4 knockout mice or mice expressing an inactive variant of TLR4 are unable to transduce LPS-promoted signals and are resistant to endotoxin-mediated shock.19,23 Hence, absence of the TLR4/MD2 complex from the surface of monocytes is directly associated with reduced secretion of pro-inflammatory cytokines, impaired innate cell activation and restricted sepsis progression after LPS stimulation. It has been recently reported that induction of HO-1 prevents the secretion of pro-inflammatory cytokines by monocytes after treatment with LPS.1,4,24 Interestingly, these studies have shown that pre-treatment with CO rendered myeloid cells resistant to LPS stimulation by decreasing the activation of the NF-jB and MAPK pathways, both in vivo and in vitro.3,4,25 Because TLR4/MD2 surface expression is required to recognize LPS, these data suggest that CO could be negatively modulating the surface expression of this complex. However, this hypothesis has not been tested. Here we show that both HO-1 induction and CO treatment decrease TLR4/MD2 expression in the surface of DC, rendering them tolerant to LPS stimulation, both in vitro and in vivo. The biological significance of the reduction in TLR4/MD2 expression was evaluated in an LPSmediated septic shock model in vivo, which is exclusively mediated by the engagement of the TLR4/MD2 complex and involves myeloid cell stimulation leading to immunopathology.14,15,19,26–28 We evaluated whether prophylactic CO administration could prevent endotoxin shock in mice due to down-regulation of TLR4/MD2 expression in myeloid cells. We observed in CO-treated mice that DC 322. and neutrophils were not recruited to blood after LPS challenge, compared with untreated mice. In addition, CO administration protected against LPS-induced hypothermia, one of the acute signs of inflammation29 and one of the final events in the switch from severe sepsis to septic shock, which is directly associated with a fatal outcome.30–32 Our findings suggest that HO-1, through the release of CO, reduces LPS-mediated inflammation by down-modulating the expression of the TLR4/MD2 complex on the surface of different myeloid cells. It is likely that such a mechanism would prevent LPS-induced activation of myeloid cells in vivo, protecting mice from the severe sepsis-to-septic shock switch and subsequent death.. Materials and methods Ethics statement Mouse handling and experimental protocols were approved by the Bioethics and Biosafety committee of the Facultad de Ciencias Biol ogicas at the Pontificia Universidad Cat olica de Chile (identification number CBB-164/ 2010), and by the Bioethics Committee of the Fondo Nacional de Desarrollo Cientıfico y Tecnol ogico de Chile (FONDECYT grant 1110397). All animal procedures used in this study are based on the Handbook for Standard Biosafety, Conicyt 2008, Chile; as well as from the Guide for the Care and Use of Laboratory Animals (NRC 2011). All procedures were performed under the supervision of a veterinarian.. Reagents and antibodies Reagents used in this study were Cobalt Protoporphyrin IX (CoPP; Frontier Scientific, Carnforth, UK), Tin Protoporphyrin IX (SnPP) (Frontier Scientific), tricarbonyldichlororuthenium (II) dimer [Ru(CO)3Cl2]2 (Sigma Aldrich, Lyon, France), DMSO, LPS from Salmonella enterica serovar typhimurium (Sigma Aldrich, St Louis, MO), granulocyte–macrophage colony-stimulating factor (GMCSF; Peprotech, Rocky Hill, NJ) and Saponin (Sigma Aldrich). Antibodies used were anti-CD11c-FITC/allophycocyanin (APC; clone HL3; BD Pharmingen, San Diego, CA), anti-CD11b-FITC/APC (clone M1/70; BD Pharmingen), anti-mouse Gr1-peridinin chlorophyll protein (PerCP)/APC (BD, clone RB6-8C5), anti-mouse CD4-PerCP (BD, clone H129.19), anti-CD3, (BD, clone MF3), anti-mouse CD8-APC (BD, clone 53-6.7), rat antiTLR4/MD2-phycoerythrin (BD, clone MTS510), antimouse HO-1 (Abcam, Cambridge, UK), goat anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad, CA), rabbit antiTLR4 (Abcam), mouse anti-MD2 (Abcam), goat anti-rabbit 647 (Invitrogen), mouse anti-HO-1 (Abcam), antiCD11c phycoerythrin-Cy7 (clone HL3, BD) and goat anti-rat AF568 (Invitrogen). ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332.
(3) CO reduces TLR4/MD2 expression on DCs Dendritic cell preparation Dendritic cells were obtained from either male or female bone marrow progenitors of C57BL/6 mice. Mice were obtained from The Jackson Laboratories (Bar Harbor, ME) and maintained and manipulated in specific pathogen-free conditions. Manipulation of the animals and the processing of the biological samples were performed according to institutional guidelines at the Pontificia Universidad Cat olica de Chile animal facility (Santiago, Chile). Bone marrow progenitors (1 9 106 to 15 9 106 cells) were seeded in 24-well plates and cultured in RPMI-1640, pH 72, containing 10% fetal bovine serum, 1 mM pyruvate, 2 mM glutamine, 1 mM nonessential amino acids, antibiotics (penicillin and streptomycin) and 10 ng/ml of recombinant murine GM-CSF (Peprotech). Cells were incubated and differentiated for 5 days, replacing medium every 2 days. DC differentiation was determined at day 5 by flow cytometry, where the expression of CD11c, CD11b, class I MHC, class II MHC and low-affinity FccRs was measured. The percentage of CD11c+ was consistently > 80%.. Treatment of DC and TLR4/MD2 expression assays To induce HO-1 expression, immature DC were treated with CoPP (Frontier Scientific), as previously described.6 In addition, to block HO-1 activity, DC were treated with SnPP. Briefly, immature DC were pulsed for 2 hr either with 50 lM CoPP or 50 lM SnPP, then washed twice and cultured for an additional 22 hr in fresh medium. To promote DC maturation, 1 hr after the last PBS wash, cells were incubated in medium containing 1 lg/ml LPS (Salmonella typhimurium; Sigma, St Louis, MO) for 22 hr. For CO treatment, immature DC were incubated for 10–15 min with 100 lM CORM2 (tricarbonyldichlororuthenium (II) dimer [Ru(CO)3Cl2]2; Sigma Aldrich, France) or its inactive form, iCORM2 (CORM2 incubated for 48 hr at 37° in a 5% CO2-humidified atmosphere to liberate CO), as a control. Maturation of treated DC was then induced for 21 hr with LPS (10 lg/ml). Supernatants and cells were subsequently harvested for cytokine and phenotypic analyses, respectively. DC were stained for TLR4/MD2, CD11c and CD11b with monoclonal antibodies (BD Biosciences, San Diego, CA) for flow cytometry analyses at different time-points.. Lentiviral transduction of DC Dendritic cells were prepared as described above. At day 5, cells were pulsed with a HO-1-expressing lentivirus (Ln-HO-1) or a GFP-expressing lentivirus (Ln-GFP) at a multiplicity of infection (MOI) equal to 35, as previously described.33 Cells were incubated for 21 hr, then washed ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332. and extracellularly stained for TLR4/MD2 and CD11c. Then, cells were fixed, permeabilized and stained intracellularly with mouse anti-HO-1 antibody. Next, cells were washed and stained with goat anti-mouse AF647 antibody. Cells were washed and analysed by flow cytometry, using an LSRII cytometer.. Confocal microscopy analysis Bone marrow progenitors were seeded in 12-mm coverslips in 24-well plates and differentiated into DC as described above. Then, DC were pulsed with CORM2 or iCORM2 and pulsed with LPS for 4 hr. Cells were stained for TLR4, MD2 and TLR4/MD2 complex overnight at 4° in a cold chamber. Then, DC were extensively washed and stained with the respective secondary antibodies. Cells were washed, fixed with 2% paraformaldehyde and mounted with Prolong-DAPI. DC were analysed in a Confocal Spectral Nikon Eclipse C2si (using Plan Apo VC60X OIL DIC N2, NA:1.4, 1024 9 1024) and each field was recorded using NIS element AR V3.2 software performing Z-stacks with a slide-wide between 02 and 025 lm per cell. Image analyses were performed using the FIJI software. To evaluate HO-1 induction by CoPP in myeloid DC, cells were seeded in coverslips at day 0 and 5 days after differentiation with GM-CSF were treated with vehicle, 50 lM of CoPP or 50 lM of SnPP for 2 hr, extensively washed and incubated for 16 hr at 37°. Then, cells were washed and fixed with 2% paraformaldehyde for 10 min at 4°. Then, cells were washed, permeabilized with 005% saponin-PBS at room temperature and coverslips were transferred to a cold chamber over a hydrophobic surface (parafilm-coated). Each coverslip was treated for 16 hr with 50 ll of a 1/200 mouse anti-HO-1, already dissolved in cold 005% saponin-PBS. Next, cells were extensively washed and stained with a secondary 1/200 goat antimouse Alexa Fluor 488 for 3 hr at 4°. Cells were washed and mounted with DABCO for confocal microscopy analysis. Fluorescence intensity of HO-1 per cell was measured using IMAGEJ (NIH, Bethesda, MD) 1.47c software.. In vivo TLR4/MD2 expression determination C57BL/6 mice were intraperitoneally (i.p.) treated with 30 mg/kg of iCORM2 or CORM2 every 48 hr for 1 week. Then, mice were anaesthetized with isoflurane 2% and bled from the cheek using a 21G needle. Blood was mixed with 100 µl Heparin, centrifuged and the plasma was harvested. Cell fractions were treated twice with red blood cell lysis buffer (ACK) and then washed with cold PBS. Cells suspensions were stained for CD11c, CD11b, Gr1 and TLR4/MD2 using the monoclonal antibodies described above. Samples were analysed in a FACScalibur cytometer. 323.
(4) S. A. Riquelme et al. Septic shock assays C57BL/6 mice were i.p. treated with 30 mg/kg of iCORM2 or CORM2 every 48 hr during 1 week. Then, mice were i.p. challenged with 15 mg/kg of S. typhimurium LPS. As controls, mice treated either with iCORM2 or CORM2 were injected with PBS (vehicle). Using an infrared thermometer, body temperature was recorded (VeraTemp; Brooklands Inc., Boca Raton, FL). Survival was evaluated on a daily basis. At day 2 and between days 2 and 3 after LPS/PBS challenge, blood was obtained from surviving mice and DC (CD11c+ CD11b+) and neutrophils (Gr1high+ CD11bhigh+) cells were stained and analysed by flow cytometry, as described above. In addition, 15 hr after LPS/PBS challenge each mouse was recorded and the mean velocity of displacement was measured. In addition, mice that survived after LPS/PBS challenge at day 4 were weighted and the fold change with respect to their initial weight was calculated. At day 4 post-challenge, a group of mice was killed and spleens were removed to study the distribution of innate and adaptive immune cells. In parallel, to test the effect of CO treatment for ongoing sepsis, mice were treated either with PBS or 15 mg/kg of LPS. Two hours later mice received either 30 mg/kg or 60 mg/kg of CORM2/iCORM2. Survival was evaluated daily.. Results CO decreases the expression of TLR4/MD2 on the surface of DC To evaluate whether CO administration could modulate TLR4/MD2 expression on the surface of DC, murine bone marrow-derived DC were treated with CORM2, a non-toxic CO-releasing molecule, which in solution produces CO gas.2 As controls, DC were treated either with vehicle (DMSO) or iCORM2, an inactive form of CORM2 that does not release CO.2,4 After 10 min of treatment, DC were washed and cultured during several time-points. As shown in Fig. 1(a), the population of TLR4/MD2+ CD11c+ CD11b+ (identified using an antibody anti-TLR4/MD2 complex, clone MTS510) was reduced over time in CO-treated cells, but not in control cells, which showed no changes in TLR4/MD2 expression. Interestingly, reduction of TLR4/MD2 complex expression was observed as early as 4 hr post treatment with CO, reaching a 50% decrease at 21 hr post treatment (Fig. 1b). The same result was observed by confocal microscopy (Fig. 1c,f). Importantly, low expression of the TLR4/MD2 complex was not associated with reduced amounts of individual TLR4 or MD2 over the DC surface (Fig. 1c–e). To corroborate that activation of HO-1 also reduced the expression of TLR4/MD2 in 324. CD11c+ CD11b+ cells, we treated bone-marrow-derived DC with an HO-1 inducer known as Cobalt Protoporphirin IX, which promotes a strong induction of HO-1 after 21 hr of treatment (CoPP; see Supporting information, Fig. S1a). As shown in Fig. S1(b,c), HO-1 induction in CD11c+ CD11b+ cells also reduced the expression of TLR4/MD2+ to 50%. Consistent with these findings, cells treated with SnPP, an inhibitor of the HO-1 activity, showed no significant reduction in TLR4/ MD2 expression, supporting the notion that TLR4 expression was specifically modulated by the activity of HO-1. In agreement with these results, transduction of DC with an HO-1-expressing lentiviral vector also reduced the expression of surface TLR4/MD2 complex (Fig. S1f–j). Upon LPS engagement, the TLR4/MD2 complex is activated, modifies its structure and triggers secretion of pro-inflammatory cytokines.18 This conformation change prevents recognition by MTS510 antibody. For this reason, we tested whether reduced staining of DC by MTS510 antibody after CO pulse was associated with increased secretion of pro-inflammatory cytokines. As shown in Fig. 1(g), secretion of IL-12p70 by DC treated with CORM2 or iCORM2 was similar to that by control DC, suggesting that TLR4/MD2 reduction from the surface of DC due to CO treatment did not alter IL-12p70 secretion. However, when DC were first treated with CORM2 and then challenged with LPS, they produced significantly less IL-12p70 than did control cells (iCORM2-treated and untreated DC, Fig. 1g), supporting the notion that CO rapidly renders DC unresponsive to LPS stimulation. In this case, we observed that after 20 min of CO treatment and a cold PBS wash, the expression of TLR4/MD2 was about 30% lower than DC treated with iCORM2 (see Supporting information, Fig. S2). Hence, when CO-treated DC were pulsed with LPS, the fraction of cells able to respond to endotoxin was significantly lower than control cells. In agreement with the results described above, the pattern of IL-12p70 secretion of DC treated with CoPP was similar to CORM2-treated cells. As shown in Fig. S1(d), CoPP-treated DC secreted less IL-12p70 in response to LPS challenge compared with control DC. The SnPPtreated cells (HO-1 inhibited) showed higher secretion of IL-12p70 than CoPP-treated DC (Fig. S1d). As previously reported,24 the secretion of IL-10 was not affected, although we observed that CoPP-treated cells pulsed with LPS secreted less IL-12p70 (Fig. S1e). The production of other pro-inflammatory cytokines by DC, such as interferon-c, was not detected after LPS priming34 (see Supporting information, Fig. S3). These data corroborate the anti-inflammatory role associated with HO-1 in DC and how this enzyme can impair the onset of the intracellular inflammatory pathway after receptor modulation.4 ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332.
(5) CO reduces TLR4/MD2 expression on DCs. 50. MD2 TLR4 Nuclei. (e). TLR4/MD2-PE. (f). 100. 3. 10. TLR4/MD2-PE. 4. R2. R2. R1. TLR4/MD2 Nuclei. TLR4 Nuclei. S. S. LP + 2. 0. 150 R2. 2. MD2 TLR4 Nuclei. R2. R2 R1. R1. 2. 0. 5. 10. 15. Time (hr). 20. 25 TLR4 Nuclei. TLR4/MD2 Nuclei. % IL-12p70 secretion (respect to vehicle + LPS). + CORM2 + LPS. CORM. iCORM. 0. MD2 Nuclei. TLR4 + MD2 TLR4/MD2. Vehicle. TLR4. 50. ns. (g). R1. R1 100. R2. MD2. % TLR4/MD-2+CD11c+CD11b+ (respect to vehicle at t0). (b). R1. 100. S. 10. M. 2. 200. R. 10. MD2 TLR4 Nuclei. ns. O. 1. R1. *** ***. 300. iC O R. 10. MD2 Nuclei. R2. TLR4 + MD2 TLR4/MD2. 0 0 10. R1. TLR4. 25. R2. 400. iC. 50. + iCORM2 + LPS. 75. MFI TLR4/MD2 per cell. MD2. 21 hr. LP. TLR4/MD2 Nuclei. TLR4 Nuclei. +. 104. 2. 103. R. 102. TLR4/MD2-PE. O. 101. 0 M. R1. 100. R. R1. 200. O. R2. ns. iC. R2. 25 0 100. % total events. MD2 TLR4 Nuclei. iC. + CORM2. 50. MD2 Nuclei. TLR4 + MD2 TLR4/MD2. % total events. 17 hr. 75. TLR4. 100. 300. 2. R1. 4. M. 10. R. 3. S. 10. M. 2. LP. 10. R. 1. +. 10. 2. 0 0 10. ns ns. M. R1. 400. O. R2. MFI MD2 per cell. R2. C. TLR4/MD2 Nuclei. TLR4 Nuclei. 25. 2. 50. MD2. % total events. 4 hr. 75. 0. M. R2. LP. R2. 100. 100. R. R1. +. R1. 2. TLR4/MD2-PE. MD2 Nuclei. M. 104. O R M. 103. 200. O R M C 2 O R O M R M 2 C 2 +L O PS R M 2 + LP S. 102. ns. iC. 101. ns. iC. 100. TLR4 + MD2 TLR4/MD2. 0. + iCORM2. 25. ns 300. C. R2. 2. R2. 400. O. 75. (d). R2. TLR4. % total events. R1. MD2. R1. C. R1. 0 hr. 2. (c). C O. iCORM2. CORM2. Vehicle. 100. MFI TLR4 per cell. (a). ** **. 100 Vehicle 80. CORM. 2. iCORM. 2. 60 40 20 0. Untreated. + 1 µg/ml LPS. Figure 1. Carbon monoxide (CO) decreases Toll-like receptor 4 (TLR4)/MD2 expression in dendritic cells (DC). (a) DC were treated for 10 min with Vehicle (grey filled), CO-releasing molecules (CORM2; black filled) or inactive CO-releasing molecules (iCORM2; green line), washed and then cultured for different periods. After incubation, DC were detached, stained for CD11c, CD11b and TLR4/MD2 and analysed by flow cytometry. (b) TLR4/MD2 expression by CD11c+ CD11b+ cells after CO treatment. Each value was standardized against Vehicle at time equal to t0. (c) Confocal microscopy images showing surface MD2 (green), TLR4 (red) and TLR4/MD2 complex (magenta) in DC. Before the staining, cells were treated with iCORM2 or CORM2 and then left untreated or treated with lipopolysaccharide (LPS). (d) Microscopy mean fluorescence intensity (MFI) quantification for surface TLR4 expression per cell. (e) Microscopy MFI quantification for surface MD2 expression per cell. (f) Microscopy MFI quantification for surface TLR4/MD2 expression per cell. (g) Interleukin-12p70 (IL-12p70) secretion by DC treated with Vehicle, CORM2 or iCORM2. To corroborate effective IL-12p70 secretion by the TLR4/MD2 pathway, DC were challenged after CO treatment with 1 lg/ml of LPS. For (b) and (c) data are a pool of triplicates from three independent experiments. Data in (d–f) are the mean values of two independent experiments. Data are shown as mean SEM. Data were analysed by one-way analysis of variance. **P < 001, ns not significant.. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332. 325.
(6) S. A. Riquelme et al. Carbon monoxide reduces expression of TLR4/MD2 on the surface of peripheral myeloid immune cells Next we evaluated whether CO treatment could decrease TLR4/MD2 expression in myeloid DC in vivo. For this purpose, mice were i.p. injected with either CORM2 or iCORM2 and the population of TLR4/MD2+ CD11c+ CD11b+ cells was quantified in peripheral blood. In cells selected from gate R1 (Fig. 2a), we observed a 30% reduction in the amount of DC expressing TLR4/MD2, relative to control cells (Fig. 2b,c). Further, we evaluated whether CO administration could modulate surface expression of. Blood CD11c+CD11b+ Dendritic cells. (a). TLR4/MD2 in neutrophils, another cell type contributing to the exacerbated inflammation induced by LPS during endotoxic shock. Although no significant changes were observed in the frequency of blood neutrophils after CORM2 treatment, (Fig. 2d) (region R2, CD11bhigh+ Gr1high+), the expression of TLR4/MD2 on the surface of these cells was significantly decreased in comparison to iCORM2-treated mice (Fig. 2e,f). Hence, CO administration not only modulates TLR4/MD2 surface expression on myeloid DC, but also can modulate the expression of this receptor in peripheral neutrophils. These data suggest that CO administration in vivo can reduce the expression of. Blood CD11bhigh+Gr1high+ Neutrophils. (d). 104. 104. 101 100 100. 103 101 102 CD11b-PerCP. 103 Gr1-PerCP. CD11c-FITC. R1. 102. 102 101 100 100. 104. 101. 102. 103. 104. CD11b-FITC. (b) 100. (e) 100 Auto. 75. % total events. % total events. Total white blood cells. Total white blood cells. R2 103. iCORM2. 50. CORM2. 25 0 100. 101. 102 103 TLR4/MD2-PE Gated on R1. Auto 75. iCORM2. 50. CORM2. 25 0 100. 104. 101. 102 103 TLR4/MD2-PE Gated on R2. 104. (f). (c) iCORM2. iCORM2. *. CORM2 0. 20. 40. 60. 80. % TLR4/MD-2+CD11c+CD11b+ (respect to iCORM2). 100. *. CORM2 0. 20. 40. 60. 80. 100. % TLR4/MD-2+CD11bhigh+Gr1high+ (respect to iCORM2). Figure 2. Carbon monoxide (CO) decreases Toll-like receptor 4 (TLR4)/MD2 expression in both peripheral dendritic cells (DC) and neutrophils. (a) C57BL/6 mice were intraperitoneally injected with 30 mg/kg of either CO-releasing molecules (CORM2) or inactive CO-releasing molecules (iCORM2) each 48 hr for 1 week, bled, and cells were stained for TLR4/MD2, CD11b and CD11c markers. (a) Representative density plot with the gating region (R1) from which the percentage of TLR4/MD2+ CD11c+ CD11b+ cells was evaluated. (b) Histograms showing the distribution of TLR4/MD2+ DC gated from R1. Grey-filled histogram: Auto; black-filled histogram: CORM2-treated mice; lined histogram: iCORM2-treated mice. (c) Bar graph showing the fraction of cells expressing TLR4/MD2 on their surface with respect to the major value obtained for iCORM2-treated mice. Five mice from two independent experiments were analysed. Data are shown as mean SEM. Data were analysed by a Student’s t-test. *P < 005. (d) C57BL/6 mice were intraperitoneally injected with 30 mg/kg of either CORM2 or iCORM2 each 48 hr for 1 week, bled and cells were stained for TLR4/MD2, CD11b and Gr1 markers. (d) Representative density plot with the gating region (R1) from which was evaluated the percentage of TLR4/MD2+ CD11bhigh+ CD11bhigh+ cells. (e) Histograms showing the distribution of TLR4/MD2+ neutrophils gated from R1. Grey-filled histogram: Auto; black-filled histogram: CORM2-treated mice; green-lined histogram: iCORM2-treated mice. (f) Bar graph showing the fraction of cells expressing TLR4/MD2 on their surface with respect to the major value obtained for iCORM2-treated mice. Five mice from two independent experiments were analysed. Data are shown as mean SEM. Data were analysed by a Student’s t-test. *P < 005.. 326. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332.
(7) CO reduces TLR4/MD2 expression on DCs TLR4/MD2 in CD11c+ CD11b+ DC and CD11bhigh+ Gr1high+ neutrophils, which could contribute to reducing mouse sensitivity to LPS-mediated inflammation.15. of around 10° in mice that received iCORM2 or vehicle and then LPS. These mice showed trembling and reduced mobility (see Supporting information, Video S1). Hence, we monitored each mouse for lapses of 60 seconds and measured their mean displacement velocity (Fig. 3b,c). We observed that before CO treatment, mice showed a mean velocity of 10 cm/second (Fig. 3c, left panel). After CO treatment, mice that received PBS maintained their ability to move with no significant changes in their velocity (Fig. 3c, right panel). On the contrary, for the groups of mice that received LPS, only those that were pre-treated with CORM2 were able to keep moving at approximately 80% of their initial velocity. However, those that received iCORM2 and LPS showed a significant drop in their mobility, with values falling > 70% compared with the values observed before LPS challenge. The body weight of each mouse was measured during these experiments to monitor the severity of the response to LPS stimulus. Control mice that received either. Carbon monoxide protects mice from severe sepsis To validate our previous findings, we used a mouse model of endotoxin-mediated shock. Because CO decreased TLR4/MD2 expression on the surface of innate cells, it would be expected that CO treatment could reduce the susceptibility of mice to the endotoxin-mediated inflammation, as reported for TLR4 / mice.19 Hence, we treated mice with either active CORM2 or iCORM2 and then a challenge with LPS was performed. As shown in Fig. 3(a), CO prevented mice from having severe hypothermia caused by the administration of a lethal LPS dose.32,35 After 15 hr of LPS challenge and under hypothermic conditions, we observed a decrease in body temperature (b). Challenge iCORM2 + PBS CORM2 + PBS iCORM2 + LPS CORM2 + LPS. 38 36 34 32 30 28 26 24 22 20. Tf – Ti. R = Ri T = Ti. ns ns. ns ns. *** ***. 15 10 5 0. (d) Fold change weight (with respect to control). Mean velocity (cms/seconds). Rf – Ri. =. R = Rf. Time post i.p. challenge (hr). 20. Δtime. Distance. –10 1 2 3 4 5 6 7 14 19 24 42. (c). ΔDistance. Mean velocity =. ns 1·1. ns. T = Tf. ***. 100. **. 1·0 0·9 0·8. + PBS + LPS. Post-challenge. iCORM2 + PBS CORM2 + PBS iCORM2 + LPS CORM2 + LPS. 80 60. * *. 40 20 0. 0·7. + PBS + LPS Pre-challenge. Time. (e). ns. Percent survival. Body temperature (°c). (a). Pre-challenge. Post-challenge. Control groups:. iCORM2. CORM2. Control groups:. iCORM2. CORM2. Experimental groups:. iCORM2. CORM2. Experimental groups:. iCORM2. CORM2. 0. 2. 4. 6. 8. Days post i.p. challenge. Figure 3. Carbon monoxide (CO) treatment prevents lipopolysaccharide (LPS) -mediated septic shock in mice. C57BL/6 mice were intraperitoneally injected with 30 mg/kg of either CO-releasing molecules (CORM2) or inactive CO-releasing molecules (iCORM2) each 48 hr for 1 week and then challenged either with PBS or 15 mg/kg of LPS. (a) Body temperature was measured during different time-points after PBS/LPS challenge. (b) Time lapse for mice displacements. Each mouse was monitored for displacement during 60 seconds at 1 day (pre-challenge) and +1 day (15 hr, post challenge). Ri, initial position; Rf, final position; Ti, initial time; Tf, final time. Using the parameters measured here, the mean velocity of displacement for each mouse was calculated using the equation described above the image. (c) Mean velocity (cm/second) for each mouse is shown as described in (b). (d) Surviving mice at day +4 post PBS/LPS were weighed and their body weights were standardized against their initial value ( 2 days from challenge). (e) Curve for surviving mice after PBS/LPS challenge. For (a), a representative experiment out of three is shown (three mice per curve). For (c–e) data are from at least six mice pooled, out of three independent experiments. Data are shown as mean SEM. Data were analysed by one-way analysis of variance. **P < 001; ***P < 0001; ns: non-significant.. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332. 327.
(8) S. A. Riquelme et al. in the blood frequency of DC (Fig. 4a, upper panel; b, left panel). Importantly, CO treatment was able to prevent the LPS-induced increase of DC in the blood, compared with control mice receiving iCORM2 (Fig. 4a, lower panel; b, right panel). Hence, CO administration prevented LPS-driven blood mobilization of DC during sepsis. Further, we evaluated whether CO treatment could prevent neutrophil recruitment (CD11bhigh+ Gr1high+) to peripheral blood in response to LPS challenge. We observed that before a PBS/LPS challenge, the frequency of neutrophils in peripheral blood was equivalent for all the experimental groups (Fig. 5b, left panel). Control mice that were challenged with PBS did not show any significant change in blood neutrophil frequency, independently of either iCORM2 or CORM2 pre-treatment (Fig. 5a, upper panel; b, right panel). However, mice treated with iCORM2 and then challenged with LPS showed a drastic increase in the blood fraction of neutrophils, up to 25-fold (average of 10%). Such an increase in blood neutrophils was not observed in mice challenged with LPS that were pre-treated with CORM2 (Fig. 5a, lower panel; b, right panel). Hence, treatment with CO protected mice from the harmful increase of neutrophils in blood, which is a prominent symptom of septic inflammation associated with organ dysfunction.26 In addition, we evaluated whether CO administration could modulate the recruitment of DC and neutrophils to other immune tissues, such as spleen. As shown in Fig. S5(a,b) (see Supporting information), CO was not able to alter the frequency of these cells in the spleens of PBStreated or LPS-treated mice.. iCORM2 or CORM2 and that were challenged with vehicle (PBS) did not show significant changes in their body weight (Fig. 3d, right panel). However, surviving mice that were pre-treated with iCORM2 and then challenged with LPS suffered a drastic decrease of body weight (almost 18%) compared with their pre-challenge weights (Fig. 3d, right panel). Moreover, mice that received CORM2 showed a significant increase in survival to LPS challenge, as compared with mice receiving iCORM2 (Fig. 3e). Thus, i.p. administration of CO prevented mice from suffering hypothermia, mobility loss, body weight loss and death by endotoxin-induced shock. However, when mice were first challenged with LPS and then treated with CO, no significant protection could be observed (see Supporting information, Fig. S4). These results suggest that during sepsis, LPS induces a strong effect on TLR4/MD2 complexes that cannot be reverted by CO.. Carbon monoxide prevents the recruitment of blood DC and neutrophils after LPS challenge in vivo Dendritic cells play a major role in LPS-mediated sepsis.14,15,27 During this process, these cells are transported by either blood or lymph vessels to secondary lymphoid tissues.15,27 We monitored whether CO could modulate the proportion of DC in the blood after the LPS challenge at the time of mortality of control mice (Fig. 3e). Before LPS or PBS challenge, all mice showed equivalent blood frequencies of CD11c+ CD11b+ DC (Fig. 4b, left panel). After control PBS challenge, mice that received either iCORM2 or CORM2 did not display significant changes. 4. iCORM2 + PBS. 3. 10. 2. 1·81%. 10. 101. 4. 10. 0. 3. 10. 2. 10. 101 10 0 1 2 3 4 10 10 10 10 10. CD11b-FITC. 6·03%. 101 100 0 10 101 102 103 104. CD11b-FITC. 104. CD11c-APC. CD11c-APC. 10. 102. CD11b-FITC. iCORM2 + LPS. 3. 1·74%. 0. 10 0 1 2 3 4 10 10 10 10 10. 104. CORM2 + PBS. CORM2 + LPS. ns. 8 ns. ***. **. 6 4 2 0 + PBS + LPS. 3. 10. 102. *. (b). % CD11c+CD11b+cells. CD11c-APC. 10. CD11c-APC. (a). 4·46%. Pre-challenge. 101 100 0 10 101 102 103 104. CD11b-FITC. Post-challenge. Control groups:. iCORM2. CORM2. Experimental groups:. iCORM2. CORM2. Figure 4. Carbon monoxide (CO) administration restricts lipopolysaccharide (LPS) -mediated increase of dendritic cells (DC) in blood during sepsis. (a) Representative density plots at 3–4 days post challenge. Upper panel shows inactive CO-releasing molecules (iCORM2)/ CO-releasing molecules (CORM2) -treated mice that were challenged with PBS (control groups). Lower panel shows iCORM2/CORM2-treated mice that were challenged with LPS (experimental groups). R1 shows the population of cells that were CD11c+ CD11b+ in blood. (b) Bar graph showing the percentage of CD11c+ CD11b+ cells (R1) in blood pre-challenge ( 2 days from PBS/LPS challenge) and post-challenge (+3–4 days post PBS/LPS challenge). Control groups were challenged intraperitoneally (i.p.) with PBS and experimental groups were i.p. challenged with LPS. Data are from five mice of two independent experiments. Data are shown as mean SEM. Data were analysed by one-way analysis of variance. *P < 005; **P < 001; ***P < 0001; ns: non-significant.. 328. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332.
(9) CO reduces TLR4/MD2 expression on DCs. Gr1-PerCP. iCORM2 + PBS 3·04%. 103 2. 10. 1. 10. 104. 0. 103 2. 10. 1. 10. 10 0 1 2 3 4 10 10 10 10 10. CD11b-FITC. Gr1-PerCP. 103. CD11b-FITC. iCORM2 + LPS 8·45%. 102 1. 10. 0. 10 0 1 2 3 4 10 10 10 10 10. CD11b-FITC. 4. 10. Gr1-PerCP. 4. 4·04%. 0. 10 0 1 2 3 4 10 10 10 10 10. 10. CORM2 + PBS. 103. CORM2 + LPS. (b). ns 12. % CD11bhighGr1high. 4. 10. Gr1-PerCP. (a). ns ns. 4. 0 + PBS + LPS Pre-challenge. 1. 10. 0. 10 0 1 2 3 4 10 10 10 10 10. **. 8. 4·11%. 102. **. Post-challenge. Control groups:. iCORM2. CORM2. Experimental groups:. iCORM2. CORM2. CD11b-FITC. Figure 5. Carbon monoxide (CO) administration suppresses lipopolysaccharide (LPS) -mediated increase of neutrophils in blood during severe sepsis. (a) Representative density plots at 3–4 days post challenge. Upper panel shows inactive CO-releasing molecules (iCORM2)/ CO-releasing molecules (CORM2)-treated mice that were challenged with PBS (control groups). Lower panel shows iCORM2/CORM2-treated mice that were challenged with LPS (experimental groups). R1 shows the population of cells that were CD11bhigh+ Gr1high+in blood. (b) Bars graph showing the percentage of CD11bhigh+ Gr1high+ cells (R1) in blood pre-challenge ( 2 days from PBS/LPS challenge) and post-challenge (+3–4 days post PBS/ LPS challenge). Control groups were challenged intraperitoneally (i.p.) with PBS and experimental groups were i.p. challenged with LPS. Data are from five mice of two independent experiments. Data are shown as mean SEM. Data were analysed by one-way analysis of variance. **P < 001; ns: non-significant.. Discussion Recently, it has been shown that CO, produced by HO-1 activity, can inhibit the pro-inflammatory functions of TLR in innate cells.1–3,24,25,36 In DC4,24 and macrophages3,25 this gas impairs the pro-inflammatory function of TLR4/MD2, the surface receptor complex that recognizes lipopolysaccharide and triggers innate immunity.18,19 Either treatment of monocytes with CO or induction of HO-1 expression by haem homologues, such as CoPP, can impair the intracellular activation of the interferon regulatory factor 3, NF-jB and MAPK pathways.3,4,25 The ablation of these routes renders monocytes unable to secrete inflammatory cytokines. Hence, it has been suggested that CO increases the resistance of these cells to an endotoxin challenge, as described before for both TLR4 knockout cells18,19 and cells blocked with an anti-TLR4/MD2 antibody.21 However, whether this increased resistance to endotoxin challenge is due to a decreased expression of the TLR4/MD2 surface complex had not been addressed before. In this study, we have shown that either HO-1 induction or CORM2 treatment decreases the surface expression of the TLR4/MD2 complex in both myeloid DC and neutrophils. In vitro experiments revealed that significant removal of this complex from the surface of DC takes place as soon as 20 min after CO treatment. Further, either HO-1 induction or CORM2 treatment reduced DC sensitivity down to 50%, as shown by three different experiments. These data support the notion that CO (produced by HO-1) renders DC unable to respond to endotoxin challenge by decreasing the surface expression of TLR4/MD2 complex. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332. Interestingly, reduced detection of the TLR4/MD2 complex by the MTS510 antibody was not associated with reduced expression of individual TLR4 or MD2 on the surface of DC. As MTS510 antibody recognizes the native structure of the receptor before LPS binding,37 it is likely that CO could affect the interaction between these two proteins on the DC surface. Supporting this notion is the observation that TLR4 and MD2 are transported to the cell surface upon the LPS stimulus only after TLR4 glycosylation in an MD2-dependent manner.38 Our data on DC suggest that CO impairs the interaction between TLR4 and MD2, so decreasing the TLR4/MD2 plasma membrane turnover and depleting this complex from the cell surface. It has been reported that binding of LPS to TLR4/MD2 hides the epitope recognized by the MTS510 antibody in TLR4.37 It is because the complex suffers a conformational change. We corroborated these data because LPS reduced the signal provided by MTS510 without altering both TLR4 and MD2 surface expression at 4 hr (Fig. 1c–e and Fig. S3k). Interestingly, CO did not modify the levels of TLR4/MD2 on LPS-primed DC at 4 hr post treatment (Fig. 1c,f). Altogether, our result suggests that HO-1 induction and CO production cause a conformational change in the TLR4/MD2 complex leading it to lose its native geometry, which naturally enables the recognition of LPS. An alternative explanation is that CO destabilizes the interaction between both proteins on the DC surface. This could cause low affinity of the complex for LPS, being the cause for impaired triggering of inflammation. Further research is required to determine whether the trafficking and turnover of the endotoxin receptor in DC differs from previous studies in other cell types, such as macrophages. 329.
(10) S. A. Riquelme et al. Consistent with our findings, it was recently reported that biliverdin, another HO-1 by-product, mediates the reduction of TLR4/MD2 surface expression in macrophages in a nitric oxide (NO) -dependent manner.39 Interestingly, NO and CO have been shown to promote equivalent physiological responses, such as vasodilatation and anti-inflammation.40,41 Because CO and NO are similar in structure, it is possible that they could exert equal effects on immune cells. Consistent with this notion is the observation that CO and NO can modulate the activity of soluble guanylate cyclase,42 an enzyme with antiinflammatory activity in human DC that suppresses their response to LPS.43 Nevertheless, it is also possible that CO and NO could be targeting independent molecules on myeloid cells that reduce their inflammatory capacity. Although further research would be required to elucidate these questions, the similarities and potential synergy between CO and NO could be beneficial to reduce tissue damage by inducing an anti-inflammatory mechanism in myeloid cells. Recently, it has been shown that in macrophages, LPS induces the recruitment of TLR4 and MD2 from intracellular compartments to lipid rafts located on the cell surface, which favours the interaction with adaptor molecules and triggers intracellular signalling pathways in response to endotoxin.44 It was also shown that CO impaired the increment of TLR4 surface expression due to LPS treatment.44 Consistently with this notion, COtreated macrophages show an increased interaction between TLR4 and Caveolin-1 in caveolae domains, which sequesters TLR4 and suppresses the response to LPS, even at 16 hr post endotoxin treatment.45 Our data suggest that the effect of CO might differ between macrophages and DC, as in these latter cells LPS causes no increase of TLR4 surface expression even at 4 and 21 hr post-treatment. However, on DC CO reduced the surface expression of the TLR4/MD2 complex, leading to unaltered expression of individual TLR4 and MD2 molecules. Furthermore, CO did not promote significant expression changes in TLR4/MD2 when cells were primed with LPS. However, it is also possible that alterations in the expression of TLR4/MD2 induced by CO could be masked by the LPS-driven conformational changes of the receptor, which could prevent recognition by the MTS510 antibody. Endotoxin-mediated septic shock is an acute inflammatory condition that renders the host unable to control body temperature and is associated with exacerbated activation of the innate immune response after pathogen-associated molecular pattern recognition. In the context of Gram-negative sepsis, the TLR4/MD2 complex mediates both the priming and mobilization of DC and neutrophils into the blood and other tissues. Activated myeloid cells release massive amounts of inflammatory cytokines causing endorgan damage and death.15,26,28,46,47 In only 1 year in the USA, the proportion of patients suffering sepsis reaches 330. around 12% of total ICU patients, with a total cost of $167 billion ($22 100/treatment/patient).48 Similar epidemiological data have been reported in Europe and Latin America,49–51 indicating that endotoxic shock can be a major public health problem. Hence, because TLR4/MD2 is required for the development of sepsis,14,18–20 the identification of molecular strategies to modulate the expression of this receptor on the surface of DC and neutrophils is an important biomedical goal. Here we have shown that CO reduces sepsis susceptibility in mice by down-modulating TLR4/MD2 expression on myeloid immune cells. Our in vivo experiments showed that administration of CO-releasing molecules decreased the TLR4/MD2 expression on the surface of both DC and neutrophils. Further, CO treatment prevented the increment in the blood of these two immune cell types, suggesting that the lack of TLR4/MD2 expression reduced their sensitivity to endotoxin challenge. These data are in agreement with the observation that TLR4 knockout monocytes show a null response to LPS with significantly reduced secretion of pro-inflammatory cytokines, such as IL-6, tumour necrosis factor-a and IL-12.14,18,19,26,46 Lack of or reduced TLR4 expression prevents progression from sepsis to severe sepsis and the occurrence of septic shock. Here we show that protection against endotoxic shock can be accomplished by a CO-induced reduction in TLR4/MD2 expression on the surface of myeloid immune cells. Further, CO protected mice from hypothermia and mobility loss that, in septic patients, is a clear sign of end-organ damage and a high risk for death.30,31,35 Notably, CO failed to protect from septic shock those mice that had been previously treated with LPS (CO post-challenge). Because LPS promotes rapid changes in the TLR4/MD2 structure triggering intracellular inflammatory pathways, it is possible that CO is not able to compete with these conformational molecular changes. Furthermore, the modification of TLR4/MD2 by LPS is stronger than that produced by the concentrations of CO used in this study. This notion is supported by our in vitro data. In addition, in vivo administration of LPS could be modifying other pro-septic components, such as different immune cell types that cannot be counteracted by CO. In addition, the use of increasing CO concentrations can compromise blood pressure in hypothermic mice.2,41 Hence, in vivo CO-mediated TLR4/MD2 reduction and priming impairment of innate immunity could be relevant to prevent sepsis and endotoxic shock in susceptible patients or in local tissues where DC will promote adaptive immunity in a TLR4/MD2-dependent manner.52 In the context of innate cells that support exacerbated inflammation during sepsis, DC have been shown to be the main producers of soluble mediators. Along these lines, it has been reported that mice with reduced circulating fractions of DC are resistant to lethal LPS challenge.14,15,27,36 Our data suggest that reduced TLR4/MD2 ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332.
(11) CO reduces TLR4/MD2 expression on DCs expression on the surface of either CO-treated or HO1-induced DC was associated with a decreased secretion of IL-12, a pro-inflammatory cytokine. In contrast, CO or HO-1 activity did not alter secretion of the antiinflammatory cytokine IL-10.1,4,24 These results suggest an increased IL-10/IL-12 ratio as a possible explanation for the induction of an endotoxin-resistant state by CO. In addition to DC, blood neutrophils are increased during endotoxin-mediated sepsis.26 These cells can migrate to different organs by extravasation and lead to an excessive degranulation of several molecules that cause tissue damage.26,28,53–55 Here we have shown that CO prevented the increment of peripheral neutrophils during LPS challenge. Our data suggest that the endotoxin-resistant profile induced by CO on neutrophils was the result of a decreased surface expression of the TLR4/MD2. However, during LPS-driven sepsis it has been described that neutrophils are recruited from bone marrow, causing increased numbers and survival of these cells in the blood.26,54 It is likely that, by reducing TLR4/MD2 expression, CO could also interfere with the process by which LPS increases the viability of these cells. As a result of CO exposure, neutrophils would not accumulate in the blood nor interact with LPS-primed endothelial cells, which would reduce extravasation into organs and tissue damage.26 In summary, we have found that HO-1 induction/CO treatment modified the interaction between TLR4 and MD2 and can be considered as a new approach to protect from shock by reducing the expression of endotoxin receptors on critical cells of the innate immune system. Such a novel therapeutic strategy could contribute to preventing acute host stress, hypothermia, reduced mobility, weight loss, exacerbated inflammation, organ dysfunction and death in patients with clinical conditions associated with sepsis due to ailments such as appendicitis, intestinal perforation and acute peritonitis.. Acknowledgements We are grateful to Dr Marıa Olga Bargsted for her support in animal care and manuscript proofreading. Also, we are grateful to Kristianne Galpin for manuscript proofreading. This work was supported by funding from the Millennium Institute on Immunology and Immunotherapy from Chile (P09/016-F for AMK and SB), La Region Pays de la Loire through the ‘Chaire d’excellence programme’ for AMK and Grant ‘Nouvelles Equipes-nouvelles thematiques’ (to AMK and SMB), INSERM CDD grant, the ECOS France-Chile grant, FONDECYT no 1070352, FONDECYT no 1050979, FONDECYT no 1040349, FONDECYT no 1100926, FONDECYT no 1110397, FONDECYT no 1140010, FONDECYT no 1110604 and Biomedical Research Consortium CTU06. SAR is a CONICYT-Chile fellow. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332. Disclosures The authors declare no conflict of interest.. References 1 Blancou P, Tardif V, Simon T, Remy S, Carreno L, Kalergis A, Anegon I. Immunoregulatory properties of heme oxygenase-1. Methods Mol Biol 2011; 677:247–68. 2 Motterlini R, Haas B, Foresti R. Emerging concepts on the anti-inflammatory actions of carbon monoxide-releasing molecules (CO-RMs). 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Supporting Information. Otterbein LE. Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase. Proc Natl Acad Sci USA 2011; 108:18849–54. 40 Xue L, Farrugia G, Miller SM, Ferris CD, Snyder SH, Szurszewski JH. Carbon monoxide and nitric oxide as coneurotransmitters in the enteric nervous system: evidence from genomic deletion of biosynthetic enzymes. Proc Natl Acad Sci USA 2000; 97:1851–5. 41 Motterlini R, Clark JE, Foresti R, Sarathchandra P, Mann BE, Green CJ. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ Res 2002; 90:E17–24. 42 Sharma VS, Magde D. Activation of soluble guanylate cyclase by carbon monoxide and nitric oxide: a mechanistic model. Methods 1999; 19:494–505. 43 Tsai IF, Lin CY, Huang CT, Lin YC, Yang CM, Liao CH. 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Figure S4. Carbon monoxide treatment after lipopolysaccharide challenge did not protect mice from ongoing sepsis. Figure S5. Carbon monoxide administration does not modulate innate cells in spleen during lipopolysaccharidemediated sepsis. Video S1. Mice treated with carbon monoxide are resistant to endotoxin shock.. with caveolin-1. J Immunol 2009; 182:3809–18.. 332. ª 2014 John Wiley & Sons Ltd, Immunology, 144, 321–332.
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