IMMUNOLOGY
ORIGINAL ARTICLE
Carbon monoxide exposure improves immune function in lupus-prone mice Juan P. Mackern-Oberti,1 Carolina Llanos,1,2 Leandro J. Carre~ no,1 Sebastian A. Riquelme,1 Sergio H. Jacobelli,1,2 Ignacio Anegon3 and Alexis M. Kalergis1,2,3 1 Millennium Institute on Immunology and Immunotherapy, Pontificia Universidad Catolica de Chile, 2Departamento de Inmunologıa Clınica y Reumatologıa, Facultad de Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile, and 3UMR 1064-Centre for research in Transplantation and Immunology, INSERM, Nantes, France
doi:10.1111/imm.12124 Received 26 February 2013; revised 23 April 2013; accepted 16 May 2013. Correspondence: Dr Alexis M. Kalergis, Millennium Institute on Immunology and Immunotherapy, Departamento de Genetica Molecular y Microbiologıa, Facultad de Ciencias Biol ogicas, Departamento de Inmunologıa Clınica y Reumatologıa, Facultad de Medicina, Pontificia Universidad Cat olica de, Portugal #49, Santiago 8331010, Chile. Email:
[email protected] Senior author: Dr Alexis M. Kalergis
Summary Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple alterations affecting the normal function of immune cells, such as lymphocytes, dendritic cells (DCs) and monocytes. Although the understanding of autoimmunity has significantly increased, the breakthrough in effective therapies has been modest, making necessary the development of new therapeutic strategies. Here we propose that a new potential target for therapy is haem oxygenase-1 (HO-1), an enzyme that catalyses the degradation of the haem group into biliverdin, carbon monoxide (CO) and Fe2+. These products exhibit immunosuppressive and anti-inflammatory effects, which can contribute to improving tolerance during organ transplantation. Because HO-1 is highly expressed by immune cells involved in SLE pathogenesis, such as monocytes and DCs, we evaluated whether induction of HO-1 expression or the administration of CO could ameliorate disease in the FccRIIb knockout (KO) mouse model for SLE. We found that CO administration decreased the expansion of CD11b+ cells, prevented the decline of regulatory T cells and reduced anti-histone antibodies observed in untreated FccRIIb KO mice. Furthermore, CO-treated animals and HO-1 induction showed less kidney damage compared with untreated mice. These data suggest that HO-1 modulation and CO administration can ameliorate autoimmunity and prevent the lupus symptoms shown by FccRIIb KO mice, highlighting HO-1 as a potential new target for autoimmune therapy. Keywords: carbon monoxide; haem oxygenase-1; systemic lupus erythematosus.
Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nuclear antigens, such as dsDNA, nucleosomes and ribonucleoproteins.1 Inflammatory systemic compromise is thought to be the consequence of immune complex deposition, which leads to inflammation and tissue damage through the activation of monocytes, macrophages and complement.2–4 Symptoms of SLE can be hetero-
geneous, varying from mild disease to life-threatening kidney injury. Most components of the immune system contribute to SLE pathogenesis, including elements of both the innate and adaptive immune response.5–7 Monocytes contribute to the initiation and perpetuation of the autoimmune response and organ damage observed during SLE.8 In a similar way, the impaired clearance of apoptotic fragments by monocytes is thought to allow the priming of self-reactive lymphocytes by nuclear autoantigens.8–10 Furthermore, the number of
Abbreviations: APC, allophycocyanin; CO, carbon monoxide; CoPP, cobalt protoporphyrin; DCs, dendritic cells; GM-CSF, granulocyte–macrophage colony-stimulating factor; HO-1, haem oxygenase 1; IL-6, interleukin-6; PE, phycoerythrin; SLE, systemic lupus erythematosus; SnPP, tin protoporphyrin; TNF, tumour necrosis factor; Tregs, regulatory T cells; WT, wild-type ª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
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J. P. Mackern-Oberti et al. circulating monocytes expands in both people with SLE and in mice suffering from an SLE-like disorder. Expansion of monocytes is thought to be promoted by an unbalanced expression of activating/inhibitory Fcc receptors.11–15 Further, lupus nephritis has been associated with tissue infiltration by monocytes expressing an inflammatory phenotype.16,17 Hence, an unbalanced expression of activator/inhibitory molecules on monocytes and dendritic cells (DCs) could contribute to autoimmune inflammation during SLE. Although the understanding of autoimmunity has greatly increased, these new findings have not led to a significant breakthrough in the development of more efficient or safer therapies. Unfortunately, current available therapies for SLE are mainly based on unspecific immunosuppressive agents, such as corticoids.18 Haem oxygenase 1 (HO-1) is an enzyme that catalyses the degradation of the haem group into biliverdin, free iron and carbon monoxide (CO).19 Although enzymatic products derived from HO-1 activity, such as biliverdin and bilirubin, possess antioxidant and immunosuppressive activity,19 CO has been shown to down-modulate immune responses in a variety of pathogenic processes and it is thought to mediate most of the immunomodulatory effects attributed to HO-1.20–24 It remains to be determined whether the modulation of HO-1 activity could prevent the immune alterations that are characteristic of SLE. Only one report has shown that the administration of an HO-1 inducer reduces nephritis severity as well as anti-dsDNA antibody levels in the MRL-Faslpr mouse model of lupus.25 Importantly, HO-1-deficient mice, as well as individuals with HO-1 deficiency, display immunological alterations, such as chronic inflammation, that are thought to be mediated by myeloid immune cells.26,27 In this context, we have recently shown that patients with SLE display a decreased expression of HO-1 on peripheral blood monocytes, which might be related to SLE pathogenesis.28 These data suggest that HO-1 induction, as well as the anti-inflammatory capacity of CO as an HO-1 product, could be considered a potential therapy to improve SLE progression. Here, we have evaluated the effects of HO-1 induction and CO exposure in the onset and development of autoimmunity, using FccRIIb-deficient mice as an animal model for SLE. This strain of mice was chosen because an FccRIIb deficiency leads to increased susceptibility to myeloid cell activation in response to stimulation with immune complexes.15,29,30 Remarkably, we found that FccRIIb-deficient mice displayed a significant reduction in the expression of HO-1 in the spleen. In this organ these mice showed progressive immunological alterations such as the expansion of CD11b+ cells and the contraction of the CD4+ Foxp3+ cell [regulatory T (Treg) cell] population. When FccRIIbdeficient mice were exposed to CO, the expansion of CD11b+ cells was partially prevented and the frequency of 124
Treg cells was restored. This was not seen for CoPP-treated mice. In addition, CO treatment decreased anti-histone IgG levels in FccRIIb-deficient mice. Importantly, HO-1 induction and exposure to CO ameliorated the renal damage leading to proteinuria. Taken together, these data suggest that CO treatment, and to a lesser extent HO-1 induction, may contribute to preventing the immunological alterations observed in lupus mice. Our results support the notion that HO-1 activity could play a crucial role in immune system homeostasis by inducing pleiotropic antiinflammatory effects, which could be considered as a potential therapy target for SLE patients.
Materials and methods Antibodies and reagents FITC-conjugated anti-mouse HO-1 monoclonal antibody (clone HO-1-2) and anti-mouse HO-1 monoclonal antibody (clone HO-1-1) were purchased from Abcam (Cambridge, UK). Anti-mouse CD11c-phycoerythrin (PE)/FITC/allophycocyanin (APC) (clone HL3), antimouse CD11b-PE/FITC (clone M1/70), anti-mouse Gr1 (clone RB6-8C5), anti-mouse CD4-FITC/PE (clone GK1.5), anti-mouse CD4-Peridinin chlorophyll protein (PerCP) (clone H129.19), anti-mouse Foxp3-AlexaFluor 647, (clone MF3), anti-mouse CD25-APC, anti-mouse CD8-APC (clone 53-6.7), anti-mouse B220-FITC (clone RA3-6B2) and anti-mouse CD16/32-PE (clone 2.4G2) were all purchased from BD Biosciences (San Jose, CA). Anti-mouse CD25PE-Cy7 (clone PC61.5) was purchased from eBioscience (San Diego, CA). Anti-actin (clone C4) was purchased from Chemicon–Millipore (Billerica, MA).
Mice C57BL/6J, 129SJ and B6;129S4-Fcgr2btm1Ttk/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice used in this study were sex-matched and age-matched in all the experiments. Female and male mice were used. The FccRIIb / (Fcgr2b targeted mutagenesis) phenotype were confirmed by double staining with anti-mouse CD16/32-PE and anti-mouse B220-FITC. All mice were kept under specific pathogen-free conditions at the animal facility of the Pontificia Universidad Cat olica de Chile. All animal work was performed according to institutional guidelines and supervised by a veterinarian.
Assessment of urinary protein excretion Proteinuria was estimated by examining fresh urine with Combur Test sticks for urinalysis (Roche, Basel, Switzerland) using a scale of 0–3, where 0/trace = negative, 1 = 30 mg/dl, 2 = 100 mg/dl, and 3 = 500 mg/dl. ª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
Carbon monoxide reduces lupus symptoms Proteinuria scores > 2 were considered to represent moderate glomerulonephritis.
Assessment of antinuclear antibodies Antinuclear antibody levels in serum were determined with HEp-2 cells in 12-well slides (BioRad, Hercules, CA). HEp-2 cells were incubated for 30 min with 1 : 40 diluted serum derived from FccRIIb-deficient mice, followed by staining for 30 min with FITC-conjugated antimouse IgG (Invitrogen, Life Technologies, Carlsbad, CA).
CO exposure Mice were exposed to compressed CO at a concentration equal to 250 ppm for 2 hr per day from weeks 12–15 to week 52.23 A CO analyser was used to measure CO levels in the chambers and maintain a controlled CO concentration. Anti-coagulated cardiac blood samples (04 ml) were taken immediately after CO exposure and carboxyhaemoglobin levels were measured using a haemoximeter (ROCHE cobas b 221Blood gas system, Mannheim, Germany). Carboxyhaemoglobin levels were 23–27% in all experiments using CO gas.
Pharmacological modulation of HO-1 expression Either CoPP or tin protoporhyrin (SnPP) (Frontier Scientific, Logan, UT) was used either to induce HO-1 expression or to inhibit HO-1 activity, respectively. The CoPP and SnPP were dissolved in 01 M NaOH. Treatments consisted of a weekly intraperitoneal dose of CoPP (30 mg/ kg)31 or SnPP (35 mg/kg)32 from 12–15 weeks of age to 52 weeks.
Flow cytometry Spleen or inguinal lymph nodes from 1-year-old mice from different groups were harvested and minced in PBS supplemented with 10% fetal calf serum, until a homogeneous cell suspension was achieved. Cells were washed with PBS–1% BSA, re-suspended at 2 9 106 cells/ml (50 ll/tube) and incubated with FITC-, PE-, PerCP-, PE-Cy7- and APC-conjugated antibodies for 30 min at 4°. For HO-1 and Foxp3 intracellular staining, fixed cells were incubated with a FITC-conjugated anti-HO-1 or Alexa Fluor 647-conjugated anti-Foxp3 antibody in permeabilization buffer (PBS/BSA 3%–Saponin 05%) for 4 hr. Cells were washed with permeabilization buffer and analysed using a FACS Canto II flow cytometer (BD Biosciences).
Real-time RT-PCR RNA was extracted from spleens and kidneys using Trizol (Invitrogen), according to the manufacturer’s instrucª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
tions. RT-PCR and cDNA synthesis were performed using random primers (ImProm-II; Promega, Madison, WI). Real-time PCR were carried out using a StepOne plus thermal cycler (Applied Biosystems, Foster City, CA). Briefly, cDNAs amplified out of total RNA were tested for HO-1 amplification using the following primers: mouse HO-1forward 5′-CCTCTGACGAAGTGACGCC-3′ and reverse 5′-CAGCCCCACCAAGTTCAAA-3′. PCR amplification of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an internal control using the following primers: mouse HPRT forward 5′-ATCCAGCAGGTCAGCAAAGA-3′ and reverse 5′-CGTGATTAGCGATGATGAACC-3′. To corroborate amplification specificity, PCR products were subjected to a melting curve program. Abundance of HO-1 mRNA was determined by relative expression to HPRT by the 2 DDCt method.
ELISA Serum samples from different groups of mice were obtained during the treatment and total IgG antibodies against DNA (Invitrogen) and histones (Calbiochem, EMD Millipore, Bellerica, MA) were quantified by ELISA. Briefly, ELISA plates were coated overnight at 4° with 2 µg/ml DNA or 2 µg/ml histones in PBS, washed and then blocked with PBS/BSA 10% for 2 hr at room temperature. After washing three times, serum samples were diluted in PBS/BSA 1% starting at 1 : 100 and incubated for 2 hr at room temperature. IgG was detected with a goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Invitrogen). After washing three times, horseradish peroxidase substrate was added (3, 30, 5, 50tetramethylbenzidine; Sigma, St Louis, MO) and optical density (OD) at 450 nm was measured on a microplate reader.
Western blot Kidneys were harvested from CoPP 3 mg/kg-treated C57BL/6J mice after 48 hr. Kidney tissue was prepared for Western blot analysis as follows: 100 mg of tissue was lysed in radioimmunoprecipitation assay lysis buffer. The protein concentration of each sample was determined using the bicinchoninic acid assay (Thermo Scientific, Rockford, IL). Equal amounts of protein (20 µg) were loaded onto 12% SDS–polyacrylamide gels. After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (HybondC-Extra; Amersham Biosciences, Piscataway, NJ). Membranes were blocked with 4% non-fat dried milk in PBS for 2 hr and incubated with anti-mouse HO-1 mAb (clone HO-1-1) or anti-actin antibody (clone C4) in 2% non-fat dried milk in PBS for 12 hr at 4°. Immunoreactive bands were detected using horseradish peroxidase-linked antibody 125
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Statistics Data and statistical analyses were performed using PRISM 5 software (Graph Pad Software, Inc., San Diego, CA). For statistical analyses, analyses of variance and Student’s t-test were used. P-values < 005 were considered statistically significant. Flow cytometry data were analysed using FACS DIVA software (BD Biosciences).
Results HO-1 expression is decreased in the lupus mouse model It has been previously shown that an HO-1 deficiency in mice may promote an inflammatory state.27 Hence, we decided to evaluate the levels of HO-1 mRNA in the spleens of 12-month-old FccRIIb-deficient mice by RT-PCR. As shown in Fig. 1(a), HO-1 expression was significantly decreased in splenocytes from FccRIIb-deficient mice compared with wild-type (WT) animals (Fig. 1a). To better assess HO-1 expression in immune cells, splenocytes from FccRIIb-deficient and WT mice were stained with specific surface markers for monocytes, DCs and CD4+ T cells, in addition to anti-HO-1-specific antibodies, and analysed by FACS. Monocytes, CD4+ T cells and DCs from FccRIIb-deficient mice all showed decreased expression of HO-1 compared with WT mice (Fig. 1b–d). To evaluate 126
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To evaluate CoPP-driven HO-1 induction in murine myeloid cells (see Supplementary material, Fig. S5), we produced CD11c+ CD11b+ bone marrow-derived DCs by using granulocyte–macrophage colony-stimulating factor (GM-CSF) differentiation culture as previously described.33 DCs were seeded on coverslips at day 0 and at day 5 after differentiation, cells were treated either with vehicle or with 50 µM CoPP for 2 hr, then extensively washed and incubated for 16 hr at 37°. Then, cells were washed and fixed with 2% p-formaldehyde for 10 min at 4°. Next, cells were washed, permeabilized with 005% saponin-PBS at room temperature and coverslips were transferred to a cold chamber over a hydrophobic surface (paraffin-coated). Coverslips were treated with 50 µl of a 1/200 mouse antiHO-1 already dissolved in 005% saponin-PBS for 16 hr. Next, cells were extensively washed and stained with a 1/ 200 goat anti-mouse Alexa Fluor 488 for 3 hr at 4°. Cells were washed and mounted with DABCO for confocal microcopy analysis. HO-1-associated fluorescence intensity per cell was measured using IMAGEJ 1.47c software (NIH, Bethesda, MD).
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Figure 1. Expression of haem oxygenase 1 (HO-1) is reduced in FccRIIb-deficient mice. Single cell suspensions of splenocytes from FccRIIb-deficient and C57BL/6 wild type (WT) mice at 12 months were obtained. (a) Total mRNA was purified and HO-1 mRNA levels were evaluated by real-time PCR. *P < 005 by unpaired t-test. The data are presented as relative expression to HPRT by 2 DDCt mean SEM of three independent experiments with nmice/ group = 5. (b–d). Spleen cell suspensions from FccRIIb-deficient and WT mice were stained with specific anti-CD11c phycoerythrin (PE), -CD11b PE and -CD4 peridinin chlorophyll protein monoclonal antibodies, fixed, permeabilized, stained with an anti-HO-1 FITC monoclonal antibody, and evaluated by FACS. The data are presented as HO-1 mean fluorescence intensity (MFI) Geo mean expression ratio FccRIIb-deficient/WT mice of CD11c+ cells (b), CD11b+ cells (c) and CD4+ cells (d). FccRIIb / (FccRIIb-deficient). *P < 005, **P < 001 by unpaired t-test.
HO-1 levels in non-lymphoid tissues, we measured HO-1 expression in kidneys and lungs by real-time PCR. In contrast, in other tissues where HO-1 is abundant, such as the kidneys, or is scarce, such as the lung, it showed similar levels of HO-1 mRNA expression in FccRIIb-deficient mice compared with WT mice (data not shown). These results suggest that HO-1 metabolism is mainly affected in immune cells from FccRIIb-deficient mice.
CO reduces the expansion of CD11b+ cells in the spleen of lupus mice Because monocytes and granulocytes play a relevant role in the pathogenesis of several autoimmune diseases by infiltrating tissues during inflammation, we evaluated the presence of CD11b+ cells during the progression of auª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
Carbon monoxide reduces lupus symptoms toimmunity in FccRIIb-deficient mice. Interestingly, we found an age-dependent expansion of CD11b+ cells in the spleens of FccRIIb-deficient mice (Fig. 2a,b), which is consistent with the development of autoimmunity as indicated by the emergence of circulating anti-nuclear antibodies (see Supplementary material, Fig. S1). No major changes were seen for CD4+, CD8+, CD11c+ and B220+ cells from FccRIIb-deficient mice compared with C57BL/6 or C57BL/6 9 129S WT mice (see Supplementary material, Fig. S2a–e). Next, whether exposure to CO could ameliorate the increase in CD11b+cells in FccRIIb-
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deficient mice was evaluated. Accordingly, mice were exposed to 250 ppm CO for 2 hr daily for 9 months. To corroborate the efficacy of CO treatment, carboxyhaemoglobin levels were determined in peripheral blood. CO treatment resulted in 23–27% saturation of carboxyhaemoglobin after 2 hr of exposure (see Supplementary material, Fig. S3). Remarkably, CO treatment decreased the expansion of spleen CD11b+ cells in 1-year-old mice (Fig. 2d,e). The effect of CO treatment in this CD11b+ population was mainly observed in CD11b+ Gr1+ cells (Fig. 2f,h). However, although not statistically significant,
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Figure 2. Study of CD11b+ cells in spleen from FccRIIb-deficient mice. Spleen from FccRIIb / , C57129S wild-type and C57BL/6 wild-type (WT) mice (n = 5/group) from 4 to 12 months-old were harvested and minced in PBS until a homogeneous cell suspensions were reached, then red blood cells were removed using hypotonic lysis buffer. (a) Spleen cell suspension from FccRIIb-deficient (4–12 months), C57129S (12 months) and C57BL/6 (4–12 months) mice were stained with specific anti-CD11b phycoerythrin-conjugated monoclonal antibodies and evaluated by FACS. The graph represents the percentage of CD11b+ cells in spleens for three independent experiments. nmice/group = 6. *P < 005, ***P < 0001 by one-way analysis of variance test. (b) Total cell counts from spleen cell suspension from FccRIIb-deficient (12 months) and C57BL/6 (12 months) mice were measured by cell counting with Turk solution in a Neubauer chamber. The data are presented as cells 9 106per spleen. *P < 005 by unpaired t-test. (c) Messenger RNA was purified from spleen cell suspension of cobalt protoporphyrin (CoPP) -treated FccRIIb-deficient and untreated FccRIIb-deficient mice (12 months), haem oxygenase 1 (HO-1) mRNA levels were evaluated by real-time PCR (CoPP administration from 12 to 52 week of age). *P < 005 by unpaired t-test. nmice/group = 5. (d–g) Spleen cell suspension from untreated and +CO groups of C57BL/6 (WT), and untreated, +CO, vehicle (intraperitoneal PBS, 500 µl), +CoPP (intraperitoneal 30 mg/kg in PBS, 500 µl), + tin protoporphyrin (SnPP) (intraperitoneal 35 mg/kg in PBS, 500 µl) of FccRIIb-deficient mice were stained with specific antiCD11b-phycoerythrin and anti-Gr1-allophycocyanin monoclonal antibodies and evaluated by FACS. All mice were killed at 12 months-old. Graphs represent the percentage (d) and total cell count (e) of CD11b+ cells, and total cell count of CD11b+ Gr1+ (f) and CD11b+ Gr1 (g) cells in spleens for two independent experiments. nmice/group = 7–8. ***P < 0001, **P < 001 and *P < 005 by unpaired t-test, for untreated WT versus untreated FccRIIb-deficient mice; untreated WT versus +CO WT; and untreated FccRIIb-deficient mice untreated versus +CO FccRIIbdeficient mice. (h) Representative dot plots of CD11b and Gr1 expression in spleens of untreated and CO-treated groups of C57BL/6 (WT) and FccRIIb-deficient mice of 12 months.
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J. P. Mackern-Oberti et al. CO treatment also caused a decrease in the CD11b+ Gr1 population in FccRIIb-deficient mice when compared with untreated controls (Fig. 2g,h). Because HO-1 induction leads to CO production in cells, we evaluated whether CO effects could also be observed after treating 3-month-old FccRIIb-deficient mice for 9 months with either CoPP (3 mg/kg) or SnPP (35 mg/kg); an HO-1 inductor and an HO-1 inhibitor, respectively. HO-1 induction in spleen, kidney and DCs was evaluated using RT-PCR (Fig. 2c), Western blot and confocal microscopy (see Supplementary material, Fig. S5). Unlike CO treatment, HO-1 induction by CoPP had no effect on the total number of splenic CD11b+ cells (Fig. 2d,e). As in CoPP-treated mice, FccRIIb-deficient animals that received SnPP showed no changes in
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It has been reported that Treg cells are decreased in peripheral blood from patients with SLE and that they can show different alterations in their phenotype.34–36 Accordingly, the frequency of Treg cells in the spleens of FccRIIbdeficient mice was determined by using flow cytometry. As shown in Fig. 3, an age-dependent reduction of CD4+ Foxp3+ cells was observed for FccRIIb-deficient mice, which was not seen in WT mice (Fig. 3a,b). Frequency of Treg cells was also decreased in lymphatic
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Figure 3. Study of FOXP3+ cells in spleen from FccRIIb-deficient mice. (a) Spleen cell suspension from FccRIIb-deficient (4–12 months), C57129S (12 months) and C57BL/6 (4–12 months) were stained with specific anti-CD4 FITC, fixed, permeabilized and stained with FOXP3 Alexa 647 monoclonal antibodies and evaluated by FACS. nmice/group = 6. *P < 005, ***P < 0001 by one-way analysis of variance test. (b) Spleen cell suspensions from FccRIIb-deficient (12 months) and C57BL/6 (12 months) mice were stained with anti-CD4 FITC, anti-CD25 phycoerythrin-Cy7, fixed, permeabilized and stained with FOXP3 Alexa 647 monoclonal antibodies and evaluated by FACS. nmice/group = 6. (c) Inguinal lymphatic nodes from FccRIIb-deficient (12 months) and C57BL/6 (12 months) mice were harvested and minced until a cell suspension was reached. Then cells were stained with anti-CD4 FITC and anti-CD25 PE, fixed, permeabilized and incubated with anti-FOXP3 Alexa Fluor 647. (d) Messenger RNA was purified from spleen cell suspensions of wild-type (WT) and FccRIIb-deficient mice (12 months), FOXP3 mRNA levels were evaluated by real-time PCR. *P < 005 by unpaired t-test. (e) Spleen cell suspensions from FccRIIb-deficient (12 months) and C57BL/6 (12 months) mice were stained with anti-CD4 FITC, anti-CD25 phycoerythrin-Cy7 and analysed by FACS. (f–h) Spleen cell suspension from untreated and +CO groups of C57BL/6 (WT), and untreated, +CO, vehicle (intraperitoneal PBS, 500 µl), + cobalt protoporphyrin (CoPP) (intraperitoneal 30 mg/kg in PBS, 500 µl), + tin protoporphyrin (SnPP) (intraperitoneal 35 mg/kg in PBS, 500 µl) of FccRIIb-deficient mice were stained with anti-CD4 FITC, fixed, permeabilized and stained with FOXP3 Alexa Fluor 647 monoclonal antibodies and evaluated by FACS. Graphs represent the percentage of CD4+ FOXP3+ (CD4 gated) cells (f) and the total cell count (h) of CD4+ FOXP3+ cells. nmice/group = 7–8. ***P < 0001 and *P < 005 by unpaired t-test, untreated WT versus untreated FccRIIb-deficient mice; untreated WT versus +CO WT; untreated FccRIIb-deficient mice untreated versus +CO FccRIIb-deficient mice. (g) Representative dot plots of CD4 and FOXP3 expression in spleens of untreated and treated CO groups of C57BL/6 (WT) and FccRIIb-deficient mice of 12 months.
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Carbon monoxide reduces lupus symptoms
HO-1 induction and CO exposure ameliorate renal damage of lupus mice To evaluate the therapeutic effect of HO-1 in renal manifestations, mice were treated with either CoPP or CO from week 12 to week 52 of age. The urinary protein levels were determined using reactive strips. As shown in Fig. 5, this weekly CoPP treatment or CO exposure delayed the onset of proteinuria and reduced the incidence of proteinuria at the end of the study when compared with untreated FccRIIb-deficient mice (Fig. 5a). To determine whether CoPP treatment could induce HO-1 in kidney and so promote its effects locally, we quantified HO-1 mRNA levels in the kidney. As shown in Fig. 5, HO-1 was induced in the kidney of CoPP-treated mice, indicating that protection may be mediated locally ª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
DO (IgG anti histone 1/800)
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The pathogenesis of SLE is intimately linked to the activation of auto-reactive B cells and the production of nephritogenic autoantibodies against dsDNA.37 Although FccRIIb-deficient mice in the B6;129S4 genetic background show low levels of antinuclear antibodies, they develop mild renal symptoms. When IgG autoantibody levels were determined by ELISA, CO-treated animals exhibited decreased anti-histone but not anti-DNA autoantibody levels compared with untreated mice (Fig. 4a,b). CoPP treatment did not alter anti-histone or anti-DNA antibodies. On the contrary, in SnPP-treated mice antihistone and anti-DNA antibody levels were both increased compared with control mice treated with vehicle. These findings suggest that CO treatment may ameliorate lupus renal damage by decreasing autoantibody levels. On the other hand, HO-1 inhibition by SnPP, as observed in HO-1 deficiency, may promote inflammation leading to autoantibody production and auoimmunity.27
IgG anti histone
1·5
0·0
DO (IgG anti DNA 1/800)
CO decreases autoantibody levels in lupus mice
(a)
U
nodes at 12 months in FccRIIb-deficient mice compared with WT animals (Fig. 3c). Furthermore, Foxp3 mRNA expression was also decreased in the spleens of FccRIIbdeficient mice compared with WT mice (Fig. 3d). On the contrary, no significant differences were observed between FccRIIb-deficient and WT mice when the frequency of CD4+ CD25+ cells was determined in spleens (Fig. 3e). Whether CO, CoPP or SnPP treatment could restore CD4+ Foxp3+ cell numbers in the spleens of FccRIIbdeficient mice was evaluated. As shown in Fig. 3, CO treatment indicated that the total number of CD4+ Foxp3+cells in FccRIIb-deficient mice remained equivalent to that in WT mice (Fig. 3f–h). However, FccRIIb-deficient animals treated with CoPP and SnPP showed no restoration of CD4+ Foxp3+ cells. Hence, CO exposure but not CoPP administration could prevent the contraction of the Foxp3+ cell population.
FcγRIIb–/– 12 m
Figure 4. Therapeutic effects of CO and cobalt protoporphyrin (CoPP) treatment on autoantibody production in FccRIIb-deficient mice. SerumIgG anti-dsDNA and IgG anti-histone antibody levels in wild-type (WT) and FccRIIb-deficient mice treated with CO, vehicle, CoPP, tin protoporphyrin (SnPP) and untreated were determined by ELISA. (a) SerumIgG anti-histone antibody levels at 12 months of age from all groups studied. nmice/group = 7–8. (b) Serum IgG antiDNA antibody levels at 12 months of age from all groups studied. nmice/group = 7–8. ***P < 0001 and *P < 005 by unpaired t-test, untreated WT versus untreated FccRIIb-deficient mice; untreated versus +CO FccRIIb-deficient mice; vehicle versus + tin protoporphyrin (SnPP) FccRIIb-deficient mice. The data shown are the mean SEM.
(Fig. 5b). Therapeutic effects of CoPP may be related to a local decrease in inflammatory mediators, reactive oxygen species or cytoprotection. These results support the notion that HO-1 modulation could be exploited as a potential therapeutic target for lupus nephritis.
Discussion Our results show that HO-1 expression is significantly reduced in the spleen of FccRIIb-deficient mice compared with WT mice. These differences are specific for lymphoid tissue because no significant differences were found in kidneys or lungs. We also found immunological alterations, such as expansion of CD11b+ cells and the contraction of 129
J. P. Mackern-Oberti et al. (a)
Proteinuria
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(b) 6 HO-1 mRNA (relative expression)
*** 4
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oP C +
Ve
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Figure 5. Therapeutic effects of CO and cobalt protoporphyrin (CoPP) treatment on proteinuria in FccRIIb-deficient mice. Presence of urinary protein excretion was evaluated throughout the study (from 10 to 52 weeks) by examining fresh urine with Combur Test sticks for urinalysis (Roche). Urinary protein above 100 mg/dl was considered as proteinuria. (a) Presence of proteinuria in FccRIIbdeficient mice treated with ♦ CO, ▲ CoPP or ■ untreated over time. nmice/group = 7–8. The data shown are the Kaplan–Meier survival fractions profile. (b) Messenger RNA was purified from kidney from untreated and +CoPP FccRIIb-deficient mice at 12 months of age. Messenger RNA haem oxygenase 1 (HO-1) levels were evaluated by real-time PCR. *P < 005 by unpaired t-test. The data are presented as relative expression of HO-1 mRNA to HPRT by 2 DDCt method. Mean SEM of two independent experiments with nmice/ group = 4.
CD4+ Foxp3+ cells. To our knowledge this study is the first to report that CO exposure reduces immunological alterations seen in FccRIIb-deficient mice, including prevention of CD11b+ cell expansion and the depletion of Treg cells. Furthermore, treatment with CO decreased histone-specific autoantibodies in FccRIIb-deficient animals. Although it has been reported that during acute inflammatory states there is an increase in HO-1 expression that may lead to an anti-inflammatory response, there are no reports evaluating HO-1 expression in murine models for lupus.38 Along these lines, we have previously reported for the first time that peripheral blood CD14+ cells from SLE patients showed a reduced expression of HO-1 on peripheral blood monocytes.28 Here, 130
FccRIIb-deficient mice (12 months old) showed a decreased HO-1 expression in spleen. Defective expression of FccRIIb on immune cells may promote deregulated chronic activation via stimulatory FccRs in response to immune complex unbalancing HO-1 homeostasis. Hence, FccRIIb deficiency may affect HO-1 expression, so contributing to autoimmunity. The fact that HO-1 deficiency leads to chronic inflammation but not autoimmunity strongly suggests that an HO-1 deficiency is unlikely to directly cause autoimmunity.26,27 Further research is required to elucidate whether HO-1 deregulation may lead to an inflammatory condition or an exacerbated inflammatory response that could in turn contribute to the development of autoimmunity in susceptible hosts. This latter notion is supported by our recent observation that monocytes derived from SLE patients showed reduced HO-1 expression.28 As mentioned above, this is the first report showing that CO exposure modulates spleen CD11b+ cell expansion and Treg cell contraction. Expansion of CD11b+ cells, seen in the spleens of FccRIIb-deficient mice, is most likely a result of the lack of FccRIIb expression, which could lead to monocyte/granulocyte activation in response to immune complexes. In addition, this activation could promote a deregulated cytokine production, including GM-CSF, interleukin-6 (IL-6) and tumour necrosis factor-a (TNFa), resulting in the subsequent inhibition of HO-1 expression.39–41 We found that CO exposure could restore the number of monocyte/granulocyte cells in the spleens of FccRIIb-deficient mice to levels equivalent to WT mice. The effect of CO could be exerted by inhibiting signalling pathways triggered during FccRIIb deficiency as it has been reported that CO could modulate p38 mitogen-activated protein kinase, mitogen-activated protein kinase kinase 3 and caspase 3 signalling.42 The nature of expanded splenic CD11b+ Gr1+ and CD11b+ Gr1 cell populations remains to be determined as several myeloid sub-populations have been described, including inflammatory monocytes, granulocytes and suppressor myeloid cells.43,44 However, it is remarkable that CO exposure could both limit the expansion of CD11b+ subpopulations to normal levels as well as maintaining Treg cell numbers. Furthermore, the mechanism responsible for regulatory CD4+ Foxp3+ T-cell reduction in FccRIIb-deficient mice has not yet been determined. One explanation may be that pro-inflammatory cytokine production by expanded CD11b+ cells may affect regulatory T-cell population homeostasis and survival. Based on recent reports, it is probable that monocytes may act as an important source of pro-inflammatory cytokines such as IL-6 and TNF-a, and that these cytokines, mainly IL-6, may affect Treg cell development and function.42,45,46 Moreover, it has been recently reported that CO can suppress the secretion of pro-inflammatory cytokines without altering IL-10 production by myeloid DCs.47 Hence, it is likely that by ª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
Carbon monoxide reduces lupus symptoms promoting IL-10 production, CO exposure would prevent monocyte expansion and activation, reducing the secretion of pro-inflammatory cytokines, such as IL-6, and sustaining the numbers of regulatory T cells.47 On the contrary, the effect of CO in the CD11b+ or CD4+ Foxp3+ cell populations could not be reproduced by CoPP treatment. This lack of response in CoPP-treated mice could have been because of differences in final CO concentrations in lymphoid tissues between our two approaches. As can be seen in the Supplementary material, Fig. S4, CoPP treatment failed to induce enough CO to be measured by blood carboxyhaemoglobin. It remains to be elucidated whether physiological CO levels derived from haem group degradation by HO-1 could play a relevant role in immune cell suppression. Another possible explanation for the reduced effect of CoPP on immune cells compared with CO therapy could be the occurrence of secondary side effects due to chronic HO-1 induction. The subsequent increase of haem degradation products after HO-1 induction may be harmful to cells, limiting the beneficial immunosuppressive effect of CoPP-induced CO release.48,49 Decreased IgG anti-histone levels and normal regulatory T-cell numbers following CO exposure may be secondarily associated with CD11b+ cell modulation. When the number of CD11b+ cells was decreased by CO, proinflammatory mediators may also be decreased, limiting autoantibody production and the contraction of the Treg cell population. The improvement of renal status in COtreated mice may be associated with a decreased level of autoantibodies, as immune complex deposition in the kidney, which activates local monocyte populations. Consistent with this notion, it has been observed that hemin treatment prevented glomerulonephritis development in MRL lupus mice without modifying the total cell number in spleens.25 Similarly, we found that CoPP treatment ameliorates renal symptoms (urinary protein secretion) but failed to prevent CD11b+ cell expansion or Treg cell contraction. However, we could not see any reduction in anti-DNA or anti-histone antibodies in response to CoPP treatment, which indicates that the HO-1 modulation effect on proteinuria may be independent of a reduction in self-reactive IgG. Consistent with the importance of HO-1 for the modulation of the inflammatory response during lupus, inhibition of HO-1 by SnPP led to increased levels of anti-DNA and histone autoantibodies in FccRIIb-deficient mice. As lupus nephritis is known to be partially mediated by nitric oxide production, it is possible that the underlying mechanism of CoPP in renal protection observed in our study could be associated with inhibition of local nitric oxide synthase in kidneys, whereas CO therapy would be mediated mainly by modulation of immune cells.25,50 It has been recently reported that biliverdin and bilirubin are able to protect mice from developing diabetic nephropathy by reducing NAD(P)Hdependent reactive oxygen species production. It is thereª 2013 John Wiley & Sons Ltd, Immunology, 140, 123–132
fore possible that the local effect of HO-1 in the kidney in terms of protection from damage, could be mediated by an equivalent mechanism.51 To our knowledge, these are the first results characterizing the anti-inflammatory properties of CO in FccRIIbdeficient mice suffering from a lupus-like syndrome. Our results suggest that an impaired immune cell function can be the result of reduced HO-1 expression in FccRIIb-deficient cells, resulting in a bias to deregulated inflammation and autoimmunity. The anti-inflammatory effects of CO could be the result of a blockade of the expansion of immune cells, such as monocytes, which would in turn reduce inflammation. It remains to be determined whether CO exposure mediates anti-inflammatory effects in lupus through inhibition of production of pro-inflammatory cytokines such as IL-6, TNF-a or GM-CSF. Nonetheless, the data shown here suggest that HO-1 could work as a potent regulator of systemic homeostasis through pleiotropic mechanisms and that the understanding of these pathways is of therapeutic relevance in autoimmunity and inflammation research.
Acknowledgements This work was supported by grants FONDECYT no 1070 352, FONDECYT no 1050979, FONDECYT no 1040349, FONDECYT no 1100926, FONDECYT no 1110397, FONDECYT no 1100971, FONDECYT no 1110604, Proyecto de Inserci on de Capital Humano Avanzado en la Academia 2011 No 791100015, Vicerrectorıa de Investigacio´n PUC Posdoctorado no 4/2010 and Millennium Institute on Immunology and Immunotherapy (P09-016-F). LJC is a Pew Latin American Fellow. We would like to thank Camila Schmidt for her skilful technical assistance. AMK is a Chaire De La Region Pays De La Loire De Chercheur Etranger D’excellence and the collaboration between AMK and IA is supported by a Grant ‘Nouvelles Equipes-nouvelles thematiques’ from the La Region Pays De La Loire and by an INSERM CDD grant.
Disclosures The authors declare that they have no financial or commercial conflict of interest.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Study of antinuclear antibodies in FccRIIbdeficient mice. Figure S2. Study of immune cells in spleen from FccRIIb-deficient mice. Figure S3. Blood carboxyhaemoglobin level after CO exposure. Figure S4. Blood carboxyhaemoglobin level after cobalt protoporphyrin (CoPP) treatment and CO exposure. Figure S5. Haem oxygenase 1 (HO-1) protein induction by cobalt protoporphyrin (CoPP) determined by in vivo and in vitro assays.
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