Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP‐1 and p38
2014; Springer Nature; Volume: 33; Issue: 10 Linguagem: Inglês
10.1002/embj.201386369
ISSN1460-2075
AutoresAmelia Escolano, Sara Martı́nez-Martı́nez, Arántzazu Alfranca, Katia Urso, Helena M. Izquierdo, Mario Delgado, Francisco Martı́n, Guadalupe Sabio, David Sancho, Pablo Gómez‐del Arco, Juan Miguel Redondo,
Tópico(s)Galectins and Cancer Biology
ResumoArticle4 March 2014free access Source Data Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP-1 and p38 Amelia Escolano Amelia Escolano Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Sara Martínez-Martínez Sara Martínez-Martínez Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Arántzazu Alfranca Arántzazu Alfranca Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Área de Biología Celular y del Desarrollo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Katia Urso Katia Urso Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Helena M Izquierdo Helena M Izquierdo Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Mario Delgado Mario Delgado Instituto de Parasitología y Biomedicina "López-Neyra", CSIC, Granada, Spain Search for more papers by this author Francisco Martín Francisco Martín Human DNA variability Department and Oncology Department, Pfizer-Universidad de Granada-Junta de Andalucía, Centre for Genomics and Oncological Research (GENYO), Granada, Spain Search for more papers by this author Guadalupe Sabio Guadalupe Sabio Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author David Sancho David Sancho Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Pablo Gómez-del Arco Pablo Gómez-del Arco Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Juan Miguel Redondo Corresponding Author Juan Miguel Redondo Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Amelia Escolano Amelia Escolano Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Sara Martínez-Martínez Sara Martínez-Martínez Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Arántzazu Alfranca Arántzazu Alfranca Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Área de Biología Celular y del Desarrollo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain Search for more papers by this author Katia Urso Katia Urso Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Helena M Izquierdo Helena M Izquierdo Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Mario Delgado Mario Delgado Instituto de Parasitología y Biomedicina "López-Neyra", CSIC, Granada, Spain Search for more papers by this author Francisco Martín Francisco Martín Human DNA variability Department and Oncology Department, Pfizer-Universidad de Granada-Junta de Andalucía, Centre for Genomics and Oncological Research (GENYO), Granada, Spain Search for more papers by this author Guadalupe Sabio Guadalupe Sabio Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author David Sancho David Sancho Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Pablo Gómez-del Arco Pablo Gómez-del Arco Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Juan Miguel Redondo Corresponding Author Juan Miguel Redondo Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain Search for more papers by this author Author Information Amelia Escolano1, Sara Martínez-Martínez1, Arántzazu Alfranca1,2, Katia Urso1, Helena M Izquierdo1, Mario Delgado3, Francisco Martín4, Guadalupe Sabio1, David Sancho1, Pablo Gómez-del Arco1,5 and Juan Miguel Redondo 1 1Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain 2Área de Biología Celular y del Desarrollo, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain 3Instituto de Parasitología y Biomedicina "López-Neyra", CSIC, Granada, Spain 4Human DNA variability Department and Oncology Department, Pfizer-Universidad de Granada-Junta de Andalucía, Centre for Genomics and Oncological Research (GENYO), Granada, Spain 5Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain *Corresponding author. Tel: +34 91 453 1200; Fax: +34 91 453 1265; E-mail: [email protected] The EMBO Journal (2014)33:1117-1133https://doi.org/10.1002/embj.201386369 See also: JL Schultze (May 2014). PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti-inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN-targeted macrophages or direct injection of LxVP-encoding lentivirus has anti-inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP-1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN-inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti-inflammatory phenotype of CN-targeted macrophages, and mice with defective p38-activation were resistant to the anti-inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti-inflammatory status. Synopsis Targeting of calcineurin in macrophages unveils their anti-inflammatory activity, which bears significant potential for therapeutic exploitation. Calcineurin deletion or inhibition with LxVP peptide, but not the immunosuppressive drugs CsA or FK506, induces anti-inflammatory macrophages. p38 activity mediates the anti-inflammatory activation induced by specific calcineurin targeting in macrophages. Calcineurin targeting releases p38 from MKP-1-mediated repression. In gene or cell therapy approaches, specific calcineurin targeting in macrophages confers resistance to inflammation associated with arthritis and contact hypersensitivity. Introduction The phosphatase calcineurin (CN) couples calcium-mobilizing signals to cell responses and is the target of the immunosuppressive (IS) drugs cyclosporin A (CsA) and FK506 (Liu et al, 1991). Each of these drugs forms a complex with a specific immunophilin (IP) (cyclophilin A and FK506 binding protein, respectively), and it is these IS/IP complexes that bind and inhibit CN (Schreiber & Crabtree, 1992). These drugs have been widely used in molecular studies of CN function and are used therapeutically to prevent transplant rejection and treat inflammatory diseases such as atopic dermatitis (Lee et al, 2004), severe asthma (Niven & Argyros, 2003), and rheumatoid arthritis (Tugwell et al, 1995). However, IS/IP complexes have CN-independent effects on other signaling pathways (Matsuda et al, 2000) and many side effects, including hepatotoxicity, nephrotoxicity, and high blood pressure (Kiani et al, 2000; Martinez-Martinez & Redondo, 2004). Many CN-dependent processes described in mammals involve the regulation of the nuclear factor of activated T cells (NFAT) family of transcription factors (Crabtree & Olson, 2002; Hogan et al, 2003; Aramburu et al, 2004). Structural and functional analyses of NFAT proteins have identified PxIxIT and LxVP motifs as docking sites involved in the interaction with CN (Li et al 2011; Liu et al, 2001; Martinez-Martinez et al, 2006; Park et al, 2000). We recently showed that a peptide based on LxVP interferes with the CN-NFAT interaction by binding to the same docking site on CN as the IS/IP complexes (Rodriguez et al, 2009). In addition to inhibiting the binding of CN to LxVP-containing substrates, LxVP inhibits the phosphatase activity of CN (Martinez-Martinez et al, 2006; Rodriguez et al, 2009). In contrast, the PxIxIT peptide does not affect CN activity and only inhibits its binding to PxIxIT-containing substrates (Aramburu et al, 1998). A central role for the CN-NFAT pathway in adaptive immune responses has been documented through extensive studies in T cells (Macian, 2005); however, much less is known about its role in cells of the innate immune system such as macrophages. Macrophages not only constitute the first line of defense to an inflammatory insult, but also regulate the specific immune response by conditioning the cytokine milieu (Mantovani et al, 2004). Macrophages are reciprocally influenced by the surrounding environment and adopt pro-inflammatory or anti-inflammatory properties in response to it. Anti-inflammatory macrophages contribute to the suppressive microenvironment during tumorigenesis (Mantovani et al, 2002), maintain adipose tissue homeostasis (Lumeng et al, 2007), and promote resolution of inflammation in atherosclerosis (Mantovani et al, 2009) and myocardial infarction (Frangogiannis, 2012). Here, we reveal a novel role for CN in macrophage activation and show that specific targeting of CN in macrophages confers resistance to inflammation by preventing MKP-1-mediated suppression of p38 MAPK activity. We moreover report a macrophage-based anti-inflammatory treatment that specifically targets CN through mechanisms unrelated to those of conventional IS drugs. Results Constitutive deletion of CN, but not inhibition with CsA or FK506, drives macrophages toward an anti-inflammatory phenotype To study the influence of CN activity on macrophage phenotype, we generated Cnb1Δ/flox LysM-Cre mice, in which CN is constitutively deleted specifically in the myeloid lineage. CN expression was efficiently suppressed in peritoneal macrophages (Fig 1A), and CN-deficient cells showed increased expression of several anti-inflammatory markers, such as IL-10, Arg1, and Mrc1 (Fig 1B–D), and decreased levels of the pro-inflammatory marker iNOS (Fig 1E). To test the impact of CN-negative macrophages in vivo, we examined two models of acute inflammation: oxazolone-induced contact hypersensitivity in the ear and zymosan-induced inflammation in the paws. In both models, Cnb1Δ/flox LysM-Cre mice were resistant to inflammation (Fig 1F and G). These results suggest that suppression of CN activity induces anti-inflammatory properties in macrophages. However, this phenotype was not reproduced by treatment of wild-type peritoneal macrophages with the classical CN inhibitors CsA or FK506; indeed, these inhibitors reduced the levels of the Arg1, Mrc1, and IL-10 anti-inflammatory markers (Fig 1H). These results suggest either that the phenotype of macrophages constitutively deficient for CN is not a direct result of CN-deletion or that CsA and FK506 have off-target effects that mask the effect of CN inhibition. Figure 1. Constitutive CN deficiency, but not IS treatment, confers anti-inflammatory properties to macrophages A. Western blot confirming deletion of CnB and destabilization of CnA in macrophages from Cnb1Δ/flox LysMCre+ mice (CN KO) compared with Cnb1flox/flox LysMcre− mice (Control). B. ELISA analysis of IL-10 protein in supernatants of CN-deleted and control macrophages (mean ± s.d.; n = 3). C–E. mRNA levels of Arg1 (C) and Mrc1 (D) and iNOS protein (E) in peritoneal macrophages from CN KO and control mice. F, G. In vivo imaging analysis of inflammation in CN KO and control mice after (F) sensitization with oxazolone in the right ear and (G) zymosan inoculation in the right hindpaw (n = 9 animals per genotype and condition). H. mRNA levels of IL-10, Arg1, Mrc1, and iNOS in peritoneal macrophages treated with CsA or FK506 (mean ± s.e.m.; n > 3). Data information: *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embj201386369-SourceData-Fig1.pdf] Download figure Download PowerPoint Previous reports showed that CsA and FK506 inhibit iNOS expression in macrophages and other cell types (Hortelano et al, 1999; Hamalainen et al, 2002). We confirmed that high doses of CsA (3–10 μg/ml) inhibit iNOS gene expression in macrophages (Supplementary Fig S1A). However, this effect is not associated with the inhibition of the CN activity, since lower CsA doses (200 ng/ml) that efficiently inhibit CN-NFAT signaling (Supplementary Fig S1B) not only failed to inhibit iNOS expression but significantly increased it (Supplementary Fig S1A). Inducible CN deletion results in a population of anti-inflammatory macrophages with therapeutic effects in collagen-induced arthritis To bypass potential indirect effects of constitutive CN deficiency on macrophages, we deleted CN ex vivo by transducing CnB1Δ/flox macrophages with CRE-encoding lentivirus (Fig 2A). These CN-deleted macrophages, but not control CRE-transduced cells, upregulated IL-10, Arg1, and Mrc1, and downregulated iNOS (Fig 2B–E and Supplementary Fig S2A–D), mirroring the phenotype of constitutively CN-deficient macrophages. To further examine the anti-inflammatory properties of induced CN-deleted macrophages, we transferred them to a mouse model of collagen-induced arthritis (CIA), in which disease is triggered in the susceptible DBA1J mouse strain by two intradermal injections of collagen (Supplementary Fig S3). Local transfer of CN-deleted macrophages into the footpads of arthritic mice before the second collagen injection significantly reduced disease severity compared with mice inoculated with control macrophages (Fig 2F). Consistent with this, paws inoculated with CN-deleted macrophages expressed higher levels of the anti-inflammatory markers Mrc1 and IL-10 (Fig 2G). Figure 2. Inducible CN deletion generates anti-inflammatory macrophages that protect against CIA A. Western blot confirming CnB deletion and CnA destabilization in macrophages from Cnb1Δ/flox mice transduced ex vivo with CRE-encoding lentivirus. Tubulin expression is shown as a loading control. B. ELISA analysis of IL-10 protein in supernatants of CN-deleted and control macrophages (mean ± s.d.; n = 3). C, D. mRNA expression of Arg1 (C) and Mrc1 (D) in control and CN-deleted macrophages. E. Representative flow cytometry analysis of iNOS protein in CN-deleted and control macrophages. F. CIA score in arthritic mice inoculated in the footpad with CN-deleted or control macrophages. Data are means ± s.e.m. from a representative experiment; n = 10 mice per group. G. Immunofluorescence staining (red) of Mrc1 (top) and IL-10 (bottom) in sections from paws inoculated with CN-deleted or control macrophages. Nuclei are stained blue. Data information: *P < 0.05; **P < 0.01. Source data are available online for this figure. Source Data for Figure 2 [embj201386369-SourceData-Fig2.pdf] Download figure Download PowerPoint Specific CN targeting by LxVP induces anti-inflammatory properties in macrophages The failure of CsA and FK506 treatment to reproduce the consistent results obtained with both CN-deletion strategies indicated that IS drugs may have off-target effects. To clarify this issue, we tested the effect of an alternative strategy for CN inhibition with the peptide LxVP, which inhibits CN independently of IPs and therefore could avoid many of the side effects of IS drugs. Macrophages were transduced with lentivirus encoding LxVP or the mutant version AxAA (mutLxVP), both fused to GFP. Expression of LxVP in peritoneal macrophages strongly inhibited the phosphatase activity of CN (Fig 3A). Moreover, LxVP-transduced macrophages isolated from transgenic mice expressing an NFAT-luciferase reporter showed decreased NFAT activity under basal conditions and in response to the NFAT-inducer zymosan (Fig 3B; Goodridge et al, 2007). Figure 3. LxVP expression induces an anti-inflammatory phenotype in macrophages. CN activity in total protein extracts of peritoneal macrophages isolated 5 days after i.p. injection with LxVP or mutLxVP lentivirus (mean ± s.d.; n = 3). NFAT transcriptional activity (relative luciferase units) in untreated and zymosan-stimulated (Zym) macrophages from NFAT-luc transgenic mice i.p. injected with LxVP or mutLxVP lentivirus (mean ± s.d.; n = 5). IL-10 protein levels in culture supernatants of macrophages isolated from mice 5 days after i.p. injection with LxVP or mutLxVP lentivirus. Real-time PCR analysis of Arg1 and iNOS mRNA in isolated LxVP- and mutLxVP-transduced macrophages (mean ± s.d.; n = 3). Flow cytometry analysis of Mrc1 and SIGNR1 expression in isolated LxVP- and mutLxVP-transduced macrophages. Multiplex analysis of pro-inflammatory cytokines in culture supernatants of macrophages from mice i.p. injected with LxVP or mutLxVP lentivirus. Presentation of antigen (ovalbumin) to B3Z T-cell hybridoma cells by macrophages expressing LxVP or mutLxVP. Phagocytosis of opsonized red blood cells by LxVP- or mutLxVP-expressing macrophages. Osteoclast differentiation, recorded as the number of multinucleated TRAP+ cells. CIA score in arthritic mice inoculated in the footpads with LxVP- or mutLxVP-transduced macrophages. Macrophages were injected on day 20 to ensure their exposure to the booster collagen treatment. Data are means ± s.e.m. of three independent experiments; n = 30 mice per group. Data information: *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Like the CN-deleted macrophages, LxVP-transduced macrophages had a typical anti-inflammatory expression profile, including upregulated expression of IL-10 and Arg1, downregulated iNOS, and increased cell-surface expression of Mrc1 and SIGNR1 (Fig 3C–E). Moreover, LxVP-transduced macrophages had downregulated expression of pro-inflammatory cytokines including IL-17, TNF-α, IFN-γ, and IL-6 (Fig 3F). LxVP also conferred anti-inflammatory functions, such as reduced antigen-presentation capacity, increased phagocytic activity, and impaired differentiation to osteoclasts (Fig 3G–I and Supplementary Fig S4A and B), three hallmarks of the resolution of inflammation. Consistent with the ability of the VIVIT peptide (a high-affinity PxIxIT-derived peptide) to interfere with CN-NFAT interaction, lentiviral-mediated VIVIT expression in macrophages inhibited zymosan-induced NFAT-dependent transcription (Supplementary Fig S5A). However, VIVIT failed to induce the anti-inflammatory phenotype displayed by the LxVP-transduced macrophages (Supplementary Fig S5B–D). To evaluate the anti-inflammatory potential of LxVP-transduced macrophages in vivo, we performed cell therapy assays in the CIA model, in which transduced macrophages were injected into the footpads of arthritic mice. Paws injected with LxVP-transduced macrophages had significantly lower arthritic scores than those injected with control-transduced macrophages (Fig 3J). These results, together with the cell therapy experiments with CN-deleted macrophages, indicate that macrophages with suppressed CN-activity are competent to mediate anti-inflammatory effects in vivo. The anti-inflammatory actions of CN-targeted macrophages are mediated by releasing p38 MAPK from MKP-1-mediated repression Since CN modulates p38 activity in other cell types (Lim et al, 2001; Braz et al, 2003), we analyzed whether the anti-inflammatory macrophage phenotype triggered by CN gene deletion and LxVP administration was mediated by p38 activity, and whether p38 activation was implicated in the differences from the effect of IS drugs. LxVP-transduced and CN-deficient macrophages, but not control or IS-drug-treated macrophages, showed sustained activation of p38 (Fig 4A–D). Treatment of LxVP-transduced or CN-deficient macrophages with the p38-chemical inhibitor SB203580 (SB) reduced the expression of the anti-inflammatory markers Mrc1, Arg1, and IL-10 to basal levels (Fig 4E and F and Supplementary Fig S6A and B). Since IS drugs have been reported to activate p38 activation in macrophages (Kang et al, 2007), we again determined whether this was dependent on the IS doses used. As with iNOS expression, p38 was only activated at very high concentrations of CsA (50 μg/ml) and FK506 (10 μg/ml), but not at pharmacological doses of the drugs able to inhibit CN activity (Supplementary Fig S7A and B). Figure 4. p38 MAPK activity mediates the induction of anti-inflammatory macrophages upon specific CN targeting A. Representative Western blot showing expression of phosphorylated P-p38 and P-ERK in peritoneal macrophages treated with CsA or FK506 (FK) or transduced with LxVP or control lentivirus in vitro. p38 and tubulin were used as loading controls. B. Quantification of P-p38 expression in the experimental conditions mentioned in (A) (mean ± s.e.m.; n = 3). C. P-p38 (top) and CNB (center) protein expression in peritoneal macrophages from Cnb1 flox/flox LysMCre− (control) and Cnb1 Δ/flox LysMCre+ (CN KO) mice treated with CsA or FK506. Tubulin was used as a loading control (Tub). D. Quantification of P-p38 expression in the experimental conditions in (C) (mean ± s.e.m.; n = 3). E, F. Effect of p38 inhibition by SB203580 (SB) treatment on Mrc1, Arg1, and IL-10 mRNA levels in (E) LxVP-transduced macrophages and (F) CN KO macrophages. Data information: *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 4 [embj201386369-SourceData-Fig4.pdf] Download figure Download PowerPoint p38 activation in macrophages is regulated by the phosphatase MKP-1 (Perdiguero et al, 2011; Comalada et al, 2012; Liu et al, 2013). Consistent with the p38-activation induced by CN targeting, both gene deletion and LxVP-mediated CN inhibition reduced the expression of MKP-1 (Fig 5A and B). In MKP-1-deficient macrophages, p38 activation was enhanced to a similar extent as in LxVP-treated wild-type macrophages (Fig 5C and D), and LxVP treatment did not further activate p38 in MKP-1-deficient macrophages (Fig 5D). These results implicate MKP-1 in the CN-mediated regulation of p38 activation and indicate that p38 contributes to the anti-inflammatory phenotype of CN-targeted macrophages. Figure 5. MKP-1 links CN targeting with p38 MAPK activation MKP-1 protein expression in control and mutLxVP- or LxVP-transduced macrophages (n = 5). MKP-1 protein expression in control and CN KO macrophages (n = 3). P-p38 expression in wild-type and MKP-1-deficient macrophages (n = 3). P-p38 expression in wild-type and MKP-1-deficient macrophages transduced with mutLxVP or LxVP lentivirus (n = 2). Data information: Quantifications (relative units) are shown next to the corresponding Western blots in all panels. *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure. Source Data for Figure 5 [embj201386369-SourceData-Fig5.pdf] Download figure Download PowerPoint Systemically delivered LxVP targets macrophages, which migrate to sites of inflammation The above results establish the potential of LxVP as an anti-inflammatory treatment based on cell therapy. To test the therapeutic potential of LxVP in a gene therapy approach, we assessed the anti-inflammatory effects of LxVP lentivirus in the CIA model after systemic administration by intraperitoneal (i.p.) injection. To determine lentivirus tropism after i.p. injection, we examined the peritoneal exudate 5 days after inoculation of naive mice. At this time, all detected GFP+ cells are transduced by the lentivirus, and we can exclude false positives due to phagocytosis of GFP molecules or lentiviral particles. Although peritoneal exudates were enriched in cell populations such as T and B cells, GFP was undetectable in CD4+, CD8+, or B220+ cells, and all GFP+ cells were triple positive for F4/80, CD11b, and CD11c and also expressed Mertk (Fig 6A) clearly identifying these cells as macrophages. Figure 6. Systemically delivered LxVP lentivirus transduces macrophages, which then migrate to inflammatory foci Top, Flow cytometry analysis of GFP expression in gated CD4+, CD8+, or B220+ cells from peritoneal exudate obtained 5 days after i.p. injection with GFP-encoding lentivirus. Bottom, F4/80, CD11b, CD11c, and MertK expression in gated GFP+ cells from peritoneal exudate. Data are from a representative experiment (n ≥ 3). In vivo tracking of i.p.-injected DIR-labeled non-transduced macrophages in mice with zymosan-induced acute inflammation in the right hindpaw. In vivo images (top) and quantification (bottom). Zym, zymosan. (means ± s.d.; n = 3). Time profile of DIR signal in inflamed paws after i.p. injection of DIR-labeled non-transduced macrophages (means ± s.d.; n ≥ 3). In vivo imaging analysis of the capacity of DIR-labeled LxVP- or mutLxVP-transduced macrophages to migrate to inflamed paws. In vivo tracking of i.p.-injected DIR-labeled non-transduced macrophages in mice with oxazolone-induced contact hypersensitivity in the right ear. OXA, oxazolone (means ± s.d.; n ≥ 3). Data information: *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Since macrophages transduced upon i.p. administration of LxVP lentivirus were located in the peritoneal cavity, and therefore far from the site of action in the inflamed paw, we tested whether these macrophages migrated toward the inflammation focus to have their anti-inflammatory effects. CIA affects all four limbs, so we used a model of zymosan-induced acute paw inflammation to generate a single inflammation focus in one paw, leaving the contralateral paw as a non-inflammation control. We inoculated mice with zymosan in the right hind footpad and subsequently injected (i.p.) donor macrophages labeled ex vivo with the fluorescent tracer DIR. Macrophages migrated selectively from the peritoneal cavity to the inflamed paw, with no signal detected in control paws (Fig 6B). Migration increased progressively from 2 h to 2 days after macrophage transfer, and migrated cells were detectable for at least 13 days (Fig 6C). LxVP- and control-transduced macrophages had the same migratory capacity, as revealed by in vivo analysis of DIR signal in inflamed paws at 24 h post-injection (Fig 6D) and by GFP staining in tissue sections (Supplementary Fig S8). Homing of DIR-labeled macrophages to the inflammation site was also seen in the model of oxazolone-induced contact hypersensitivity in mouse ears, suggesting that peritoneal macrophages can migrate to different inflamed locations (Fig 6E). Gene therapy with LxVP lentivirus resolves remote inflammation in a p38-dependent manner Systemic i.p. treatment with LxVP lentivirus prevented the progression of CIA, maintaining the low arthritic scores recorded at the time of treatment (Fig 7A). Histological analysis of joint sections confirmed the near absence of inflammation in LxVP-treated mice, whereas joints of control-transduced animals showed abundant inflammatory cell infiltrates and severe bone and cartilage damage (Fig 7B). Injection of control lentivirus did not exacerbate disease symptoms (Supplementary Fig S9). A clear anti-inflammatory effect was also observed after systemic treatment with LxVP lentivirus in the oxazolone-induced contact hypersensitivity model in mouse ears (Fig 7C). Figure 7. Systemically delivered LxVP lentivirus protects against CIA and contact hypersensitivity in a p38-dependent manner The scheme shows the CIA protocol, indicating the time of lentivirus injection. The chart shows the time profile of arthritic score in mice inoculated i.p. with LxVP or mutLxVP lentivirus at disease onset (day 28). Data are means ± s.e.m. of three independent experiments; n = 20 mice per group. Masson's trichrome staining in paw joints of animals inoculated i.p. at disease onset with mut
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