Neutralization of pro‐inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis
2016; Springer Nature; Volume: 35; Issue: 6 Linguagem: Inglês
10.15252/embj.201592649
ISSN1460-2075
AutoresLiraz Shmuel‐Galia, Tegest Aychek, Avner Fink, Ziv Porat, Batya Zarmi, Biana Bernshtein, Ori Brenner, Steffen Jung, Yechiel Shai,
Tópico(s)Immunotherapy and Immune Responses
ResumoArticle16 February 2016free access Transparent process Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis Liraz Shmuel-Galia Liraz Shmuel-Galia Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tegest Aychek Tegest Aychek Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Avner Fink Avner Fink Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ziv Porat Ziv Porat Department of Biological Services, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Batya Zarmi Batya Zarmi Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Biana Bernshtein Biana Bernshtein Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ori Brenner Ori Brenner Department of Veterinary Resources, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Steffen Jung Corresponding Author Steffen Jung Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yechiel Shai Corresponding Author Yechiel Shai Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Liraz Shmuel-Galia Liraz Shmuel-Galia Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Tegest Aychek Tegest Aychek Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Avner Fink Avner Fink Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ziv Porat Ziv Porat Department of Biological Services, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Batya Zarmi Batya Zarmi Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Biana Bernshtein Biana Bernshtein Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Ori Brenner Ori Brenner Department of Veterinary Resources, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Steffen Jung Corresponding Author Steffen Jung Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Yechiel Shai Corresponding Author Yechiel Shai Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel Search for more papers by this author Author Information Liraz Shmuel-Galia1,‡, Tegest Aychek2,‡, Avner Fink1, Ziv Porat3, Batya Zarmi1, Biana Bernshtein2, Ori Brenner4, Steffen Jung 2 and Yechiel Shai 1 1Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel 2Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel 3Department of Biological Services, The Weizmann Institute of Science, Rehovot, Israel 4Department of Veterinary Resources, The Weizmann Institute of Science, Rehovot, Israel ‡These authors contributed equally to the work *Corresponding author. Tel: +972 8 934 2787; Fax: +972 8 934 4141; E-mail: [email protected] *Corresponding author. Tel: +972 8 934 2711; Fax: +972 8 934 4112; E-mail: [email protected] The EMBO Journal (2016)35:685-698https://doi.org/10.15252/embj.201592649 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 Monocytes have emerged as critical driving force of acute inflammation. Here, we show that inhibition of Toll-like receptor 2(TLR2) dimerization by a TLR2 transmembrane peptide (TLR2-p) ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C+ monocytes without affecting their recruitment to the colon. We report that TLR2-p directly interacts with TLR2 within the membrane, leading to inhibition of TLR2–TLR6/1 assembly induced by natural ligands. This was associated with decreased levels of extracellular signal-regulated kinases (ERK) signaling and reduced secretion of pro-inflammatory cytokines, such as interleukin (IL)-6, IL-23, IL-12, and IL-1β. Altogether, our study provides insights into the essential role of TLR2 dimerization in the activation of pathogenic pro-inflammatory Ly6Chi monocytes and suggests that inhibition of this aggregation by TLR2-p might have therapeutic potential in the treatment of acute gut inflammation. Synopsis Here, we utilize a novel strategy to neutralized TLR2 activation by inhibiting its dimerization by TLR2 transmembrane-derived peptide (TLR2-p). We show that TLR-2 peptide ameliorated DSS-induced colitis by interfering specifically with the activation of Ly6C+ monocytes without affecting their recruitment to the colon. The TLR2 transmembrane-derived peptide (TLR2-p) inhibits TLR2 signaling by interacting with its reciprocal receptors within the membrane. TLR2-p inhibits the dimerization of TLR2–TLR6/1 induced by natural ligands, resulting in attenuation of pro-inflammatory downstream signaling. Inhibition of TLR2 dimerization ameliorates acute colitis. TLR2-p inhibits TLR2 signaling in pathogenic pro-inflammatory Ly6Chi monocytes without affecting their recruitment to the inflamed gut. Introduction Intestinal immune cells play a fundamental role in the maintenance of gut homeostasis and gut defense against pathogen invasion (Kabat et al, 2014). Gut health relies on the establishment of quiescent coexistence of immune cells and the commensal bacteria. Dysregulation of the response toward commensals results in the development of inflammatory bowel disease (IBD) (Casellas et al, 1998; Strober et al, 2002; Cho, 2008; Sun et al, 2011; Zhang & Li, 2014). Human IBD includes Crohn's disease (CD) and ulcerative colitis (UC), which differ in the location of the maladies. Ulcerative colitis is characterized by pronounced inflammation of the intestinal mucosa, generally restricted to the colon. Until recently, UC pathogenesis was thought to be largely driven by an aggressive adaptive immune response against luminal bacterial antigens. However, more recent studies and genomewide association studies (GWAS) suggest uncontrolled reactivity of innate immune cells as prime cause of chronic inflammation in UC (Jostins et al, 2012; Corridoni et al, 2014). Specifically, mouse UC models have highlighted a prominent of influx of Ly6Chi blood monocytes into the tissue. In the healthy lamina propria, these cells maintain the intestinal macrophage compartment (Varol et al, 2009; Bain et al, 2014) and differentiate into quiescent intestinal macrophages (Rivollier et al, 2012; Zigmond et al, 2012). Under pathological conditions however, the same cells fail to be appeased, but respond to bacterial stimuli by acquiring pro-inflammatory activities and, as a result, actively promote disease (Rivollier et al, 2012; Zigmond et al, 2012). Toll-like receptors (TLR) are membrane pattern recognition receptors (PRRs) that recognize a wide range of microbial products (Kang & Lee, 2011). TLR activation by certain ligands induces the critical formation of TLR dimers with unique specificities (Botos et al, 2011). Upon binding to di- and tri-acylated lipoproteins, TLR2 dimerizes, for instance, with TLR6 and TLR1, respectively (Ozinsky et al, 2000; Takeuchi et al, 2002). TLR dimerization is coordinated through ligand binding to the extracellular domain of one receptor, leading to their lateral movement toward the partner molecule resulting in conformational changes throughout the proteins (Akira & Takeda, 2004). Once in the complex, TLR2 interacts with co-receptors allowing recruitment of adaptor proteins, such as MyD88 or TRIF (Deguine & Barton, 2014; Narayanan & Park, 2015) and stimulating signaling pathways (Kawai & Akira, 2011), which ultimately induce a range of inflammatory cytokines and chemokines (Kaisho & Akira, 2006; Fukata & Arditi, 2013). During acute and chronic inflammation, TLR engagement by commensal-derived products triggers the production of pro-inflammatory agents. The latter notion is supported by abounding evidence of TLR over-expression and activity in tissues obtained from IBD patients (Hausmann et al, 2002; Canto et al, 2006). Specifically, TLR2 was shown to be significantly more active in peripheral mononuclear cells from UC patients than those obtained from healthy individuals (Toiyama et al, 2006; Frolova et al, 2008). Moreover, the dimerization of TLR2 with TLR6 was shown to play an important role in colitis, as compared to TLR2–TLR1 dimerization (Morgan et al, 2014). The critical role of TLR2–TLR6 dimers in disease development is further supported by the finding that TLR6-deficient mice are resistant to colitis (Morgan et al, 2014). Collectively, these results highlight the importance of TLR2 and particularly its assembly with TLR6 in UC development. Ly6Chi monocytes that enter inflamed gut tissue upregulate expression of TLR2, TLR6, and NOD2 (Zigmond et al, 2012) that are involved in the induction of pro-inflammatory cytokines, such as IL-6, IL-23, and IL-1β (Zigmond et al, 2012; Neurath, 2014). Here, we investigated the role of TLR signaling in monocytes in the development of acute murine colitis. We show that TLR2 heterodimerization plays a fundamental role in the activation of the pro-inflammatory potential of Ly6Chi monocytes. Specifically, inhibition of TLR2 dimer formation using the TLR2 transmembrane domain (TMD)-derived peptide, TLR2-p (Fink et al, 2013), ameliorated DSS-induced acute colitis by interfering with Ly6Chi monocyte activation without affecting monocyte recruitment. Altogether, our data reveal that TLR2 heterodimerization plays a fundamental role in the activation of pathogenic pro-inflammatory Ly6Chi monocytes in UC and that its inhibition might have therapeutic potential in the treatment of acute gut inflammation. Results The TLR2 TMD-derived peptide, TLR2-p, inhibits TLR2 signaling by interacting with its reciprocal receptors within the membrane Hetero- and homodimerization of TLR are critical steps in the activation of these PRRs (Akira & Takeda, 2004; Kaisho & Akira, 2006; Botos et al, 2011; Kang & Lee, 2011; Irvine et al, 2013). To specifically target TLR2 and modulate its activities, we synthesized a peptide derived from the C-terminus of the TLR2 TMD region (TLR2-p) and established that it impaired induced TLR2 signaling in a sepsis model (Fink et al, 2013). To investigate whether interference with TLR2 activation by the TLR2-p peptide is due to peptide–protein interactions within the membrane, we analyzed its co-localization with TLR2 and TLR1 using confocal microscopy. Bone marrow (BM) culture-derived macrophages were probed with an antibody against TLR2, followed by staining with a secondary APC-labeled antibody (Fig 1A) or with an antibody against TLR1, followed by staining with a secondary FITC-labeled antibody (Fig 1B). For membrane staining, cells were labeled with the lipid dye DiD (Fig 1C). The stained cells were then treated with either a rhodamine-labeled TLR2-p peptide or scrambled TLR2 peptide (scrTLR2-p), as control. Merge images revealed that TLR2-p localized to the cell membrane and in the same regions as TLR2 and TLR1, unlike the scrTLR2-p peptide that was found throughout the cell (Fig 1A and B). The co-localization of the TLR2-p with TLR2, TLR1, or the membrane was significantly higher, than with the control peptide. Figure 1. TLR2 inhibitor peptide co-localized with its corresponding TLR A–C. Representative images of cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti-TLR2 antibody followed by staining with APC-labeled secondary antibody or (B) with anti-TLR1 antibody followed by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labeled with lipid dye, DiD (C). Then, rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars, 10 μm. The mean Pearson correlation coefficient observed for TLR2-p or scrTLR2-p peptides with TLR2, TLR1, and DiD is presented as mean of two independent experiments ± SEM (***P < 0.001, n = 19–61). Download figure Download PowerPoint The specific interaction of the TLR2-p with its native reciprocal TLR was further investigated by co-immunoprecipitation assay. BM macrophage lysates were incubated with either rhodamine-labeled TLR2-p or control peptide and then subjected to immunoprecipitation using specific TLR antibodies bound to protein G beads. Following SDS–PAGE, proteins bound to rhodamine-labeled peptides were detected by a fluorescent scanner (Fig 2A). TLR2-p efficiently precipitated with TLR2, TLR1, and TLR6 displaying preference for the latter, while the scrTLR2-p peptide showed only low interaction. To validate the specificity of TLR2-p peptide co-precipitation with its reciprocal receptor, we probed TLR2-p interactions with TLR4 that does not bind TLR2. TLR2-p showed low affinity to TLR4, similar to the levels observed with the control peptide. TLR2-p peptide was further tested for its inhibitory activity in vitro. Primary murine CD115+ monocytes cells treated with lipoteichoic acid (LTA) (TLR2-6 ligand) and PAM3CSK (TLR2-1 ligand) expressed reduced levels of the pro-inflammatory cytokines TNF-α and IL-6 following treatment with TLR2-p, while the control peptide showed no inhibition (Fig 2B). The same cells, when treated with LPS (TLR4 ligand), showed no differences in their secreted pro-inflammatory cytokines following treatment with TLR2-p. Collectively, this establishes the specific interaction of the TLR2-p peptide with its corresponding receptors, TLR1, TLR2, and TLR6, and provides critical mechanistic insights into the action of TLR2-p and its potential to block its dimerization and activation (Figs 1 and 2). Figure 2. TLR2 inhibitor peptide physically interacts with its corresponding TLR Biochemical analyses of peptide interaction with TLR. BM-derived macrophages were incubated with 1 μM fluorescently labeled rhodamine (Rho)-TLR2-p or its mutant Rho-ScrTLR2-p peptide. After 1 h of incubation at 37°C, the cells were lysed. Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies. Protein samples were resolved by SDS–PAGE, and the presence of the labeled peptide was detected with a fluorescent scanner. Subsequently, the gel was transferred to a membrane and subjected to Western blotting for TLR6, TLR1, TLR2, and TLR4 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. The results are presented as the mean fluorescent intensity from two independent experiments. Non-specific binding of the peptides to G protein beads was subtracted. The results are mean ± SEM of at least two independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). Protein analysis of secreted pro-inflammatory cytokines, IL-6 and TNF-α from primary CD115+ cells. Cells were incubated with 20 μM of TLR2-p or scrTLR2-p peptide for 0.5 h and then washed and incubated with LTA (5 μg/ml), PAM3CSK (0.5 μg/ml), or LPS (0.2 μg/ml) at 37°C for 5 h (for TNF-α detection) and 22 h (for IL-6 detection). The results are mean ± SEM of two independent experiments (***P < 0.001, n = 8). Download figure Download PowerPoint TLR2-p peptide inhibits the dimerization of TLR2 and TLR6, resulting in attenuation of downstream signaling To test whether the TLR2-p peptide inhibits TLR2–TLR6 dimerization, we performed a fluorescence resonance energy transfer (FRET) assay using the ImageStreamX imaging flow cytometer. BM macrophages were decorated with anti-TLR6 antibodies conjugated to PE, used as a donor (yellow, middle column) and antibodies against TLR2 followed by staining with a secondary APC-labeled antibody, acting as acceptor (see scheme in Fig 3A). LTA exposure induced an APC-FRET signal, indicating TLR6–TLR2 dimerization. Cells pre-incubated with TLR2-p for half an hour prior to LTA activation showed reduced levels of FRET signal similar to non-stimulated cells (Fig 3B). Note that in accordance with earlier reports, we observed weak association between TLR2 and TLR6 before LTA stimulation (Triantafilou et al, 2006). In contrast, control peptide had no effect on dimerization and showed the same FRET signal as LTA-stimulated cells. To verify that the signal observed was due to direct TLR2–TLR6 interactions, we measured FRET between TLR2 and another surface receptor expressed by macrophages, the surface glycoprotein EMR1 (recognized by the F4/80 antibody). No increase of FRET signal was observed in this setting upon LTA stimulus (Fig 3C). Addition of TLR2-p to BM macrophages also efficiently blocked LTA-induced ERK phosphorylation (Fig 3D). Activation of cells pre-treated with scrTLR2-p was indistinguishable from LTA-stimulated cells. Collectively, these results establish that TLR2-p blocks TLR2 signaling in in vitro cultures by inhibiting critical TLR2–TLR6 dimerization following LTA stimulation. Figure 3. TLR2-p peptide directly inhibits the dimerization of TLR2 and TLR6 after LTA treatment resulting in reduced levels of ERK1/2 signaling A. A scheme showing the FRET reaction. B, C. Representative images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by fluorescence resonance energy transfer (FRET) using ImageStreamX. Scale bars, 10 μm. Cells were incubated with 20 μM of TLR2-p or scrTLR2-p peptide for 0.5 h and then washed and incubated with 500 ng/ml LTA for another 0.5 h at 37°C. Cells were probed with anti-TLR6-PE- or anti-F4/80-PE-conjugated antibody (donor) and anti-TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreamX imaging flow cytometer. The FRET intensity is shown as mean ± SEM of two independent experiments (***P < 0.001, n = 2,579–15,567). D. Representative image of ERK1/2 phosphorylation levels in CD115+ BM monocytes upon LTA exposure. Cells were pre-treated for 0.5 h with 20 μM of TLR2-p peptide, scrTLR2-p peptide or untreated and then washed and incubated with 500 ng/ml LTA for the indicated times. ERK1/2 phosphorylation levels and total ERK1/2 levels were detected by Western blotting. Equal loading was detected by measuring tubulin. Results are representative data of two independent experiments. The band intensity was quantified for p-ERK levels after 15 min of LTA exposure. Results are normalized to tubulin levels and are the mean of two experiments ± SD (***P < 0.001). Download figure Download PowerPoint Inhibition of TLR2 dimerization ameliorates acute colitis Signals that emanate from TLR2 after its obligatory pairing with TLR6 or TLR1 are thought to contribute to gut inflammation. Accordingly, strategies interfering with TLR2 activation improve disease scores in UC animal models, although the exact mechanisms of action and cell types targeted by these regimen remain mostly undefined (Hausmann et al, 2002; Pierik et al, 2006; Toiyama et al, 2006; Heimesaat et al, 2007; Frolova et al, 2008). To investigate the therapeutic potential of TLR2 dimerization inhibition, we tested the effect of TLR2-p in an acute colitis model, in which mice are exposed to an oral dextran sodium sulfate (DSS) regimen (Okayasu et al, 1990). Mice were exposed to DSS-containing drinking water for 7 days, with or without intraperitoneal (IP) injections of TLR2-p every second day (see scheme, Fig 4A). Mice treated with DSS only, or with DSS and the scrambled control peptide (scrTLR2-p), exhibited weight loss and severe colitis, as evaluated by colonoscopy (Becker et al, 2006) (Fig 4B–D). In contrast, TLR2-p-treated mice exhibited significant mitigation in the severity of colitis upon DSS challenge. Moreover, TLR2-p-treated mice also displayed milder mucosal pathology when compared to scrTLR2-p/DSS or DSS only-treated mice, as evidenced by decreased luminal blood and reduced edema and lesion formation (Fig 4E and F). Furthermore, analysis of supernatants of colon explant cultures showed that TLR2-p-treated animals displayed reduced levels of IL-6, IL-1β, and IFN-λ (Fig 5A). The latter finding was supported by results obtained by qRT–PCR analysis of the respective colonic tissue (Fig 5B). Collectively, these results establish that inhibition of TLR2 dimerization by TLR2-p reduces acute inflammation in DSS-challenged animals. Figure 4. TLR2-p inhibitor peptide ameliorated DSS-induced colitis A scheme showing peptide and DSS administration regime. Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5 mg/kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one. A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide, and untreated mice (n = 19, 21, 22 and 19, respectively). Results represent the mean ± SD of three independent experiments (***P < 0.001). A graphical summary of changes in body weight (day 6) of the indicated groups. Results are the mean ± SD of two independent experiments (n = 6–8) (***P < 0.001). Representative H&E histological images for distal colon section (day 7). Black asterisk indicates large collection of blood in the lumen, arrowheads indicate widespread mucosal collapse and ulceration, and arrows indicate edema in the sub mucosa. Scale bars, 100 μm (lower row) and 1,000 μm (upper row). A graphical summary of histological severity score of the indicated treatments. Results are the mean ± SD of two independent experiments (n = 9–10) (**P < 0.01). Download figure Download PowerPoint Figure 5. TLR2-p inhibitor peptide downregulates pro-inflammatory cytokines in the colonic mucosa A, B. Protein and RNA samples of colon tissue were extracted from mice with the indicated treatments. (A) Cytokine levels of IL-6, IL-1β, and IFN-λ were determined by ELISA (**P < 0.01, ***P < 0.001, n = 4–7) and (B) mRNA expression levels of IL-6, IL-12p35, and IL-23p19 were assessed by quantitative RT–PCR and normalized to TBP (*P < 0.05, **P < 0.01, n = 4–6). Results represent two or more independent experiments. Download figure Download PowerPoint TLR2-p peptide inhibits TLR2 signaling in Ly6Chi monocytes without affecting their recruitment to the inflamed gut DSS colitis is associated with a massive tissue infiltration of Ly6Chi monocytes that acquire a pronounced pro-inflammatory signature (Zigmond et al, 2012). Ablation or sequestration of Ly6Chi monocytes ameliorates acute colitis establishing these cells or their derivatives as critical drivers of gut inflammation (Zigmond et al, 2012; Getts et al, 2014). Monocyte infiltrates display prominent expression of TLR2 and TLR6 (Zigmond et al, 2012). We therefore reasoned that the TLR2-p regimen might improve the DSS colitis scores by targeting Ly6Chi monocytes and inhibiting their pro-inflammatory response. First, we evaluated colonic monocyte infiltration. With DSS, the frequency of Ly6Chi cells within the total CD11b+ pool increased in the colon, corroborating earlier studies (Waddell et al, 2011; Zigmond et al, 2012). Monocyte infiltrates of DSS/TLR2-p- and DSS/scrTLR2-p-treated mice were comparable to non-peptide-treated controls, establishing that TLR2-p does not affect monocyte recruitment (Fig 6A and B). Next, we investigated whether TLR2-p blocks the pro-inflammatory activity of Ly6Chi monocytes. To compensate for in vivo peptide degradation and to increase binding probability of the peptides to Ly6Chi monocytes, we performed an additional injection on day 5 and sorted Ly6Chi monocytes on day 6 of DSS challenge (see scheme, Fig 7A). Also this modified protocol improved colitis scores of DSS-treated animals (Fig 7B). To probe for a direct effect of the TLR2-p on Ly6Chi monocytes, we isolated the latter from the different animal groups (Fig EV1) and subjected the cells to qRT–PCR analysis for IL-6, IL-12, and IL-23 production. As shown in Fig 7C, TLR2-p treatment significantly impaired monocyte production of pro-inflammatory cytokines. Taken together, this provides a mechanistic explanation for the fact that TLR2-p treatment ameliorates acute colitis development. Specifically, we show that the agent interferes with the TLR2-triggered activation of Ly6Chi monocytes that infiltrate the gut tissue, thereby curbing the pro-inflammatory reaction. Figure 6. TLR2 inhibitor peptide does not have an effect on the recruitment of pro-inflammatory monocytes Flow cytometry analysis of colonic lamina propria CD11c− CD11b+Ly6Chi MHCII-monocytes from steady state and DSS day 7 treated with TLR2 peptide or scrTLR2 peptide, showing comparable monocyte infiltrates into the colitis colon. Plots were pre-gated on live CD45+ cells. Graphical summary of monocytes presented as % out of CD11b + cells. Results are the mean ± SEM of three independent experiments (n = 3 per group). Download figure Download PowerPoint Figure 7. TLR2-p inhibitor peptide downregulates pro-inflammatory cytokines expressed by pro-inflammatory monocytes A scheme showing peptide and DSS administration regime. Weight loss of DSS-challenged mice on day 6 (***P < 0.001). Results are the mean ± SD of three independent experiments (n = 4–5). Real-time PCR analysis showing mRNA expression levels of IL-6, IL-12p35, and IL-23p19 of FACS-isolated monocytes collected from mice with the indicated treatments. Results are the mean ± SEM of two to three independent experiments (*P < 0.05, **P < 0.01, n = 6–7 mice pooled per group). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Flow cytometry sorting strategy of colonic lamina propria pro-inflammatory monocytes A, B. Flow cytometry analysis of colonic lamina propria CD11b+Ly6Chi monocytes (A) pre-sort and (B) post-sort. Download figure Download PowerPoint Discussion Monocytes have emerged as critical drivers of acute gut inflammation. Here, we show that neutralization of the pro-inflammatory activities of Ly6Chi monocytes by targeting TLR signaling ameliorated DSS-induced acute colitis. Specifically, we used peptide-based interference with specific TLR dimerization to manipulate the differentiation of recruited monocytes. Collectively, we establish the critical role of TLR2 dimer formation in the local generation of pro-inflammatory cells in acute gut inflammation. Ly6Chi monocytes entering the healthy colon acquire a non-inflammatory gene expression profile (Zigmond et al, 2012). In contrast, when entering inflamed tissue, the differentiation of these cells into the quiescent, non-inflammatory CX3CR1hi macrophages is blocked and diverted to a distinct fate (Rivollier et al, 2012; Zigmond et al, 2012). Ly6Chi monocytes then respond to the bacterial products they encounter with the production of pro-inflammatory cytokines, such as IL-23 and IL-6 (Zigmond et al, 2012). These effector monocytes actively promoted gut inflammation, as their ablation using an anti-CCR2 regimen (Zigmond et al, 2012), or microparticle-mediated sequestration (Getts et al, 2014) ameliorates DSS colitis. Given their plasticity and role as macrophage precursors, Ly6Chi monocytes, and their equivalent in the human, the classical CD14+ CD16+/− monocytes, have emerged as attractive targets for cellular therapy. However, approaches interfering with monocyte recruitment, such as ablation or sequestration, or targeting of critical chemokine receptors (Leuschner et al, 2011), also inherently prevent the re-establishment of non-inflammatory CX3CR1hi macrophages and thus by themselves compromise gut homeostasis. Manipulations that specifically interfere with the local development of pro-inflammatory effector monocytes are hence to be favored. Here, we highlight the potential of such an approach by showing that specific neutralization of TLR2 signaling by inhibiting critical TLR dimerization blocks monocyte differentiation into pro-inflammatory cells. Interference with TLR2 signaling has been proposed to have therapeutic potential in the treatment of gut inflammation and UC (Hausmann et al, 2002; Pierik et al, 2006; Toiyama et al, 2006; Heimesaat et al, 2007; Frolova et al, 2008). To transmit a signal upon ligand engagement, TLR2 needs to form heterodimers with TLR6 or TLR1 (Irvine et al, 2013; Ozinsky et al, 2000; Fukata & Arditi, 2013; Botos et al, 2011; Morgan et al, 2014). TLR assembly is partially mediated by interactions of their TMDs (Reuven et al, 2014). Accordingly, exogenously added peptides derived from receptor TMD regions can inhibit assembly and activation (Gerber et al, 2004; Yin et al, 2007; He et al, 2011; Reuven et al, 2014). Here, we utilized this strategy to modulate TLR2 signaling in the context of gut inflammation by directly abrogating TLR2 dimerization within the membrane, using a TLR2 TMD-derived peptide. The inhibitory activity of T
Referência(s)