Pleiotropic antifibrotic actions of aspirin-triggered resolvin D1 in the lungs

Introduction: Pulmonary fibrosis is a destructive, progressive disease that dramatically reduces life quality of patients, ultimately leading to death. Therapeutic regimens for pulmonary fibrosis have shown limited benefits, hence justifying the efforts to evaluate the outcome of alternative treatments. Methods: Using a mouse model of bleomycin (BLM)-induced lung fibrosis, in the current work we asked whether treatment with pro-resolution molecules, such as pro-resolving lipid mediators (SPMs) could ameliorate pulmonary fibrosis. To this end, we injected aspirin-triggered resolvin D1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid; ATRvD1; i.v.) 7 and 10 days after BLM (intratracheal) challenge and samples were two weeks later. Results and discussion: Assessment of outcome in the lung tissues revealed that ATRvD1 partially restored lung architecture, reduced leukocyte infiltration, and inhibited formation of interstitial edema. In addition, lung tissues from BLM-induced mice treated with ATRvD1 displayed reduced levels of TNF-α, MCP-1, IL-1-β, and TGF-β. Of further interest, ATRvD1 decreased lung tissue expression of MMP-9, without affecting TIMP-1. Highlighting the beneficial effects of ATRvD1, we found reduced deposition of collagen and fibronectin in the lung tissues. Congruent with the anti-fibrotic effects that ATRvD1 exerted in lung tissues, α-SMA expression was decreased, suggesting that myofibroblast differentiation was inhibited by ATRvD1. Turning to culture systems, we next showed that ATRvD1 impaired TGF-β-induced fibroblast differentiation into myofibroblast. After showing that ATRvD1 hampered extracellular vesicles (EVs) release in the supernatants from TGF-β-stimulated cultures of mouse macrophages, we verified that ATRvD1 also inhibited the release of EVs in the bronco-alveolar lavage (BAL) fluid of BLM-induced mice. Motivated by studies showing that BLM-induced lung fibrosis is linked to angiogenesis, we asked whether ATRvD1 could blunt BLM-induced angiogenesis in the hamster cheek pouch model (HCP). Indeed, our intravital microscopy studies confirmed that ATRvD1 abrogates BLM-induced angiogenesis. Collectively, our findings suggest that treatment of pulmonary fibrosis patients with ATRvD1 deserves to be explored as a therapeutic option in the clinical setting.Fil: Guilherme, Rafael F.. Universidade Federal do Rio de Janeiro; BrasilFil: Silva, José Bruno N.F.. Universidade Federal do Rio de Janeiro; Brasil. Universidade Federal do Tocantins; BrasilFil: Waclawiack, Ingrid. Universidade Federal do Rio de Janeiro; BrasilFil: Fraga Junior, Vanderlei S.. Universidade Federal do Rio de Janeiro; BrasilFil: Nogueira, Thaís O.. Universidade Federal do Rio de Janeiro; BrasilFil: Pecli, Cyntia. Universidade Federal do Rio de Janeiro; BrasilFil: Araújo Silva, Carlla A.. Universidade Federal do Rio de Janeiro; BrasilFil: Magalhães, Nathalia S.. Ministerio de Salud de Brasil. Fundación Oswaldo Cruz. Instituto Oswaldo Cruz;Fil: Lemos, Felipe S.. Ministerio de Salud de Brasil. Fundación Oswaldo Cruz. Instituto Oswaldo Cruz;Fil: Bulant, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil; ArgentinaFil: Blanco, Pablo Javier. Laboratório Nacional para Computação Científica; BrasilFil: Serra, Rafaela. Universidade Federal do Rio de Janeiro; BrasilFil: Svensjö, Erik. Universidade Federal do Rio de Janeiro; BrasilFil: Scharfstein, Júlio. Universidade Federal do Rio de Janeiro; BrasilFil: Moraes, João A.. Universidade Federal do Rio de Janeiro; BrasilFil: Canetti, Claudio. Universidade Federal do Rio de Janeiro; BrasilFil: Benjamim, Claudia F.. Universidade Federal do Rio de Janeiro; Brasi

CCR4 Controls the Suppressive Effects of Regulatory T Cells on Early and Late Events during Severe Sepsis.

Sepsis is a deadly disease characterized by an overwhelming release of inflammatory mediators and the activation of different types of cells. This altered state of cell activation, termed leukocyte reprogramming, contributes to patient outcome. However, the understanding of the process underlying sepsis and the role of regulatory T cells (Tregs) in sepsis remains to be elucidated. In this study, we investigated the role of CCR4, the CCL17/CCL22 chemokine receptor, in the innate and acquired immune responses during severe sepsis and the role of Tregs in effecting the outcome. In contrast with wild-type (WT) mice subjected to cecal ligation and puncture (CLP) sepsis, CCR4-deficient (CCR4-/-) septic mice presented an increased survival rate, significant neutrophil migration toward the infection site, a low bacterial count in the peritoneum, and reduced lung inflammation and serum cytokine levels. Thus, a better early host response may favor an adequate long-term response. Consequently, the CCR4-/- septic mice were not susceptible to secondary fungal infection, in contrast with the WT septic mice. Furthermore, Tregs cells from the CCR4-/- septic mice showed reduced suppressive effects on neutrophil migration (both in vivo and in vitro), lymphocyte proliferation and ROS production from activated neutrophils, in contrast with what was observed for Tregs from the WT septic mice. These data show that CCR4 is involved in immunosuppression after severe sepsis and suggest that CCR4+ Tregs negatively modulate the short and long-term immune responses

Role of Chemokine Receptor CCR4 and Regulatory T Cells in Wound Healing of Diabetic Mice

International audienceWound healing is a well-coordinated process that involves inflammatory mediators and cellular responses; however, if any disturbances are present during this process, tissue repair is impaired. Chronic wounds are one of the serious long-term complications associated with diabetes mellitus. The chemokine receptor CCR4 and its respective ligands, CCL17 and CCL22, are involved in regulatory T cell recruitment and activation in inflamed skin; however, the role of regulatory T cells in wounds is still not clear. Our aim was to investigate the role of CCR4 and regulatory T cells in cutaneous wound healing in diabetic mice. Alloxan-induced diabetic wild-type mice (diabetic) developed wounds that were difficult to heal, differently from CCR4 e/e diabetic mice (CCR4 e/e diabetic), and also from anti-CCL17/22 or anti-CD25einjected diabetic mice that presented with accelerated wound healing and fewer regulatory T cells in the wound bed. Consequently, CCR4 e/e diabetic mice also presented with alteration on T cells population in the wound and draining lymph nodes; on day 14, these mice also displayed an increase of collagen fiber deposition. Still, cytokine levels were decreased in the wounds of CCR4 e/e diabetic mice on day 2. Our data suggest that the receptor CCR4 and regulatory T cells negatively affect wound healing in diabetic mice

Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo.

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4+ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund's Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated

Tregs from CCR4^-/- post-septic mice exhibit attenuated suppressive activity.

<p>(A) WT naïve mice (n = 5–6 mouse per group) were challenged with 5 x 10⁷<i>A</i>. <i>fumigatus</i> conidia or saline (SAL) or co-injected with 5 x 10⁴ of CD4⁺CD25⁺ Tregs obtained from WT (sham or L) or CCR4^-/- mice (sham or L). Tregs were purified from spleens and mesenteric lymph nodes at 4 days after surgery and were then used for co-injection. After 24 h, BAL was performed to evaluate neutrophil population/migration. The results are expressed as the number of neutrophils in the BAL. * <i>P</i><0.05 compared to the <i>A</i>. <i>fumigatus</i> challenged group (second bar) and to the sham-WT group. (B) Bone marrow-derived neutrophils were pre-incubated with CD4⁺CD25⁺ Tregs (ratio 39:1) obtained from L-WT or L-CCR4^-/- mice for 2 h at 37°C on day 4 after surgery. After pre incubation, the cells were incubated in 24-transwell plates for 2 h at 37°C. Neutrophil migration was evaluated by cell accumulation in the bottom chamber. *<i>P</i><0.05 compared with the group challenged with conidia that received Tregs from WT-sham group. (C) Bone marrow-derived neutrophils were incubated with supernatants (1:20) from WT CD4⁺CD25⁺ Tregs (sham or L) or CCR4^-/- CD4⁺CD25⁺ Tregs (sham or L) for 2 h at 37°C. After incubation, the cells were stimulated with PMA or PBS for 1 h. They were then centrifuged, and a ROS detection probe (CM-H₂DCFDA) was used to measure ROS production by flow cytometry. The results are expressed as MFI. The data are representative of two experiments, and each experiment was performed with neutrophil pools from 3–4 mice. Statistical analysis was not conducted because the data were generated from pooled cells.</p

Presence of CD4+Foxp3+ Tregs in lymphoid tissue after surgery.

<p>WT and CCR4^-/- mice were subjected to sham or CLP surgery and antibiotic treatment with ertapenem (75 mg/kg) at 6, 24 and 48 h after surgery. On day 1 or 4 after surgery, mice were sacrificed, and blood (left two columns), spleen (middle two columns) and mesenteric lymph node (right two columns) samples were collected and fixed. The samples were stained for CD4 and Foxp3 and analyzed by flow cytometry. (A) The dot plot data show one representative mouse each from the WT and CCR4^-/- sham and L-groups. (B) Absolute numbers of CD4⁺Foxp3⁺ Tregs in the blood, spleen and MLN on days 1 and 4 after surgery. The graphs display the mean ± SEM for each group (n = 3–4 per group).</p

CD4⁺Foxp3⁺ Tregs in the peritoneal cavity and dendritic cells, NK cells, and macrophages in the lungs of WT and CCR4^-/- mice.

<p>(A) Percentages of CD4⁺Foxp3⁺ Tregs (left panel) and their absolute numbers (right panel) in the peritoneal cavity at 24 h after sham and CLP surgery in the WT and CCR4^-/- mice. (B) The percentages of CD11c⁺ (dendritic cells), NK1.1⁺ (natural killer cells), and F4/80⁺ cells (macrophages) (left panels) and their absolute numbers (right panels) were determined in the lungs of the WT and CCR4^-/- mice at 24 h after sham and CLP surgery. The data are representative of two experiments. *<i>P</i><0.05 between the L-WT and sham-WT groups.</p

CCR4^-/- mice show increased resistance to severe sepsis.

<p>(A) WT (open symbols) and CCR4^-/- (filled symbols) mice underwent sham (square) or L-CLP (circle) surgery. All groups received antibiotic therapy of ertapenem (75 mg/kg) at 6, 24 and 48 h after surgery. The survival of these mice was followed until day 7. The data are representative of four experiments (n = 7–11 mice per group). *<i>P</i><0.05 between the CLP CCR4^-/- and CLP WT groups. (B) Neutrophil migration to the peritoneal cavity in the sham, Non-Lethal CLP (NL) and Lethal-CLP (L) groups evaluated at 6 h after surgery. *<i>P</i><0.05 between the NL-CLP and sham groups of both strains (WT and CCR4^-/-); **<i>P</i><0.05 between L-CLP and NL-CLP groups of WT mice; and #<i>P</i><0.05 between the L-CLP WT and L-CLP CCR4^-/- mice. (C) Bacterial counts in peritoneal exudate and (D) blood at 6 h after NL- or L-CLP surgery of the WT and CCR4^-/- mice. *<i>P</i><0.05 between the L-CLP WT and NL-CLP WT groups; and #<i>P</i><0.05 between the L-CLP CCR4^-/- and L-CLP WT groups. (E) Percentages of neutrophils in the blood of naïve WT and CCR4^-/- mice. (F) Absolute numbers of neutrophils in the bone marrow of naïve WT and CCR4^-/- mice.</p

L-CCR4^-/- mice have reductions in TNF-α and KC levels and an increased production of IL-10 in the peritoneal cavity and do not develop a systemic inflammatory response.

<p>(A) TNF-α, KC and IL-10 levels were detected in peritoneal exudates at 6 h after surgery by ELISA, as described in the Methods section. *<i>P</i><0.05 between the L-WT and sham-WT groups; and #<i>P</i><0.05 between the L-CCR4^-/- and L-WT groups. (B) Absolute numbers of neutrophils were determined in the lung tissue (left panel) and BAL (right panel) at 6 h after surgery by MPO assay or cell counts, respectively. *<i>P</i><0.05 between the L-WT and NL-WT groups; and #<i>P</i><0.05 between the L-CCR4^-/- and L-WT groups. (C) TNF-α and IL-10 levels were detected at 6 h after surgery in blood from WT and CCR4^-/- mice in the sham and L-groups. *<i>P</i><0.05 between the L-WT and NL-WT groups; and #<i>P</i><0.05 between the L-CCR4^-/- and L-WT groups. (D) TNF-α, KC and IL-10 levels were detected at 6 h after surgery in BAL by ELISA. *<i>P</i><0.05 between the L-WT and sham-WT groups; and #<i>P</i><0,05 between the L-CCR4^-/- and L-WT groups.</p

CCR4^-/- post-septic Tregs showed reduced suppressive effects on T effector cell proliferation.

<p>WT and CCR4^-/- mice were subjected to sham or CLP surgery, and on day 4 after surgery, the spleen and mesenteric lymph nodes were collected for Tregs purification. Splenocytes from naïve mice were stained with CFSE for proliferation assay. Tregs and splenocytes were co-incubated (1:4) for 96 h in the presence of anti-CD3 and anti-CD28, and the cells were analyzed by flow cytometry. These data are representative of two different experiments (n = 3 for each experiment).</p