Antiflammin-1 attenuates bleomycin-induced pulmonary fibrosis in mice

Background

Antiflammin-1 (AF-1), a derivative of uteroglobin (UG), is a synthetic nonapeptide with diverse biological functions. In the present study, we investigated whether AF-1 has a protective effect against bleomycin-induced pulmonary fibrosis.

Methods

C57BL/6 mice were injected with bleomycin intratracheally to create an animal model of bleomycin-induced pulmonary fibrosis. On Day 7 and Day 28, we examined the anti-inflammatory effect and antifibrotic effect, respectively, of AF-1 on the bleomycin-treated mice. The effects of AF-1 on the transforming growth factor-beta 1 (TGF-β1)-induced proliferation of murine lung fibroblasts (NIH3T3) were examined by a bromodeoxycytidine (BrdU) incorporation assay and cell cycle analysis.

Results

Severe lung inflammation and fibrosis were observed in the bleomycin-treated mice on Day 7 and Day 28, respectively. Administration of AF-1 significantly reduced the number of neutrophils in the bronchoalveolar lavage fluid (BALF) and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in the lung homogenates on Day 7. Histological examination revealed that AF-1 markedly reduced the number of infiltrating cells on Day 7 and attenuated the collagen deposition and destruction of lung architecture on Day 28. The hydroxyproline (HYP) content was significantly decreased in the AF-1-treated mice. In vitro, AF-1 inhibited the TGF-β1-induced proliferation of NIH3T3 cells, which was mediated by the UG receptor.

Conclusions

AF-1 has anti-inflammatory and antifibrotic actions in bleomycin-induced lung injury. We propose that the antifibrotic effect of AF-1 might be related to its suppression of fibroblast growth in bleomycin-treated lungs and that AF-1 has potential as a new therapeutic tool for pulmonary fibrosis.     

Characterization of chromosomal instability in murine colitis-associated colorectal cancer

Background

Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men.

Methods

The AOM/DSS model (n = 23) and IL-10^−/− mice (n = 8) were applied to monitor malignancy development via endoscopy and to analyze premalignant and malignant stages of CACs. CIN was assessed using DNA-image cytometry. Protein expression of p53, beta-catenin and Ki67 was evaluated by immunohistochemistry. The degree of inflammation was analyzed by histology and paralleled to local interferon-γ release.

Results

CIN was detected in 81.25% of all murine CACs induced by AOM/DSS, while all carcinomas that arose in IL-10^−/− mice were chromosomally stable. Beta-catenin expression was strongly membranous in IL-10^−/− mice, while 87.50% of AOM/DSS-induced tumors showed cytoplasmatic and/or nuclear translocation of beta-catenin. p53 expression was high in both models and Ki67 staining revealed higher proliferation of IL-10^−/−-induced CACs.

Conclusions

AOM/DSS-colitis, but not IL-10^−/− mice, could provide a powerful murine model to mechanistically investigate CIN in colitis-associated carcinogenesis.     

MIF participates in Toxoplasma gondii-induced pathology following oral infection

Background

Macrophage migration inhibitory factor (MIF) is essential for controlling parasite burden and survival in a model of systemic Toxoplasma gondii infection. Peroral T. gondii infection induces small intestine necrosis and death in susceptible hosts, and in many aspects resembles inflammatory bowel disease (IBD). Considering the critical role of MIF in the pathogenesis of IBD, we hypothesized that MIF participates in the inflammatory response induced by oral infection with T. gondii.

Methodology/Principal Findings

Mif deficient (Mif⁻/⁻) and wild-type mice in the C57Bl/6 background were orally infected with T. gondii strain ME49. Mif⁻/⁻ mice had reduced lethality, ileal inflammation and tissue damage despite of an increased intestinal parasite load compared to wt mice. Lack of MIF caused a reduction of TNF-α, IL-12, IFN-γ and IL-23 and an increased expression of IL-22 in ileal mucosa. Moreover, suppressed pro-inflammatory responses at the ileal mucosa observed in Mif⁻/⁻ mice was not due to upregulation of IL-4, IL-10 or TGF-β. MIF also affected the expression of matrix metalloproteinase-9 (MMP-9) but not MMP-2 in the intestine of infected mice. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in Mif⁻/⁻ mice compared to wild-type mice.

Conclusion/Significance

In conclusion, our data suggested that in susceptible hosts MIF controls T. gondii infection with the cost of increasing local and systemic inflammation, tissue damage and death.     

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose

Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice.

Methods

The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8 ^+/− mice expressing ß-galactosidase. Aged Mfge8 ^+/− and Mfge8 ^−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV.

Results

rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8^−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch''s membrane (BM) was slightly but significantly thicker in Mfge8^−/− mice as compared to controls.

Conclusions

Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8^−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD.     

The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

Background

Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47^phox -/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway.

Methods

Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.

Results

BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47^phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.

Conclusions

These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis.     

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Background

Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.

Methods

The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.

Results

We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt⁺ γδ T cells and to a lesser extent by CD4αβ⁺ T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.

Conclusions

Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.     

Puerarin suppresses invasion and vascularization of endometriosis tissue stimulated by 17β-estradiol

Background

Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17β-estradiol (E2) and producing an anti-estrogenic effect. This study was to investigate whether puerarin could suppress the invasion and vascularization of E2-stimulated endometriotic tissue.

Methodology/Principal Findings

The endometriotic stromal cells (ESCs) were successfully established and their invasive ability under different treatments was assessed through a Transwell Assay. Simultaneously, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by western blotting. Vascularization of endometriotic tissues was observed by chicken chorioallantoic membrane (CAM) assay. The staining of MMP-9, intercellular adhesion molecule 1 (ICAM-1), TIMP-1, and vascular endothelial growth factor (VEGF) in grafted endometriotic tissues was examined using immunohistochemistry analysis. The purity of ESCs in isolated cells was >95%, as determined by the fluoroimmunoassay of vimentin. E2 (10⁻⁸ mol/L) promoted the invasiveness of ESCs by increasing MMP-9 accumulation and decreasing TIMP-1 accumulation. Interestingly, puerarin (10⁻⁹ mol/L) significantly reversed these effects (P<0.01). The CAM assay indicated that puerarin (10⁻⁹ mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (10⁻⁸ mol/L) treatment (P<0.05). Accordingly, immunohistochemistry showed that the accumulation of MMP-9, ICAM-1, and VEGF was reduced whereas that of TIMP-1 increased in the combination treatment group compared with the E2 treatment group.

Conclusions/Significance

This study demonstrated that puerarin could suppress the tissue invasion by ESCs and the vascularization of ectopic endometrial tissues stimulated by E2, suggesting that puerarin may be a potential drug for the treatment of endometriosis.     

BAFF promotes Th17 cells and aggravates experimental autoimmune encephalomyelitis

Background

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.

Methodology/Principal Findings

Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff^−/− mice. Th17 cells in B6.Baff^−/− mice bearing a BAFF Tg (B6.Baff^−/−.BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff^−/− T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4⁺ cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff^−/− mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff^−/− cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff^−/− mice and correlated with MOG_35–55 peptide-induced Th17 cell responses.

Conclusions/Significance

Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases.     

Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis

Background

Various signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis.

Methods

Mice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated.

Results

Phospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis.

Conclusions

The present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0261-z) contains supplementary material, which is available to authorized users.     

Particulate matter-induced lung inflammation increases systemic levels of PAI-1 and activates coagulation through distinct mechanisms

Background

Exposure of human populations to ambient particulate matter (PM) air pollution significantly contributes to the mortality attributable to ischemic cardiovascular events. We reported that mice treated with intratracheally instilled PM develop a prothrombotic state that requires the release of IL-6 by alveolar macrophages. We sought to determine whether exposure of mice to PM increases the levels of PAI-1, a major regulator of thrombolysis, via a similar or distinct mechanism.

Methods and Principal Findings

Adult, male C57BL/6 and IL-6 knock out (IL-6^−/−) mice were exposed to either concentrated ambient PM less than 2.5 µm (CAPs) or filtered air 8 hours daily for 3 days or were exposed to either urban particulate matter or PBS via intratracheal instillation and examined 24 hours later. Exposure to CAPs or urban PM resulted in the IL-6 dependent activation of coagulation in the lung and systemically. PAI-1 mRNA and protein levels were higher in the lung and adipose tissue of mice treated with CAPs or PM compared with filtered air or PBS controls. The increase in PAI-1 was similar in wild-type and IL-6^−/− mice but was absent in mice treated with etanercept, a TNF-α inhibitor. Treatment with etanercept did not prevent the PM-induced tendency toward thrombus formation.

Conclusions

Mice exposed to inhaled PM exhibited a TNF-α-dependent increase in PAI-1 and an IL-6-dependent activation of coagulation. These results suggest that multiple mechanisms link PM-induced lung inflammation with the development of a prothrombotic state.     

Quantification of lung fibrosis and emphysema in mice using automated micro-computed tomography

Background

In vivo high-resolution micro-computed tomography allows for longitudinal image-based measurements in animal models of lung disease. The combination of repetitive high resolution imaging with fully automated quantitative image analysis in mouse models of lung fibrosis lung benefits preclinical research. This study aimed to develop and validate such an automated micro-computed tomography analysis algorithm for quantification of aerated lung volume in mice; an indicator of pulmonary fibrosis and emphysema severity.

Methodology

Mice received an intratracheal instillation of bleomycin (n = 8), elastase (0.25U elastase n = 9, 0.5U elastase n = 8) or saline control (n = 6 for fibrosis, n = 5 for emphysema). A subset of mice was scanned without intervention, to evaluate potential radiation-induced toxicity (n = 4). Some bleomycin-instilled mice were treated with imatinib for proof of concept (n = 8). Mice were scanned weekly, until four weeks after induction, when they underwent pulmonary function testing, lung histology and collagen quantification. Aerated lung volumes were calculated with our automated algorithm.

Principal Findings

Our automated image-based aerated lung volume quantification method is reproducible with low intra-subject variability. Bleomycin-treated mice had significantly lower scan-derived aerated lung volumes, compared to controls. Aerated lung volume correlated with the histopathological fibrosis score and total lung collagen content. Inversely, a dose-dependent increase in lung volume was observed in elastase-treated mice. Serial scanning of individual mice is feasible and visualized dynamic disease progression. No radiation-induced toxicity was observed. Three-dimensional images provided critical topographical information.

Conclusions

We report on a high resolution in vivo micro-computed tomography image analysis algorithm that runs fully automated and allows quantification of aerated lung volume in mice. This method is reproducible with low inherent measurement variability. We show that it is a reliable quantitative tool to investigate experimental lung fibrosis and emphysema in mice. Its non-invasive nature has the unique benefit to allow dynamic 4D evaluation of disease processes and therapeutic interventions.     

Serine protease inhibitor attenuates ovalbumin induced inflammation in mouse model of allergic airway disease

Background

Serine proteases promote inflammation and tissue remodeling by activating proteinase-activated receptors, urokinase, metalloproteinases and angiotensin. In the present study, 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) a serine protease inhibitor was evaluated for prophylactic and therapeutic treatment in mouse model of airway allergy.

Methods

BALB/c mice were sensitized by i.p route and challenged with ovalbumin. They were treated i.n. with 2, 10 and 50 µg of AEBSF, one hour before or after challenge and euthanized to collect BALF (bronchoalveolar lavage fluid), blood and lungs. Proteolytic activity, total cell/eosinophil/neutrophil count eosinophil peroxidase activity (EPO), IL-4, IL-5, IL-10, IL-13, cysteinyl leukotrienes and 8-isoprostane were determined in BALF and immunoglobulins were measured in serum. H&E and PAS stained lung sections were examined for cellular infiltration and airway inflammation.

Results

Mice exposed to ovalbumin and treated with PBS showed increased cellular infiltration in lungs and higher serum IgE, IgG1 and IgG2a levels as compared to sham mice. Treatment with AEBSF reduced total cells/eosinophil/neutrophil infiltration. Both prophylactic and therapeutic AEBSF treatment of 10 or 50 µg reduced serum IgE and IgG1 significantly (p<0.05) than control. AEBSF treatment reduced the proteolytic activity in BALF. IL-4 IL-5 and IL-13 levels decreased significantly (p<0.05) after AEBSF treatment while IL-10 levels increased significantly (p<0.05) in BALF. Airway inflammation and goblet cell hyperplasia reduced as demonstrated by lung histopathology, EPO activity and cysteinyl leukotrienes in BALF after treatment. AEBSF treatment also suppressed oxidative stress in terms of 8-isoprostane in BALF. Among the treatment doses, 10 or 50 µg of AEBSF were most effective in reducing the inflammatory parameters.

Conclusions

Prophylactic and therapeutic treatment with serine protease inhibitor attenuates the airway inflammation in mouse model of airway allergy and have potential for adjunct therapy.     

Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β

Background

Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease.

Methods

C57BL/6 wild-type (WT) and SP-D−/− mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D.

Results

Dp-challenged SP-D−/− mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D−/− mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D−/− mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D−/− mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D−/− mice.

Conclusion

These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0143-9) contains supplementary material, which is available to authorized users.     

Rac2 is involved in bleomycin-induced lung inflammation leading to pulmonary fibrosis

Background

Pulmonary fibrotic diseases induce significant morbidity and mortality, for which there are limited therapeutic options available. Rac2, a ras-related guanosine triphosphatase expressed mainly in hematopoietic cells, is a crucial molecule regulating a diversity of mast cell, macrophage, and neutrophil functions. All these cell types have been implicated in the development of pulmonary fibrosis in a variety of animal models. For the studies described here we hypothesized that Rac2 deficiency protects mice from bleomycin-induced pulmonary fibrosis.

Methods

To determine the role of Rac2 in pulmonary fibrosis we used a bleomycin-induced mouse model. Anesthetized C57BL/6 wild type and rac2 ^-/- mice were instilled intratracheally with bleomycin sulphate (1.25 U/Kg) or saline as control. Bronchoalveolar lavage (BAL) samples were collected at days 3 and 7 of treatment and analyzed for matrix metalloproteinases (MMPs). On day 21 after bleomycin treatment, we measured airway resistance and elastance in tracheotomized animals. Lung sections were stained for histological analysis, while homogenates were analyzed for hydroxyproline and total collagen content.

Results

BLM-treated rac2 ^-/- mice had reduced MMP-9 levels in the BAL on day 3 and reduced neutrophilia and TNF and CCL3/MIP-1α levels in the BAL on day 7 compared to BLM-treated WT mice. We also showed that rac2 ^-/- mice had significantly lower mortality (30%) than WT mice (70%) at day 21 of bleomycin treatment. Lung function was diminished in bleomycin-treated WT mice, while it was unaffected in bleomycin-treated rac2 ^-/- mice. Histological analysis of inflammation and fibrosis as well as collagen and hydroxyproline content in the lungs did not show significant differences between BLM-treated rac2 ^-/- and WT and mice that survived to day 21.

Conclusion

Rac2 plays an important role in bleomycin-induced lung injury. It is an important signaling molecule leading to BLM-induced mortality and it also mediates the physiological changes seen in the airways after BLM-induced injury.     

Phenotypic overlap between MMP-13 and the plasminogen activation system during wound healing in mice

Background

Proteolytic degradation of extracellular matrix is a crucial step in the healing of incisional skin wounds. Thus, healing of skin wounds is delayed by either plasminogen-deficiency or by treatment with the broad-spectrum metalloproteinase (MP) inhibitor Galardin alone, while the two perturbations combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-edge keratinocytes of skin wounds, which may account for this phenotypic overlap between these classes of proteases.

Methodology

To further test that hypothesis we generated Mmp13;Plau and Mmp13;Plg double-deficient mice in a cross between Mmp13- and Plau-deficient mice as well as Mmp13- and Plg-deficient mice. These mice were examined for normal physiology in a large cohort study and in a well-characterized skin wound healing model, in which we made incisional 20 mm-long full-thickness skin wounds.

Principal Findings

While mice that are deficient in Mmp13 have a mean healing time indistinguishable to wild-type mice, wound healing in both Plau- and Plg-deficient mice is significantly delayed. Histological analysis of healed wounds revealed a significant increase in keratin 10/14 immunoreactive layers of kerationcytes in the skin surface in Mmp13;Plau double-deficient mice. Furthermore, we observe, by immunohistological analysis, an aberrant angiogenic pattern during wound healing induced by Plau-deficiency, which has not previously been described.

Conclusions

We demonstrate a phenotypic overlap, defined as an additional delay in wound healing in the double-deficient mice compared to the individual single-deficient mice, between MMP-13 and the plasminogen activation system in the process of wound healing, but not during gestation and in postnatal development. Thus, a dual targeting of uPA and MMP-13 might be a possible future strategy in designing therapies aimed at tissue repair or other pathological processes, such as cancer invasion, where proteolytic degradation is a hallmark.     

IL-17RA signaling amplifies antibody-induced arthritis

Objective

To investigate the role of IL-17RA signaling in the effector phase of inflammatory arthritis using the K/BxN serum-transfer model.

Methods

Wild-type and Il17ra^−/− mice were injected with serum isolated from arthritic K/BxN mice and their clinical score was recorded daily. Mice were also harvested on days 12 and 21 and ankles were analyzed for cytokine and chemokine mRNA expression by qPCR on day 12 and for bone and cartilage erosions by histology on day 21, respectively. The induction of cytokine and chemokine expression levels by IL-17A in synovial-like fibroblasts was also analyzed using qPCR.

Results

Il17ra^−/− mice were partially protected from clinical signs of arthritis and had markedly fewer cartilage and bone erosions. The expression of several pro-inflammatory mediators, including the chemokines KC/CXCL1, MIP-2/CXCL2, LIX/CXCL5 MIP-1γ/CCL9, MCP-3/CCL7, MIP-3α/CCL20, the cytokines IL-1β, IL-6, RANKL and the matrix metalloproteinases MMP2, MMP3, and MMP13 were decreased in the ankles of Il17ra^−/− mice compared to wild-type mice. Many of these proinflammatory genes attenuated in the ankles of Il17ra^−/− mice were shown to be directly induced by IL-17A in synovial fibroblasts in vitro.

Conclusions

IL-17RA signaling plays a role as an amplifier of the effector phase of inflammatory arthritis. This effect is likely mediated by direct activation of synovial fibroblasts by IL-17RA to produce multiple inflammatory mediators, including chemokines active on neutrophils. Therefore, interrupting IL-17RA signaling maybe a promising pharmacological target for the treatment of inflammatory arthritis.     

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis

Background

Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis.

Methods

Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY^−/−) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice.

Results

DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY^−/− as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS^−/− and NPY^−/−/nNOS^−/− mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY –treated rat enteric neurons in vitro exhibited increased nitrite and TNF-α production.

Conclusions

NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.     

Rapamycin combined with anti-CD45RB mAb and IL-10 or with G-CSF induces tolerance in a stringent mouse model of islet transplantation

Background

A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3⁺Treg and IL-10–producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell–associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent.

Methodology/Principal Findings

Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3⁺Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4⁺IL-10⁺IL-4⁻ T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4⁺IL-10⁺IL-4⁻ T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4⁺IL-10⁺IL-4⁻ T cells.

Conclusions/Significance

The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic.