Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Inhibitor as a Novel Therapeutic Tool for Lung Injury

Abstract

Pulmonary fibrosis is a progressive disease characterized by lung remodeling due to excessive deposition of extracellular matrix. In this study, the bleomycin experimental model of pulmonary fibrosis was employed to investigate the anti-fibrotic and immunomodulatory activity of the inhibition of MALT1 protease activity. Mice received a single intra-tracheal administration of bleomycin (1 mg/kg) in the presence or absence of MI-2, a selective MALT1 inhibitor, (a dose of 30 mg/kg administered intra-peritoneally 1 h after bleomycin and daily until the end of the experiment). Seven days after bleomycin instillation mice were sacrificed and bronchoalveolar lavage fluid analysis, measurement of collagen content in the lung, histology, molecular analysis and immunohistochemistry were performed. To evaluate mortality and body weight gain a subset of mice was administered daily with MI-2 for 21 days. Mice that received MI-2 showed decreased weight loss and mortality, inflammatory cells infiltration, cytokines overexpression and tissue injury. Moreover, biochemical and immunohistochemical analysis displayed that MI-2 was able to modulate the excessive production of reactive oxygen species and the inflammatory mediator upregulation induced by bleomycin instillation. Additionally, MI-2 demonstrated anti-fibrotic activity by reducing transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA) and receptor associated factor 6 (TRAF6) expression. The underlying mechanisms for the protective effect of MI-2 bleomycin induced pulmonary fibrosis may be attributed to its inhibition on NF-κB pathway. This is the first report showing the therapeutic role of MALT1 inhibition in a bleomycin model of pulmonary fibrosis, thus supporting further preclinical and clinical studies.

1. Introduction

Persistent airway stimulation from nonantigenic and antigenic sources leads to pulmonary fibrosis, producing progressive remodeling of lung architecture by deposition of collagen [1,2,3]. At early stages tissue repair has positive effects on the histological alteration induced by pathology, but prolonged connective tissue deposition produces scarring, fibrosis and loss of organ functionality [4].

Fibrotic lungs show enriched area of proliferating myofibroblasts and fibroblasts with excessive extracellular matrix deposition (ECM), which in turn produce respiratory failure and compromise gaseous exchange [5]. Thanks to its pulmonary toxicity, administration of bleomycin is a widely used model of pulmonary fibrosis. This model is characterized by increased inflammatory cell response, collagen deposition and T lymphocyte activation. In particular, seven days after bleomycin-induced injury inflammation is accompanied with fibroproliferative activity. Moreover, it modifies the antioxidant/oxidant balance and increased lipid peroxidation. Up-regulated production of reactive oxygen species (ROS) leads to nitrosative stress and DNA damage. While new therapeutic strategies continue to be developed, enhancing our understanding of the immunologic foundation responsible for pulmonary fibrosis remains a crucial point. Recently, mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) has been described as a crucial modulator of immune cell function [6] thanks to its proteolytic activity [7,8]. Many MALT1 substrates are implicated in the regulation of the inflammatory answers, its protease activity is emerging as therapeutic tool [9,10].

Recently, different studies present MALT1 as modulator of T cell activation. In particular, MALT1 proteolytic activity is activated after T cell stimulation [10]. Additionally, MALT1 together with caspase-recruitment domain containing membrane-associated guanylate kinase protein 1 (CARMA1) and B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10) forms a complex which manages the recruitment of (receptor associated factor 6) TRAF6 and induce the activation of several transcription factors such as NF-kB, which is involved in the bleomycin-induced lung injury [8,11].

Recent evidences underline the important role of MALT1 in alveolar macrophages and its ability to manage their functions in respiratory disease [12], suggesting that pharmacological inhibition of MALT1 protease activity may be an effective strategy for preventing/treating sepsis associated lung injury. Moreover, compound MI-2 has been indicated as a MALT1 inhibitor, binding the active site of MALT1 [13]. In particular, it has been described that the administration of MI-2 can selectively enhance the expression of myeloid monocyte chemotactic protein-inducible protein 1 (MCPIP1) and protected mice from LPS-induced inflammation, lung injury and death [14].

In order to evaluate the role of MALT1 in the pulmonary fibrosis pathway, we used an animal model of bleomycin induced lung injury and the MALT1 inhibitor MI-2.

2. Results

2.1. MI-2 Improved Histopathological Score and Reduced Mortality and Weight Loss Bleomycin-Induced

Seven days after bleomycin administration, histological analysis of lung samples harvested from vehicle-treated mice showed tissue injury and extracellular matrix deposition (Figure 1B,E), as compared to sham animals (Figure 1A,E). MI-2 (Figure 1C,E) and mepazine (Figure 1D,E) treatment reduced lung damage, as showed by hematoxylin and eosin staining, and pulmonary edema, as showed by wet/dry weight ratio (Figure 1F). Additionally, MI-2 and mepazine were able to reduce neutrophils activity, as showed by MPO assay (Figure 1G). A subset group of mice was than observed for twenty-one days after bleomycin instillation. MI-2 and mepazine administration reduced mortality (Figure 1H) and body weight loss (Figure 1I) associated with pulmonary fibrosis.

2.2. MI-2 Reduced Mast Cells Infiltration and Degranulation Bleomycin-Induced

Seven days after bleomycin injection an augmented number of infiltrating mast cells was detected in lung samples harvested from vehicle treated mice (Figure 2B,D), as compared to the sham tissue group (Figure 1A,D). MI-2 injections reduced mast cells recruitment in lung tissue (Figure 2C,D). Additionally, it was able to decrease chymase activity (Figure 2E), which is the major proteins secreted by mast cells and promote inflammation and matrix destruction.

2.3. MI-2 Suppressed T Lymphocyte Infiltration Bleomycin-Induced

Seven days after bleomycin instillation vehicle-treated animals showed an increased number of CD4 and CD8 positive cells (Figure 3B,D,F,H), as compared to sham animals (Figure 3A,D,E,H). In MI-2 treated animals these numbers were decreased (Figure 3C,D,G,H).

2.4. MI-2 Reduced Inflammatory Cells Recruitment and Cytokines Overexpression Bleomycin-Induced

Seven days after bleomycin instillation vehicle treated mice showed inflammatory cells recruitment in the bronchoalveolar lavage fluid, as compared to the sham group (Figure 4A).

In particular, we evaluated neutrophils, macrophages, eosinophils and lymphocytes detecting a significant rise in cell numbers (Figure 4B). MI-2 reduced the inflammatory cells infiltration in the bronchoalveolar lavage fluid (Figure 4A,B). Moreover, it was able to reduce IL-1β, TNF-α and IL-6 over-expressions in both bronchoalveolar lavage fluid (Figure 4C–E) and lung tissues (Figure 4F–H) associated with pulmonary fibrosis.

2.5. MI-2 Reduced Oxidative Stress Bleomycin-Induced

To test whether MI-2 may modulate the oxidative stress induced by bleomycin administration antioxidant enzymes activity were measured. Vehicle-treated mice showed decreased SOD (Figure 5A) and GSH (Figure 5B) activity, as compared to sham mice. Additionally, they showed increased lipid peroxidation (Figure 5C) and NO content (Figure 5D). MI-2 restored SOD and GSH levels, while decreased lipid peroxidation and NO production in the bronchioalveolar lavage fluid. In order to evaluate nitrosative stress and DNA damage immunohistochemical analysis for nitrotyrosine and PARP expression were performed. No positive staining of both antibodies was detected in sham group (Figure 5E,H,I,L). Vehicle treated mice showed nitrotyrosine (Figure 5F,H) and PARP (Figure 5J,L) increased expression, while MI-2 reduced them immunostaining (Figure 5G,H,K,L).

2.6. MI-2 Reduced Lung Fibrotic Changes Bleomycin-Induced

Pulmonary injury produced by bleomycin is characterized by increased collagen deposition and severe fibrosis. Masson trichrome staining showed increased blue area in tissues from vehicle-treated mice (Figure 6B), as compared to sham tissue (Figure 6A). MI-2 reduced fibrosis and collagen deposition as showed by masson trichrome (Figure 6C) and sircol soluble collagen assay (Figure 6D). In order to evaluate fibrosis, we also performed immunohistochemical analysis for TGF-1β and α-sma expression. No positive staining of both antibodies was detected in sham group (Figure 6E,H and Figure 7A,D). Vehicle treated mice showed TGF-1β (Figure 6F,H) and α-sma (Figure 7B,D) increased expression, while MI-2 reduced them immunostaining (Figure 6G,H and Figure 7B,C).

The levels of total TGF-β in BAL fluid also significantly increased in the vehicle treated mice as compared with sham mice; however, treatment with MI-2 did not decrease these levels (Figure 6I). The levels of active TGF-β in the BAL fluid were also significantly elevated in the vehicle treated mice as compared with the sham group. Interestingly, treatment with MI-2 significantly reduced these levels (Figure 6J).

Western blot analysis confirmed the increased expression of α-sma in vehicle treated animals and its reduced levels following MI-2 treatment (Figure 7E). Additionally, vehicle-treated mice showed increased TRAF6 expression, as compared to the sham animals. Samples harvested from MI-2 mice displayed reduced TRAF6 expression (Figure 7F).

2.7. MI-2 Managed the MCPIP1 and NF-kB Pathways Bleomycin-Induced

To further investigate the MALT1 proteolitic activity we evaluated the MCPIP1 protein degradation in the lungs. Western blot analysis showed that the MCPIP1 protein levels were markedly decreased in lungs from vehicle-treated mice compared with those from control mice. MI-2 treatment restored MCPIP1 protein levels (Figure 8A), while the MALT1 protein levels was not changed by any of these treatments (Figure 8B).

In order to evaluate the pathway behind inflammation bleomycin-induced we evaluated the Ikb-α/NF-kB expressions by western blot analysis. Vehicle-treated mice showed increased reduced Ikb-α expression, as compared to sham animals (Figure 8C). Additionally, samples from vehicle group showed increased NF-kB expression into the nucleus (Figure 8D). MI-2 administration restored Ikb-α expression in cytoplasm and reduced NF-kB expression into the nucleus.

3. Discussion

Our experiments have demonstrated, for the first time, that MI-2, a MALT-1 inhibitor, can protect from bleomycin-induced pulmonary fibrosis based on the the following results: MI-2 reduced lung damage, pulmonary edema, inflammatory cells infiltration in both bronchoalveolar lavage fluid and lung tissues, cytokines over-expression, production of free oxygen radicals, collagen deposition, fibrosis, changes in α-sma, an EMT related gene, and TGF-β expression. Crucially, those effects were accompanied with MI-2-mediated suppression on infiltration of T lymphocyte and modulation of TRAF6, Ikb-α/NF-kB pathways. Bleomycin administration induces alveolar epithelial cell injury and pan-alveolitis [15], modifying of lung architecture [16]. MI-2 administration for seven consecutive days ameliorated the histological changes produced by pulmonary fibrosis and counteracted the body weight loss and mortality related with this pathology. Additionally, it reduced the inflammatory cells recruitment in the injured airways, both in lung tissues and bronchoalveolar lavage fluid. In particular, we provided some side-by-side comparison with an alternative MALT1 inhibitor, mepazine, to support better the aim and interpretation of the study. However, it did not rule out that off-target activities of MI-2 might have contributed to its overall in vivo efficacy.

Seven days from bleomycin administration MI-2 reduced MPO and chymase activity, showing a decreased infiltration of neutrophils and mast cells in the lung tissues. Infiltration of T lymphocyte, a key cellular inflammatory response, plays a key role in the pathophysiology of pulmonary fibrosis [17]. MI-2 reduced lung tissue sequestration of T lymphocytes, as showed by the reduced number of CD4 and CD8 positive cells. Another prominent characteristic of the bleomycin induced pulmonary fibrosis is the cytokines and chemokines overexpression [18] and particular relevance has been attributed to TNF-α, IL-1β and IL6 [19]. Seven days from bleomycin instillation bronchoalveolar lavage fluid and lung tissues showed increased expression of these inflammatory mediators and MI-2 treatment reduced them overexpression. Several evidences reported important modification in the cellular redox state in animal models and patients affected by pulmonary fibrosis. Bleomycin administration produced increased of oxidative stress and free oxygen radicals (ROS) production [20]. MI-2 restored homeostasis in the anti-oxidant/oxidant balance, increasing SOD and GSH activity and reducing lipid peroxidation. Animals subjected to pulmonary fibrosis also exhibited NO over-production [21]. NO combined with ROS produce of highly reactive mediators such as peroxynitrite and other stress nitrosative derivatives. Nitrotyrosine formation is a widely used marker for nitrosative stress. MI-2 was able to reduce NO content of bronchoalveolar lavage fluid and to reduce nitrotyrosine expression in lung tissues. ROS and nitrogen species mediate DNA damage activating DNA repair mechanisms such as nuclear enzyme PARs. MI-2 reduced DNA damage as showed by the reduced expression of PARP in lung tissues of treated mice. There is a complex relationship between chronic inflammation, lung injury and epithelial to mesenchymal transition (EMT). Bleomycin administration mediates fibrogenic events as showed by the increased collagen deposition and fibrosis in lungs. EMT contribute to collagen-producing fibroblasts in pulmonary fibrosis [22,23]. TGF-β1 is a crucial mediator in the trans-differentiation of epithelial cells into cells with myofibroblast or fibroblast properties, a process involved into fibroproliferative disease [24]. In particular, myofibroblast differentiation is characterized by α-smooth muscle actin (α-SMA) expression [25]. Injured lung cells showed increased expression of TGF-β [26] which drives differentiation of neighboring cells into collagen-producing fibroblast. Several studies show the key role of TRAF6 in the TGF-β induced EMT [27]. MI-2 reduced fibrosis, TGF-β, α-SMA and TRAF6 expression.

In order to evaluate the pharmacological inhibition of MALT1 by MI-2 we evaluated the MCPIP1 protein levels in lung tissues from different groups. Bleomycin administration reduced MCPIP1 expression in lung tissues from vehicle treated mice, as compared to the sham mice. MI-2 treatment by inhibiting MALT1 activity increase MCPIP1 protein levels. Taken together, these results demonstrate that the MCPIP1 protein levels in the lungs are preserved by pharmacological inhibition of MALT1 protease. Administration of MI-2 protects mice from lung injury and fibrosis.

The increased expression of cytokines, chemokines and inflammatory mediators produce changes in transcription factors [28]. In particular, pulmonary inflammation and fibrosis has been linked with the activation of the NF-kB pathway. It enhance the epithelial–mesenchymal transition and fibrosis and in response to chronic inflammation [29]. NF-kB is usually stored in cytoplasm firmed to the IκBα inhibitor. Chronic inflammatory stimuli induce the phosphorylation of the inhibitor and release NF-kB which translocates into the nucleus [30]. MI-2 administration reduced NF-kB nuclear expression and IκBα cytoplasmic expression.

4. Materials and Methods

4.1. Animals

Male CD1 mice (Envigo, Milan, Italy) were housed in a controlled location, with food and water ad libitum. The University of Messina Review Board for animal care (OPBA) approved the study (9 February 2017, 137/2017-pr). All in vivo experiments followed the new directives of USA, Europe, Italy and the ARRIVE guidelines.

4.2. Induction of Lung Injury

Bleomycin administration was performed as previously described [19]. Bleomycin sulphate (0.1 lU per mouse) was delivered by an intratracheal administration [31]. One hour after surgery MI-2 (30 mg/kg in DMSO) was given intraperitoneally [12]. The treatment was repeated daily. Seven days from bleomycin administration animals were euthanized and tissues were harvested for further analysis.

4.3. Experimental Groups

Mice were randomized into experimental groups (n = 10):

• Bleomycin + vehicle group. Mice received bleomycin administration and were treated daily with vehicle (DMSO);

• Bleomycin + MI-2 group. Mice received bleomycin administration and were treated daily with MI-2 (30 mg/kg);

• Bleomycin + mepazine group. Mice received bleomycin administration and were treated daily with Mepazine (30 mg/kg);

• Sham + mepazine group. Identical to the bleomycin + Mepazine group but animals received intratracheal instillation of saline (0.9% w/v) instead of bleomycin, and were treated daily with mepazine (30 mg/kg);

• Sham + vehicle group. Identical to the bleomycin + vehicle group but animals received intratracheal instillation of saline (0.9% w/v) instead of bleomycin, and were treated daily with vehicle (DMSO).

• Sham + MI-2 group. Identical to the bleomycin + MI-2 group but animals received intratracheal instillation of saline (0.9% w/v) instead of bleomycin and were treated daily with MI-2 (30 mg/kg).

The dose of MI-2 was selected based on previous experiments [32].

4.4. Survival Rate and Body Weight Gain

Mortality and body weight were assessed daily up to 21 days.

4.5. Bronchoalveolar Lavage (BAL)

Seven days after bleomycin instillation, mice were sacrificed, the tracheas cannulated and the lavage performed as previously described [33]. 0.5 mL Dulbecco’s phosphate-buffered saline (PBS) (GIBCO, Paisley, UK) was employed. The BAL fluid recovered was spun, the pelleted cells collected and the supernatants stored at −20 °C. Using a hemocytometer total BAL cells were counted in presence of trypan blue [34].

4.6. Measurement of Lung Edema

After 7 days from injection of bleomycin wet lung weights were recorded. Subsequently, they were dried for 48 h at 180 °C and then weighed again. Water content of the lungs was calculated as the ratio of wet: dry weight of the tissue.

4.7. Histological Examination

Lung tissues samples were collected 7 days from bleomycin injection. Tissues were fixed in buffered formaldehyde solution (10% in PBS), histological sections were stained with haematoxylin and eosin and Masson Trichrome and evaluated using a Leica DM6 microscope (Leica Microsystems SpA, Milan, Italy) associated with Leica LAS X Navigator software (Leica Microsystems SpA). The severity of lung fibrosis was scored on a scale from 0 to 8 as already published [19]. Lung sections were stained with toluidine blue to enumerate mast cells [35].

4.8. Measurement of Chymase Activity

Lung tissues were homogenized and the chymase activity was measured in the supernatant according the method described by Pasztor et al. [36,37].

4.9. MPO Assay

MPO activity was determined as already described [19,38]. It was defined as the quantity of enzyme degrading 1 μmol of peroxide per min at 37 °C and was expressed in units per gram wet tissue weight.

4.10. Soluble Collagen Assay

The Sircol Soluble Collagen Assay (Biocolor, Newtownabbey, Northern Ireland), was performed following the manufacturer’s instructions [39].

4.11. Enzyme-Linked Immunosorbent Assays (ELISA)

The bronchoalveolar fluid lavage (BALF) IL,6 IL-1β, TNF-α and TGF-β levels were detected by using ELISA kits (Dakewe, Shenzhen, China; Biosource International, Camarillo, CA, USA; R&D, Minneapolis, MN, USA) [40,41,42]. Levels of IL6 IL-1β and TNF-α in lung tissues were measured by using ELISA kits (Calbiochem-Novabiochem Corporation, San Diego, CA, USA) [43,44].

4.12. Nitric Oxide (NO) Analysis

The pulmonary production of NO in the BALF was analyzed with a nitrate/nitrite colorimetric assay [45]. The absorbance was measured at 550 nm using a plate absorbance reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).

4.13. Measurement of Oxidative Stress

The malondialdehyde (MDA) [46], glutathione (GSH) and superoxide dismutase (SOD) levels in the lung tissues were measured using activity assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) [40].

4.14. Western Blot Analysis

Western blots were performed as described from our previous studies [47,48]. Specific primary antibody: anti-IkBα (Santa Cruz Biotechnology, Dallas, TX, USA) or anti-NF-kB p65 (Santa Cruz Biotechnology) or anti-TRAF6 (Santa Cruz Biotechnology) or anti-αsma (Santa Cruz Biotechnology) or anti-MCPIP1 (Santa Cruz Biotechnology) or anti-MALT1 (Santa Cruz Biotechnology) were mixed in 1× PBS, 5% w/v nonfat dried milk, 0.1% Tween-20, and incubated at 4 °C, overnight. After, blots were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA, USA) for 1 h at room temperature. To verify that membranes were loaded with equal amounts of protein, they were also incubated with the antibody against laminin (1:1000; Santa Cruz Biotechnology) and GADPH (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent according to manufacturer’s instructions (Super- Signal West Pico Chemiluminescent Substrate, Pierce, Altrincham, UK). The relative expression of the protein bands was quantified by densitometry with ChemiDoc XRS software (Bio-Rad, Perth, UK) and standardized tob-actin levels. Images of blot signals (8-bit/600-dpi) were imported to analysis software (Image Quant TL, v2003, Altrincham, UK).

4.15. Immunohistochemical Localization of Transforming Growth Factor-B (TGF-β), α-Smooth Muscle Actin (α-sma), Poly(ADP-Ribose Polymerase) (PARP), Nitrotyrosine, CD4 and CD8

Immunohistochemical analysis was performed as already described [49]. The sections were incubated overnight with primary antibodies: anti-TGF-β antibody (1:250, Santa Cruz Biotechnology), anti-(α-sma antibody (1:250, Santa Cruz Biotechnology), anti-nitrotyrosine antibody (1:500, Millipore), anti-PARP antibody (1:250, Santa Cruz Biotechnology), anti-CD4 (1:250, Santa Cruz Biotechnology) and anti-CD8 (1:250, Santa Cruz Biotecnology). All sections were washed with PBS and then treated as previously reported [50]. Stained sections from each mouse were scored in a blinded fashion and observed using a Leica DM6 microscope (Leica Microsystems SpA) following a typical procedure [51]. The histogram profile is related to the positive pixel intensity value obtained [52]. The number of positive cells was counted in three sections per animal and presented as the number of positive cells per high-power field.

4.16. Materials

All compounds used in this study were purchased from Sigma-Aldrich Company Ltd. (Milan, Italy) and were of the highest commercial grade available. MI-2 were purchased from DBA (Milan, Italy).

4.17. Statistical Evaluation

All values in the figures and text are expressed as mean ± standard error of the mean (SEM) of n number of animals. Results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value < 0.05 was considered significant. * p < 0.05 vs. sham, ^# p < 0.05 vs. vehicle, ** p < 0.01 vs. sham, ^## p < 0.01 vs. vehicle, *** p < 0.001 vs. sham, ^### p < 0.001 vs. vehicle.

5. Conclusions

In conclusion, our data demonstrate that inhibition of the protease activity of MALT1 may a viable strategy for the treatment of pulmonary fibrosis, suggesting MALT1 as potential therapeutic target against lung injury.

Author Contributions

Conceptualization, S.C. and R.D.P.; Methodology, D.I.; Software, M.C.; Validation, M.C., R.S. and E.G.; Formal Analysis, R.C. and T.G.; Investigation, A.F.P.; Resources, R.F.; data curation, R.D.; writing—original draft preparation, R.F.; writing—review and editing, R.D.P.; visualization, E.G.; supervision, R.D.P.; project administration, S.C.; funding acquisition, S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

BAL	Bronchoalveolar lavage
BALF	Bronchoalveolar fluid lavage
GSH	Glutathione
H&E	Haematoxylin and eosin
MALT1	Mucosa-associated lymphoid tissue lymphoma translocation 1
MDA	Malondialdehyde
MPO	Myeloperoxidase
NFkB	Nuclear factor-κB
NO	Nitric oxide
PMN	Polymorphonuclear leukocytes
ROS	Reactive oxygen species
SOD	Superoxide dismutase

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Figures

Figure 1. Effect of MI-2 on histological changes, lung edema, MPO activity, survival rate and body weight: hematoxylin and eosin staining of sham (A), vehicle (B), MI-2 (C), Mepazine (D), histological injury score (E), wet/dry lung weight ratio (F), MPO activity (G), survival rate (H), body weight gain (I). For each analysis, n = 10 animals from each group were employed.

Figure 2. Effect of MI-2 on mast cells analysis: sham (A), vehicle (B), MI-2 (C), mast cells count (D); chymase activity (E). For each analysis, n = 10 animals from each group were employed.

Figure 3. Effect of MI-2 on CD4 and CD8 expression: Immunohistochemical analysis of CD4: sham (A), vehicle (B), MI-2 (C), number of positive cells/field (D), immunohistochemical analysis of CD8: sham (E), vehicle (F), MI-2 (G), number of positive cells/field (H). For each analysis, n = 10 animals from each group were employed.

Figure 4. Effect of MI-2 on cells infiltration and proinflammatory cytokines over-expression: total cells (A); macrophages, neutrophils, lymphocytes and eosinophils (B); cytokines expression in bronchoalveolar lavage fluid: TNF-α (C), IL-1β (D), IL6 (E); cytokines expression in lung tissues: TNF-α (F), IL-1β (G), IL6 (H). For each analysis, n = 10 animals from each group were employed.

Figure 5. Effect of MI-2 on oxidative stress: SOD (A), GSH (B), MDA (C), NO content (D), immunohistochemical analysis of nitrotyrosine expression: sham (E), vehicle (F), MI-2 (G), % of positive pixels (H), immunohistochemical analysis of PARP: sham (I), vehicle (J), MI-2 (K), % of positive pixels (L). For each analysis, n = 10 animals from each group were employed.

Figure 6. Effect of MI-2 on collagen deposition and TGF-β expression: Masson trichrome staining: sham (A), vehicle (B), MI-2 (C), sircol soluble collagen assay (D), immunohistochemical analysis of TGF-1β expression: sham (E), vehicle (F), MI-2 (G), % of positive pixels (H), concentration of total TGF-β (I) and active TGF-β (J) in BAL fluids. For each analysis, n = 10 animals from each group were employed.

Figure 7. Effect of MI-2 on α-sma and TRAF6 expression: Immunohistochemical analysis of α-sma: sham (A), vehicle (B), MI-2 (C), % of positive pixels (D). Western blot analysis of α-sma (E) and TRAF6 (F). For each analysis, n = 10 animals from each group were employed.

Figure 8. Effect of MI-2 on MCPIP1, Ikb-α, NF-kB expression: Western blot analysis of MCPIP (A), MALT1 (B), Ikb-α (C), and NF-kB (D). For each analysis, n = 10 animals from each group were employed.