Targeting TLR2 Attenuates Pulmonary Inflammation and Fibrosis by Reversion of Suppressive Immune Microenvironment¹

Pulmonary fibrosis, defined as a wound-healing response that has gone out of control after lung injury, is a high-mortality and progressive disease characterized by the loss of alveolar structure through the apoptosis of epithelial and endothelial cells, proliferation of fibroblasts, and excessive deposition of extracellular matrix (1). Although pulmonary fibrosis may result from a wide variety of insults, including infection, oxidative stress, radiation, and chemotherapeutic agents, the pathogenesis of pulmonary fibrosis remains an enigma. Accumulated evidence indicates that persistent or repeated insulting stimuli sustain the tissue wound-healing process (2). The host’s response to these insults, which is primarily mediated by the innate immune system, is intricately directed toward the recognition of pathogen-associated molecular patterns (PAMPs),⁴ produced by pathogenic organisms, or damage-associated molecular patterns (DAMPs) released from damaged tissues through interactions with pattern recognition receptors (i.e., TLRs) (3, 4). This host response to tissue damage leads to the recruitment of inflammatory cells and the subsequent accumulation of extracellular matrix as a consequence of chronic epithelial injury and failure of repair due to aberrant epithelial-mesenchymal interactions (4). The several immune and nonimmune cell types in the damaged tissue contribute to the chronic inflammatory response and tissue remodeling through secretion of growth factors, cytokines, chemokines, and activation of extracellular matrix-synthesizing processes (2). If PAMPs and DAMPs are effectively cleared, the affected tissue often heals appropriately and the inflammatory process resolves. However, if PAMPs and DAMPs persist in the host tissue, the Th1 immune response pattern is often skewed toward the Th2 response pattern that recruits more inhibitory immune cells (e.g., regulatory T cells (Tregs) and M2 cells) to produce inhibitory immune factors in the damaged tissue and to establish an immunosuppressive tissue microenvironment during the progression of wound healing (5). The resulting immunosuppressive microenvironment in turn facilitates the persistence of PAMPs as well as DAMPs, thereby allowing this vicious cycle to continue (6).

Obviously, these observations strongly argue the rationale for current treatments of pulmonary fibrosis using a group of anti-inflammatory agents and immunosuppressive agents, including corticosteroids and cytotoxical agents (7). Indeed, a great number of studies indicate that suppressing the immune response is unlikely to be a good approach to treat the fibrotic tissue damage that occurs during chronic inflammation (8). Alternatively, strategies directed toward eliminating immunosuppressive cells and cytokines might prove highly beneficial in the context of tissue fibrosis during chronic inflammation, as their elimination would presumably diminish the concomitant chronic inflammatory and fibrotic mechanisms (9). For example, we recently found that either the vaccine bacillus Calmette-Guérin or adjuvant administration of TLR4 agonist can protect experimental hypertensive mice from pressure overload-induced cardiovascular fibrosis via regulation of cardiovascular immune microenvironment (10).

Based on Razonable’s recent work (11) and our recent findings that bleomycin (BLM) is an agonist of TLR2, which mediates BLM-induced inflammation and lung injury, we tested the hypothesis that TLR2 not only mediates BLM-induced systemic inflammation and lung injury but also mediates BLM-induced suppressive immune microenvironments, which is critical to the development of pulmonary fibrosis. We found that TLR2 deficiency or targeting TLR2 not only prevents BLM-induced inflammation, but that it also protects from and reverses progressive pulmonary fibrosis through a reversion of the immunosuppressive microenvironments in the BLM-caused fibrotic tissue. Our results demonstrate that TLR2 is a promising target for the prevention and treatment of pulmonary fibrosis and that targeting immunosuppressive microenvironments using immunostimulants such as TLR2 antagonist is a novel therapeutic strategy for the life-threatening illness of pulmonary fibrosis.

Male C57BL/6J mice (17 ± 1 g, 6–8 wk) were obtained from the Vital River Laboratory Animal Technology. TLR2^−/− and corresponding wild-type (WT) mice were purchased from The Jackson Laboratory. Ultra-pure Ec-LPS (from Escherichia coli) and Pam3Cys were obtained from InvivoGen. FITC-, PE-, or PE-cy5-conjugated anti-mouse CD11c, MHCII, CD40, CD80, CD86, TLR2 Abs (mAb), and Stat3 inhibitory peptide were purchased from eBioscience. The neutralizing TLR2 mAb was purchased from R&D System. The ELISA kits for IFN-γ and TGF-β1 were purchased from eBioscience. The ELISA kit for HMGB1 was purchased from Adlitteram Diagnostic Laboratories. The BLM was purchased from Nippon Kayaku. Smad 3 inhibitor (SIS3) was from Merck-Calbiochem. The endotoxin level in the solutions of BLM and neutralizing Abs was <0.01 ng/ml tested by the Limulus amebocyte lysate assay (BioWhittaker). All other materials were purchased from standard commercial resources.

The mice were anesthetized with 50 mg/kg i.p. pentobarbital (Merck,). Using an insulin syringe, 50 μl of LPS-free saline or clinical grade BLM (3.0 U/kg) was directly injected into the trachea as previously described (12). The neutralizing anti-TLR2 or isotype-matched Ab (2 μg/mouse) in 200 μl saline was injected i.v. 1 day before, or on days 7 and 14 after BLM instillation. Mice were then sacrificed by excessive anesthesia for the collection of single-cell suspensions, bronchoalveolar lavage fluid (BALF), and lungs at different times. The lungs were excised and fixed or frozen for morphological evaluation or for the measurement of hydroxyproline content. The lung index was determined by lung weight (mg)/body weight (g).

Mouse immature DCs (CRL-11904; ATCC) were maintained in α-MEM, supplemented with 20% FBS (BioWhittaker), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.05 mM 2-ME, 100 U/ml penicillin and streptomycin, and 5 ng/ml mouse recombined GM-CSF as previously described (13). To evaluate the regulatory effects of BLM on the phenotype and activity of DCs, immature DCs were cultured in 6-well plate in a density of 5 × 10⁵ cell/ml and BLM (20 μg/ml) was added. To investigate the role of TLR2 in BLM-induced DC maturation, anti-TLR2 or isotype-matched Ab (10 μg/ml) was added 1 h before BLM addition. The neutralizing effects of anti-TLR2 Ab were previously proven (14).

Single-cell suspensions were prepared from murine lungs as previously described (7) with minor modification. Briefly, lungs were inflated with dispase II, allowed to collapse, and then placed in 1 ml of dispase II, while gently agitating at room temperature for 45 min. Lungs were minced to ∼1-mm pieces and resuspended in 2 ml of dispase II containing collagenase IV (2 μg/ml) and DNase (50 μg/ml). Digested lungs were resuspended in DMEM supplemented with 10% FBS and sequentially filtered through 200-μm filters. The expression of various surface molecules and the number of immune cells, such as marrow-derived DCs (mDCs), plasmocytoid DCs (pDCs), M1 type of macrophages (M1 cells), and Treg cells were then analyzed using the lung single-cell suspensions.

Surface molecule expression of lung cells and cultured DCs were analyzed using multicolor flow cytometry as described previously (15). In brief, lung cells and cultured DCs were first harvested, washed, and suspended in cold PBS containing 3% FBS and 0.02% NaN₃. The cells were then incubated with a mixture of rat and mouse IgG (1:1) to reduce nonspecific binding followed by serial incubations with saturating concentrations of FITC-conjugated mAb, PE-conjugated mAb, and/or PE-cy5-conjugated mAb for 1 h at 4°C. Isotype-matched mAb were used in control samples. After incubation, at least 20,000 stained cells were analyzed using CellQuest software (BD Biosciences). In addition, the levels of various cytokines, such as IL-6, IL-10, IL-13, and TGF-β1, were determined by an intracellular staining method as previously described (8). To prevent cytokine secretion, Monensin (1.7 μg/ml) was added for the final 4 h. The cells were then fixed (2% paraformaldehyde), permeabilized (0.5% saponin or methanol), and stained with FITC-, PE-, and/or PE-cy5-conjugated mAb or isotype-matched mAb. The fluorescence data was collected and analyzed as described above.

The concentrations of HMGB1, IFN-γ, and TGF-β1 in BALF were detected using ELISA kits in accordance with the manufacture’s instructions.

At the end of the experiment, the lungs were rapidly excised, fixed with 4% paraformaldehyde, and embedded in paraffin for histo-pathological examination. Tissue sections (5-μm thick) were prepared and stained with H&E or Masson’s Trichrome. The grades of pulmonary inflammation and fibrosis were analyzed by a professional researcher of pathology, who was blinded for groups. Additionally, expressions of IFN-γ, TGF-β1, IL-13, MCP-1, and the phosphorylative activity of Stat3 or Smad3 in the lungs were stained with corresponding Abs and were semiquantitatively assessed according to the methods described (9). The average integrated OD (IOD) of the collagen deposition was determined by Image-Pro Plus image analysis software (Media Cybernetics) in 10 randomly chosen regions per tissue sample at a magnification of ×200.

Collagen deposition was determined by assaying total hydroxyproline content of the lung according to revised Reddy GK’s method (16). In brief, the total lungs were hydrolyzed with the 2.5 N NaOH at 120°C 0.1 kPa for 40 min. After neutralization with hydrochloric acid, the hydrolyzates were diluted with distilled water. Hydroxyproline in hydrolyzates was assessed calorimetrically at 550 nm with p-dimethylaminobenzaldehyde. Results were represented as μg per lung.

Data was represented as mean ± SE. Statistical analysis was performed with one-way ANOVA, in which α value was set at 0.05, followed by Tukey-Kramer’s or Dunnett’s post hoc multiple comparison test. The survival rates were analyzed by the Kaplan-Meier method.

Using an in vitro DC functional screening system, we investigated the effects of BLM on DC maturation and function. The precursor DCs were exposed to BLM (20 μg/ml), or TLR2 specific agonist Pam3Cys (100 ng/ml), or pg-LPS (data not shown). The morphological phenotypes, such as CD11c, MHC, and costimulatory molecules, were analyzed by flow cytometry. The TLR2 agonist Pam3Cys significantly stimulated the maturation of DCs by enhancing the surface expression of CD11c, MHC class I, MHC class II, CD40, CD80, and CD86. BLM treatment not only significantly enhanced the percentage of CD11c⁺MHCII⁺ DCs (p < 0.001, Fig. 1,A) and CD11c⁺MHCI⁺ DCs (p < 0.001, Fig. 1,B), but also up-regulated the expression of CD40, CD80, and CD86 (p < 0.001, Fig. 1, C and D). However, this BLM-induced expression of surface molecules was markedly abrogated by a TLR2-neutralizing Ab (Fig. 1, A–D). Moreover, BLM treatment significantly increased the levels of IL-6 (p < 0.01, Fig. 1,E) and IL-12 (p < 0.001, Fig. 1,G) but did not change the level of IL-10 (p > 0.05, Fig. 1,F). Treatment of these cells with Pam3Cys also significantly stimulated the release of IL-6 and IL-10 but not IL-12 (Fig. 1, E–G). Blocking TLR2 evidently attenuated BLM-stimulated release of IL-6 and IL-12 (p < 0.01, Fig. 1,E; p < 0.001, Fig. 1 G). These results indicated that BLM is a specific agonist of TLR2 in DCs.

To determine the relationship between TLR2 and the immune response properties of “BLM lungs,” the time-dependent in vivo effects of BLM on tissue-infiltrating immune cells and cell surface molecule expression were examined in TLR2-deficient and WT mice. In the WT mice, intratracheal administration of BLM resulted in a significant increase in expression of CD11c (p < 0.01, Fig. 2,A) and MHC class II (p < 0.01, Fig. 2,B) on day 1, but was shortly followed by a time-dependent decrease from day 1 to 28. TLR2 deficiency, however, noticeably dampened the BLM-induced expression of CD11c (p < 0.01, Fig. 2,A) and MHC class II (p < 0.01, Fig. 2,B). Additionally, the expression of CD40 was significantly up-regulated from days 1 (10.3 ± 1.71%, p > 0.05) to 7 (15.3 ± 1.54%, p < 0.01) and down-regulated from days 7 to 28 (10.9 ± 0.48%, p < 0.05) in the WT mice but did not change in the TLR2-deficient mice (Fig. 2,C). The expression of CD80 on lung cells was inhibited on day 7 after BLM treatment (p < 0.05) in the WT mice but not in the TLR2-deficient mice (Fig. 2,D). Interestingly, the expression of CD86 was significantly up-regulated from days 1 to 7 after BLM administration in WT mice, but TLR2 deficiency almost completely inhibited the BLM-induced expression of CD86 throughout the experimental period (p < 0.001, Fig. 2,E). The M1 cells (CD11b⁺F4/80⁺CD206⁻ cells) were increased from 4.45 ± 0.37% on day 0 to 13.0 ± 2.25% (p < 0.01) on day 3, and were sustained at 9.32 ± 1.23% (p > 0.05) on days 28 in the BLM-treated WT mice. TLR2 deficiency, however, resulted in decreased tissue-infiltrating M1 macrophages throughout the length of the experiment (Fig. 2,F). Notably, the administration of BLM did not change the number of tissue-infiltrating suppressive immune cells, including M2 cells (CD11b⁺F4/80⁺CD206⁺ cells) (Fig. 2,G), pDCs (PDCA-1⁺ cells, Fig. 2,H), and FoxP3⁺ Tregs (Fig. 2,I) during the early stages of exposure (days 1 and 3). However, as the BLM-induced inflammatory responses declined, these suppressive cells significantly increased from days 7 to 28. TLR2 deficiency markedly blocked the BLM-increased infiltration of pDCs, M2 cell, and FoxP3⁺ Tregs from the days 7 to 28 (Fig. 2, G–I). These results indicate that BLM initiated the recruitment of proinflammatory cells at the early stages of BLM treatment (days 1 to 7) and promoted an infiltration of the suppressive immune cells into the lungs at later stages of BLM treatment (days 7 to 28) in a TLR2 activity-dependent manner.

HMGB1 is a DAMPs molecule and plays a critical role in the development of inflammation (17). We found that BLM significantly up-regulated the expression of HMGB1 over 10-fold by day 3 (p < 0.001) and remained at a higher level than sham animals by 3-fold on days 28 post-BLM treatment (p < 0.001) (Fig. 2,J). The extracellular level of HMGB1 in BALF was also remarkably enhanced from days 1 to 28 following BLM treatment (Fig. 2,K). In contrast, TLR2 deficiency significantly blocked the BLM-stimulated accumulation and release of HMGB1 (Fig. 2, J and K). BLM administration resulted in a TGF-β1 production peak on days 7 and sustained a higher level of TGF-β1 in the BALF from days 1 to 28 after BLM treatment (Fig. 2,L), whereas IFN-γ production peaked on day 1 and was markedly inhibited in the BALF between days 5 and 28 post-BLM treatment (Fig. 2,M). TLR2 deficiency significantly attenuated BLM-enhanced TGF-β1 production and BLM-reduced IFN-γ production in BALF. The time-dependent alterations in the tissue-filtrating proinflammatory cells (e.g., M1 cells and CD11c⁺ DCs), immune inhibitory cells (e.g., Tregs and M2 cells), and collagen deposition are illustrated in Fig. 2,N. BLM exposure was also found to induce an influx of inflammatory cells, which peaked between days 3 and 7 post-treatment and was followed by an influx of immunosuppressive cells, peaking between days 14 and 28. Simultaneously, the collagen deposition peaked from days 14 to 28 after BLM treatment (Fig. 2,N). Additionally, BLM administration caused a persistent apoptosis in the lungs from days 1 to 28 post-BLM treatment, while TLR2 deficiency significantly protected from the BLM-induced apoptosis (Fig. 3).

We further investigated the role of TLR2 in BLM-induced pulmonary inflammation and fibrosis using the TLR2-deficient and corresponding WT mice. BLM exposure (3 U/kg) resulted in a 60% death rate (p < 0.001, Fig. 4,A) and significantly increased pulmonary inflammation and fibrosis (p < 0.01, Fig. 4, B–E) in WT mice. TLR2 deficiency clearly decreased this BLM-induced animal death (the death rate 13.2%, p < 0.001) and significantly decreased the BLM-increased lung index (p < 0.01, Fig. 4,B), pulmonary inflammatory score (p < 0.01, Fig. 4, C and E), and collagen deposition (p < 0.01, Fig. 4, C and D). Moreover, TLR2 deficiency significantly decreased the infiltrating FoxP3⁺ Tregs from 18.3 ± 3.85% to 5.22 ± 0.45% (p < 0.05, Fig. 4,F), pDCs from 18.1 ± 2.25% to 15.5 ± 2.1% (p < 0.05, Fig. 4,G), M2 cells from 9.82 ± 1.05% to 5.73 ± 1.50% (p < 0.05, Fig. 4,H), and M1 cells from 6.28 ± 1.26% to 3.90 ± 0.90% (p < 0.05, Fig. 4 I).

These results indicate that TLR2 activity has a critical role in the BLM induction of pulmonary inflammation and fibrosis and that targeting TLR2 is a potential strategy for the prevention and treatment of BLM-induced pulmonary inflammation and fibrosis. Indeed, treatment of animals with a TLR2-neutralizing Ab beginning from the day 0, 7, or 14 after BLM administration markedly improved BLM-induced animal survival (Figs. 5,A and 6, A and B). Importantly, the TLR2-neutralizing Ab not only prevented BLM-induced animal death at the early stage of BLM administration (Fig. 5,A) but also therapeutically attenuated BLM-induced animal death at the later stages following BLM administration (Figs. 5,A and 6, A and B). The preventive effects of anti-TLR2 Ab on BLM-induced animal death was not only associated with the inhibition of BLM-stimulated pulmonary inflammation and injury (p < 0.05, Fig. 5, B, C, and E) but also related to the attenuation of BLM-induced pulmonary fibrosis (Fig. 5, C, D, F, and G). The TLR2 Ab’s reduction of pulmonary fibrosis was associated with a reduced collagen deposition (p < 0.05, Fig. 5, C and D), a decreased lung index (p < 0.05, Fig. 5,F), a reduced hydroxyproline content (p < 0.05, Fig. 5,G), and a down-regulated expression of HMGB1 in the lungs (p < 0.001, Fig. 5,H). Moreover, treatment of animals with the anti-TLR2 Ab beginning on days 7 or 14 after BLM administration significantly decreased the tissue-infiltrating inflammatory cells and inflammation score (Fig. 6, C and E), reduced the collagen deposition (Fig. 6, C and D) and hydroxyproline content (Fig. 6,F) in the lung, and improved the lung index (Fig. 6 G). These results suggest that targeting TLR2 could attenuate pulmonary inflammation and reverse the established pulmonary fibrosis.

To determine the mechanism of TLR2 inhibition in the attenuation of pulmonary fibrosis, we investigated the effects of targeting TLR2 on the regulation of a BLM-induced immunosuppressive microenvironment in the lungs. Inhibition of TLR2 was found to decrease the BLM-enhanced infiltration of anti-inflammatory cells, including FoxP3⁺ Tregs (p < 0.01, Fig. 7,A), pDCs (p < 0.01, Fig. 7,B), and M2 cells (p < 0.01, Fig. 7,B) and increased the infiltration of proinflammatory cells including CD11c⁺ mDCs (p < 0.01, Fig. 7,C) and M1 cells (p < 0.05, Fig. 7 E). The anti-TLR2 Ab decreased the number of FoxP3⁺ Tregs while increasing the number of mDCs.

The Th1/Th2 balance is a critical factor in the determination of immune microenvironment. Although BLM instillation did not affect the level of Th1 cytokine IFN-γ (p > 0.05, Fig. 7,F), BLM significantly polarized the immune paradigm toward Th2 by enhancing in vivo production of the Th2 cytokines, such as TGF-β1 (p < 0.01, Fig. 7,G) and IL-13 (p < 0.05, Fig. 7,H). Blockage of TLR2 significantly elevated the level of IFN-γ by more than 2-fold (p < 0.01, Fig. 7,F) and reduced the levels of TGF-β1 (p < 0.05, Fig. 7,G) and IL-13 (p < 0.05, Fig. 7,H). Additionally, anti-TLR2 Abs also reduced the BLM-induced expression of MCP-1, a critical chemokine responsible for the recruitment of immune cells to the tissue (p < 0.01, Fig. 7 I).

Previous work indicates that the expression of suppressive cytokines (such as IL-6 and TGF-β1) and chemokines (MCP-1) are controlled by the transcription factors Stat3 and Smad3 (18). We found that BLM instillation significantly enhanced phosphorylation of Stat3 and Smad3 in vivo (p < 0.01, Fig. 8, A and B) and that blocking TLR2 evidently inhibited this BLM-stimulated phosphorylation of Stat3 (p < 0.01, Fig. 8,A) and Smad 3 (p < 0.01, Fig. 8,B). In addition, blocking TLR2 reduced the nucleus translocation of Smad3, which is normally induced by BLM administration (Fig. 8,C). Interestingly, inhibiting the activity of either Stat3 with specific Stat3 I or Smad 3 with SIS3 (a Smad3 inhibitor) significantly reduced the in vitro production of IL-6 by BLM-stimulated DCs (p < 0.01, Fig. 8 D).

Accumulated evidence suggests that inflammation disassociates from tissue fibrosis and that ongoing inflammation is needed to reverse established and progressive tissue fibrosis (19). Indeed, tissue fibrosis might be a consequence of the actions of unique cells, such as the extracellular matrix component-synthesizing myofibroblast, and immunosuppressive cells, such as immature DCs, M2 macrophage, and Tregs, which produces inhibitory cytokines and chemokines (2). Many types of fibrotic tissue share the common feature of a demonstrable skewing of the cytokine response toward a Th2-type cytokine and chemokine profile. This leads to the accumulation and activation of fibroblasts derived from resident cells, bone marrow-derived cells, and M2 cells or epithelial-mesenchymal transition, which leads to recurring infiltration of immunosuppressive cells into the damaged tissue (2). Thus, the immunosuppressive cells, cytokines, and chemokines favor a tissue microenvironment for fibrotic progression. TLRs are expressed in the respiratory epithelial cells and many types of immune cells including mast cells, DCs, T cells, B cells, endothelium, fibroblasts, and vascular smooth muscle (20, 21). Following PAMPs or DAMPs binding, TLRs or non-TLRs are able to regulate the function of these cells and skew specific immune responses toward the Th1, Th2, Th17 or Tregs phenotype according to the antigenic stimulation involved (22). In the family of TLRs, TLR2 is a unique member that mediates a TH1 response on one hand (23) and TH2-biased response on the other (24). For instance, it can be activated by injury through a vitamin D-dependent mechanism and is also involved in Th2-biased immune responses (24, 25, 26). Thus, targeting TLR2 signaling provides substantial new opportunities for the prevention and treatment of chronic lung diseases (11, 20, 27).

Our current study demonstrates that TLR2 plays a critical role in the BLM-induced inflammatory and fibrotic responses (Fig. 8,E). During the early exposure to BLM, a strong Th1-dominant inflammatory response is caused by activation of TLR2 (Figs. 1,G and 2, F and M). This BLM-induced inflammatory response can be induced either by the interactions between T cells and BLM-pulsed DCs or by BLM effects independent of APCs. In addition, the IFN-γ-producing Th1-cell response triggered by BLM stimulates the acute phase reactions that cause tissue damage, cell death, and apoptosis. Tissue injury can enhance the TLR2-mediated functions via a Vitamin D dependent mechanism (25) or via the release of DAMPs, such as HMGB1, heat shock protein 60, and hyaluronan, all of which have been identified as endogenous ligands of TLR2 (28). For instance, HMGB1 is a critical NF that mediates the response to infection, injury, and inflammation (17). TLR2-mediated HMGB1 is closely associated to the inflammation induced by BLM. In contrast, an anti-inflammatory Th2 response may be established by a direct interaction between TLR2 and BLM or an indirect interaction between TLR2 and BLM-induced apoptotic cells (29, 30, 31). The TLR2-mediated Th2 responses, induced by IL-4 as well as IL-5 and IL-13, are strongly linked with the process of fibrogenesis by up-regulating the transcription and expression of several genes involved in wound healing and fibrosis, such as procollagen I, procollagen III, matrix metallopeptidase (MMP) 2, and MMP9 in the lungs of BLM-treated mice (32). The Th2 responses also function as a double-edged sword, facilitating wound healing while simultaneously contributing to tissue remodeling by reducing the ratio of tissue inhibitor of metallopeptidase 2/MMP2. Thus, TLR2 activation induces an immunosuppressive tissue microenvironment by increasing the infiltration of immunosuppressive cells and up-regulating suppressive cytokines during the later stage of BLM exposure, which contributes to the pathogenesis of BLM-induced fibrosis (Fig. 8 E).

Thus, the mechanisms by which targeting TLR2 attenuates BLM-induced pulmonary fibrosis may be largely attributable to a reduction of BLM-induced inflammation and a reversion of the BLM-induced immunosuppressive microenvironment. On one hand, blocking TLR2 attenuates BLM-mediated inflammatory responses and protects the lungs from the TLR2-mediated recruitment of proinflammatory cells, such as M1 cells and mDCs, into the lung tissue. On the other hand, targeting TLR2 attenuates TLR2-mediated recruitment of a large number of immunosuppressive cells (e.g., FoxP3⁺ Tregs, pDCs, and M2 cells) and immunosuppressive cytokines (e.g., IL-6, IL-13, and TGF-β1) in the lungs. Estes et al. (33) also found that TLR2-meditated expansion and function of Tregs induces the tissue fibrosis by producing TGF-β1 and that pDCs have a unique function in tolerance induction and development of Tregs (34). However, adaptive transfer of Tregs suppresses the efferent phase of Th1 immune response and the subsequent pulmonary interstitial fibrosis (35). Therefore, the increase in Tregs initially following BLM-exposure may contribute to resolution by inhibition of inflammatory cells. In contrast, the increase in Tregs during the later stages of BLM-exposure may result in the aggravation of pulmonary fibrosis due to the production of the suppressive cytokine TGF-β1. Misson et al. (36) reported that the development of fibrosis is associated with a shift from a M1 activation to a M2 polarization by overexpression of NO synthase-2 and arginase. Indeed, the M2 macrophages are emerging as the potential target for Th2-related diseases, such as tissue fibrosis and tumor (36, 37).

The observed reversion of the immunosuppressive microenvironment, via the targeting of TLR2, may also be the result of inhibition of transcription factors Smad3 and Stat3 (Fig. 8,E). TGF-β1 is the most powerful physiological immunosuppressor in mammals (38), and the TGF-β1/Smad3 pathway is critical for damaged tissue to switch from a Th1- to Th2- predominated response. Smad3 mediates most effects of TGF-β1, including epithelial-to-mesenchymal transition and tissue fibrosis (18). Since Smad3 appears to be a key player in mediating fibrosis, independent of the agonist (BLM or TGF-β1) or cell type (fibroblasts or other mesenchymal), it offers a cleaner target to reverse tissue fibrosis in a safe predictable manner. Stat3 is initially identified as an acute phase response gene in the liver and has a pivotal role in directing inflammatory responses by inducing the gene expression of cytokines, chemokines, and adhesion molecules (39). Although prior studies suggest that Stat3 has a largely anti-inflammatory role in innate immune responses, elucidation of the role of Stat3 in biologic responses in general has been hindered by the fact that deletion of the Stat3 gene results in embryonic lethality (40). However, accumulated evidence suggests that the activation of Stat3 contributes to the establishment of an immunosuppressive tumor microenvironment (39). Also, evidence is emerging for the role of Stat3 in the pathogenesis of fibrotic diseases. For example, Ogata et al. (41) found that TGF-β1 is a target gene of Stat3 and that Stat3 enhances hepatic fibrosis through the up-regulation of TGF-â1 expression. Indeed, we recently found that TLR2 agonists significantly activate Stat3 and that blockade of the basal activity of TLR2 inhibits the constitutive activation of Stat3 and reverses immunosuppressive microenvironment in tumor tissue (unpublished data). Taken together, our studies provide further evidence to suggest that the activation of transcription factors Smad3 and Stat3 is associated with the development of BLM-established immunosuppressive microenvironment, which contributes to the BLM-induced pulmonary fibrosis (Fig. 8 E).

In summary, the identification of TLR2 as a critical receptor molecule for mediating BLM-stimulated pulmonary inflammation and fibrosis by Razonable’s recent work (11) and by this study is an important observation that highlights TLR2 as a promising target for the development of therapeutic agents against BLM lung injury and many fibroproliferative diseases. Also, our studies indicate that the combination therapy of BLM with the specific anti-TLR2 Ab or TLR2 antagonists will improve the anti-cancer efficacy and reduce the life-threatening side effects of BLM.

We thank Daniel Martinez (Moores Cancer Center, University of California-San Diego) for critical reading of the manuscript.

The authors have no financial conflict of interest.