TNF-α-dependent lung inflammation upregulates PD-L1 in monocyte-derived macrophages to contribute to lung tumorigenesis.

Chronic inflammation, which is dominated by macrophage-involved inflammatory responses, is an instigator of cancer initiation. Macrophages are the most abundant immune cells in healthy lungs, and associated with lung tumor development and promotion. PD-L1 is a negative molecule in macrophages and correlated with an immunosuppressive function in tumor environment. Macrophages expressing PD-L1, rather than tumor cells, exhibits a critical role in tumor growth and progression. However, whether and how PD-L1 in macrophages contributes to inflammation-induced lung tumorigenesis requires further elucidation. Here, we found that higher expression of PD-L1 in CD11b+ CD206+ macrophages was positively correlated with tumor progression and PD-1+ CD8+ T cells population in human adenocarcinoma patients. In the urethane-induced inflammation-driven lung adenocarcinoma (IDLA) mouse model, the infiltration of circulating CD11bhigh F4/80+ monocyte-derived macrophages (MoMs) was increased in pro-tumor inflamed lung tissues and lung adenocarcinoma. PD-L1 was mainly upregulated in MoMs associated with enhanced T cells exhaustion in lung tissues. Anti-PD-L1 treatment can reduce T cells exhaustion at pro-tumor inflammatory stage, and then inhibit tumorigenesis in IDLA. The pro-tumor lung inflammation depended on TNF-α to upregulate PD-L1 and CSN6 expression in MoMs, and induced cytokines production by alveolar type-II cells (AT-II). Furthermore, inflammatory AT-II cells could secret TNF-α to upregulate PD-L1 expression in bone-marrow driven macrophages (BM-M0). Inhibition of CSN6 decreased PD-L1 expression in TNF-α-activated macrophage in vitro, suggesting a critical role of CSN6 in PD-L1 upregulation. Thus, pro-tumor inflammation can depend on TNF-α to upregulate PD-L1 in recruited MoMs, which may be essential for lung tumorigenesis.

Lung adenocarcinoma-related TNF-α-dependent inflammation upregulates MHC-II on alveolar type II cells through CXCR-2 to contribute to Treg expansion.

MHC-II on alveolar type-II (AT-II) cells is associated with immune tolerance in an inflammatory microenvironment. Recently, we found TNF-α upregulated MHC-II in AT-II in vitro. In this study, we explored whether TNF-α-mediated inflammation upregulates MHC-II on AT-II cells to trigger Treg expansion in inflammation-driven lung adenocarcinoma (IDLA). Using urethane-induced mice IDLA model, we found that IDLA cells mainly arise from AT-II cells, which are the major source of MHC-II. Blocking urethane-induced inflammation by TNF-α neutralization inhibited tumorigenesis and reversed MHC-II upregulation on tumor cells of AT-II cellular origin in IDLA. MHC-II-dependent AT-II cells were isolated from IDLA-induced Treg expansion. In human LA samples, we found high expression of MHC-II in tumor cells of AT-II cellular origin, which was correlated with increased Foxp3+ T cells infiltration as well as CXCR-2 expression. CXCR-2 and MHC-II colocalization was observed in inflamed lung tissue and IDLA cells of AT-II cellular origin. Furthermore, at the pro-IDLA inflammatory stage, TNF-α-neutralization or CXCR-2 deficiency inhibited the upregulation of MHC-II on AT-II cells in inflamed lung tissue. Thus, tumor cells of AT-II cellular origin contribute to Treg expansion in an MHC-II-dependent manner in TNF-α-mediated IDLA. At the pro-tumor inflammatory stage, TNF-α-dependent lung inflammation plays an important role in MHC-II upregulation on AT-II cells.

High mobility group box-1 protects against Aflatoxin G1-induced pulmonary epithelial cell damage in the lung inflammatory environment.

Aflatoxin G1 (AFG1) is a member of the carcinogenic aflatoxin family. Our previous studies indicated that oral administration of AFG1 caused tumor necrosis factor (TNF)-α-dependent inflammation that enhanced oxidative DNA damage in alveolar epithelial cells, which may be related to AFG1-induced lung carcinogenesis. High mobility group box-1 (HMGB1) is a nuclear DNA-binding protein; the intracellular and extracellular roles of HMGB1 have been shown to contribute to DNA repair and sterile inflammation. The role of HMGB1 in DNA damage in an aflatoxin-induced lung inflammatory environment was investigated in this study. Upregulation of HMGB1, TLR2, and RAGE was observed in AFG1-induced lung inflamed tissues and adenocarcinoma. Blocking AFG1-induced inflammation by neutralization of TNF-α inhibited the upregulation of HMGB1 in mouse lung tissues, suggesting that AFG1-induced TNF-α-dependent inflammation regulated HMGB1 expression. In the in vitro human pulmonary epithelial cell line model, Beas-2b, AFG1 directly enhanced the cytosolic translocation of HMGB1 and its extracellular secretion. The addition of extracellular soluble HMGB1 protected AFG1-induced DNA damage through the TLR2/NF-κB pathway in Beas-2b cells. In addition, blockade of endogenous HMGB1 by siRNA significantly enhanced AFG1-induced damage. Thus, our findings showed that both extracellularly-released and nuclear and cytosolic HMGB1 could protect the cell from AFG1-induced cell damage in a TNF-α-dependent lung inflammatory environment.

Aflatoxin G1 induced TNF-α-dependent lung inflammation to enhance DNA damage in alveolar epithelial cells.

Aflatoxin G1 (AFG1 ), a member of the AF family with cytotoxic and carcinogenic properties, could cause DNA damage in alveolar type II (AT-II) cells and induce lung adenocarcinoma. Recently, we found AFG1 could induce chronic lung inflammation associated with oxidative stress in the protumor stage. Chronic inflammation plays a critical role in cigarette smoke or benzo[a]pyrene-induced lung tissues damage. However, it is unclear whether and how AFG1 -induced lung inflammation affects DNA damage in AT-II cells. In this study, we found increased DNA damage and cytochrome P450 (CYP2A13) expression in AFG1 -induced inflamed lung tissues. Furthermore, we treated the mice with a soluble tumor necrosis factor (TNF)-α receptor and AFG1 and found that TNF-α neutralization inhibited the AFG1 -induced chronic lung inflammation in vivo, and then reversed the CYP2A13 expression and DNA damage in AT-II cells. The results suggest that AFG1 induces TNF-α-dependent lung inflammation to regulate 2A13 expression and enhance DNA damage in AT-II cells. Then, we treated the primary mice AT-II cells and human AT-II like cells (A549) with AFG1 and TNF-α and found that TNF-α enhanced the AFG1 -induced DNA damage in mice AT-II cells as well as A549 cells in vitro. In AFG1 -exposed A549 cells, TNF-α-enhanced DNA damage and apoptosis were reversed by CYP2A13 small interfering RNA. Blocking NF-κB pathway inhibited the TNF-α-enhanced CYP2A13 upregulation and DNA damage confirming that the CYP2A13 upregulation by TNF-α plays an essential role in the activation of AFG1 under inflammatory conditions. Taken together, our findings suggest that AFG1 induces TNF-α-dependent lung inflammation, which upregulates CYP2A13 to promote the metabolic activation of AFG1 and enhance oxidative DNA damage in AT-II cells.

Inflammation-mediated SOD-2 upregulation contributes to epithelial-mesenchymal transition and migration of tumor cells in aflatoxin G1-induced lung adenocarcinoma.

Tumor-associated inflammation plays a critical role in facilitating tumor growth, invasion and metastasis. Our previous study showed Aflatoxin G1 (AFG1) could induce lung adenocarcinoma in mice. Chronic lung inflammation associated with superoxide dismutase (SOD)-2 upregulation was found in the lung carcinogenesis. However, it is unclear whether tumor-associated inflammation mediates SOD-2 to contribute to cell invasion in AFG1-induced lung adenocarcinoma. Here, we found increased SOD-2 expression associated with vimentin, α-SMA, Twist1, and MMP upregulation in AFG1-induced lung adenocarcinoma. Tumor-associated inflammatory microenvironment was also elicited, which may be related to SOD-2 upregulation and EMT in cancer cells. To mimic an AFG1-induced tumor-associated inflammatory microenvironment in vitro, we treated A549 cells and human macrophage THP-1 (MΦ-THP-1) cells with AFG1, TNF-α and/or IL-6 respectively. We found AFG1 did not promote SOD-2 expression and EMT in cancer cells, but enhanced TNF-α and SOD-2 expression in MΦ-THP-1 cells. Furthermore, TNF-α could upregulate SOD-2 expression in A549 cells through NF-κB pathway. Blocking of SOD-2 by siRNA partly inhibited TNF-α-mediated E-cadherin and vimentin alteration, and reversed EMT and cell migration in A549 cells. Thus, we suggest that tumor-associated inflammation mediates SOD-2 upregulation through NF-κB pathway, which may contribute to EMT and cell migration in AFG1-induced lung adenocarcinoma.