Benzo[a]pyrene immunogenetics and immune archetype reprogramming of lung

Abstract

Overexposure to carcinogenic precursor, benzo[a]pyrene [BaP], modulates the lung immune microenvironment. The present review seeks to elucidate novel pathways behind the tumor effect of BaP in the lungs, emphasizing immunomodulatory mediators and immune cells. In this review, BaP reprograms lung immune microenvironment through modulating transforming growth factor-beta (TGF-β), programmed cell death 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), Interleukin 12 (IL-12), indoleamine 2,3 dioxygenase (IDO), forkhead box protein P3 (FOXP3) and interferon-gamma (IFN-γ) levels. Moreover, BaP modulated lung immune cellular architecture such as dendritic cells, T cells, Tregs, macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs). All mentioned changes in immune architecture and mediators lead to the induction of lung cancer.

Introduction

Lung cancer is the leading cause of cancer-related deaths worldwide, and its prognosis is strongly correlated to radon, ultraviolet radiation, arsenic, chromium, and polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) is an example, exposure (Bray et al., 2018; de Groot et al., 2018; Dela Cruz et al., 2011). Exposure to BaP induces abnormalities in the liver, adipose tissue, spleen, immune system, bone marrow, and thymus (Nebert et al., 2013).

Besides smoking, PAHs are found in grilled meat, vegetables, oils, grains, fruits, and smoked fish, making it a critical risk factor for lung cancer due to its high distribution pattern between different dietary products (Kazerouni et al., 2001). Polluted air is the main source of BaP in aboveground tissues. When BaP was strictly controlled in both air and quartz sand culture medium, the background values of BaP in rice plant tissues were uniformly very low. There was no significant difference of BaP contents of rice grain between control and treatments of BaP in controlled air quality trials. This indicated that the source of BaP in the rice grains is not from any BaP in the root culture media (Li et al., 2009). Factor analysis revealed a positive correlation of BaP with elements usually emitted in coal combustion processes, such as Tl and V. This observation fosters the hypothesis of a historical and indelible pollution fingerprint in soils whose sources, characteristics and potential environmental and health concerns deserve further attention. All things considered, caution should be taken when using soil screening levels in regions associated with coal exploitation and heavy industry (Boente et al., 2020).

Besides Lung cancer, Bap also induce hepatocellular carcinoma (HCC). After 52 weeks, BaP, besides phenobarbital, induced tumors including HCC in rats (Kitagawa et al., 1980). Su et al. (2014) mentioned that the high level of BPDE-DNA adducts in blood is associated with HCC and that environmental exposure to B[a]P may increase the risk of HCC, especially among drinkers and populations with hepatitis B virus infection. Ge et al. (2021) findings reveal a possible adverse outcome pathway of SOCS3/JAK/STAT3 regulation in B[a]P-induced HCC progress which provide a better understanding of the adverse effects of chronic exposure to B[a]P on human health.

The liver metabolizes BaP to BaP-7,8-diol-9,10-epoxide (BPDE), which activates the aryl hydrocarbon receptor (AHR), a cytoplasmic enzyme that moves to the nucleus and induces CYP1A2, CYP1B1, and CYP1A1 gene expression. BPDE also binds covalently to N6 of adenine, producing the minor N6-deoxyadenosine adduct or N2 of guanine producing the major BPDE-dG adduct, which is a well-established risk factor for lung cancer through induction of mutation in the tumor suppressor gene p53 (Ajayi et al., 2019; Saxena et al., 2018; Madeen et al., 2019; Kaur et al., 2019; Jimma et al., 2019; Alexandrov et al., 2010).

The BPDE-dG adduct induces the conversion of p53 G: C to T: The lack of normal cellular growth-control mechanisms and subsequent cancer development due to a transversion and mutation in the ras and myc oncogenes (Alexandrov et al., 2010). In addition, BPDE-dG adduct induces mutations associated with disruption of cell-cycle checkpoints, chromosomal instability, and other changes (Osada and Takahashi, 2002). Carcinoembryonic antigen level and Bcl-2 expression were significantly increased in BaP-induced lung cancer-bearing mice compared to the control group, which supports the evidence of tumorigenic role BaP and its metabolite BPDE through its anti-apoptotic effect (Ravichandran et al., 2014; El-Ashmawy et al., 2016, 2017; Arrieta et al., 2013).

Benzo[a]pyrene-induced oxidative stress in lung tissue aids in tumor progression. Activities of the mitochondrial antioxidant enzymes SOD, CAT, GPx, GST, and GR were lowered in BaP-treated animals (Anandakumar et al., 2008). BaP treatment also dramatically reduced citric acid cycle enzymes, which could be attributed to cancer cell shape, ultrastructural changes, and mitochondria's ability to undergo metabolic modifications (Anandakumar et al., 2008).

BaP and its metabolite BPDE also enhances tumor invasion and metastasis. BaP-treated Hep-G2 cells showed increased expression of the phosphorylated extracellular regulated kinase protein, thus enhancing cancer cell invasion and metastasis compared to the control group (Wang et al., 2018). Hep-G2 cells treated with BaP moved to wounds faster than cells treated with DMSO. According to the findings of this investigation, Hep-G2 cells treated with BaP (2 and 4 M) had a more vital ability to penetrate through the Matrigel matrix than cells treated with DMSO. All of the above findings support the carcinogenic role of BaP and indicate the BaP also supports tumor invasion and metastasis (Wang et al., 2018).

The above data support that BaP and its metabolite BPDE induces cancer through different mechanisms such as apoptosis, proliferation, oxidative stress, and metastasis. Besides the tumorigenic effect of BaP and its metabolite BPDE on lung tissue, we investigated the immunomodulatory effect of BaP and its metabolite BPDE on the lung tumor microenvironment (Immune mediators and immune cells), which adds to the multiple carcinogenic mechanisms of BaP.

Transforming growth factor-beta is a cytokine that promotes cell proliferation, suppresses the immune system, and promotes cancer development (Jeon and Jen, 2010; Youssef et al., 2021). Tumor development and metastasis are aided by increased TGF-β gene expression in the tumor microenvironment (Li et al., 2018). TGF-β regulates the generation and effector functions of many immune cell types. It inhibits the formation and function of effector T cells, dendritic cells and blocks Th1 development by directly encouraging the proliferation of T regulatory (Treg) cells, an inhibitory T cell (Batlle and Massagué, 2019).

During the priming phase, TGF-β silences the expression of IL-2, the cytokine that elicits subsequent CD4+ T cell proliferation. TGF-β similarly controls the innate immune system, suppressing natural killer (NK) cells and controlling the complicated activity of macrophages and neutrophils, producing a network of negative immunological regulatory inputs (Saito et al., 2018).

Activation of AHR induces dioxin response elements, increasing TGF-β gene expression (Li et al., 2014). Gramatzki et al. (2009) found thatinhibition of AHR with CH-223191 lowered TGF-β1 and TGF-β2 in malignant glioma cells. AHR inhibition with CH-223191, AHR antagonist, or AHR gene silencing showed that constitutive AHR activity positively impacts TGF-β1 and TGF-β2 in malignant glioma cells (Gramatzki et al., 2009). AHR-null astrocytes, hepatocytes, and glioblastoma cells were less sensitive to integrin inhibition-mediated changes in TGF-β signaling, demonstrating that AHR controls integrin regulation of the TGF-β pathway (Silginer et al., 2016). The previous findings support that BaP increases TGF-β level with its multiple pathogenic effects on the tumor microenvironment as an immunosuppressive agent and thus worse prognosis (Fig. 1).

Programmed cell death one and its ligands (PD-L1 and PD-L2) stimulate molecules of the immune checkpoint pathway with the primary function to dampen the effector phase of activated T-cells in peripheral tissues and limit inflammatory response and autoimmunity. PD-1 is a transmembrane protein, expressed on blood cell types such as B cells, T cells, NK cells, dendritic cells, and Treg, and is upregulated in the effector phase of the immune response (Guzik et al., 2019).

The binding of PD-1 to PD-L1 inhibits kinase signaling paths and thus carries suppressive messages for T-cells, downregulating them by causing their exhaustion or apoptosis and preventing the autoimmune attack. PD-1 was identified as an immunosuppressive checkpoint pathway that tumor cells may exploit to evade immune surveillance. Many cancer forms, including melanoma, gastric, renal, glioblastoma, and non-small cell lung cancer (NSCLC), have expressed PD-L1 (Tsoukalas et al., 2019).

Because of recent practical uses in several advanced malignancies, the discovery of immune checkpoint inhibitor drugs targeting PD1 or PDL1 has sparked much interest in immunotherapy (Schoenfeld et al., 2020). Because of its role in CD8+ T cell exhaustion, therapeutic monoclonal antibodies that target PD1 or PDL1 have shown to be effective in treating a variety of advanced malignancies (Zhang et al., 2020).

Activation of TGF-β1 boosts PD-L1 gene expression, which boosts lung cancer cell proliferation. TNF-activation promotes TGF-β1, IKK/NF-B signaling pathways through demethylation of PD-1/PD-L1 promoters (Sow et al., 2019; Asgarova et al., 2018). BaP promotes the expression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50, which could explain why the PD-L1 gene is upregulated (Asgarova et al., 2018) (Fig. 1). In the A549 cell line, NF-κB inhibition downregulates PD-L1 gene expression (Asgarova et al., 2018). Furthermore, inhibiting the NF-κB pathway reduces PD-L1 gene expression in a human breast cancer cell line MDA-MB-231 (Asgarova et al., 2018). AHR is required for BaP-induced skin cancer and mediates BaP-induced chemokine CXCL13 synthesis by lung epithelial cells (Shimizu et al., 2000). While BaP boosted PD-L1 promoter-driven luciferase activity in the wild-type condition, deletion of one XRE-like region (5′-GCGTC-3′) dramatically reduced this activity (Wang et al., 2019).

Interleukin 12 is a cytokine that acts as an antitumor aid by activating natural killer cells (NK) and CD8+ T cells. Interferon-gamma (IFN-γ) induces proliferation and activation of NK cells, cytotoxic T cells and enhances antibody-mediated cellular cytotoxicity (Yue et al., 2016).

After being pulsed with tumor antigen, DC treated with the IL-12 gene showed good lymphocyte proliferation, cytotoxic T cells activation with specific antitumor activity. Treatment with IL-12 caused NK cells to release IFN-γ, which inhibited tumor angiogenesis and lung tumor growth, allowing lung cancer-bearing animals to live for a long time (Yue et al., 2016). Benzo[a]pyrene inhibits the expression of the IL-12 gene, which could be attributed to a reduction in RelB activity. Inhibition of RelB reduces myeloid DC differentiation and, as a result, IL-12 gene expression (Wagage et al., 2014). In AHR-null mice, AHR-/- cells produced more IFN-gamma and IL-12 than AHR+/+ cells (Rodríguez-Sosa et al., 2005). In addition, intravenous IL-12 treatment reduced AHR, pulmonary eosinophilia, T lymphocyte infiltration, serum IgE, IL-4, and IL-13 levels in lung tissue (Kuribayashi et al., 2002) (Fig. 1).

Transcription factor forkhead box protein P3 is a critical biological marker for immunosuppressive Treg cells. Immunosuppressive Treg cells block other leukocytes from activating and carrying out their tasks. In animal tests, therapy with foxp3-knockout-Treg cells diminishes tumors, and foxp3 plays a crucial role in lung carcinogenesis (Peng et al., 2018).

Overexpression of the foxp3 gene boosts the proliferation and invasiveness of cervical tumor cells, resulting in cancer development and dissemination. In NSCLC patients, Treg cell infiltration in tumor tissue worsens prognosis (Pircher et al., 2014).

Foxp3 inhibition, on the other hand, inhibits cell proliferation, migration, and invasion, as well as the release of inhibitory cytokines, suggesting that foxp3 may be a tumor suppressor gene in lung adenocarcinoma development. The quantity of foxp3+Treg cells was associated with the prognosis of various malignancies (Hu et al., 2019).

Benzo [a]pyrene has been shown to form adducts at CpG dinucleotides, affect histone acetylation status, modulates global DNA methylation, and inhibit DNMT function (Pacheco, 2012). BaP increases FOXP3 levels, which AHR activation may interpret causes demethylation of the FOXP3 gene's promoter region and CpG islands, which upregulates FOXP3 gene expression (Lu et al., 2017) (Fig. 1). In BaP-exposed B6 mice, FOXP3 implicated in atherosclerosis is expressed at higher levels (1.1 fold) than in B6.D2 mice (Kerley-Hamilton et al., 2012).

Cytotoxic T-lymphocyte associated antigen 4is a CD28 homolog with much higher binding affinity for CD80 and CD86 expressed on APC; however, binding of CTLA-4 to B7 acts as a competitive antagonist which prevents binding of CD28. This competitive binding can prevent the costimulatory signal generally provided by CD28: CD80 and CD86 binding. The amount of CD28 determines a T cell's activation or energy: CD80 and CD86 binding compared to CTLA-4: CD80 and CD86 binding (Perez-Ruiz et al., 2019).

Furthermore, CTLA-4 binding to CD80 and CD86 generates inhibitory signals that counteract the stimulatory signals generated by CD28:CD80 and CD86, as well as TCR: MHC binding. Direct inhibition at the TCR immunological synapse, suppression of CD28 or its signaling pathway, or increased mobility of T cells resulting in reduced ability to engage with APCs are all potential mechanisms for such inhibitory signals (Perez-Ruiz et al., 2019).

T cell receptor attachment to its stimulant CD80 and CD86 is prevented by the release of CTLA-4, which has a higher affinity for both CD80 and CD86 than CD28, inhibiting cytotoxic T cell activation and resulting in a drop in the CD8+ cell population (Perez-Ruiz et al., 2019). BaP enhanced CTLA-4 gene expression, which could be due to Tr1 cell activation (Mascanfroni et al., 2015) (Fig. 1).

Kynurenine is a tryptophan metabolite that has an AHR agonist effect that leads to immunosuppression. Kynurenine causes the AHR to translocate to the nucleus, allowing it to activate its target genes. The rate-limiting enzymes for metabolizing tryptophan to kynurenine are IDO-1, IDO-2, and tryptophan-2,3-dioxygenase 2 (TDO-2) (Vogel et al., 2008).

The AHR regulates the expression of IDO1 and TDO-2. During tumor development, such enzymatic activity causes tryptophan fatigue in the local milieu, suppresses T-cell responses, and promotes the formation of Treg cells. In many types of malignancies, elevated IDO expression induces immune evasion and cancer-related inflammation. Activation of AHR by TCDD, an AHR agonist similar to BO, causes a rise in DC IDO1 and IDO2, which leads to an increase in the Treg marker Foxp3 in TCDD-treated C57BL/6 mice's spleen (Vogel et al., 2008) (Fig. 1).

Interferon is a cytokine that acts as an antiviral, anticancer, and immunomodulator. Interferon functions by coordinating both the innate and adaptive immune responses. IFN activates the immune response by initiating apoptosis in tumor cells through its cytotoxic effect besides granzyme B and perforin. IFN also inhibits immune over-activation and tissue damage by enabling the production of immune checkpoint inhibitory molecules and IDO (Gonzalez et al., 2018).

IFN production is controlled by natural killer (NK), T (NKT), CD8+, and CD4+ T-cells. IFN secretion is triggered by a positive feedback system including cells, in situ produced interleukins, tumor- or pathogen-secreted antigens, and IFN itself (Schoenborn and Wilson, 2007).

In the tumor microenvironment, secreted pro-inflammatory cytokines connect to their receptors on IFN-producing cells, causing transcription elements including signal transducer and activator of transcription (STAT) and activator protein 1 (AP-1) to trigger more IFN production. Antigen-presenting cells (APCs) such as macrophages, dendritic cells (DCs), and B cells generate the interleukin IL-12, which activates STAT4 in CD4+ T-cells. When the IL-12 receptor is active, Janus (JAK) kinases such as JAK2 and TYK2, which phosphorylate STAT4, increase (Shekhar et al., 2014).

In response to cytokines generated during inflammation, naive CD4+ T-cells differentiate into Th1 and Th2 T-cells. Apart from IL-12, IL-2, IFN, and TGF-β expression, CD4+ Th1 cells are the primary source of IFN. Ambrosio, Insfran (Ambrosio et al., 2019) found that the activation of the AHR receptor resulted in a significant reduction in the proportion and absolute quantity of CD8 + TSKB20/Kb + cells and CD4+ splenocytes generating IFN-γ, and therefore in IFN- secretion, leading to tumor growth.

Macrophages are tissue-resident innate immune cells derived from peripheral circulating monocytes. There are critical drivers of chronic inflammation at every stage of cancer development, including early carcinogenesis, metastatic progression, and treatment resistance (Sarode et al., 2020).

Macrophages support tumor growth in various ways, including enhancing angiogenesis, encouraging proliferation and EMT, remodeling the extracellular matrix (ECM), increasing immunosuppression, and suppressing antitumor cytotoxic actions. In NSCLC patients, the density of tumor-infiltrating macrophages is linked significantly and favorably with intratumor microvessel counts and adversely with patient survival (Mei et al., 2016).

Most invading immune cells in tumors are tumor-associated macrophages (TAMs), and their existence is often unrelated to the clinical outcome. Higher TAM density is linked to a worse patient survival rate in lung cancer (Sumitomo et al., 2019). According to their activity, activated macrophages are classified as either M1 (pro-inflammatory type) or M2 (anti-inflammatory type). M1 macrophages are involved in the removal of immunogenic tumor cells during early carcinogenesis. In contrast, fewer immunogenic tumor cells remain and bias macrophages against the M2 phenotype, promoting tumor survival, angiogenesis, EMT, and immune evasion (Ostuni et al., 2015; Zhou et al., 2020).

M1- macrophages secrete Microbicidal and pro-inflammatory activities-related reactive oxygen species (ROS) and cytokines like IL-6, IL-12, IL-23, and TNF-alpha. As a result, they are known as "fighting" macrophages, linked to a better prognosis in cancer patients (Lin et al., 2019).

The tumorigenic effect of BaP and its metabolite BPDE was correlated to an increase in TGF-β gene expression, which decreased TLR agonist-induced IFN-gamma and thus decreasing M1 macrophage (Eriksson et al., 2006; Salem et al., 2021). IFN-γ, lysozyme, and IgM gene expression were downregulated by BaP in primary macrophage cells, indicating that BaP has a suppressive role in reducing M1 macrophages (Hur et al., 2013) (Fig. 2).

On the other hand, M2-like macrophages are characterized as "repair" or "fix" macrophages because they aid tissue healing through immunological tolerance, tissue remodeling, debris scavenging, and immune regulation (Guo et al., 2019; Vinogradov et al., 2014). M2-like macrophages promote angiogenesis in cancer by secreting vascular epithelial growth factors (VEGFs) and generating immunosuppressive molecules such as IL10, PD-L1, and TGF-β (Hu et al., 2016) (Fig. 2).

Because M2 to M1 remodeling enhances the effectiveness of T cell antitumor activity, M2 macrophages reduce immunological response in TME, as indicated by reduced PD-L1 expression and lower amounts of IL-6 and TGF-β (Najafi et al., 2019). Chronic NF-κB signaling activation resulted in chronic inflammation and adenoma, which increased M2 macrophage infiltration and promoted Treg differentiation by producing IL-10 and TGF-β (Lee et al., 2019).

Due to the effect of BaP and its metabolite BPDE on TGF-β, TGF-β induced IκB-α phosphorylation followed by NF-κB p65 subunit nuclear translocation and increased NF-κB activity, which increases M2 macrophage infiltration and thus tumor progression (Giridharan and Srinivasan, 2018).

Inflamed tissues release chemotactic molecules such as IL-8, which aid in the recruitment of neutrophils. Tumor-associated neutrophils (TANs) promote cancer growth by infiltrating the tumor with neutrophils, and the neutrophil/lymphocyte ratio negatively affects prognosis in various cancers, including lung cancer (Eruslanov, 2017).

There are two types of neutrophils: "N1" and "N2." As N2 neutrophils induce angiogenesis, immunosuppression, and the generation of ROS, N2 neutrophils are pro-tumorigenic. TANs can take on an anticancer N1 phenotype under certain situations, such as TGF-β blockade (Fridlender et al., 2009).

Neutrophils and platelets are quickly drawn to damaged areas. Platelets, neutrophils, and endothelial cells all participate in this trafficking, mediated by coordinated ligand-receptor interactions. PSGL-1 and CD24, which are expressed on endothelial cells, detect and activate P-selectin expressed on platelets. Endothelial expression of ICAM-1 is enhanced by activated platelets, resulting in greater neutrophil adherence (Giese et al., 2019).

Inhibiting neutrophil-platelet interactions thus protected mouse models from acute lung damage. PSGL-1 promotes a redistribution of receptors that govern neutrophil recruitment and trafficking when it is activated on neutrophils. In organ injury models, inhibiting PSGL-1, a docking location for active platelets during inflammation, was found to be protective (Faget et al., 2017).

Activated neutrophils have antitumor properties. They can lyse tumor cells directly and destroy them by antibody-dependent cell-mediated cytotoxicity (Carus et al., 2013). Mittendorf et al. (2012) showed that breast cancer cells absorbed neutrophil elastase (NE) produced by TAN, rendering them more susceptible to CTL lysis. Despite neutrophils' antitumor properties, granulocytic cell proliferation and accumulation in the tumor microenvironment lead to tumor growth and dissemination (Singel and Segal, 2016).

In addition to pathogen killing via oxidant damage produced by NOX2/MPO activation, NOX2 can improve host defense by activating granular proteases and producing neutrophil extracellular traps (NETosis). NETosis is a type of neutrophil mortality characterized by membrane breakdown and extracellular release of DNA, histones, and granular components (Grecian et al., 2018).

NETosis can be caused by bacterial and fungal infections, as well as non-infectious stimuli that mimic infection. NOX2-dependent and NOX2-independent mechanisms are both possible. Neutrophil apoptosis results in non-inflammatory cell death, whereas NETs produce substances that boost antimicrobial host defense and cause tissue injury. In the tumor microenvironment, the same activities that tear down and alter the extracellular matrix may enhance tumor cell motility and invasion (Cools-Lartigue et al., 2014).

When mice were given BaP, the mouse IL-8 and keratinocyte chemoattractant (KC), was overexpressed in the lungs. It also induced neutrophil recruitment in bronchoalveolar lavage (BAL) fluids (Podechard et al., 2008). IL-8 also depleted intracellular glutathione (GSH), resulting in higher levels of unmetabolized BaP and higher quantities of the metabolite BaP-7,8-diol (Shi et al., 2017).

Release of tumor necrosis factor (TNF)-α and IL-10 was increased following BaP exposure which recruits TANs to the TME. While BaP exposure reduced the synthesis of pro-inflammatory cytokines, it increased the secretion of IL-10. Furthermore, BaP exposure elevated the expression of MHC-II, CD14, Fc receptor I (FcRI/CD64), and CD86, as well as NO generation and phagocytosis. All of the previous findings may be advantageous for phagocytosis and the elimination of microbial pathogens (Fueldner et al., 2018).

By stimulating angiogenesis, altering the ECM, producing ROS, and modulating immunity, TANs have a role in tumor initiation and progression. Neutrophils also produce neutrophil elastase (NE), which activates protein kinase B signaling and promotes lung cancer growth (Kim et al., 2019). Neutrophils promote tumor cell extravasation and metastasis by secreting chemokines, NE, collagenase IV, heparanase, and MMPs (MMP-9). All of which causes VEGF release, break down the ECM, and aid tumor cell extravasation and thus metastasis (Mishalian et al., 2013).

Myeloid-derived suppressor cells are immature myeloid cells that decrease immunological responses in malignancy and chronic inflammation. MDSCs are divided into two types: polymorphonuclear (PMN-MDSCs) and mononuclear (MDSCs) (M-MDSCs) (Yang et al., 2020). MDSCs boost MDSC proliferation by activating T cells and stromal cells with tumor-secreted GM-CSF, IL-1, IL-6, TGF-β, and TNF (Srivastava et al., 2012).

The key mediators of MDSC immunosuppressive effects, which directly limit T cell activity, are Arg1, iNOS, TGF-β, IL-10, COX2, and IDO. MDSCs generate oxidative stress, preventing T cells from becoming activated (Ma et al., 2018). IFN-γ and IL-4 receptors stimulation are associated with MDSC development and immunosuppressive activity (Sinha et al., 2012).

Increased Th2 cytokines, such as IL-13, are connected to MDSC and M2 macrophage polarization, as well as CD163 + M2 macrophage infiltration into cancer tissues, encouraging the creation of an immunosuppressive microenvironment in patients with esophageal cancer (Gao et al., 2014). TNF gene expression was upregulated by BaP through the AHR pathway, while IL-6 and IFN gene expression was upregulated through a calcium-dependent pathway that activated MDSCs (Hur et al., 2013).

In C3H/HeJ mice, BaP raised the amounts of IL-13, which activates MDSCs (Yanagisawa et al., 2016). Mucin 5AC expression, after AHR activation, was highly correlated with increased-expression cyclooxygenase-2 and IL-1β, which activates MDSCs (Wong et al., 2010; Elkabets et al., 2010). AHR activation increases the TGF-β level that induces phosphorylation of mammalian target of rapamycin, which subsequently activated hypoxia-inducible factor-1 (HIF-1) and subsequently enhanced CD39/CD73 expression on MDSCs in NSCLC patients (Li et al., 2017).

MDSCs secretes IDO, which polarizes APCs to a tolerant phenotype. MDSCs also make PD-L1, an immunological checkpoint molecule that binds to PD-1 on T cells and causes T cells to become exhausted. MDSCs have a role in tumor development through interacting with other immune cells. They produce IL-10 to lower DC activity, polarise macrophages toward M2, and recruit Tregs through IL-10 and TGF-β (Yamauchi et al., 2018).

T cells known as regulatory T cells suppress the immune system, avoiding autoimmune illness, and penetrate the tumor microenvironment, causing it to progress. Tregs are CD4+CD25+ cells that express FoxP3. In a mouse Lewis lung cancer model, Tregs suppress NK cell-mediated cytotoxicity in a TGF-β dependent way, and Treg removal restored NK cell anti-metastatic activity (Verma et al., 2019).

In comparison to non-tumor lung tissue, NSCLC specimens revealed a significant increase in Tregs. Treg levels have also been linked to a higher risk of recurrence and a shorter life expectancy in NSCLC tumors and peripheral blood (Singh et al., 2019). The fact that AHR activation by BaP increases demethylation of the FOXP3 gene's promoter region, CpG islands, and hence boosts FOXP gene expression could explain the overexpression of the FOXP3 gene (Salem et al., 2021) (Fig. 2).

Tregs have two ways of suppressing the immune system: contact-dependent and contact-independent suppression. CTLA-4, PD-1, PD-L1, lymphocyte-activation protein 3 (LAG-3), and CD39/73 are examples of contact-dependent co-inhibitory molecules. Contact-independent mechanisms create immunosuppressive chemicals such as IL-10, TGF-β, adenosine, prostaglandin E2 (PGE2), and IL-35 (Verma et al., 2019). CTLA-4 gene expression was enhanced by BaP, possibly due to Tr1 cell activation (Mascanfroni et al., 2015). In lung tumors, Tregs are also associated with the expression of COX-2, suggesting Tregs are involved in tumor cell dissemination.

The most prevalent antitumor cells are CD8 + T cells, which, when activated, transform into CTLs, release perforin and granzyme-containing granules and exert antitumor effects. CD4+ Th1 cells secrete IL-2 and IFN, which promote T cell priming, activation, and CTL cytotoxicity, resulting in antitumor activity. T cell-based antitumor immunity requires both cytotoxic CD8 + T cells and Th1 cells to improve the efficacy of the antitumor response. Infiltrates of CD8 + T and Th1 cells have been associated with a better prognosis in cancer patients. TME, MDSCs, and DCs directly suppress T cell function (van der Leun et al., 2020).

Perforin and granzyme levels were lower in TILs from lung cancer patients, indicating that they were dysfunctional. As shown by a low PD-1 to CD8 ratio, fewer tired T cells establish a favorable immunological environment, which helps patients survive post-resection and react to immunotherapy in advanced NSCLC (An et al., 2019).

Dendritic cells are antigen-presenting cells (APCs) found in peripheral tissues and immunological organs like the thymus, bone marrow, spleen, lymph nodes, and Peyer's patches (DCS). Their job is to search peripheral tissues for pathogens, which they recognize, take up, and process, and then present pathogen-derived antigenic peptides to naive T lymphocytes in lymphoid organs in the context of major histocompatibility molecules (MHCs) (El-Ashmawy et al., 2021). DCs serve as a vital link between innate and adaptive immunity and are required to form antigen-specific immune responses due to these processes (Lu et al., 2012) (Fig. 2).

Immature DCs in peripheral tissues can detect exogenous PAMP-bearing bacteria due to their high expression of cell surface and vesicular PRRs. DCS recognizes pathogens and phagocytoses, breaking them down into peptide bits. Because innate immunity does not permanently eliminate illnesses, an adaptive immune response targeting pathogen-associated antigenic epitopes may be necessary to address the immunological threat (Lu et al., 2012). Antigenic peptide fragments derived from the processed pathogen bind to MHC molecules and present on the DC surface. These MHC molecules can generate an adaptive immune response by exposing antigenic peptides to naive T-cell receptors (Lu et al., 2012).

An immature DC that has digested a pathogen develops and migrates to lymphoid regions in the presence of pro-inflammatory cytokines, where it can present the antigen peptide to naive T cells (Lu et al., 2012). MHC class II, CD40, CD80, CD86, OX40 L, and CCR7 are upregulated during maturation, while CCR6 is downregulated (Stevens et al., 2021).

Following LPS stimulation, AHR-activated BMDCs showed enhanced messenger RNA expression of the regulatory gene IDO2 and upregulation of IDO1, IDO2, TGF-β1, and TGF-β3 gene expression. Furthermore, in vitro, AHR-activated BMDCs promoted CD4+ CD25+ FoxP3+ Tregs in an IDO-dependent manner. In vivo, however, AHR-activated BMDCs had little effect on antigen-specific T-cell activation. Overall, TCDD-induced AHR activation affects the differentiation, activation, innate, and immunoregulatory functions of steady-state BMDCs, but not their ability to activate T cells (Simones and Shepherd, 2011).

TCDD decreased LPS and CpG-induced NF-κB p65 levels in BMDCs while producing a tendency toward increased RelB levels. AHR activation by TCDD controlled the absorption of soluble and particulate antigens by BMDCs. Induction of indoleamine-2,3-dioxygenase (IDO) and TGF-β3 after AHR activation is linked to Tregs generation (Bankoti et al., 2010).

Besides the anti-apoptotic, proangiogenic, proliferative, and oxidative stress-inducing properties of BaP, it changes the tumor immune microenvironment through its immunosuppressive action. It suppresses immune-stimulatory cells as cytotoxic T cells, DCs, M1 macrophage, and neutrophils while induces immunosuppressive cells as Treg, tolerogenic DCs, MDSCs, and M2 macrophage. BaP also induces immunosuppressive mediators such as TGF-β, FOXP3, PD-L1, CTLA-4, and IDO while inhibiting immuno-stimulatory mediators such as IFN-γ and IL-12. All these changes support its carcinogenic role in the induction of lung cancer.