Accumulating evidence suggests that Toll-like receptor 4 (TLR4), a sensor for danger signals, is expressed not only in immune cells, but also in resident epithelial cells, and appears to participate in tissue homeostasis. To explain the premetastatic microenvironment created by the newly discovered endogenous TLR4 ligands, I propose a hypothesis of homeostatic inflammation that includes the classical danger hypothesis.
To accomplish hematogenous metastasis, tumor cells must undergo detachment from their primary sites, intravasation, migration through circulation, extravasation, resettlement and regrowth in metastatic organs. This complicated biological process can be partly explained by the concept of epithelial mesenchymal transition, in which primary tumor cells lose E-cadherin-mediated contacts and degrade the extracellular matrix with matrix metalloproteases (MMPs) to invade the surrounding tissues.1 This migration is reminiscent of the chemokine-regulated mobilization of immune cells to remote tissues. We have found that metastasis-promoting chemokines S100A8/S100A9 (two proteins that can form homo- or heterodimeric structures, which are described here as S100A8/S100A9 unless otherwise indicated) and serum amyloid A3 (SAA3) are endogenous ligands for Toll-like receptor 4 (TLR4), one of the first defense sensors for danger signals in the innate immune system.2 S100A8 and S100A9 were identified in 1987 in the synovial fluid of rheumatoid-arthritis patients (reviewed in Ref. 3). Whereas knockout of the S100A8 gene is embryonic lethal at E9.5, S100A9 appears to stabilize S100A8 because knockout of S100A9 abolishes the expression of S100A8. SAA3 belongs to the acute phase reactant SAA family. Proposed candidate receptors for SAA1 and SAA2 include the receptor for advanced glycation end-products and TLR2.4
Metastasis has been studied from the standpoint of intrinsic tumor cell properties and host responses, including angiogenesis and immune surveillance at primary sites. On the one hand, given that adaptive antitumor immunity of endogenous origin, in general, fails to work efficiently in clinical settings, much effort has been made to abrogate immunoediting in tumor cells by modifying naturally occurring tumor antigens and manipulating dendritic cells (DCs), which bridge innate and adaptive immunity.5 On the other hand, there have been controversies involving the correlation between T-cell infiltration within tumor tissues and patient prognosis. The recently revealed functional diversity of CD4+ T cells, for example, as represented by Th17 and regulatory T cells (Tregs) in addition to the classical Th1 and Th2 cells, demonstrated that these cells communicate with other immune cells in the mononuclear phagocyte system, including monocytes, macrophages and DCs (myeloid DCs in this text, hereafter simply called DC) that originate from bone marrow.6
In this minireview, I focus on the possible biological significance of endogenous TLR4 ligands in tumor immunology. Because S100A8/S100A9 and SAA3 expression is induced in the lungs, which are destined to allow metastasis, I first briefly review the immunological events that occur in primary tumors and then extend the discussion to metastatic sites. Finally, I explain the premetastatic microenvironment and propose the hypothesis of homeostatic inflammation.7
Acquisition of metastatic potential in primary tumors
Stromal gene expression profiles of 53 breast tumors have shown results associated with Th1-type immune responses.8 A protective role of Th17 cells in antitumor immunity, in which these cells induce the expression of Th1-type chemokines, such as CXCL9 and CXCL10, has been shown in 201 ovarian cancer patients.9 Another study of 104 ovarian tumors has shown that Treg is recruited to tumors by the tumor-derived chemokine CCL22, acts to suppress growth and interferon (IFN)-γ production of T cells and is associated with poor survival.10 Thus, it is likely that the tumor microenvironment that favors Th1 responses results in good antitumor immunity, and this favorable condition is affected by tumors.
Immunoediting may undergo epithelial mesenchymal transition. This event could be accompanied by a loss of tumor antigen and MHC molecules through unknown mechanisms.11 However, that is not sufficient to explain immunosuppression. One of the major and initially proven factors involved in epithelial mesenchymal transition is transforming growth factor (TGF)-β derived from mesenchymal cells in tumor tissues or from tumor cells themselves.1 As shown in mouse experiments with T-cell-specific deletion of TGF-β receptor II, TGF-β not only autonomously inhibits CD4+ T-cell differentiation into Th1, but also maintains Tregs12 (Figure 1). Another tumor-derived immunosuppressive growth factor is vascular endothelial growth factor (VEGF). Activation of VEGF receptor 2 (VEGFR2) in endothelial cells plays an essential role in their proliferation and vascular permeability by promoting structures, such as fenestration in the endothelium, through which primary tumor cells extravasate. Tumor cells almost always produce abundant VEGF. Thus, a decrease in VEGF may allow T-cell infiltration into tumors. Tumor-necrosis factor (TNF)-α-induced nuclear factor (NF)-κB activation in DC maturation was shown to be blocked by VEGFR1 on DCs.13 In addition to VEGF, maturation of DCs is also blocked by tumor-producing colony-stimulating factor (CSF)-1, IL-10 and TGF-β (Figure 1). Both CSF-1 and IL-10 promote macrophage differentiation and growth. Immature DCs and macrophages are thought to be distributed throughout tumor tissue, but mature DCs are found in peritumorous regions. Given the unique ability of DCs to present tumor antigens to T cells, their link to adaptive immunity is also abrogated by tumors.
Macrophages are functionally versatile and have a spectrum of functional states between M1 and M2, whose polarization is shown to be regulated by cytokines derived from subsets of CD4+ T cells.14 Conventionally, Th1 cytokines, such as IFN-γ and microbial lipopolysaccharide (LPS), are thought to activate M1 macrophages, which in turn produce cytokines, including TNF-α, IL-6 and IL-12, and chemokines, such as CXCL9 and CXCL10. This event establishes a danger signal that not only mobilizes Th1 cells by the chemokines, but also kills invading pathogens by production of NO and ROS (Figure 1). Although detailed expression analysis of S100A8/S100A9 and SAA3 in primary tumor cells awaits further study, other endogenous TLR4 ligand candidates derived from dying tumor cells, such as high-mobility group box 1 and heat shock cognate protein 70, or from destroyed extracellular matrix and vessels, including hyaluronan fragments and fibrin, could substitute for LPS.7 However, tumor-associated macrophages (TAM) manifest M2 features, and are less cytotoxic to tumor cells. M2-polarized macrophages express IL-10 and CCL22, which recruits Tregs.15 TAM are frequently found in avascular areas and, therefore, express VEGF in a hypoxia inducible factor-1-dependent manner. A variety of tumor cells have been found to express TLR4, which, once activated, secrete CCL20 to recruit immature DCs that show a low level of antigen presentation even after TLR4 activation.16
Then, what reinforces M2 polarization in TAM? In the mouse mammary tumor virus-polyoma middle T breast cancer model, it was shown that Th2-cell-derived IL-4 not only polarizes CD11b+Gr1-F4/80+ TAM to M2, but also works in concert with CSF-1 to induce TAM to secrete epidermal growth factor (EGF).17 Activation of EGF receptor in tumor cells results in CSF-1 expression, amplifying bidirectional signaling between M2 macrophages and tumor cells. EGF receptor-activated tumor cells obtain a greater ability to accomplish lung metastasis without changing VEGF expression levels and angiogenic density at primary sites. Given that both RAG-null and CD4-null mice display a decreased number of circulating tumor cells and attenuated lung metastasis, cells of acquired immunity can actively participate in metastasis. Because IL-4 cannot directly activate NF-κB, whose inhibition can switch polarization from M2 to M1 by targeting IκB kinase-β activity,18 other factors should be involved in the polarization and/or maintenance of the M2 status.
Gr-1+CD11b+ myeloid cells, which were initially found in tumor-bearing mice, can promote antigen-specific immunosuppressive Tregs in a CD80-mediated manner.19 In the mouse ovarian surface epithelial cell 1D8 model of ovarian cancer, both Tregs and Gr-1+CD11b+ cells are required for the suppression of human papillomavirus type 16 virus-like particle-induced release of IFN-γ by CD25-depleted splenocyte cultures. In addition, they not only favor IL-10 production, but also suppress IL-12 secretion in macrophages, providing Th2-like responses.20 Moreover, Gr-1+CD11b+ myeloid cells directly suppress T-cell immunity by Nox-2-mediated generation of ROS.21
Carcinoma-associated fibroblasts may also participate in cancer immunology. These cells produce CXCL12 (stromal cell-derived factor-1), VEGF and MMP9. CXCL12 can induce chemotaxis of Tregs.22 Correlation between Treg infiltration and CXCL12 expression has been found in cervical cancer.23 Classical Tregs inhibit T cells in lymphoid organs. Although CXCL12 is expressed in bone marrow, G-CSF has been reported to reduce CXCL12 in bone marrow. These changes may mobilize Tregs from the bone-marrow reservoir to the periphery.22
Primary tumors affect premetastatic tissues
Although metastatic outgrowth of tumor cells is abrogated in CD4-null conditions, including the RAG knockout, which attenuated EGF receptor-mediated activation of metastatic potential of primary tumor cells, we still do not know the mechanisms of the last three steps in metastasis: extravasation, recruitment and regrowth in the lungs.24 Less than 1% of recruited tumor cells appear to achieve regrowth. How metastasizing tumor cells survive and start outgrowth in a given organ is still an unanswered question, but this event has recently been reported to be regulated by VEGFR1 in the lungs,25 Src-mediated signaling of CXCL12 in bone,26 and transcription factor HoxB9 and Lef1-dependent Wnt/Tcf signaling in brain and bone.27 Even in RAG-null mice, normal breast cells can be recruited to the lungs without oncogene activation.28 Experimental activation of oncogenes after homing results in metastatic colonization. This finding suggests that tumor cells may travel to potential metastatic sites at any stage of the multistep malignant transformation sequence. Once regrowth starts, involvement of T cells that facilitate regrowth through bidirectional signaling between macrophages and regrowing tumor cells may be accomplished, as in the case of primary tumors described above.
Premetastatic organs can be affected by primary tumors before metastasizing tumor cells actually arrive. Microarray-based gene expression analysis of premetastatic lungs of B16 melanoma-bearing mice revealed the upregulation of Angpt2, MMP3 and MMP10, but not VEGF, with a concomitant lung-specific fibrin deposit, suggesting vascular destabilization.29 Primary tumor-derived TNF-α and TGF-β were assumed to be responsible for the upregulation of these three genes. In primary tumors, tumor cells express a tissue factor that activates the extrinsic coagulation pathway, and a fibrin deposit is observed on the surface of TAM. LPS-stimulated TLR4 can not only promote tissue factor expression through cytokines, such as TNF-α, but also induce the expression of plasminogen activator inhibitor-1 through activating transcription factor 3.7 Therefore, TLR4 activation by endogenous agonists in the tumor microenvironment may facilitate coagulation and block fibrinolysis. Fibrin and fibrinogen have been claimed to activate TLR4. We have performed similar experiments with B16 and Lewis lung carcinoma (LLC) and found that the chemokines S100A8/S100A9 and SAA3 are upregulated in premetastatic lungs in a primary tumor-derived growth-factor-dependent fashion, including TNF-α, TGF-β and VEGF.24 Both chemokines were identified as strong candidates for endogenous TLR4 agonists, as judged by surface plasmon resonance analysis. Less efficient, but appreciable binding of SAA3 to TLR2 was also found.2 Principally, chemokine receptor-expressing cells migrate toward regions abundant in the corresponding chemokine. Serum levels of SAA3 increase in the course of primary tumor development (Figure 2a). A peak concentration of 0.15 μg/ml was observed 8 days after tumor challenge, which was two magnitude less (one hundredth, ten to the minus second) than that of SAA1 in the same tumor-bearing mice or in cancer patients. The numbers of CD11b+ cells as well as endothelial cells with induced expression of SAA3 are also increased. Therefore, TLR4-expressing cells in the mononuclear phagocyte system could travel to the lungs. The upregulation of SAA3 that we observed in premetastatic lungs might be mediated by the vascular instability induced by the MMPs in a TNF-α-dependent manner. However, in our study, fibrinogen failed to induce SAA3 and S100A8 in macrophages and endothelial cells (Ishibashi and Maru, unpublished results). On the contrary, S100A8/S100A9 was shown to dissociate cell–cell contacts in both endothelial and epithelial cells, promoting leukocyte infiltration.30 SAA3 was shown to induce several species of MMPs in chondrocytes.31 Whichever event comes first, vascular destabilization, if any, or upregulation of endogenous TLR4 agonists may serve as a danger signal even before the eventual arrival of the true danger, metastasizing tumor cells. Injection of LLC or 3LL, a highly metastatic variant of LLC, into the tail vein of mice to block pulmonary homing and spontaneous metastasis by inhibition of the S100A8–SAA3–TLR4 system, respectively, demonstrates that the fourth and fifth steps in metastasis and, as a consequence, the final regrowth step, are regulated by TLR4.2 In metastasis induced by tumor-derived versican, TLR2 plays an essential role in driving the production of bone marrow-derived myeloid cells.32 In both cases, cross-talk between TLR and the TNF receptor exists. SAA3 is capable of inducing TNF-α expression in the lungs,33 and versican-induced metastasis is abrogated by knocking out TNF-α.32
TLR4 is expressed in granulocyte-macrophage progenitors through differentiated myeloid cells. This differentiation usually occurs upon the action of cytokines, such as G-CSF, but LPS-stimulated granulocyte-macrophage progenitors do not require cytokine action.33 We do not know the exact properties of recruited Gr-1+CD11b+ myeloid cells, whose number is increased in premetastatic lungs,7 which are inhibited in TLR4 knockout mice. However, their number is increased in the peripheral blood, possibly by the action of G-CSF produced from primary tumors.34 These cells are shown to make direct contact with CD8+ T cells and render them unresponsive to antigen stimulation by abolishing CD8 and TCR through the production of NO and ROS.35 IFN-γ and LPS were shown to sustain Gr-1+CD11b+-cell expansion.36 Interestingly, the endogenous TLR4 ligand S100A9, but not S100A8, was revealed to induce Gr-1+CD11b+-cell accumulation and inhibit DC differentiation, as judged by cytokine- and LPS-mediated in vitro maturation assays of hematopoietic progenitor cells from S100A9 transgenic mice.37 Conversely, transplanted tumors are efficiently rejected in S100A9 knockout mice, a phenotype which was rescued by anti-CD8 blocking antibody. Collectively, lung recruitment of TLR4-expressing cells that may be included in the Gr-1+CD11b+ population of myeloid cells38 is likely to prepare an immunosuppressive microenvironment to allow metastasizing tumor cells to establish themselves.
T cells may also be directly involved in premetastatic lungs. The production of CD4+ T cells is slightly increased in premetastatic lungs and is enhanced 24 h after injection of tumor cells into the tail vein of mice (Sakurai and Maru, unpublished results). In the 4T1 breast cancer-bearing mouse model, premetastatic lungs were also shown to express CCL17 and CCL22, which mobilize CCR4-expressing Tregs and tumor cells to the lungs.39 Successful killing of natural killer cells by CCR4+ Tregs in vitro suggests immune escape that may also occur in the lungs.
Presence of concrete inflammation in the lungs facilitates metastasis. An established ovalbumin sensitization/aerosol challenge model of asthma, combined with a metastasis assay, has shown more than threefold enhancement in metastasis that is abrogated by CD4+ T-cell depletion.40 An LPS-induced danger signal emitted by either resident macrophages or DCs results in M1-like responses to eliminate the pathological microbes. A danger signal provoked by induced expression of endogenous TLR4 agonists may initially provide M1-like responses. Recruited immature DCs in the lungs may differentiate into mature DCs, but with no antigen to present in the absence of microbes or tumor cells. This abortive maturation expands the field of bone marrow-derived myeloid cells that respond to primary tumor-producing growth factors to generate more TLR4 agonists, presumably with the recruitment of immunosuppressive Tregs. TLR4-expressing tumor cells can reach the immunocompromised lungs to start colonization.
The aforementioned idea that TLR4 favors metastasis is seemingly in disagreement with the report that the frequency of post-operation metastasis is higher in patients that possess TLR4 with a loss-of-function mutation than that without.41 The report lacks information on organotropism, and not only immune cells, but breast cancer cells themselves also lack TLR4, which could result in a poor metastatic microenvironment in the lung and less tumor cell mobilization. Therefore, it is difficult to precisely interpret the results for the biological significance of TLR4.
The concept of homeostatic inflammation explains the premetastatic microenvironment
When the radiosensitivity of different TLR4-expressing cells is controlled in the LPS-induced asthma model, intratracheal administration of LPS results in an increased cellular influx of Gr-1+CD11b+ cells and MHC II+CD11c+ DCs in the lungs.42 Production of CCL20 in a TLR4-dependent fashion in lung structural cells attracts CCR6+ cells. Upregulation of the CCL2–CCR2 system also recruits CCR2+Gr-1+ myeloid cells, which are precursors for CD11b+ DCs. TLR4 in lung epithelial cells was shown to be essential for triggering asthma. TLR4-expressing Clara cells, which play a xenobiotic role in terminal bronchiolar epithelium, could be supplied from bone marrow when the epithelium is injured.43 Although it is hard to administer S100A8 or SAA3 preferentially in the lungs, a similar experiment to ours with LPS demonstrated SAA3 induction in the lungs (Tomita and Maru, unpublished results). These data suggest that TLR4 that is expressed not only in mononuclear phagocyte system, but also in epithelial cells may serve as a sensor to defend against constant assaults by air-borne bacteria or chemicals in a homeostatic manner. Notably, TLR4 knockout mice display emphysema with activation of Nox3.44 In the bleomycin-induced acute lung-injury model, transepithelial migration of leukocytes is impaired in TLR4/TLR2 double-knockout mice; in this system, an endogenous TLR4 candidate hyaluronan fragment induced CXCL2 (MIP-2).45 However, before tissue destruction, high molecular weight hyaluronan contributes to tissue architecture maintenance and repair via the TLR4/TLR2 system by regulating the basal activation of NF-κB. Displacement of the high molecular weight form by its fragment may switch homeostasis to a danger signal.
Then, what is the role of endogenous TLR4 ligands in the lung? Both S100A8 and SAA3 can activate NF-κB signaling.7, 24 At least three modes of regulation in the signaling of those endogenous TLR4 agonists suggest their participation in homeostasis:
The autoregulatory loop: S100A8 induces the expression of SAA3, and SAA3 amplifies the expression of its own mRNA.2 In the inflammation-associated liver carcinogenesis model, S100A8/S100A9 was shown as a NF-κB target.46
The negative feedback loop: both agonists activate activating transcription factor 3, resulting in a negative feedback on LPS-stimulated NF-κB activation.7
Oscillatory activation: sustained TLR4 activation, which is required for cytokine expression, is consistent with oscillation in NF-κB signaling due to coordinated degradation and resynthesis of IκB proteins (conceptually reviewed in Ref. 47).
Accumulation of distinct signaling exerted at different time points may reach a threshold for gene expression. How endogenous ligand-induced signaling contributes to the summation of the oscillatory activation of NF-κB awaits further study. As expected from the positive results on surface plasmon resonance analysis between purified TLR4/MD-2 complex and SAA3, SAA3 bound to the immobilized membrane fraction of human monocytic cells that express TLR4; this binding was inhibited by an anti-SAA3 antibody in a dose-dependent manner (Figure 2b). Pre-treatment of these cells with SAA3 followed by LPS stimulation showed neither an increase nor a decrease in phosphorylated IκB signals in the presence of proteasome inhibitors (Tsukahara and Maru, unpublished results), suggesting that endogenous ligands might interfere with exogenous LPS. An anti-S100A8 blocking antibody abrogated transepithelial migration of macrophages and neutrophils into the alveolar space.48 In addition, S100A8 is capable of inducing NOS2,49 and TLR4 signaling is closely linked with ROS generation.50, 51, 52 Thus, endogenous ligand may serve to balance TLR4-mediated NF-κB signaling that is sensitive to redox.
Furthermore, in the gastrointestinal tract system, TLR4-expressing mucosal cells communicate with DCs in intestinal homeostasis. A granulocyte-macrophage progenitor comprises a common precursor for macrophages and DCs that express CX3CR1.53 Antigens are sampled in gastrointestinal tract by DCs that extend transepithelial dendrites while maintaining the integrity of the epithelial barrier in a CX3CR1-dependent manner. In Drosophila, regulated NF-κB signaling through dynamically balanced ROS production by Nox family members may play a role in gastrointestinal tract homeostasis, which needs to discriminate between commensal and pathogenic microbes.54 In dextran sodium sulfate-induced colitis, TLR4 activation was shown to play a protective role in the regulation of colonic repair.55 SAA3 is expressed in colon epithelial cells possibly as a consequence of NF-κB signaling exerted by LPS from commensal bacteria.56 SAA3 induces IL-6 expression in macrophage RAW cells.33 In the colitis-associated cancer model, both IL-6 and its signaling via Stat3 are required for the survival of epithelial cells.57
In premetastatic lungs, endogenous TLR4 ligands are upregulated without tissue destruction. This upregulation alone contributes to lung homeostasis in the absence of primary tumors. However, this event mobilizes tumor cells from the primary site as well as a variety of immune cells that could facilitate immune escape. Once bidirectional signal amplification between macrophages and tumor cells starts, as exemplified by CD4+ T-cell-induced EGF expression in macrophages in primary tumors, metastasis is achieved to produce a classical danger signal. These sequential events, from physiological host defense, its aberration by remote control of primary tumors and eventually to the production of a danger signal after the arrival and regrowth of tumor cells, are what I propose as homeostatic inflammation (Figure 2).
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I thank Dr Y. Sakurai, Dr T. Tomita and Dr F. Tsukahara, and Ms S. Ishibashi for help with experiments. This work was partly supported by Grants-in-Aid for Scientific Research from the Japanese government (No. 21117008) (homeostatic inflammation study group) to YM.
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Maru, Y. Premetastatic milieu explained by TLR4 agonist-mediated homeostatic inflammation. Cell Mol Immunol 7, 94–99 (2010). https://doi.org/10.1038/cmi.2009.113
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