Imiquimod Induces Apoptosis of Squamous Cell Carcinoma (SCC) Cells via Regulation of A20

Imiquimod, a nucleoside analogue of the imidazoquinoline family, is being used to treat various cutaneous cancers including squamous cell carcinoma (SCC). Imiquimod activates anti-tumor immunity via Toll-like receptor 7 (TLR7) in macrophage and other immune cells. Imiquimod can also affect tumor cells directly, regardless of its impact on immune system. In this study, we demonstrated that imiquimod induced apoptosis of SCC cells (SCC12) and A20 was involved in this process. When A20 was overexpressed, imiquimod-induced apoptosis was markedly inhibited. Conversely, knockdown of A20 potentiated imiquimod-induced apoptosis. Interestingly, A20 counteracted activation of c-Jun N-terminal kinase (JNK), suggesting that A20-regulated JNK activity was possible mechanism underlying imiquimod-induced apoptosis of SCC12 cells. Finally, imiquimod-induced apoptosis of SCC12 cells was taken place in a TLR7-independent manner. Our data provide new insight into the mechanism underlying imiquimod effect in cutaneous cancer treatment.     

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod‐induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c‐Jun‐N‐terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA‐like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca²⁺ release, degradation of calpain and subsequent cleavage of caspase‐4. Moreover, imiquimod triggers the activation of NF‐κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X‐linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase‐9, caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod‐induced apoptosis. They demonstrate that inhibition of NF‐kB by the inhibitor of nuclear factor kappa‐B kinase (IKK) inhibitor Bay 11‐782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.     

Imiquimod Suppresses Propagation of Herpes Simplex Virus 1 by Upregulation of Cystatin A via the Adenosine Receptor A1 Pathway

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A(1) and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.     

Identification and characterization of pDC-like cells in normal mouse skin and melanomas treated with imiquimod

Among the different subsets of dendritic cells (DC) described in humans and mice, epidermal Langerhans cells and dermal DCs represent the only DC populations resident in normal skin. In this study we describe a population of CD4(+)CD3(-) plasmacytoid DC (pDC)-like cells that accumulate in the dermis and spleens of mice topically treated with imiquimod, a low m.w. immune response modifier with potent antiviral and antitumor activities. These CD4(+)CD3(-) cells coexpress GR-1, B220, MHC class II, and, to a lesser extent, CD11c and display the phenotypic features of pDCs described in lymphoid organs. The accumulation of pDC-like cells after imiquimod treatment was detected not only in normal skin, but also in intradermally induced melanomas. Imiquimod treatment leads either to complete regression or to a significant reduction of the tumors. The number of pDCs correlates well with the clinical response of the tumors to the drug, suggesting that the antitumor effects of imiquimod could be mediated at least in part by the recruitment of pDC-like cells to the skin. Therefore, strategies aimed at activating and directing these cells into neoplastic tissues may be a promising and novel approach for the immunotherapy of various types of cancer.     

5-Lipoxygenase Activating Protein (FLAP) Dependent Leukotriene Biosynthesis Inhibition (MK591) Attenuates Lipid A Endotoxin-Induced Inflammation

The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.     

The TLR-7 agonist, imiquimod, enhances dendritic cell survival and promotes tumor antigen-specific T cell priming: relation to central nervous system antitumor immunity

Immunotherapy represents an appealing option to specifically target CNS tumors using the immune system. In this report, we tested whether adjunctive treatment with the TLR-7 agonist imiquimod could augment antitumor immune responsiveness in CNS tumor-bearing mice treated with human gp100 + tyrosine-related protein-2 melanoma-associated Ag peptide-pulsed dendritic cell (DC) vaccination. Treatment of mice with 5% imiquimod resulted in synergistic reduction in CNS tumor growth compared with melanoma-associated Ag-pulsed DC vaccination alone. Continuous imiquimod administration in CNS tumor-bearing mice, however, was associated with the appearance of robust innate immune cell infiltration and hemorrhage into the brain and the tumor. To understand the immunological mechanisms by which imiquimod augmented antitumor immunity, we tested whether imiquimod treatment enhanced DC function or the priming of tumor-specific CD8+ T cells in vivo. With bioluminescent, in vivo imaging, we determined that imiquimod dramatically enhanced both the persistence and trafficking of DCs into the draining lymph nodes after vaccination. We additionally demonstrated that imiquimod administration significantly increased the accumulation of tumor-specific CD8+ T cells in the spleen and draining lymph nodes after DC vaccination. The results suggest that imiquimod positively influences DC trafficking and the priming of tumor-specific CD8+ T cells. However, inflammatory responses induced in the brain by TLR signaling must also take into account the local microenvironment in the context of antitumor immunity to induce clinical benefit. Nevertheless, immunotherapeutic targeting of malignant CNS tumors may be enhanced by the administration of the innate immune response modifier imiquimod.     

The immune response modifier resiquimod mimics CD40-induced B cell activation

Members of the imidazoquinoline molecule family, including imiquimod and resiquimod (R-848), have potent antiviral and antitumor activities. Imiquimod cream (5%) (Aldara) is currently indicated for treatment of external genital and perianal warts. Previous characterization of these compounds has focused upon their ability to activate monocytes and dendritic cells, but recent studies have shown that resiquimod also stimulates B lymphocytes to proliferate and express an activated phenotype. This suggests that resiquimod could potentially serve as an effective vaccine adjuvant in stimulating a humoral immune response. This study shows that resiquimod mimics effects of the T-dependent CD40 signal in both mouse and human B cell lines. Resiquimod, like CD40, stimulates antibody secretion, cytokine production, protection from apoptosis, and CD80 upregulation. In addition, it shows synergy with signals delivered by the B cell antigen receptor and heightens CD40-mediated B cell activation, demonstrating that resiquimod can enhance antigen-specific responses in B lymphocytes.     

The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes

Imiquimod and R-848 are members of a family of immune response modifiers that stimulate cytokine production in monocyte/macrophages and dendritic cell cultures. This study evaluated the effects of the imidazoquinolines, imiquimod and R-848, on B lymphocyte activation. Both agents induced proliferation of murine T-cell-depleted and highly purified splenic B cell preparations as well as purified human B cells. Resting and activated B cells responded to these agents, with activated cells responding more efficiently. B cells from the LPS-hyporesponsive C3H/HeJ mice and guanosine-hyporesponsive SJL mice proliferated in response to imiquimod and R-848, indicating a different mechanism of action than lipopolysaccharide and guanine nucleosides. B cells were also stimulated by imiquimod and R-848 to produce increased immunoglobulin levels. Increased expression of a number of B cell activation markers were seen following imiquimod or R-848 stimulation. Finally, R-848 was shown to act as a vaccine adjuvant enhancing OVA-specific IgG2a levels while suppressing total IgE. These results indicate that R-848 and imiquimod are potent activators of B lymphocytes and are capable of augmenting antigen-specific immunoglobulin production.     

Autophagy-mediated anti-tumoral activity of imiquimod in Caco-2 cells

Imiquimod (IMQ) is recognized as a topical immune response modifier compound that enhances immune responses with anti-viral and anti-tumoral activities. Its anti-tumoral effects have been previously demonstrated in a variety of cancer cells, and were identified as indirect responses mediated by the immune modulation of cutaneous dendritic cells. Recently, the pro-apoptotic activities of IMQ occurring via the modulation of bcl-2 family have been reported in several tumor cells. In this study, we first observed IMQ-initiated autophagy determined by vesicular organelle formation and the generation of LC3-II in Caco-2 human colonic adenocarcinoma cells, which expressing functional TLR7. Additionally, IMQ-induced autophagy resulted in cell death occurring independently of molecular changes of apoptotic markers. Loxoribine also induced autophagy and autophagy-induced cell death at less potent than IMQ. Moreover, the activation of autophagy by rapamycin induced enhanced cell death in TNF-alpha-treated Caco-2 cells, which were autophagy and cell death-resistant. Our results led us to conclude that IMQ exerts a direct effect on the anti-tumoral activity of Caco-2 cells via autophagy-induced cell death. In conclusion, the modulation of autophagy might be applied in a potential cancer therapy for the treatment of colon cancer cells.     

Roles of TLR7 in Activation of NF-κB Signaling of Keratinocytes by Imiquimod

Imiquimod is known to exert its effects through Toll-like receptor 7 (TLR7) and/or TLR8, resulting in expression of proinflammatory cytokines and chemokines. Keratinocytes have not been reported to constitutively express TLR7 and TLR8, and the action of imiquimod is thought to be mediated by the adenine receptor, not TLR7 or TLR8. In this study, we revealed the expression of TLR7 in keratinocytes after calcium-induced differentiation. After addition of calcium to cultured keratinocytes, the immunological responses induced by imiquimod, such as activation of NF-κB and induction of TNF-α and IL-8, were more rapid and stronger. In addition, imiquimod induced the expression TLR7, and acted synergistically with calcium to induce proinflammatory cytokines. We confirmed that the responses induced by imiquimod were significantly inhibited by microRNAs suppressing TLR7 expression. These results suggest that TLR7 expressed in keratinocytes play key roles in the activation of NF-κB signaling by imiquimod, and that their modulation in keratinocytes could provide therapeutic potential for many inflammatory skin diseases.     

Divergent trophoblast responses to bacterial products mediated by TLRs

Intrauterine infections have been associated with pregnancy complications that are also linked with increased trophoblast apoptosis. TLRs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. We hypothesize that intrauterine infections may cause the excessive trophoblast cell apoptosis observed in abnormal pregnancies and that TLR may provide a mechanism of pathogenesis. In this study we describe the expression and function of TLR-2 and TLR-4 in first trimester trophoblast cells. Although ligation of TLR4 induced cytokine production by trophoblast cells, TLR-2 activation induced apoptosis. TLR-2 mediated apoptosis was dependent upon the Fas-associated death domain, the inactivation of the X-linked inhibitor of apoptosis, and the activation of caspases 8, 9, and 3. These results suggest that certain intrauterine infections may directly induce trophoblast cell death through TLR-2. Our findings provide a novel mechanism of pathogenesis for certain pregnancy complications in which there is engagement of the innate immune system.     

Carbon monoxide promotes Fas/CD95-induced apoptosis in Jurkat cells

A properly functioning immune system is dependent on programmed cell death/apoptosis at virtually every stage of lymphocyte development and activity. Carbon monoxide (CO), an enzymatic product of heme oxyenase-1, has been shown to possess anti-apoptotic effects in a number of different model systems. The purpose of the present study was to expand on this knowledge to determine the role of CO in the well established model of Fas/CD95-induced apoptosis in Jurkat cells, and to determine the mechanism by which CO can modulate T-cell apoptosis. Exposure of Jurkat cells to CO resulted in augmentation in Fas/CD95-induced apoptosis, which correlated with CO-induced up-regulation of the pro-apoptotic protein FADD as well as activation of caspase-8, -9, and -3 while simultaneously down-regulating the anti-apoptotic protein BCL-2. These effects of CO were lost with overexpression of the small interfering RNA of FADD. CO, as demonstrated previously in endothelial cells, was also anti-apoptotic in Jurkat cells against tumor necrosis factor and etoposide. We further demonstrate that this pro-apoptotic effect of CO was independent of reactive oxygen species production and involved inhibition in Fas/CD95-induced activation of the pro-survival ERK MAPK. We conclude that in contrast to other studies showing the anti-apoptotic effects of CO, Fas/CD95-induced cell death in Jurkat cells is augmented by exposure to CO and that this occurs in part via inhibition in the activation of ERK MAPK. These data begin to elucidate specific differences with regard to the effects of CO and cell death pathways and provide important and valuable insight into potential mechanisms of action.     

Adjuvant activities of immune response modifier R-848: comparison with CpG ODN

R-848 and imiquimod belong to a class of immune response modifiers that are potent inducers of cytokines, including IFN-alpha, TNF-alpha, IL-12, and IFN-gamma. Many of these cytokines can affect the acquired immune response. This study examines the effects of R-848 on aspects of acquired immunity, including immunoglobulin secretion, in vivo cytokine production, and Ag-specific T cell cytokine production. Results are compared with those of Th1 CpG ODN. R-848 and CpG ODN are effective at skewing immunity in the presence of Alum toward a Th1 Ab response (IgG2a) and away from a Th2 Ab response (IgE). R-848 and CpG ODN are also capable of initiating an immune response in the absence of additional adjuvant by specifically enhancing IgG2a levels. Both R-848 and imiquimod showed activity when given subcutaneously or orally, indicating that the compound mechanism was not through generation of a depot effect. Although CpG ODN behaves similarly to R-848, CpG ODN has a distinct cytokine profile, is more effective than R-848 when given with Alum in the priming dose, and is active only when given by the same route as the Ag. The mechanism of R-848's adjuvant activity is linked to cytokine production, where increases in IgG2a levels are associated with IFN-alpha, TNF-alpha, IL-12, and IFN-gamma induction, and decreases in IgE levels are associated with IFN-alpha and TNF-alpha. Imiquimod also enhances IgG2a production when given with Ag. The above results suggest that the imidazoquinolines R-848 and imiquimod may be attractive compounds for use as vaccine adjuvants and in inhibiting pathological responses mediated by Th2 cytokines.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.     

Efficacy of Imiquimod-Based Transcutaneous Immunization Using a Nano-Dispersed Emulsion Gel Formulation

Background

Transcutaneous immunization (TCI) approaches utilize skin associated lymphatic tissues to elicit specific immune responses. In this context, the imidazoquinoline derivative imiquimod formulated in Aldara applied onto intact skin together with a cytotoxic T lymphocyte (CTL) epitope induces potent CTL responses. However, the feasibility and efficacy of the commercial imiquimod formulation Aldara is limited by its physicochemical properties as well as its immunogenicity.

Methodology/Principal Findings

To overcome these obstacles, we developed an imiquimod-containing emulsion gel (IMI-Gel) and characterized it in comparison to Aldara for rheological properties and in vitro mouse skin permeation in a Franz diffusion cell system. Imiquimod was readily released from Aldara, while IMI-Gel showed markedly decreased drug release. Nevertheless, comparing vaccination potency of Aldara or IMI-Gel-based TCI in C57BL/6 mice against the model cytotoxic T-lymphocyte epitope SIINFEKL, we found that IMI-Gel was equally effective in terms of the frequency of peptide-specific T-cells and in vivo cytolytic activity. Importantly, transcutaneous delivery of IMI-Gel for vaccination was clearly superior to the subcutaneous or oral route of administration. Finally, IMI-Gel based TCI was at least equally effective compared to Aldara-based TCI in rejection of established SIINFEKL-expressing E.G7 tumors in a therapeutic setup indicated by enhanced tumor rejection and survival.

Conclusion/Significance

In summary, we developed a novel imiquimod formulation with feasible pharmaceutical properties and immunological efficacy that fosters the rational design of a next generation transcutaneous vaccination platform suitable for the treatment of cancer or persistent virus infections.     

Protein kinase C epsilon: a new target to control inflammation and immune-mediated disorders

Recent advances in understanding the molecular basis for mammalian host immune responses to microbial invasion suggest that the first line of defense against microbes is the recognition of pathogen-associated molecular patterns by a set of germline-encoded receptors: the Toll-like receptors (TLRs). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. Indeed, activation of T lymphocytes depends on their interaction with dendritic cells previously stimulated by TLR agonists such as bacterial lipopolysaccharide (LPS), a TLR-4 ligand. A novel PKC epsilon (epsilon) was recently found to be a critical component of TLR-4 signaling pathway and thereby to play a key role in macrophage and dendritic cell (DC) activation in response to LPS. Thus, controlling the kinase activity of PKC epsilon might represent an efficient strategy to prevent or treat certain inflammatory disorders of microbial origin.     

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation

Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIF(Lps2) mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.     

Regulation of pro-apoptotic leucocyte granule serine proteinases by intracellular serpins

Caspase activation and apoptosis can be initiated by the introduction of serine proteinases into the cytoplasm of a cell. Cytotoxic lymphocytes have evolved at least one serine proteinase with specific pro-apoptotic activity (granzyme B), as well as the mechanisms to deliver it into a target cell, and recent evidence suggests that other leucocyte granule proteinases may also have the capacity to kill if released into the interior of cells. For example, the monocyte/granulocyte proteinase cathepsin G can activate caspases in vitro, and will induce apoptosis if its entry into cells is mediated by a bacterial pore-forming protein. The potent pro-apoptotic activity of granzyme B and cathepsin G suggests that cells producing these (or other) proteinases would be at risk from self-induced death if the systems involved in packaging, degranulation or targeting fail and allow proteinases to enter the host cell cytoplasm. The purpose of the present review is to describe recent work on a group of intracellular serine proteinase inhibitors (serpins) which may function in leucocytes to prevent autolysis induced by the granule serine proteinases.