Fig. 2: Epidermal sheets of untreated (A) or 7 days IMQ treated (B) mouse ears stained for MHC-II expressing cells. Langerhans cells in IMQ treated ears are less in number and display an activated morphology and increased expression of MHC-II
Imiquimod (IMQ) is a synthetic immune response modifier of the family of imidazoquinolines (Fig. 1). A 5% cream formulation of IMQ, also known as Aldara, was first approved in 1997 for the treatment of genital warts caused by human papilloma virus. In 2004 Aldara was also approved for treatment of basal cell carcinoma (BCC) and actinic keratosis. Current novel indications for IMQ include superficial squamous cell carcinoma, lentigo maligna, cutaneous T cell lymphoma, warts and molluscum contagiosum .
In 2002 Toll-like receptor (TLR) 7 and 8, which are localized mainly on immune cells, were identified as receptors for IMQ. TLRs are pattern recognition receptors recognizing conserved molecular patterns derived from microbes, like lipoproteins (TLRs 1, 2 and 6) lipopolysaccharide (TLR 4), DNA (TLR 9) or RNA (TLR 3 and 7). TLR 7 and, in human, TLR 8 are sensing single stranded RNA viruses, like human immunodeficiency virus (HIV) and influenza A, and also RNA released into endosomes by bacteria like borrelia burgdorferi lysed in phagosomes. These microbial derived patterns are sensed by TLRs as “danger signals” resulting in production of proinflammatory cytokines necessary not only for innate immunity but also for boosting adaptive immune responses. A central role in this immune regulation is played by dendritic cells (DC) which are antigen presenting cells with the unique ability to prime adaptive immune responses. Cytokines produced by DC during this priming phase determine the quality of the immune response by directing T-cell differentiation into T helper (Th) type-1, Th-2 or Th-17 lineage. Plasmacytoid DC (pDC) constitutively express high levels of TLR7 and 8. Both of these receptors are signaling via the adapter molecule MyD88 thereby activating nuclear factor kappaB (NFκB), interferon (IFN)-regulatory factor-7 and the MAPK pathway. This activation results in production of proinflammatory cytokines like interleukin (IL)-1, tumor necrosis factor (TNF)-α, IL-6, IL-8 and type-I interferons.
Topical treatment with IMQ results in activation of plasmacytoid DC (pDC), which usually reside in secondary lymphoid organs, and Langerhans cells (LC), which are the resident DCs of the epidermis (Fig. 2). IMQ induces functional maturation of human and mouse LC leading to enhanced migration of LC into skin draining lymph nodes. Furthermore, after topical IMQ treatment LCs present antigen more efficiently in an animal model of contact hypersensitivity . PDC produce high amounts of type-I IFN and other cytokines after treatment with IMQ. We and others have reported that pDC are recruited to IMQ treated skin and into IMQ treated tumors and that the anti-tumor immune response correlated with the number of infiltrating pDC [3, 8]. It is however still unclear, how pDC are recruited into IMQ treated tumors. Although keratinocytes lack expression of TLR 7 and 8, treatment with imiquimod leads to production of proinflammatory cytokines like IL-6 and IL-8 in human . Furthermore it has been shown that pDC are already present in untreated BCC and that their number increases during IMQ treatment . Recruitment into IMQ-treated tumors seems to be facilitated by a massive production of chemoattractants, particularly CXCL11, CXCL4, whose cognate receptors are highly expressed on pDC, and CCL2 and CCL5 (RANTES). The exact mechanism, how IMQ acts against tumors, is not fully understood yet. Two distinct modes of action have been identified that may act in concert to fight tumor expansion. On the one hand, direct effect on tumor cells may inhibit tumor growth. On the other hand, IMQ stimulates a panel of immune cells via cell surface and intracellular receptors, like TLR 7 and 8 and adenosine receptor A2a, thereby inducing a potent anti-tumor immune response.
Anti-tumor mechanisms independent of the immune system are inhibition of proliferation and induction of apoptosis of tumor cells. For example, treatment of BCC with IMQ results in increased apoptosis of tumor cells in vivo. Moreover, in BCC cell lines, IMQ treatment leads to upregulation of opioid growth factor receptor most likely via type-I interferon . Interference with this pathway results in a retardation of cell proliferation in a number of human cancer cell lines . In vitro treatment of human and mouse skin tumor cell lines with high doses of IMQ results in induction of apoptosis by a yet unknown mechanism which, however, seems to be independent of adenosine receptor A2a or TLR 7 [5; Holcmann and Sibilia, unpublished]. However, in tumor cells adenosine receptor signaling leads to the production of proinflammatory cytokines.
A variety of immune cells may be responsible for killing of IMQ treated tumor cells, like NK-cells and cytotoxic T-cells. Human natural killer (NK) cells have been shown to be devoid of TLR7 and 8. However, type-I interferon produced by activated pDC is a potent stimulator of NK cell mediated cytotoxicity and NK cells are found in the peritumoral infiltrate of IMQ treated BCC patients . As mentioned above, dendritic cells from IMQ treated skin display an activated phenotype and have enhanced capability of stimulating T-cells. Indeed, increased numbers of activated CD8+ and CD4+ T-cells are found in the peritumoral infiltrate of IMQ treated BCC patients and in a melanoma mouse model. However, the antitumor effect of IMQ could also be observed in the absence of mature T-cells (Holcmann and Sibilia, unpublished).
Another intriguing possibility would be that tumors infiltrating inflammatory DC themselves are the effector cells in tumor killing. Both, myeloid DC as well as pDC in IMQ treated BCC lesions, were shown to express death receptor ligands . Whereas myeloid DC expressed perforin and granzymeB pDC were shown to express TNF-related apoptosis-inducing ligand (TRAIL) and both DC cell types were shown to be able of killing via these pathways in vitro in a ligand specific manner.
Given the complexity of the immune response to IMQ treatment it will be important to better understand not only which cell populations are responsible for tumor killing but also to delineate the sequential steps in recruitment and activation of the large number of distinct immune cell types to the site of IMQ treatment. This knowledge might be transferred for the use of IMQ in other tumor diseases and for the translation into vaccine science where IMQ is increasingly used as an adjuvant for tumor-protective vaccines.
1 Kono T, Kondo S, Pastore S, Shivji GM, Tomai MA, McKenzie RC, Sauder DN (1993) Effects of a novel immunomodulator, imiquimod, on keratinocytes cytokine gene expression. Lymphokine Cytokine Res 13:71-76.
3 Palamara F, Meindl S, Holcmann M, Luhrs P, Stingl G, Sibilia M (2004) Identification and characterization of pDClike cells in normal mouse skin and melanomas treated with imiquimod. J Immunol 173:3051-3061.
4 Schiller M, Metze D, Luger TA, Grabbe S, Gunzer M (2006) Immune response modifiers – mode of action. Exp Dermatol 15:331-341.
5 Schön MP, Schön M, Klotz KN (2006) The small antitumoral immune response modifier imiquimod interacts with adenosine receptor signaling in a TLR7- and TLR8-independent fashion. J Invest Dermatol 126:1338-1347.
6 Stary G, Bangert C, Tauber M, Strohal R, Kopp T, Stingl G (2007) Tumoricidal activity of TLR7/8-activated inflammatory dendritic cells. J Exp Med 204:1441-1451.
7 Urosevic M, Oberholzer PA, Maier T, Hafner J, Laine E, Slade HB et al (2004) Imiquimod treatment induces expression of opioid growth factor receptor: a novel tumor antigen induced by interferon-alpha? Clin Cancer Res 10:4959-4970.
8 Urosevic M, Dummer R, Conrad C, Beyeler M, Laine E, Burg G, Gilliet M (2005) Disease-independent skin recruitment and activation of plasmacytoid predendritic cells following imiquimod treatment. J Natl Cancer Inst 97:1143-1153.
9 Zagon IS, Donahue RN, Rogosnitzky M, McLaughlin PJ (2008) Imiquimod upregulates the opioid growth factor receptor to inhibit cell proliferation independent of immune function. Exp Biol Med (Maywood) 233:968-979.
Maria Sibilia, Ph.D.
Institute for Cancer Research
Department of Medicine I
Medical University of Vienna