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Immunotherapy of HPV infection-caused genital warts using low dose cyclophosphamide Expert Rev. Clin. Immunol. 10(6), 791–799 (2014)
Jie Zhao*1, Wanjiang Zeng1, Yuchun Cao2, Xiaoyu Liang3 and Bo Huang3 1 Department of Gynecology and Obstetrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, The People’s Republic of China 2 Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, The People’s Republic of China 3 Department of Immunology, Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, The People’s Republic of China *Author for correspondence: [email protected]
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Condylomata acuminata (CA), caused by human papillomavirus (HPV), is a common sexually transmitted disease with half a million new cases diagnosed in the United States per year and an annual increase in incidence in China. Conventional treatments, including topical cytotoxic agents, surgical excision and physical therapies, are traumatic, cause local side-effects and are invalid for subclinical or latent infection. Exploring immune approaches to eradicate HPV in CA therapeutics are highly desirable. Recent evidence shows that FOXP3+ regulatory T (Treg) cells are accumulated in genital warts where they mediate immunosuppression. Intriguingly, a low dose of cyclophosphamide (CY), a conventional chemotherapy drug, can selectively target Treg cells, thereby enhancing the function of HPV-specific T cells and NK cells leading to efficient clearance of HPV infection. These findings have important clinical significance, and could potentially lead to a therapeutic breakthrough for the treatment of CA. KEYWORDS: cyclophosphamide • HPV infection • immunotherapy • regulatory T cell
Human papillomavirus (HPV) is a member of the papovaviridae family of epitheliotropic double-stranded DNA viruses. This family includes over 200 types of species-specific viruses that are classified into 16 genera. New HPV types are also continuously being found. These ubiquitous viruses can infect almost every part of the human skin, causing a wide range of diseases from benign lesions to invasive tumors [1]. HPV can be roughly divided into three main groups according to their DNA sequence homology and tissue tropism: cutaneous, mucocutaneous and those associated with rare autosomal recessive disorders such as epidermodysplasia verruciformis [2]. The mucocutaneous group of HPV can be further subdivided into a low-risk type, mainly associated with benign warts, and a high-risk type, which is associated with low-grade and high-grade squamous intraepithelial lesions and invasive cancers. Notably, mucosal infections are more common than cutaneous HPV diseases, with the majority being asymptomatic [3–5]. In 2009, the International Agency for Research on Cancer worked on group-classified mucosal HPV
10.1586/1744666X.2014.907743
types and 15 types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were included in Group 1 for their association with cervical carcinoma [6,7], which hereafter are referred to as high-risk HPV types. In contrast to the high-risk group, HPV types 5, 6, 7, 9, 11, 42, 43, 44 and 81 are generally characterized as low-risk type. Besides these groups, 11 additional types were classified as possibly carcinogenic. The mucosal HPV types 6 and 11 are responsible for causing benign genital condylomas as well as condylomata acuminate (CA) [1,8]. In addition, low-risk-HPV types such as HPV42 and 81 are also commonly found in CA [9], and even high-risk HPV can also be detected in patients [10,11]. CA is the most prevalent sexually transmitted viral infection in the world and affects the anogenital area in both females and males [12]. Although CA is the most common manifestation of mucosal HPV infection [13], the true prevalence of CA-related HPV types in the human population is not accurately known, in part due to missed diagnosis in asymptomatic patients and the lack of standardization of
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penile cell sampling techniques for HPV DNA detection [14]. Another reason is the lack of more sensitive techniques in the diagnosis of general HPV infections, namely, visualization with acetic acid and magnification, followed by biopsy confirmation. Therefore, the epidemiology of genital warts is not well characterized or understood [15,16]. In the past two decades, the annual incidence of CA shows an increased trend worldwide. In mainland China, the formal incidental epidemiology of CA began from 1954, with CA gradually becoming more prevalent in the early 1980s after the economic reforms. Lin [17] reported that the incidence of genital warts in Hong Kong was estimated to be 203.7 per 100,000, and among them, 10.1% were new cases. In Scandinavia, it is reported that around 10% of young women develop CA before the age of 45 [18], while the overall prevalence of HPV in women with normal cytology is estimated to be 10% [19]. CA affects about 5.5 million Americans each year and is estimated to have a prevalence rate of around 20 million [20]. Although the most common mode of transmission is known to be through sexual contact, there appear to be other modes that involve nonsexual routes of transmission, such as contaminants and nonsexual contacts such as sharing baths or towels [21]. Vertical transmission of HPV from mother to infant, either in the uterus or during delivery, is an important but unfamiliar mode of spreading the virus and is associated with a 1:400 risk of secondary development of laryngeal papillomatosis or anogenital warts [22], which may only become clinically apparent months or years later. The actual appearance of CA will depend upon the sum effect of several risk factors such as sexual behavior, number of sexual partners, stress, smoking and the presence of other STDs. For example, CA is reported as the most common anorectal infection affecting homosexual men [23]. Clinical symptoms of patients with CA include excrescences, itching, discharge, bleeding, a foul odor, tenesmus, fissuring, difficult to manage perianal hygiene and painful intercourse. CA derma shows histological epithelial thickening due to severe acanthosis and papillomatosis. Primary routes of entry are the penis, pubis, perineum, urethra and perianal areas. The most infected sites in men are the glans penis and the coronal sulcus, while in women they are the labia, clitoris, vulva and vagina ectocervix [24]. Conventional treatments of CA
HPV infections tend to recover spontaneously. In recent years, some reports have described low-risk types of HPV as being associated with younger patients who would be expected to have higher rates of spontaneous clearing of the infection [25]. Approximately 70% of HPV infections resolve spontaneously in 1 year and 90% in 2 years, without treatment, while HPV persistence develops in the remainder [26]. Other studies indicate that 16–20% of women have persistent HPV infections [27,28]. Risk factors for persistent HPV include being under 21 years at first intercourse, having four or more lifetime sexual partners, pregnancy and, last but most important, having genital warts [28]. In pregnancy, subclinical lesions may increase in size and number with the onset and development of sexual 792
maturity, resulting in increased shedding of HPV from the genital tract, followed by spontaneous regression after delivery. In addition to vertical transmission, vaginal warts may occasionally grow so large that the birth canal is obstructed. CA is most likely diagnosed at the end of pregnancies [29]. Genital warts cause significant psychosocial stress and adverse impacts on quality of life, which could result from the creation of feelings of guilt and anxiety, loss of self-esteem or the embarrassment and delays in seeking treatment [30]. As a result, one cannot regard all HPV infections as transient infections. More attention is needed on subclinical or latent infections, with continual examinations over many years. Current treatment methods include topical cytotoxic medication, surgical excision and physical therapies, such as cryotherapy, carbon dioxide laser treatment and electrofulguration [31,32]. However, all these conventional treatments have limitations as discussed below. • Surgical excisions are useful for large and extensive lesions and have long been used to treat CA but with high recurrence. Surgical excisions are usually combined with other techniques such as chemical agents. One advantage of the excision approach is that it allows for pathological examination of collected specimens. • Electrocautery and cryotherapy have been used as effective ways to destroy genital warts in the past years, but electrocautery requires local anesthesia and both techniques are somewhat dependent on the skills of the operators who control the depth and width of the lesions [33]. The main side effect is a first- or second-degree burn, making it inappropriate for large or diffuse cauterization. Controlling the depth of the wound is important in preventing scarring and injury to the genitals, especially the anal sphincters. • Carbon dioxide laser therapy, first reported by Baggish in 1980 [34], is similar to other surgical techniques with various degrees of induced scars after clearing lesions. In addition, laser therapy is limited by higher equipment costs and the potential for aerosolization of viruses, which can result in laryngeal papillomatosis in the operating surgeon [35,36]. • Bichloroacetic and trichloroacetic acid, the best known and most widely available topical chemical agents, are powerful keratolytics and cauterants that have been successfully used in the management of CA [37]. Chloroacetic acid has the advantage of being easily applied and is inexpensive. Concentrations of 50% have shown to be efficacious to internal anal CA; however, its application to the normal skin around lesions can cause local skin irritations. • 5-fluorouracil (5-FU) as a cream or salicylic acid preparation has been noted to be efficacious for CA in the urethra and vulva, inhibiting the synthesis of HPV DNA and RNA. Due to undesirable side effects, this treatment is usually used as a supportive or assistant procedure with physical therapies. • Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) was first applied in dermatology in 1992 [38] and explored as a unique and effective CA treatment [39]. In addition to the direct killing effect, ALA-PDT may also have Expert Rev. Clin. Immunol. 10(6), (2014)
Immunotherapy of HPV infection-caused warts
certain antiviral actions [40,41]. The photosensitizer binds to virion surface glycoproteins and thus impedes the early steps of the infection cycle [42]. However, in vivo clinical tests have shown that in patients with thick or extensive lesions, a single ALA-PDT is not enough to achieve satisfactory results with about 20% recurrence after 3–6 months [43,44].
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Low-dose cyclophosphamide-based immunotherapy
The above treatment methods are usually invasive and cause local side effects such as ulceration, infection and scar formation [45]. More importantly, these kinds of treatments are suited for visible warts and are less than ideal for subclinical or latent infection. This might be one of the most important reasons why CA treatment is often associated with a very high recurrence rate [45,46]. Currently, there is still no cure for CA [47] and innovative approaches to eradicate HPV in CA therapeutics are highly desirable. In this regard, exploring the immune approaches to eliminate HPV infection is becoming a new hope. General antiviral pathways of the immune system
The immune system employs multiple antiviral mechanisms against both free viral particles as well as infected cells. Usually, the viral replication cycle begins with the attachment of the virus to the host cell membrane through specific receptor binding. Upon entry into the cell, the virus looses its envelope and liberates the viral genome to start the processes of viral replication and assembling. The antigenic viral capsid proteins can induce a large amount of antibodies, including IgG, IgM and IgA, which may neutralize free viral particles before viruses adhere to cells [48]. In addition, IgM may use its multiple antigen-binding sites to agglutinate viruses and reduce the number of infectious agents. In line with the effect of the antibodies, the complement components also play an important role in the clearance of free viral particles either alone or in synergy with antibodies. Both antibody and complement bind to viruses and promote their phagocytosis by macrophages through FcR and complement receptor, respectively [49]. If the free virus escapes the surveillance of the antibody or the complement and enters host cells, a second line of defense will be employed, which involves type I interferon signaling. Viral infection activates type I interferon pathway that induces the production of protein kinase R to inhibit protein synthesis via phosphorylation of eIF-2 and activates RNAse L to further reduce protein synthesis by destroying RNA of both viral and host genes [50]. Finally, the third layer of antiviral mechanism is killing by cytotoxic immune cells. NK cells are important for eliminating virus-infected cells through a nonspecific pathway. NK cells do not recognize viral antigens but can be activated by danger signals induced by viruses and expressed on the cellular membrane [51]. In contrast to NK cells, CD8+ cytotoxic T lymphocytes (CTLs) recognize viral antigens in the form of MHC class I-peptide complex on the infected cell surface. Activated CD8+ CTLs can directly kill infected cells through Fas–FasL and perforin–granzyme pathways [52]. Therefore, immunotherapy against HPV infection has been concentrated on the above three layers of targets. informahealthcare.com
Review
Imiquimod acts as an innate anti-HPV agent
To explore the anti-HPV immunotherapies, recent research has focused on immune modulators, such as imiquimod. One of the underlying mechanisms is through the generation of the inflammatory reaction by imiquimod-activated macrophages that release proinflammatory cytokines, such as IFN-g and TNF-a [53]. Another function of imiquimod is to stimulate dendritic cell maturation for T-cell-mediated anti-HPV immunity [53]. This understanding led us to speculate that the combination of imiquimod with laser therapy might improve therapeutic outcomes. However, in our clinical observation, the combination therapy did not prevent the recurrence of large CA. The reasons may be complicated and one possibility is that imiquimod-induced inflammation biases a Th2-dominated immune response under certain conditions, thus impeding antivirus cellular immunity. In addition, to maintain the immune homeostasis, local inflammation may activate certain immunosuppressive pathways to further decrease anti-HPV immunity. Other concerns include that the effect of imiquimod is transient and long-term use of it is often associated with local erythema as a common frequent side effect that is required to be treated. Therefore, alternative immunotherapeutic strategies are desirable. Currently, low-dose cyclophosphamide shows much potential in CA immunotherapy. Cyclophosphamide metabolites are cytotoxic
Cyclophosphamide (CY) is a synthetic chemotherapy drug used to treat cancers in the past 50 years [54,55]. CY is a nitrogen mustard alkylating agent and itself has no cytotoxic effects due to the chemistry and pharmacodynamics of its design. As a prodrug, CY can be transformed into the active compound in cancer cells once absorbed. Depending on the dosage, CY can also act as an immune modification agent for a lot of autoimmune diseases and transplantation surgeries [56,57]. Cytotoxic nitrogen mustard is the key component in the CY arsenal [54]. The cytotoxic activity of nitrogen ignited by mustard is due to the reactivity of the 2-chloroethy1 groups of the central nitrogen atom. When they are absorbed by cells, nitrogen mustards form intramolecular cyclizations by elimination of chloride to form a aziridinium cation, which is like a ring composed of two carbon atoms and a central nitrogen atom [58]. This highly unstable cation is ready to donate one of its carbon atoms on the ring to several nucleophiles, such as the N-7 position of DNA guanine residues. Tumor cells are highly proliferative and are less error-correcting, thus making them very sensitive to DNA damage. When massive intrastrand and interstrand DNA cross-links form, tumor cells will undergo apoptosis via the p53 pathway [59]. Multiple steps are required to convert CY to its final efficacious compounds. First, hydroxylation of the oxazaphosphorine ring by the cytochrome P450 enzyme complex within the liver is processed to generate 4-hydroxycyclophosphamide [60]. Following spontaneous interconversion between 4-hydroxycyclophosphamide and its tautomer, aldophosphamide forms a balance between the two compounds. At the same time, hepatic 793
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cells diffuse aldophosphamide into the circulation, resulting in its subsequent uptake by other cells, thereby deciding the fate of the cells and the compounds. If the cells express high levels of aldehyde dehydrogenase (ALDH), aldophosphamide will be converted into carboxyphosphamide via ALDH [61]. Carboxyphosphamide is an inactive form and causes no effect. Carboxyphosphamide and its degradation products are the major metabolites found in urine. If the cells express no apparent levels of ALDH, aldophosphamide may undergo spontaneous degradation and finally becomes the compounds, phosphoramide mustard and acrolein, which exert the needed effects. However, acrolein is toxic to the bladder and can cause hemorrhagic cystitis [62]. But this kind of toxic effect can be largely reduced by the use of aggressive hydration and/or mesna [63]. Phosphoramide mustard plays a central role in CY treatment. Depending on the different reactions of different cell lines, CY exerts different effects. Although this kind of difference is primarily decided by the levels of ALDH expression [64,65], there exist other mechanisms. The intracellular glutathione and glutathione s-transferases also play a part in detoxification of CY [66]. Different cell lines possess different concentrations of glutathione, thus causing distinct reactions when they interact with CY and cancer cells may also show different resistance toward CY depending on their DNA repair ability. Impairment of DNA repair increases the apoptotic responses to CY [67]. Based on the different reactions and the underlying mechanisms, researchers have formulated different dosages of CY therapies aimed at different cells. For cancer cells, CY is often combined with other chemo compounds to maximize their efficacy [68]. For example, CY is widely used for lymphomas and is often effective for aggressive non-Hodgkin lymphoma. Burkitt lymphoma is particularly sensitive to CY and the dose varies from 10 to 15 mg/kg (400–600 mg/m2), usually administered intravenously every 3–4 weeks [69]. In addition, hematopoietic stem cells express high levels of ALDH and as a result are relatively resistant to CY [64]. During the differentiation of hematopoietic stem cells the expression of ALDH gradually fades, thus making CY more cytotoxic to mature hematopoietic progenitors and all lymphocyte subsets, which express low levels of ALDH [70]. Leukopenia is a common manifestation after CY treatment. Another way of using CY is to administer high doses in order to suppress the immune system and control autoimmune diseases or modulate the immune microenvironment for transplantation. For example, high doses (50–200 mg/kg) of CY are used as a preconditioning agent in patients undergoing allogeneic bone marrow transplantation for refractory leukemia, aplastic anemia and lymphomas [71,72]. Another example is pediatric multiple sclerosis, in which CY was administered at 15–25 mg/kg per dose and showed some positive results [73]. Low-dose cyclophosphamide is applied to treat genital warts by targeting the Treg cell pathway
Dating back to 1970s, researchers however found an unexpected phenomenon when treating patients with low doses of CY. Contrary to the expected immunosuppression, patients exhibited 794
enhanced immune responses [74,75]. The mechanism was not clear until the identification of T regulatory cells (Treg) by Shimon Sakaguchi and colleagues in 1995 [76]. After nearly 20 years of research, Treg cells are much more than the name implies. Attributes of CD4+CD25+FOXP3+ Treg cells include their negative regulatory effects on other immune cells such as dendritic cells (DCs), NK cells, NKT cells, B cells and conventional T cells. Much evidence has demonstrated that Treg-mediated immunosuppression is one of the crucial factors that contribute to the development of many life-threatening diseases, including cancer, AIDS, hepatitis C viral infections and malaria [77–79]. In order to suppress the function of Treg many methods have been tried. Researchers have verified that low dosages of CY were associated with the ability to selectively suppress Treg cells not only in numbers but also in immunosuppressive functions, resulting in enhanced immune responses [80]. Thus, a low dose of CY has been used as a new way to manage or modulate the microenvironments of cancer and other infectious diseases. Many cases have demonstrated a promising future for application of low doses of CY. One report demonstrated that a metronomic CY regimen not only could selectively and effectively deplete CD4+CD25+ Treg by decreasing the total number of such subsets but also restore T and NK effector functions in end-stage cancer patients, resulting in a better control of tumor progression [81]. Another Phase II clinical trial also showed promising outcomes by giving patients a low daily dose of oral CY (50 mg) combined with rofecoxib (25 mg) and weekly injections of vinblastine (3 mg/m2). The results have been very promising [82]. Minimal renal, hematological and hepatic toxicities offer significant clinical benefits to patients with advanced solid tumors. While Treg cells are preferentially suppressed, low doses of CY have no apparent toxic effects toward other proliferative tissues or organs. We have extended the application of low-dose CY to target HPV infection, which is often resistant to other immune and conventional therapies described previously. This characteristic is partially due to the HPV itself. The intraepithelial life cycle of HPV has evolved and can evade the host immune system, especially through the effects produced by its interactions with APCs (DCs and Langerhans cells) [83,84]. One major evasion mechanism is the ability to replicate without inducing the death of its host cells (a nonlytic way). This result is hypoinflammatory and as a consequence, inactivated or partially activated DCs may persistently live within the lesion, with limited migrating abilities to regional lymph nodes [85]. Consistently, the number of CTLs in HPV patients is dramatically decreased and/or becomes functionally anergic. Some reports concluded that reduced and anergic CTLs may play a role in the inactivation of APCs [86]. Our study shows that the evasion of HPV is also connected with the immunosuppressive microenvironment [87]. We have recently reported that Treg accumulates within the lesions and their numbers are positively correlated with the size of the genital warts. This suggests that Treg cells play an important role in the progression of the disease. We have conducted a clinical trial using low-dose CY to selectively deplete CD4+CD25+ Treg cells in patients with large genital Expert Rev. Clin. Immunol. 10(6), (2014)
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Immunotherapy of HPV infection-caused warts
warts [88]. In this trial [89], we recruited 78 patients and treated them with laser therapy. Subsequently, 52 of them received oral 50 mg CY once daily for 7 days and 26 others took placebos. All of the patients who took low-dose CY exhibited complete clearance within the first 6 weeks. Nine of them had a recurrence later but recovered by receiving the same treatment for another week. We also set a group that received higher dosages (200 mg orally). However, 6 of these 8 patients did not exhibit such effects. This excludes the possibility of direct effects of CY on CA. Furthermore, the low-dose treatments exhibited minimal toxicity toward other organs and restored host immunity. The effects caused by CY have been observed and studied for three decades. The target cells of CY were verified nearly 10 years earlier [80]. But the detailed molecular mechanism underlying these pathways remained elusive. As previously described, the effects of CY are closely related to intracellular enzyme systems and reduction systems, while different cell types possess different qualities. The cells within highly proliferative tissues such as the liver, intestinal epithelium and hematopoietic stem cells in the bone marrow show high levels of ALDH expression and are highly resistant toward relatively low doses of CY [64]. Given the conversion of toxic aldophosphamide to inactive carboxyphosphamide by ALDH [61], lymphocytes exhibit low levels of ALDH expression, implying that the other mechanism involves detoxification of CY by Treg and other T-cell subtypes. A separate study of ours has shown that the cause is related to intracellular glutathione which is the key player reducing the reduction system, thus greatly influencing the detoxification of intracellular compounds [90]. While the synthesis process of glutathione depends on the availability of ATP, the main source of energy within the cell, we have found that Treg cells and conventional T cells express quite different intracellular ATP levels, with the former being much lower than the latter. The underlying molecular mechanism might involve two molecules miR-142–3p and ectonucleoside triphosphate diphosphohydrolase CD39. Remarked downregulation of miR-142-3p was found in Treg cells, which caused upregulation of adenyl cyclase 9 [91], an enzyme which is closely connected with the amount of intracellular ATP by converting ATP to cAMP, thus reducing the total amount of ATP. cAMP is not only a second messenger within Treg cells themselves but it can also cause the suppression of conventional T-cell functions via gap junctions [92]. CD39 is upregulated in Treg cells, catalyzing extracellular ATP and ADP, thus facilitating the efflux of intracellular ATP. The product of its catalysis, adenosine, also plays an important part in suppression of the immune milieu [93]. These two mechanisms synergistically generate a deep downregulation of intracellular ATP causing much reduced levels of glutathione synthesis in Treg cells. Glutathione is conjuncted with CY, phosphoramide mustard and other metabolites, making reactivity changes and facilitating the efflux. The ability of detoxification of CY is much more reduced, while simultaneously enhancing Treg cell hypersensitivity to it. The mechanism underlying low-dose CY selectively targeting Treg cells was summarized in a schematic (FIGURE 1). informahealthcare.com
CD39
CD39
Treg
T cell
miR-142-3p
miR-142-3p
ATP
ATP
Adenosine
Adenosine
Phosphate groups
Phosphate groups Ribose
Ribose
Treg
T cell
GSH
GSH
Sensitivity to CY
Sensitivity to CY
Figure 1. A schematic to summarize the mechanism underlying low-dose cyclophosphamide selectively targeting Treg cells. Relative to conventional T cells, Treg cells downregulate miR-142-3p and upregulate ectonucleoside triphosphate diphosphohydrolase CD39. miR-142-3p targets adenyl cyclase 9, which converts ATP to cAMP, and CD39 degrades extracellular ATP, facilitating the efflux of cytosolic ATP. As a result, ATP levels were much lower in Treg cells. The low levels of ATP attenuate the synthesis of glutathione in Treg cells, leading to a decrease in CY detoxification, thus increasing the sensitivity of Treg cells to low-dose CY. CY: Cyclophosphamide;
Expert commentary & five-year view
Anti-HPV immune responses are mainly mediated by T cells. Once HPV viruses invade the skin tissue, Langerhans cells take up, process and present viral antigens to both CD4+ and CD8+ T cells in the draining lymph node, resulting in T-cell activation. Consequently, activated T cells migrate to the infected site and eliminate viruses by lysing the infected keratinocytes. However, several pitfalls impede this process. Physiologically, keratinocytes are automatically programmed to death and desquamation. Therefore, HPV infection-induced keratinocytic death is probably more like a normal process rather than cytopathic death, leading to limited inflammation and mild immune response. Meanwhile, HPV infection itself may suppress antivirus immunity by directly downregulating the expression of IFN-a/b, two important cytokines that bridge innate 795
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and adaptive immunity by affecting DCs. Besides these two aspects, to avoid self-damage caused by over immune response, the immune system evolves multiple components to control the magnitude and type of immune responses against invading antigens. Among these components, FOXP3+ Treg cells are a key mechanism underlying immune suppression, unfortunately which is generally usurped by various types of viral infections for immune evasion, including HPV infection. However, HPV infection-induced immune tolerance is apparently reversible by low-dose CY leading to immune clearance of infectious viruses. Our clinical results indicate that a low dose of CY can prevent CA recurrence in combination with the conventional laser therapy. If we further consider the low cost and convenient oral administration, low-dose CY might represent a therapeutic breakthrough in CA treatment. However, large-scale clinical trials are required to further verify its therapeutic outcomes. Also, it would be valuable to extend the trial to patients with different genetic backgrounds. Another implication is that similar to
HPV infection, low-dose CY might also be useful in other viral infections such as hepatitis virus B infection which also involves Treg cells in pathophysiological processes. Acknowledgements
We thank Yonghong Wan from McMaster University for assistance in editing this article. Financial & competing interests disclosure
This work was partially supported by the National Natural Science Foundation of China (81102224, 30972667), the National Basic Research Program of China (2014CB542103) and the National Science Fund for Distinguished Young Scholars of China (81225021). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues • Condylomata acuminata caused by HPV type 6 or 11 is a common sexually transmitted disease with increased incidence in China. • HPV infects basal keratinocytes and causes genital warts with a variable latent period. • Spontaneous regression occurs in most HPV type 6 or 11 infection-caused lesions; however, some lesions evade anti-HPV immunity and finally develop into warts. • Multiple treatments including surgical excision and topical physical therapies are commonly associated with high recurrence rates, especially in the presence of latent HPV in the so-called clinically normal epithelium outside the treatment area. • Treg cells participate in the infection process of various viruses, including HPV type 6 or 11 infection. • Treg cells inhibit multiple components of the immune system and can mediate virus immune evasion. • Cyclophosphamide (CY) is a chemotherapeutic agent widely used in malignancies as well as autoimmune disorders. • Low-dose CY may act as immune modulator to enhance anti-HPV immune responses by targeting Treg cells, leading to clearance of infectious viruses. • Reduced intracellular ATP levels in Treg cells confer the sensitivity of Treg cells to low-dose CY.
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Review
Immunotherapy of HPV infection-caused genital warts using low dose cyclophosphamide Expert Rev. Clin. Immunol. 10(6), 791–799 (2014)
Jie Zhao*1, Wanjiang Zeng1, Yuchun Cao2, Xiaoyu Liang3 and Bo Huang3 1 Department of Gynecology and Obstetrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, The People’s Republic of China 2 Department of Dermatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, The People’s Republic of China 3 Department of Immunology, Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, The People’s Republic of China *Author for correspondence: [email protected]
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Condylomata acuminata (CA), caused by human papillomavirus (HPV), is a common sexually transmitted disease with half a million new cases diagnosed in the United States per year and an annual increase in incidence in China. Conventional treatments, including topical cytotoxic agents, surgical excision and physical therapies, are traumatic, cause local side-effects and are invalid for subclinical or latent infection. Exploring immune approaches to eradicate HPV in CA therapeutics are highly desirable. Recent evidence shows that FOXP3+ regulatory T (Treg) cells are accumulated in genital warts where they mediate immunosuppression. Intriguingly, a low dose of cyclophosphamide (CY), a conventional chemotherapy drug, can selectively target Treg cells, thereby enhancing the function of HPV-specific T cells and NK cells leading to efficient clearance of HPV infection. These findings have important clinical significance, and could potentially lead to a therapeutic breakthrough for the treatment of CA. KEYWORDS: cyclophosphamide • HPV infection • immunotherapy • regulatory T cell
Human papillomavirus (HPV) is a member of the papovaviridae family of epitheliotropic double-stranded DNA viruses. This family includes over 200 types of species-specific viruses that are classified into 16 genera. New HPV types are also continuously being found. These ubiquitous viruses can infect almost every part of the human skin, causing a wide range of diseases from benign lesions to invasive tumors [1]. HPV can be roughly divided into three main groups according to their DNA sequence homology and tissue tropism: cutaneous, mucocutaneous and those associated with rare autosomal recessive disorders such as epidermodysplasia verruciformis [2]. The mucocutaneous group of HPV can be further subdivided into a low-risk type, mainly associated with benign warts, and a high-risk type, which is associated with low-grade and high-grade squamous intraepithelial lesions and invasive cancers. Notably, mucosal infections are more common than cutaneous HPV diseases, with the majority being asymptomatic [3–5]. In 2009, the International Agency for Research on Cancer worked on group-classified mucosal HPV
10.1586/1744666X.2014.907743
types and 15 types (HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) were included in Group 1 for their association with cervical carcinoma [6,7], which hereafter are referred to as high-risk HPV types. In contrast to the high-risk group, HPV types 5, 6, 7, 9, 11, 42, 43, 44 and 81 are generally characterized as low-risk type. Besides these groups, 11 additional types were classified as possibly carcinogenic. The mucosal HPV types 6 and 11 are responsible for causing benign genital condylomas as well as condylomata acuminate (CA) [1,8]. In addition, low-risk-HPV types such as HPV42 and 81 are also commonly found in CA [9], and even high-risk HPV can also be detected in patients [10,11]. CA is the most prevalent sexually transmitted viral infection in the world and affects the anogenital area in both females and males [12]. Although CA is the most common manifestation of mucosal HPV infection [13], the true prevalence of CA-related HPV types in the human population is not accurately known, in part due to missed diagnosis in asymptomatic patients and the lack of standardization of
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penile cell sampling techniques for HPV DNA detection [14]. Another reason is the lack of more sensitive techniques in the diagnosis of general HPV infections, namely, visualization with acetic acid and magnification, followed by biopsy confirmation. Therefore, the epidemiology of genital warts is not well characterized or understood [15,16]. In the past two decades, the annual incidence of CA shows an increased trend worldwide. In mainland China, the formal incidental epidemiology of CA began from 1954, with CA gradually becoming more prevalent in the early 1980s after the economic reforms. Lin [17] reported that the incidence of genital warts in Hong Kong was estimated to be 203.7 per 100,000, and among them, 10.1% were new cases. In Scandinavia, it is reported that around 10% of young women develop CA before the age of 45 [18], while the overall prevalence of HPV in women with normal cytology is estimated to be 10% [19]. CA affects about 5.5 million Americans each year and is estimated to have a prevalence rate of around 20 million [20]. Although the most common mode of transmission is known to be through sexual contact, there appear to be other modes that involve nonsexual routes of transmission, such as contaminants and nonsexual contacts such as sharing baths or towels [21]. Vertical transmission of HPV from mother to infant, either in the uterus or during delivery, is an important but unfamiliar mode of spreading the virus and is associated with a 1:400 risk of secondary development of laryngeal papillomatosis or anogenital warts [22], which may only become clinically apparent months or years later. The actual appearance of CA will depend upon the sum effect of several risk factors such as sexual behavior, number of sexual partners, stress, smoking and the presence of other STDs. For example, CA is reported as the most common anorectal infection affecting homosexual men [23]. Clinical symptoms of patients with CA include excrescences, itching, discharge, bleeding, a foul odor, tenesmus, fissuring, difficult to manage perianal hygiene and painful intercourse. CA derma shows histological epithelial thickening due to severe acanthosis and papillomatosis. Primary routes of entry are the penis, pubis, perineum, urethra and perianal areas. The most infected sites in men are the glans penis and the coronal sulcus, while in women they are the labia, clitoris, vulva and vagina ectocervix [24]. Conventional treatments of CA
HPV infections tend to recover spontaneously. In recent years, some reports have described low-risk types of HPV as being associated with younger patients who would be expected to have higher rates of spontaneous clearing of the infection [25]. Approximately 70% of HPV infections resolve spontaneously in 1 year and 90% in 2 years, without treatment, while HPV persistence develops in the remainder [26]. Other studies indicate that 16–20% of women have persistent HPV infections [27,28]. Risk factors for persistent HPV include being under 21 years at first intercourse, having four or more lifetime sexual partners, pregnancy and, last but most important, having genital warts [28]. In pregnancy, subclinical lesions may increase in size and number with the onset and development of sexual 792
maturity, resulting in increased shedding of HPV from the genital tract, followed by spontaneous regression after delivery. In addition to vertical transmission, vaginal warts may occasionally grow so large that the birth canal is obstructed. CA is most likely diagnosed at the end of pregnancies [29]. Genital warts cause significant psychosocial stress and adverse impacts on quality of life, which could result from the creation of feelings of guilt and anxiety, loss of self-esteem or the embarrassment and delays in seeking treatment [30]. As a result, one cannot regard all HPV infections as transient infections. More attention is needed on subclinical or latent infections, with continual examinations over many years. Current treatment methods include topical cytotoxic medication, surgical excision and physical therapies, such as cryotherapy, carbon dioxide laser treatment and electrofulguration [31,32]. However, all these conventional treatments have limitations as discussed below. • Surgical excisions are useful for large and extensive lesions and have long been used to treat CA but with high recurrence. Surgical excisions are usually combined with other techniques such as chemical agents. One advantage of the excision approach is that it allows for pathological examination of collected specimens. • Electrocautery and cryotherapy have been used as effective ways to destroy genital warts in the past years, but electrocautery requires local anesthesia and both techniques are somewhat dependent on the skills of the operators who control the depth and width of the lesions [33]. The main side effect is a first- or second-degree burn, making it inappropriate for large or diffuse cauterization. Controlling the depth of the wound is important in preventing scarring and injury to the genitals, especially the anal sphincters. • Carbon dioxide laser therapy, first reported by Baggish in 1980 [34], is similar to other surgical techniques with various degrees of induced scars after clearing lesions. In addition, laser therapy is limited by higher equipment costs and the potential for aerosolization of viruses, which can result in laryngeal papillomatosis in the operating surgeon [35,36]. • Bichloroacetic and trichloroacetic acid, the best known and most widely available topical chemical agents, are powerful keratolytics and cauterants that have been successfully used in the management of CA [37]. Chloroacetic acid has the advantage of being easily applied and is inexpensive. Concentrations of 50% have shown to be efficacious to internal anal CA; however, its application to the normal skin around lesions can cause local skin irritations. • 5-fluorouracil (5-FU) as a cream or salicylic acid preparation has been noted to be efficacious for CA in the urethra and vulva, inhibiting the synthesis of HPV DNA and RNA. Due to undesirable side effects, this treatment is usually used as a supportive or assistant procedure with physical therapies. • Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) was first applied in dermatology in 1992 [38] and explored as a unique and effective CA treatment [39]. In addition to the direct killing effect, ALA-PDT may also have Expert Rev. Clin. Immunol. 10(6), (2014)
Immunotherapy of HPV infection-caused warts
certain antiviral actions [40,41]. The photosensitizer binds to virion surface glycoproteins and thus impedes the early steps of the infection cycle [42]. However, in vivo clinical tests have shown that in patients with thick or extensive lesions, a single ALA-PDT is not enough to achieve satisfactory results with about 20% recurrence after 3–6 months [43,44].
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Low-dose cyclophosphamide-based immunotherapy
The above treatment methods are usually invasive and cause local side effects such as ulceration, infection and scar formation [45]. More importantly, these kinds of treatments are suited for visible warts and are less than ideal for subclinical or latent infection. This might be one of the most important reasons why CA treatment is often associated with a very high recurrence rate [45,46]. Currently, there is still no cure for CA [47] and innovative approaches to eradicate HPV in CA therapeutics are highly desirable. In this regard, exploring the immune approaches to eliminate HPV infection is becoming a new hope. General antiviral pathways of the immune system
The immune system employs multiple antiviral mechanisms against both free viral particles as well as infected cells. Usually, the viral replication cycle begins with the attachment of the virus to the host cell membrane through specific receptor binding. Upon entry into the cell, the virus looses its envelope and liberates the viral genome to start the processes of viral replication and assembling. The antigenic viral capsid proteins can induce a large amount of antibodies, including IgG, IgM and IgA, which may neutralize free viral particles before viruses adhere to cells [48]. In addition, IgM may use its multiple antigen-binding sites to agglutinate viruses and reduce the number of infectious agents. In line with the effect of the antibodies, the complement components also play an important role in the clearance of free viral particles either alone or in synergy with antibodies. Both antibody and complement bind to viruses and promote their phagocytosis by macrophages through FcR and complement receptor, respectively [49]. If the free virus escapes the surveillance of the antibody or the complement and enters host cells, a second line of defense will be employed, which involves type I interferon signaling. Viral infection activates type I interferon pathway that induces the production of protein kinase R to inhibit protein synthesis via phosphorylation of eIF-2 and activates RNAse L to further reduce protein synthesis by destroying RNA of both viral and host genes [50]. Finally, the third layer of antiviral mechanism is killing by cytotoxic immune cells. NK cells are important for eliminating virus-infected cells through a nonspecific pathway. NK cells do not recognize viral antigens but can be activated by danger signals induced by viruses and expressed on the cellular membrane [51]. In contrast to NK cells, CD8+ cytotoxic T lymphocytes (CTLs) recognize viral antigens in the form of MHC class I-peptide complex on the infected cell surface. Activated CD8+ CTLs can directly kill infected cells through Fas–FasL and perforin–granzyme pathways [52]. Therefore, immunotherapy against HPV infection has been concentrated on the above three layers of targets. informahealthcare.com
Review
Imiquimod acts as an innate anti-HPV agent
To explore the anti-HPV immunotherapies, recent research has focused on immune modulators, such as imiquimod. One of the underlying mechanisms is through the generation of the inflammatory reaction by imiquimod-activated macrophages that release proinflammatory cytokines, such as IFN-g and TNF-a [53]. Another function of imiquimod is to stimulate dendritic cell maturation for T-cell-mediated anti-HPV immunity [53]. This understanding led us to speculate that the combination of imiquimod with laser therapy might improve therapeutic outcomes. However, in our clinical observation, the combination therapy did not prevent the recurrence of large CA. The reasons may be complicated and one possibility is that imiquimod-induced inflammation biases a Th2-dominated immune response under certain conditions, thus impeding antivirus cellular immunity. In addition, to maintain the immune homeostasis, local inflammation may activate certain immunosuppressive pathways to further decrease anti-HPV immunity. Other concerns include that the effect of imiquimod is transient and long-term use of it is often associated with local erythema as a common frequent side effect that is required to be treated. Therefore, alternative immunotherapeutic strategies are desirable. Currently, low-dose cyclophosphamide shows much potential in CA immunotherapy. Cyclophosphamide metabolites are cytotoxic
Cyclophosphamide (CY) is a synthetic chemotherapy drug used to treat cancers in the past 50 years [54,55]. CY is a nitrogen mustard alkylating agent and itself has no cytotoxic effects due to the chemistry and pharmacodynamics of its design. As a prodrug, CY can be transformed into the active compound in cancer cells once absorbed. Depending on the dosage, CY can also act as an immune modification agent for a lot of autoimmune diseases and transplantation surgeries [56,57]. Cytotoxic nitrogen mustard is the key component in the CY arsenal [54]. The cytotoxic activity of nitrogen ignited by mustard is due to the reactivity of the 2-chloroethy1 groups of the central nitrogen atom. When they are absorbed by cells, nitrogen mustards form intramolecular cyclizations by elimination of chloride to form a aziridinium cation, which is like a ring composed of two carbon atoms and a central nitrogen atom [58]. This highly unstable cation is ready to donate one of its carbon atoms on the ring to several nucleophiles, such as the N-7 position of DNA guanine residues. Tumor cells are highly proliferative and are less error-correcting, thus making them very sensitive to DNA damage. When massive intrastrand and interstrand DNA cross-links form, tumor cells will undergo apoptosis via the p53 pathway [59]. Multiple steps are required to convert CY to its final efficacious compounds. First, hydroxylation of the oxazaphosphorine ring by the cytochrome P450 enzyme complex within the liver is processed to generate 4-hydroxycyclophosphamide [60]. Following spontaneous interconversion between 4-hydroxycyclophosphamide and its tautomer, aldophosphamide forms a balance between the two compounds. At the same time, hepatic 793
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cells diffuse aldophosphamide into the circulation, resulting in its subsequent uptake by other cells, thereby deciding the fate of the cells and the compounds. If the cells express high levels of aldehyde dehydrogenase (ALDH), aldophosphamide will be converted into carboxyphosphamide via ALDH [61]. Carboxyphosphamide is an inactive form and causes no effect. Carboxyphosphamide and its degradation products are the major metabolites found in urine. If the cells express no apparent levels of ALDH, aldophosphamide may undergo spontaneous degradation and finally becomes the compounds, phosphoramide mustard and acrolein, which exert the needed effects. However, acrolein is toxic to the bladder and can cause hemorrhagic cystitis [62]. But this kind of toxic effect can be largely reduced by the use of aggressive hydration and/or mesna [63]. Phosphoramide mustard plays a central role in CY treatment. Depending on the different reactions of different cell lines, CY exerts different effects. Although this kind of difference is primarily decided by the levels of ALDH expression [64,65], there exist other mechanisms. The intracellular glutathione and glutathione s-transferases also play a part in detoxification of CY [66]. Different cell lines possess different concentrations of glutathione, thus causing distinct reactions when they interact with CY and cancer cells may also show different resistance toward CY depending on their DNA repair ability. Impairment of DNA repair increases the apoptotic responses to CY [67]. Based on the different reactions and the underlying mechanisms, researchers have formulated different dosages of CY therapies aimed at different cells. For cancer cells, CY is often combined with other chemo compounds to maximize their efficacy [68]. For example, CY is widely used for lymphomas and is often effective for aggressive non-Hodgkin lymphoma. Burkitt lymphoma is particularly sensitive to CY and the dose varies from 10 to 15 mg/kg (400–600 mg/m2), usually administered intravenously every 3–4 weeks [69]. In addition, hematopoietic stem cells express high levels of ALDH and as a result are relatively resistant to CY [64]. During the differentiation of hematopoietic stem cells the expression of ALDH gradually fades, thus making CY more cytotoxic to mature hematopoietic progenitors and all lymphocyte subsets, which express low levels of ALDH [70]. Leukopenia is a common manifestation after CY treatment. Another way of using CY is to administer high doses in order to suppress the immune system and control autoimmune diseases or modulate the immune microenvironment for transplantation. For example, high doses (50–200 mg/kg) of CY are used as a preconditioning agent in patients undergoing allogeneic bone marrow transplantation for refractory leukemia, aplastic anemia and lymphomas [71,72]. Another example is pediatric multiple sclerosis, in which CY was administered at 15–25 mg/kg per dose and showed some positive results [73]. Low-dose cyclophosphamide is applied to treat genital warts by targeting the Treg cell pathway
Dating back to 1970s, researchers however found an unexpected phenomenon when treating patients with low doses of CY. Contrary to the expected immunosuppression, patients exhibited 794
enhanced immune responses [74,75]. The mechanism was not clear until the identification of T regulatory cells (Treg) by Shimon Sakaguchi and colleagues in 1995 [76]. After nearly 20 years of research, Treg cells are much more than the name implies. Attributes of CD4+CD25+FOXP3+ Treg cells include their negative regulatory effects on other immune cells such as dendritic cells (DCs), NK cells, NKT cells, B cells and conventional T cells. Much evidence has demonstrated that Treg-mediated immunosuppression is one of the crucial factors that contribute to the development of many life-threatening diseases, including cancer, AIDS, hepatitis C viral infections and malaria [77–79]. In order to suppress the function of Treg many methods have been tried. Researchers have verified that low dosages of CY were associated with the ability to selectively suppress Treg cells not only in numbers but also in immunosuppressive functions, resulting in enhanced immune responses [80]. Thus, a low dose of CY has been used as a new way to manage or modulate the microenvironments of cancer and other infectious diseases. Many cases have demonstrated a promising future for application of low doses of CY. One report demonstrated that a metronomic CY regimen not only could selectively and effectively deplete CD4+CD25+ Treg by decreasing the total number of such subsets but also restore T and NK effector functions in end-stage cancer patients, resulting in a better control of tumor progression [81]. Another Phase II clinical trial also showed promising outcomes by giving patients a low daily dose of oral CY (50 mg) combined with rofecoxib (25 mg) and weekly injections of vinblastine (3 mg/m2). The results have been very promising [82]. Minimal renal, hematological and hepatic toxicities offer significant clinical benefits to patients with advanced solid tumors. While Treg cells are preferentially suppressed, low doses of CY have no apparent toxic effects toward other proliferative tissues or organs. We have extended the application of low-dose CY to target HPV infection, which is often resistant to other immune and conventional therapies described previously. This characteristic is partially due to the HPV itself. The intraepithelial life cycle of HPV has evolved and can evade the host immune system, especially through the effects produced by its interactions with APCs (DCs and Langerhans cells) [83,84]. One major evasion mechanism is the ability to replicate without inducing the death of its host cells (a nonlytic way). This result is hypoinflammatory and as a consequence, inactivated or partially activated DCs may persistently live within the lesion, with limited migrating abilities to regional lymph nodes [85]. Consistently, the number of CTLs in HPV patients is dramatically decreased and/or becomes functionally anergic. Some reports concluded that reduced and anergic CTLs may play a role in the inactivation of APCs [86]. Our study shows that the evasion of HPV is also connected with the immunosuppressive microenvironment [87]. We have recently reported that Treg accumulates within the lesions and their numbers are positively correlated with the size of the genital warts. This suggests that Treg cells play an important role in the progression of the disease. We have conducted a clinical trial using low-dose CY to selectively deplete CD4+CD25+ Treg cells in patients with large genital Expert Rev. Clin. Immunol. 10(6), (2014)
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Immunotherapy of HPV infection-caused warts
warts [88]. In this trial [89], we recruited 78 patients and treated them with laser therapy. Subsequently, 52 of them received oral 50 mg CY once daily for 7 days and 26 others took placebos. All of the patients who took low-dose CY exhibited complete clearance within the first 6 weeks. Nine of them had a recurrence later but recovered by receiving the same treatment for another week. We also set a group that received higher dosages (200 mg orally). However, 6 of these 8 patients did not exhibit such effects. This excludes the possibility of direct effects of CY on CA. Furthermore, the low-dose treatments exhibited minimal toxicity toward other organs and restored host immunity. The effects caused by CY have been observed and studied for three decades. The target cells of CY were verified nearly 10 years earlier [80]. But the detailed molecular mechanism underlying these pathways remained elusive. As previously described, the effects of CY are closely related to intracellular enzyme systems and reduction systems, while different cell types possess different qualities. The cells within highly proliferative tissues such as the liver, intestinal epithelium and hematopoietic stem cells in the bone marrow show high levels of ALDH expression and are highly resistant toward relatively low doses of CY [64]. Given the conversion of toxic aldophosphamide to inactive carboxyphosphamide by ALDH [61], lymphocytes exhibit low levels of ALDH expression, implying that the other mechanism involves detoxification of CY by Treg and other T-cell subtypes. A separate study of ours has shown that the cause is related to intracellular glutathione which is the key player reducing the reduction system, thus greatly influencing the detoxification of intracellular compounds [90]. While the synthesis process of glutathione depends on the availability of ATP, the main source of energy within the cell, we have found that Treg cells and conventional T cells express quite different intracellular ATP levels, with the former being much lower than the latter. The underlying molecular mechanism might involve two molecules miR-142–3p and ectonucleoside triphosphate diphosphohydrolase CD39. Remarked downregulation of miR-142-3p was found in Treg cells, which caused upregulation of adenyl cyclase 9 [91], an enzyme which is closely connected with the amount of intracellular ATP by converting ATP to cAMP, thus reducing the total amount of ATP. cAMP is not only a second messenger within Treg cells themselves but it can also cause the suppression of conventional T-cell functions via gap junctions [92]. CD39 is upregulated in Treg cells, catalyzing extracellular ATP and ADP, thus facilitating the efflux of intracellular ATP. The product of its catalysis, adenosine, also plays an important part in suppression of the immune milieu [93]. These two mechanisms synergistically generate a deep downregulation of intracellular ATP causing much reduced levels of glutathione synthesis in Treg cells. Glutathione is conjuncted with CY, phosphoramide mustard and other metabolites, making reactivity changes and facilitating the efflux. The ability of detoxification of CY is much more reduced, while simultaneously enhancing Treg cell hypersensitivity to it. The mechanism underlying low-dose CY selectively targeting Treg cells was summarized in a schematic (FIGURE 1). informahealthcare.com
CD39
CD39
Treg
T cell
miR-142-3p
miR-142-3p
ATP
ATP
Adenosine
Adenosine
Phosphate groups
Phosphate groups Ribose
Ribose
Treg
T cell
GSH
GSH
Sensitivity to CY
Sensitivity to CY
Figure 1. A schematic to summarize the mechanism underlying low-dose cyclophosphamide selectively targeting Treg cells. Relative to conventional T cells, Treg cells downregulate miR-142-3p and upregulate ectonucleoside triphosphate diphosphohydrolase CD39. miR-142-3p targets adenyl cyclase 9, which converts ATP to cAMP, and CD39 degrades extracellular ATP, facilitating the efflux of cytosolic ATP. As a result, ATP levels were much lower in Treg cells. The low levels of ATP attenuate the synthesis of glutathione in Treg cells, leading to a decrease in CY detoxification, thus increasing the sensitivity of Treg cells to low-dose CY. CY: Cyclophosphamide;
Expert commentary & five-year view
Anti-HPV immune responses are mainly mediated by T cells. Once HPV viruses invade the skin tissue, Langerhans cells take up, process and present viral antigens to both CD4+ and CD8+ T cells in the draining lymph node, resulting in T-cell activation. Consequently, activated T cells migrate to the infected site and eliminate viruses by lysing the infected keratinocytes. However, several pitfalls impede this process. Physiologically, keratinocytes are automatically programmed to death and desquamation. Therefore, HPV infection-induced keratinocytic death is probably more like a normal process rather than cytopathic death, leading to limited inflammation and mild immune response. Meanwhile, HPV infection itself may suppress antivirus immunity by directly downregulating the expression of IFN-a/b, two important cytokines that bridge innate 795
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Zhao, Zeng, Cao, Liang & Huang
and adaptive immunity by affecting DCs. Besides these two aspects, to avoid self-damage caused by over immune response, the immune system evolves multiple components to control the magnitude and type of immune responses against invading antigens. Among these components, FOXP3+ Treg cells are a key mechanism underlying immune suppression, unfortunately which is generally usurped by various types of viral infections for immune evasion, including HPV infection. However, HPV infection-induced immune tolerance is apparently reversible by low-dose CY leading to immune clearance of infectious viruses. Our clinical results indicate that a low dose of CY can prevent CA recurrence in combination with the conventional laser therapy. If we further consider the low cost and convenient oral administration, low-dose CY might represent a therapeutic breakthrough in CA treatment. However, large-scale clinical trials are required to further verify its therapeutic outcomes. Also, it would be valuable to extend the trial to patients with different genetic backgrounds. Another implication is that similar to
HPV infection, low-dose CY might also be useful in other viral infections such as hepatitis virus B infection which also involves Treg cells in pathophysiological processes. Acknowledgements
We thank Yonghong Wan from McMaster University for assistance in editing this article. Financial & competing interests disclosure
This work was partially supported by the National Natural Science Foundation of China (81102224, 30972667), the National Basic Research Program of China (2014CB542103) and the National Science Fund for Distinguished Young Scholars of China (81225021). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues • Condylomata acuminata caused by HPV type 6 or 11 is a common sexually transmitted disease with increased incidence in China. • HPV infects basal keratinocytes and causes genital warts with a variable latent period. • Spontaneous regression occurs in most HPV type 6 or 11 infection-caused lesions; however, some lesions evade anti-HPV immunity and finally develop into warts. • Multiple treatments including surgical excision and topical physical therapies are commonly associated with high recurrence rates, especially in the presence of latent HPV in the so-called clinically normal epithelium outside the treatment area. • Treg cells participate in the infection process of various viruses, including HPV type 6 or 11 infection. • Treg cells inhibit multiple components of the immune system and can mediate virus immune evasion. • Cyclophosphamide (CY) is a chemotherapeutic agent widely used in malignancies as well as autoimmune disorders. • Low-dose CY may act as immune modulator to enhance anti-HPV immune responses by targeting Treg cells, leading to clearance of infectious viruses. • Reduced intracellular ATP levels in Treg cells confer the sensitivity of Treg cells to low-dose CY.
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