Hyperthermia on skin immune system and its application in the treatment of human papillomavirus-infected skin diseases

Abstract

Hyperthermia is a condition characterized by increased body temperature as a consequence of failed thermoregulation. Hyperthermia occurs when a body produces or absorbs more heat than it dissipates. Hyperthermia also elicits various effects on the physiology of living cells. For instance, fever-range temperature (39β°C to 40β°C) can modulate the activities of immune cells, including antigen-presenting cells, T cells, and natural killer cells. Heat shock temperature (41β°C to 43β°C) can increase the immunogenicity of tumor cells. Cytotoxic temperature (>43β°C) can create an antigen source to induce an anti-tumor immune response. The immunomodulatory effect of hyperthermia has promoted an interest in hyperthermia-aided immunotherapy, particularly against tumors. Hyperthermia has also been used to treat deep fungal, bacterial, and viral skin infections. We conducted a series of open or controlled trials to treat skin human papillomavirus infection by inducing local hyperthermia. More than half of the patients were significantly cured compared with those in the control trial. A series of challenging clinical cases, such as large lesions in pregnant patients or patients with diabetes mellitus, were also successfully and safely managed using the proposed method. However, further studies should be conducted to clarify the underlying mechanisms and promote the clinical applications of hyperthermia.

Keywords

hyperthermia / HPV / immune response / virus / tumor

Introduction

Hyperthermia is a condition characterized by increased body temperature as a result of failed thermoregulation. Hyperthermia occurs when a body produces or absorbs more heat than it dissipates [1]. In a broad sense, hyperthermia can be deliberately induced using drugs or medical devices and may be systemically or locally used to treat pathological conditions. Temperature is ecologically important because this factor is pervasive and lacks spatial or temporal stability in many environments [2]. Heat-related pathologies, such as heat stroke, are considered as one of the most serious causes of mortality. Climatic shifts and other anthropogenic causes also contribute to the increase in the prevalence of hyperthermia incidents and associated deaths. In heat stroke, core body temperature>40β°C elicits acute tissue injury and multi-organ failure that is often fatal. For example, the nervous system is vulnerable to heat [3-5].

Effects of hyperthermia

Upon exposure to altered temperatures for prolonged periods, many animals adapt physiologically and biochemically; this process is termed thermal acclimatization [2]. As such, hyperthermia does not always harm organisms. In specific circumstances, fever is considered as a sign of acute inflammatory response triggered by the body as a part of host defense mechanism. In dermatology, local heating to a specific degree is applied to treat diseases.

Skin is important in the regulation of body core temperature. The skin can function as a radiator and insulator by evaporative cooling after eccrine sweating, vasodilation, and vasoconstriction, depending on the changes in temperature outside and inside the body. The excessive heating of skin may be local or systemic. In each case, innervation, circulation, and sweating are needed to dissipate heat that may cause tissue damage. In vertebrates, the “normal” core body temperature of approximately 37β°C is well conserved with minimal changes. This temperature is usually increased to 39β°C to 40 °C as a febrile response to infection or other stress factors. Under experimental conditions, temperature ranging from 41β°C to 43β°C is considered as heat shock temperature; temperature>43β°C is cytotoxic [6]. However, this classification remains controversial and is not widely applied in many studies.

Hyperthermia elicits various effects on the physiology of living cells. For instance, hyperthermia affects the fluidity and stability of cellular membranes and impedes the functions of transmembrane transport proteins and cell surface receptors. Hyperthermia also induces various changes in cytoskeletal organization (e.g., cell shape, mitotic apparatus, endoplasmatic reticulum, and lysosomes). Intracellular de novo synthesis and polymerization of RNA and DNA during protein synthesis are dose-dependently decreased in vitro at 42 °C to 45β°C. Hyperthermia dose not cause severe DNA damage; however, this condition can inhibit DNA-repair enzymes, such as DNA-polymerases [7]. Hyperthermia induces cell death via necrotic pathways [8], and specific cell types exhibit different susceptibilities to heat-induced apoptotic cell death [7].

The mechanism by which hyperthermia affects the cells at a molecular level is complicated. Such a mechanism depends on the type, developmental stage, and microenvironment of cells. Moreover, different hyperthermic conditions exhibit varied effects on subjected cells. For example, hyperthermia induces the activation of heat shock transcription factor-1 (HSF-1), which transcribes heat shock proteins (HSPs). In hepatocytes, HSF-1 activation and the subsequent expression of gene products protect cells from damage. By contrast, HSF-1 activation in spermatocytes leads to the suppression of genes involved in cellular division; as a result, spermatocytes are prone to apoptosis [9]. Prolonged mild hyperthermia can also activate HSF-1 and HSPs, as well as p38-mitogen-activated protein kinase in fibroblasts, thereby suppressing fibroblast proliferation [10].

The effect of hyperthermia on the immune system has attracted significant attention. Hyperthermia (and other stress stimuli) can induce or enhance the expression of a set of highly conserved proteins, such as HSPs. HSPs generally function as protein chaperones and regulators of protein folding; HSPs also direct the formation of protein complexes and protein degradation. Some of the HSP receptors have been characterized in antigen-presenting cells (APC; Tableβ1). This result suggested an immunological function of hyperthermia in immune responses.

HSPs and their corresponding receptors exhibit multiple functionalities. In innate immune response, HSPs directly stimulate the secretion of various inflammatory cytokines by APCs and nitric oxide by macrophages and dendritic cells (DC). HSPs also stimulate the secretion of chemokines by macrophages, the maturation of DCs, and the migration of DCs to lymphoid tissue. In adaptive immune response, an HSP, peptides from stressed cells, and major histocompatibility complexes (MHC) on APC may result in the re-presentation of a peptide on APC [11-18]. Tableβ2 shows the immunomodulatory effects of hyperthermia on immune reactions from different sources of reports. The effects are summarized as follows: (1) fever-range temperature can modulate the activities of immune cells, including APCs, Tβcells, and NK cells; (2) heat shock temperature can increase the immunogenicity of tumor cells; and (3) cytotoxic temperature can create an antigen source to induce an anti-tumor immune response.

Applications of hyperthermia

The immunomodulatory effect of hyperthermia has promoted an interest in hyperthermia-aided immunotherapy, particularly against tumors. Hyperthermia therapy as a type of medical treatment was proposed, in which the body tissue is exposed to slightly higher temperatures to damage and kill cancer cells or to enhance the sensitivity of cancer cells to the effects of radiation and anti-cancer drugs. Combined with radiation, hyperthermia can effectively increase damage to acidic, poorly oxygenated parts of tumors [19] and cells that are preparing to divide [20]. Hyperthermia treatment is highly effective when radiation is simultaneously administered. Hyperthermia treatments in conjunction with radiation have been applied in patients with early stage cancers of the breast, head and neck, and prostate. Bicher et al. [21] recorded the complete response rates of 82% for breast cancer, 88% for head and neck cancer, and 93% for prostate cancer; the projected five-year survival rates are 80% for breast cancer, 88% for head and neck cancer, and 87% for prostate cancer. Some of the clinical trials based on HSP vaccine have shown a relatively longer disease-free survival time, lower rate of recurrence, and strong immune response against tumors.

The skin also provides passive and active protection. In passive protection, the physiochemical properties of the skin resist exogeneous harmful effects. In active protection, the skin is considered as an important immune organ. In 1986, Bos et al. [22] proposed the term “skin immune system (SIS)” and described this system as the complexity of immune response-associated cells present in normal human skin. The SIS comprises Langerhans cells (LCs), APCs, keratinocytes, lymphocytes, endothelial cells, mast cells, and many humoral factors.

The effect of hyperthermia on SIS is of great importance because the skin is prone to exogeneous hyperthermia. Transient local hyperthermia promotes the migration of epidermal LCs in a temperature-dependent manner and increases the percentage of LCs with maturation markers in the emigrated portion. These phenomena are evident in human papillomavirus (HPV)-infected skin in an organotypic in vitro culture system [23]. Hyperthermia-treated (>41β°C) human skin, particularly HPV-infected skin [24], exhibits increased signals of apoptosis and induces the production of interferons in HPV-infected samples [25]. In a previous study, local hyperthermia at 44β°C promotes the infiltration of CD4⁺ and CD8⁺ T cells in the lesion of condyloma acuminate [26]. Hyperthermia-treated purified mouse LCs exhibit a stronger potency to stimulate the proliferation of CD8⁺ T cells than CD4⁺ T cells; this effect on CD4⁺ T cells is not significant (unpublished data). In a classical murine hypersensitivity reaction model, the timing of local hyperthermia applied in the sensitized sites affects the outcome at elicitation stages; local hyperthermia pre-applied to the sensitized site suppresses the subsequent intensity of hypersensitivity reaction; by comparison, local hyperthermia concurrently applied or applied at 2 d post-sensitization increases reaction intensity [27]. These studies have suggested that hyperthermia can influence the activity of SIS at cellular and molecular levels.

Hyperthermia has been applied to treat deep fungal, bacterial, and viral skin infections. Many studies are based on case reports or case series. For example, HPV is a ubiquitous family of viruses with>120 genotypes. Some of these HPV genotypes are potentially oncogenic (high-risk types) and others are the common infective causes of cervical cancers [28]. The discovery of oncogenic HPVs has resulted in the production and application of virus vaccines, which remarkably promote the prevention of such conditions in women; however, the effect of virus vaccines on the treatment of established cancer or prevention of infection by other HPV genotypes remains questionable. HPV-infected skin or mucosa is one of the most common complaints in dermatology. HPV infections are commonly treated with destructive therapy, virucidal therapy, antimitotic therapy, immunotherapy, or combinations of these methods. However, the efficacies of these therapies vary. Hence, we conducted a series of open or controlled trials to treat skin HPV infection by local hyperthermia [29-32]. Many studies have applied local hyperthermia at 44β°C for 30 min, in which the protocol requires three consecutive treatments for 3 d and with subsequent two treatments for 2 d after two weeks [29]. The effect rate was observed after three months. More than half of the treated patients were significantly cured compared with those in the control trial. This result indicated that the proposed method could be performed easily. Many patients tolerated the treatment well. A series of challenging clinical cases, such as large lesions in patients with co-morbidity of diabetes mellitus, pregnancy, lesions with cosmetic sequelae, or superimposed skin conditions, were also successfully managed using this method [30-32]. In patients with multiple lesions, target lesions and other untreated, remote lesions are successfully removed. This phenomenon is a common finding in these clinical trials. This phenomenon, along with the effect of hyperthermia on immune cells, highly suggests that local hyperthermia contributes to the development of specific immune responses against HPV-infected cells.

Summary and perspectives

In summary, hyperthermia affects numerous cellular and molecular processes in the SIS, which involves innate and adaptive immune responses. The effects of hyperthermia on immune cells depend on the dosage and level of hyperthermia, cell types, cellular environment, and developmental stage of cells. The efficacy of hyperthermia for the treatment of infectious and cancerous conditions has been validated and applied in clinical practice. However, the molecular mechanisms of hyperthermia in the immune system should be understood, but this area remains a great challenge. In clinical studies, the future directions of hyperthermia applications may rely on the following: (1) advancements in devices to deliver or measure uniform levels of heat, as well as hyperthermia combined with (2) gene therapy, (3) biologics, (4) signal transduction pathway agonists or antagonists, (5) physical or chemical factors, and (6) other auxiliary factors. To elucidate the molecular mechanisms of hyperthermia on target cells, we recommend further studies involving genomics, proteomics, and transcriptomics as well as application of genetically modified cellular and animal models.

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