Aminolevulinic acid (ALA) is an endogenous metabolite normally formed in the mitochondria from succinyl-CoA and glycine. Conjugation of eight ALA molecules yields protoporphyrin IX (PpIX) and finally leads to formation of heme. Conversion of PpIX to its downstream substrates requires the activity of a rate-limiting enzyme ferrochelatase. When ALA is administered externally the abundantly produced PpIX cannot be quickly converted to its final product - heme by ferrochelatase and therefore accumulates within cells. Since PpIX is a potent photosensitizer this metabolic pathway can be exploited in photodynamic therapy (PDT). This is an already approved therapeutic strategy making ALA one of the most successful prodrugs used in cancer treatment.

Vitamin D is well known for its classical hormonal action related to the maintenance of mineral and skeletal homeostasis. However, the discovery that vitamin D receptor (VDR) is expressed in most non-skeletal tissues points to its broad role in the human organism. Current literature emphasizes a multidirectional role of vitamin D, with a special focus on its immunomodulatory properties. As VDR and the enzyme 1-α-hydroxylase are expressed in most immune cells, vitamin D modulates the phagocytic activity of macrophages and natural killer cells. In addition, it induces the microbicidal activity of phagocytes. In contrast, vitamin D suppresses differentiation and maturation of antigen-presenting dendritic cells and B lymphocytes, and it inhibits proliferation of Th1 and Th17 cells. In this review we aimed to describe the current scientific discoveries on the role of vitamin D as immunomodulator.

Photodynamic therapy (PDT) of tumours is based on administration of a photosensitiser followed by irradiation of the tumour with visible light leading to production of reactive oxygen species that cause direct tumour cell death and vascular damage. PDT also initiates acute local inflammation, which facilitates the development of adaptive antitumour immunity. It has recently been reported that PDT can induce strong antitumour immunity towards tumours cells expressing P1A, tumour-associated antigen. Using four different tumour models, we show that antitumour immune response can be further improved when PDT is combined with a clinically approved epigenetic agent that induces expression of a silenced P1A antigen. Induction of P1A with 5-aza-2′-deoxycytidine, a methyltransferase inhibitor, resulted in potentiated antitumour effects in mice with Lewis lung carcinoma and 4T1 mammary carcinoma when combined with PDT treatment. In CT26 colon carcinoma and EMT6 mammary carcinoma models the combination therapy resulted in complete responses and long-term survival. All long-term surviving mice were resistant to re-inoculation with the same tumour cells. Antitumour efficacy of the combination treatment was severely impaired by depletion of CD8+ cytotoxic T cells, whereas adoptive transfer of CD8+ T cells from long-term surviving mice allowed for significant tumour growth delay in tumour-bearing mice. Taken together, these findings show that PDT leads to strong specific antitumour immune responses, and that epigenetic modification of tumour antigens levels may be a novel approach to further enhance the effectiveness of PDT. The present results provide a strong rationale for clinical development of this therapeutic approach.

Photodynamic therapy (PDT) of cancer is an efficient and promising therapeutic modality approved for the treatment of several types of tumors and non-malignant diseases. It involves administration of a non-toxic photosensitizer followed by illumination of the tumor site with a harmless visible light. A light activated photosensitizer can transfer its energy directly to molecular oxygen, leading to production of highly toxic reactive oxygen species (ROS). Antitumor effects of PDT result from the combination of three independent mechanisms involving direct cytotoxicity to tumor cells, destruction of tumor vasculature and induction of the acute local inflammatory response. PDT-mediated inflammatory reaction is accompanied by tumor infiltration of the leukocytes, enhanced production of pro-inflammatory factors and cytokines. Photodynamic therapy is able to effectively stimulate both the innate and the adaptive arm of the immune system. In consequence, this regimen can lead to development of systemic and specific antitumor immune response. However, there are limited studies suggesting that under some specific circumstances, PDT on its own may exert some immunosuppressive effects leading to activation of immunosuppressive cells or cytokines production. In this report we briefly review all immunological aspects of PDT treatment.

Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions. Keywords Autophagy • Neutrophil extracellular traps • Nitric oxide • Peroxynitrite • Phosphoinositide 3-kinases • Reactive nitrogen species Abbreviations 3-MA 3-Methyladenine ABAH Aminobenzoic acid hydrazide AKT Protein kinase B CGD Chronic granulomatous disease CI Calcium ionophore A23187 DHR 123 Dihydrorhodamine 123 DPI Diphenyleneiodonium ERK Extracellular signal-regulated kinases IL Interleukin LC3 protein Light chain 3 protein L-NAME N-nitroarginine methyl ester LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases MPO Myeloperoxidase NAC N-acetylcysteine NE Neutrophil elastase NEi Neutrophil elastase inhibitor GW 311616A NETs Neutrophil extracellular traps NBT Nitroblue tetrazolium NO Nitric oxide NOS Nitric oxide synthase Cellular and Molecular Life Sciences Malgorzata Wachowska and Urszula Demkow have contributed equally.