New strategy for testing efficacy of immunotherapeutic compounds for diabetes in vitro

Abstract

BackgroundThe valuable role of immunotherapy in treating autoimmune diseases is increasingly recognized by those involved in the research and clinical application of new biopharmaceuticals products. However, many aspects related to the mechanisms of immune-modulated therapies remain to be elucidated in order to explore fully the emerging opportunities. The non-obese diabetic NOD mouse develops insulin-dependent diabetes mellitus spontaneously as a consequence of an autoimmune process in the presence of pathogenic CD4+ T cells that typically exhibit Th17 cell phenotypes. The change of a Th17 phenotype into a pattern of regulatory T cells (Treg) is extremely important in controlling autoimmune diseases. Heat shock proteins (HSPs) are stress-induced proteins with immunoregulatory properties. In the current study, the capacity of Hsp65 and Hsp70 mycobacterial HSPs and a constructed DNA encoded Hsp65 (DNAhsp65) to transform the pattern of the immune response from Th17 into Treg cells has been studied in vitro using co-cultures of antigen presenting cells (APCs) and T cells in NOD mice.ResultsCells harvested from NOD mice and cultured for 48 h (without immunoregulatory compounds) presented with Th1/Th17 patterns and secretions of IL-6, IFN-γ, IL-10 and IL-17 cytokines. The cultured cells from the non-diabetic BALB/C mice exhibited a Th1 pattern and the production of IL 6 and IFN-γ secretions. An up-regulation was observed in the supernatants from the co-cultures of NOD cells that were stimulated with DNAhsp65, Hsp65 or Hsp70 through increased levels of IL-10 secretion and the suppression of IL-6, IFN-γ and IL-17 production. In addition, immunoregulation was demonstrated through IL-17 suppression in the co-culture stimulated by the specific insulin antigen. Moreover, an increase of immunoregulatory compounds were observed in the co-culture through the expression of CD11b+CD86+ activation markers on APCs, as well as the frequency of Treg cells expressing CD4+CD3+ and CD4+CD25hi.ConclusionsThe in vitro observation of Th17 cells differentiating into Tregs in NOD mice could raise the hypothesis that the immune regulatory activity of HSPs could be an efficient strategy for diabetes prevention and treatment.

The abnormal level of HSP70 is related to Treg/Th17 imbalance in PCOS patients

“…HSP70 plays an important role in antigen presentation, activation of macrophages and lymphocytes, as well as activation and maturation of dendritic cells [ 7 ]. Studies have shown that HSP70 has the ability to transform Th17 cells immune response mode into Treg cells immune response mode [ 18 ]. However, whether HSP70 is related to the ratio of Treg/Th17 in PCOS patients has not yet been reported.…”

Immunotherapeutic Activities of a DNA Plasmid Carrying the Mycobacterial hsp65 Gene (DNAhsp65)

“…The immunotherapeutic capacity of DNAhsp65 to control infectious diseases, autoimmune diseases, allergy, and tumors, which have significant differences in their immunopathology, is associated with a number of factors with distinct immunomodulatory capabilities inherent to the structure and/or composition of DNAhsp65 (4). The main factors that stand out include the presence of an HSP that has significant activities in the recognition, intracellular trafficking, antigen presentation (peptide), and interaction with receptors and signaling for activation of the innate and the adaptive immune system (17)(18)(19)(20)(21); the ability of HSPs to bind and form complexes with peptides derived from tumor or infected cells and to induce innate and/or adaptive immune response against chaperoned peptides (5,9,14); a DNA vaccine that by itself releases endogenous antigens and/or immunomodulators to stimulate innate and adaptive (cellular and humoral) immunity (9,24); the presence of immunostimulatory DNA sequences containing CpG motifs in the plasmid DNA backbone that is considered to be an important adjuvant for generating the innate and Th1 immune response (17,(78)(79)(80); the double-stranded structure of the DNA plasmid that is also thought to be an immune stimulant through non-TLR mechanisms (acting on the TBK1-STING pathway through cytosolic receptors), resulting in the generation of Type-1 interferon, which then act as an adjuvant for the generation of immune responses against the antigen(s) encoded by the plasmid DNA vaccine (87,88); the capacity for activation and regulation of adaptive immune response among Th1, Th2, Th17, Treg, and γδT pattern of lymphocytes (38,45,(64)(65)(66)71); and the properties of formulations or particulate delivery system and/or adjuvants to stimulate innate and adaptive immunity (27,28,54).…”

“…In addition, the insulitis score was decreased as a result of reduced T-CD4 + and T-CD8 + cell infiltration and Treg-IL-10 + activation ( 64 , 65 ). Moreover, when cocultures of APCs and NOD mouse T cells were incubated in the presence of DNAhsp65, Hsp65, or Hsp70 mycobacterial HSPs, the response pattern was altered from Th17 to Treg cells, showing increased IL-10 secretion and reduced IL-6, IFN-γ, and IL-17 cytokine production ( 66 ). The prime-boost strategy involving the BCG and DNAhsp565 vaccines (BCG/DNAhsp65) used as a new treatment approach reestablished blood glucose levels, reduced the inflammatory process in pancreatic islets, and promoted increased weight only in NOD mice.…”

Safety and Immunogenicity of an <em>In Vivo</em> Muscle Electroporation Delivery System for DNA-<em>hsp65</em> Tuberculosis Vaccine in Cynomolgus Monkeys

“…Since the discovery that DNA-hsp65 can have immunomodulatory and/or immunoregulatory action against TB several other works have been done by our group with other infectious diseases, autoimmune diseases, allergy, and tumors. Thus, it was demonstrated that DNA-hsp65 presents broad immunotherapeutic properties for leishmaniasis [12], schistosomiasis [9,11], paracoccidioidomycosis [13,14], chromoblastomycosis [15], helminths [10]; autoimmune diseases such as diabetes [16][17][18][19], arthritis [20,21], encephalomyelitis [22][23][24], and atherosclerosis [25], allergic diseases such as asthma and atopic dermatitis [26][27][28] and tumors in experimental models of mice and in phase I/II clinical trial in humans [29,30]. Thus, DNA-hsp65 actively participates in the activation of innate immunity, acting as an endogenous adjuvant and playing a fundamental role in the activation and control of adaptive immunity [29][30][31][32].…”