Immune responses to azacytidine in animal models of inflammatory disorders: a systematic review.

Inflammatory disorders like diabetes, systemic lupus erythematodes, inflammatory lung diseases, rheumatoid arthritis and multiple sclerosis, but also rejection of transplanted organs and GvHD, form a major burden of disease. Current classes of immune suppressive drugs to treat these disorders are never curative and side effects are common. Therefore there is a need for new drugs with improved and more targeted modes of action. Potential candidates are the DNA methyl transferase inhibitor 5-azacytidine (Aza) and its derivative 5-aza 2'deoxycitidine (DAC). Aza and DAC have been tested in several pre-clinical in vivo studies. In order to obtain an overview of disorders for which Aza and/or DAC can be a potential treatment, and to find out where information is lacking, we systematically reviewed pre-clinical animal studies assessing Aza or DAC as a potential therapy for distinct inflammatory disorders. Also, study quality and risk of bias was systematically assessed. In the 35 identified studies, we show that both Aza and DAC do not only seem to be able to alleviate a number of inflammatory disorders, but also prevent solid organ rejection and GvHD in in vivo pre-clinical animal models. Aza/DAC are known to upregulate FOXP3, a master transcription factor for Treg, in vitro. Seventeen studies described the effect on Treg, of which 16 studies showed an increase in Treg. Increasing Treg therefore seems to be a common mechanism in preventing inflammatory disorders by Aza/DAC. We also found, however, that many essential methodological details were poorly reported leading to an unclear risk of bias. Therefore, reported effects might be an overestimation of the true effect.

Intradermal injection of low dose human regulatory T cells inhibits skin inflammation in a humanized mouse model.

Recent regulatory T cell (Treg) based clinical trials support their therapeutic potential in transplantation and auto-inflammatory diseases. However, large numbers of Treg are needed to accomplish therapeutic efficacy. Local injection at the site of inflammation (targeted delivery) may lower the numbers needed for therapy. We evaluated if local delivery of low numbers of human Treg by intradermal injection was able to prevent skin inflammation, using the humanized mouse huPBL-SCID-huSkin allograft model. A dose of only 1 × 105 freshly isolated, non expanded Treg injected intradermally in close proximity to the transplanted human skin prevented inflammation of the grafted tissue induced by 4 × 107 IP injected human allogeneic PBMCs, (ratio Treg:PBMC = 1:400), as indicated by the inhibition of epidermal thickening, sustained Keratin-10 expression, the absence of Keratin-16 up regulation and prevention of human CD3+ T cell influx. A concomitant reduction of human T cells was observed in lymph nodes and spleen of the mice. Injection of Treg at the contralateral side was also shown to inhibit skin inflammation, suggesting that the inflammatory response was regulated both locally and systemically. In conclusion, local application of Treg may be an attractive way to suppress inflammation in vivo without the need for prior ex vivo expansion.

DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4+CD25high FOXP3+ Regulatory T Cells but Does Not Affect Their Suppressive Capacity.

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2'-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells.

Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for ex vivo expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize ex vivo Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that ex vivo expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the FOXP3 gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation in vivo. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the ex vivo expansion of Treg for clinical immunotherapy.

Immune activation by medium-chain triglyceride-containing lipid emulsions is not modulated by n-3 lipids or toll-like receptor 4.

BACKGROUND: Saturated medium-chain triglycerides (MCT) as part of the parenteral lipid regimen (50% MCT and 50% long chain triglycerides (LCT)) activate the immune system in vitro. Fish oil (FO)-derived n-3 fatty acids (FA) inhibit saturated FA-induced immune activation via a toll-like receptor (TLR)-4 mediated mechanism. We hypothesized that effects of parenteral MCTs on immune cells involve TLR-4 signaling and that these effects are modulated by n-3 FA that are present in FO. MATERIALS AND METHODS: To test this hypothesis we assessed effects of addition of various commercially available mixed parenteral lipid emulsions, n-3 FA and of TLR-4 inhibition on MCT-induced human immune cell activation by evaluation of the expression of leukocyte membrane activation markers and reactive oxygen species (ROS) production. RESULTS: All MCT-containing lipid emulsions activated leukocytes by inducing changes in expression of membrane markers and stimulus induced ROS production, whereas MCT-free lipid emulsions lacked this effect. Moreover, addition of n-3 FA to LCT/MCT did not prevent MCT-induced immune activation. TLR-4 inhibitors did not distinctly modulate MCT-induced changes in immune function. CONCLUSION: Taken together, these findings suggest that leukocyte activation by parenteral MCTs does not involve TLR-4 signaling and is not modulated by n-3 FA in FO-, but is exerted via different signaling pathways.