Withaferin A inhibits lymphocyte proliferation, dendritic cell maturation in vitro and prolongs islet allograft survival

Abstract

The immunosuppressive regimen for clinical allogeneic islet transplantation uses beta cell–toxic compounds such as tacrolimus that cause islet graft loss. Previously we reported that the plant-derived steroidal lactone Withaferin A (WA) can protect islet grafts by inhibiting nuclear factor-kappa B (NF-κB). Since the NF-κB signaling pathway is essential for T-cell activation, we hypothesized that long-term WA administration may also provide an immunosuppressive effect. Treatment of BALB/c donor islets and C57BL/6N recipients with WA alone resulted in 80% islet graft long-term survival vs. 40% in low-dose FK506-treated mice. In vitro, WA significantly blocked mouse and human T-cell proliferation by CD3/CD28 bead stimulation and in mixed lymphocyte reaction assay. Treatment of immature dendritic cells with WA prevented their maturation in response to inflammatory stimuli, as seen by decreased expression of CD83 and human leukocyte antigen–DR isotype. Exosomes released by islets treated with WA contained significantly fewer proinflammatory molecules interleukin-6, interleukin-8, monocyte chemoattractant protein-1, interferon-gamma-induced protein-10, inducible nitric oxide synthase, and cyclooxygenase-2. In conclusion, WA treatment not only reduced inflammation but also prolonged allograft survival, possibly through suppression of dendritic cell maturation and T-cell proliferation. WA has the potential to inhibit both the innate and adaptive immune response to prolong allograft survival.

Evaluating anticancer properties of Withaferin A—a potent phytochemical

“…Among many properties of Withaferin A, its immunomodulatory effects are also of interest. Immunosuppressive effects of Withaferin A were noted in a study investigating long-term administration of compound as a treatment post clinical allogenic islet transplantation ( Kumano et al, 2021 ). The study found significant reduction in mouse and human T-cell proliferation in a dose dependent manner and suppression of dendritic cell maturation with Withaferin A treatment; noting compound’s immunomodulatory effects ( Kumano et al, 2021 ).…”

Extracellular vesicles in β cell biology: Role of lipids in vesicle biogenesis, cargo, and intercellular signaling

“…In recognizing the potential importance of cell-to-cell communication in T1D development, much of the work characterizing β cell EVs has focused on mRNAs, miRNAs, and protein cargo. Independent studies have identified a variety of these species in β cell EVs that can impact β cell function and survival including: miR-127 which can inhibit β cell proliferation and insulin secretion [ 107 ]; mRNAs (VEGF-A, eNOS) and miRNAs (27b, 126, 130, 296) that contribute to β cell function and islet endothelial cell angiogenesis, a key for engraftment of transplanted islets [ 108 ]; miR-21–5p, which is induced during inflammation and can increase β cell apoptosis in T1D [ 109 , 110 ]; 19 miRNAs and 133 mRNAs [ 111 ] and cytokines [ 112 ] that are differentially expressed by islets under proinflammatory conditions; and accumulation of the spliced form of XBP1 mRNA, which correlates with ER stress and apoptosis [ 113 ]. It has also been suggested that islet graft rejection may be a consequence of major histocompatibility complex (MHC) molecules carried by donor islet EVs that are recognized by recipient antigen presenting cells (APCs) and immune cells [ 114 ].…”

Role of Exosomes in Islet Transplantation

“…Apart from proteins, intra-exosomal cargo contains nucleic acids, including DNA, mRNA, and miRNA. Loading of miRNA into exosomes is not a random process, as the types and diversity of miRNAs vary by cell type and cellular state at the time of sampling (40)(41)(42)(43). Following intraportal transplantation, islets are exposed to hypoxic and inflammatory conditions, leading to loss of islet mass (Figure 1).…”

“…In an allogeneic human islet transplant recipient, GAD65 antigen was detectable in donor HLA-specific exosomes at 455 days post-transplantation, with development of GAD65 autoantibodies at 1001 days post-transplantation ( 38 ). Our recently published observations revealed that human islets exposed to proinflammatory conditions released exosomes containing cytokines including IL-6, IL-8, MCP-1, and IP-10 ( 39 ). Proteomic profiling of MIN6-derived exosomes demonstrated enrichment of proteins involved in glycolysis, gluconeogenesis, citrate cycle, fructose and mannose metabolism, pyruvate metabolism, purine metabolism, and other metabolism-related pathways ( 35 ).…”

“…MIN6-derived exosomes increased CD86 expression on class II MHC-positive splenocytes and induced splenic T cell proliferation ( 36 ). After pro-inflammatory cytokine exposure, human islet-derived exosomes induced mRNA expression of NOS2 and COX2 in THP-1 cells, a macrophage cell line ( 39 ). The semidirect pathway is particularly important when recognizing donor peptides and MHC molecules by recipient immune cells.…”