Sofie Robert

1,25-Dihydroxyvitamin D3 curtails the inflammatory and T cell stimulatory capacity of macrophages through an IL-10-dependent mechanism.

The vitamin D receptor (VDR) is a hormone nuclear receptor regulating bone and calcium homeostasis. Studies revealing the expression of VDR on immune cells point toward a role for VDR-dependent signaling pathways in immunity. Here we verified the ability of the natural VDR ligand, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) to interfere in inflammatory and T cell stimulatory capacity of macrophages, in particular within a chronic inflammatory disease features of experimental type 1 diabetes (T1D). We demonstrated that VDR is constitutively expressed in macrophages and both the levels of VDR and its downstream targets, are clearly induced by 1,25(OH)(2)D(3). In control mice, macrophage programming with 1,25(OH)(2)D(3) partially abrogated the activation-provoked expression of IL-12p40, TNFα and iNOS as well as the effector T cell-recruiting chemokines, CXCL9, CXCL10 and CXCL11. Targeting VDR signaling in macrophages counteracted their T-cell stimulatory ability despite essentially unaltered expression of antigen-presenting and costimulatory molecules. Furthermore, even in non-obese diabetic (NOD) mice, where macrophages/monocytes featured a heightened responsiveness toward danger signals and a superior T cell stimulatory capacity, 1,25(OH)(2)D(3) successfully curtailed these basic macrophage-mediated functions. Interestingly, the inhibitory action of the active compound was associated with an IL-10-dependent mechanism since 1,25(OH)(2)D(3)-treatment of IL-10-deficient macrophages failed to reproduce the characteristic repression on inflammatory mediators or T cell proliferation. Combined, these results highlight the possible therapeutic applicability of this natural immunomodulator, due to its ability to counteract macrophage inflammatory and T cell-activating pathways.

Oral Delivery of Glutamic Acid Decarboxylase (GAD)-65 and IL10 by Lactococcus lactis Reverses Diabetes in Recent-Onset NOD Mice

Growing insight into the pathogenesis of type 1 diabetes (T1D) and numerous studies in preclinical models highlight the potential of antigen-specific approaches to restore tolerance efficiently and safely. Oral administration of protein antigens is a preferred method for tolerance induction, but degradation during gastrointestinal passage can impede such protein-based therapies, reducing their efficacy and making them cost-ineffective. To overcome these limitations, we generated a tolerogenic bacterial delivery technology based on live Lactococcus lactis (LL) bacteria for controlled secretion of the T1D autoantigen GAD65370–575 and the anti-inflammatory cytokine interleukin-10 in the gut. In combination with short-course low-dose anti-CD3, this treatment stabilized insulitis, preserved functional β-cell mass, and restored normoglycemia in recent-onset NOD mice, even when hyperglycemia was severe at diagnosis. Combination therapy did not eliminate pathogenic effector T cells, but increased the presence of functional CD4+Foxp3+CD25+ regulatory T cells. These preclinical data indicate a great therapeutic potential of orally administered autoantigen-secreting LL for tolerance induction in T1D.

Role of the saturated nonesterified fatty acid palmitate in beta cell dysfunction.

Sustained elevated levels of saturated free fatty acids, such as palmitate, contribute to beta cell dysfunction, a phenomenon aggravated by high glucose levels. The aim of this study was to investigate the mechanisms of palmitate-induced beta cell dysfunction and death, combined or not with high glucose. Protein profiling of INS-1E cells, exposed to 0.5 mmol/L palmitate and combined or not with 25 mmol/L glucose, for 24 h was done by 2D-DIGE, both on full cell lysate and on an enriched endoplasmic reticulum (ER) fraction. Eighty-three differentially expressed proteins (P < 0.05) were identified by MALDI-TOF/TOF mass spectrometry and proteomic results were confirmed by functional assays. 2D-DIGE analysis of whole cell lysates and ER enriched samples revealed a high number of proteins compared to previous reports. Palmitate induced beta cell dysfunction and death via ER stress, hampered insulin maturation, generation of harmful metabolites during triglycerides synthesis and altered intracellular trafficking. In combination with high glucose, palmitate induced increased shunting of excess glucose, increased mitochondrial reactive oxygen species production and an elevation in many transcription-related proteins. This study contributes to a better understanding and revealed novel mechanisms of palmitate-induced beta cell dysfunction and death and may provide new targets for drug discovery.

Recombinant Lactococcus lactis can make the difference in antigen-specific immune tolerance induction, the Type 1 Diabetes case

Especially in western civilizations, immune diseases that are driven by innocuous (auto- or allo-) antigens are gradually evolving to become pandemic threats. A particularly poignant example is type 1 diabetes, where young children are confronted with the perspective and consequences of total pancreatic β-cell destruction. Along these disquieting observations we find ourselves equipped with impressively accumulating molecular immunological knowledge on the ins and outs of these pathologies. Often, however, it is difficult to translate this wealth into efficacious medicines. The molecular understanding, the concept of oral tolerance induction, the benefit of using recombinant Lactococcus lactis therein and recent openings towards their clinical use may well enable turning all colors to their appropriate fields on this Rubiks cube.

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Antigen-based vs. systemic immunomodulation in type 1 diabetes The pros and cons

In type 1 diabetic patients insulin-producing pancreatic β-cells are destroyed by an orchestrated immune process involving self-reactive auto-antigen-specific CD4+ and CD8+ T cells. Efforts to reverse or prevent this destructive immunological cascade have led to promising results in animal models, however, the transition to the clinic has yet been unsuccessful. In addition, current clinical studies lack reliable biomarkers to circumscribe end-point parameters and define therapeutic success. Here, we give a current overview of both antigen-specific and non-specific systemic immunomodulatory approaches with a focus on the therapies verified or under evaluation in a clinical setting. While both approaches have their advantages and disadvantages, rationally designed combination therapies may yield the highest therapeutic efficacy. In order for future strategies to be effective, new well-defined biomarkers need to be developed and the extrapolation process of dose, timing and frequency from in vivo models to patients needs to be carefully reconsidered.