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 ß-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 Rubik's cube.

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.

Identification of poor prognostic joint locations in an early rheumatoid arthritis cohort at risk of rapidly progressing disease: a post-hoc analysis of the Phase III AGREE study.

BACKGROUND: Rheumatoid arthritis (RA) is a heterogeneous disease with established poor prognostic factors such as seropositivity, joint damage, and high disease activity at an early, treatment-naïve stage of disease. However, few studies have examined if specific joint locations are correlated with these factors in such a population. This analysis explored the potential correlation of individual swollen and erosive joints with other disease characteristics at baseline and with remission rates in a post-hoc analysis of the Phase III randomized AGREE study. METHODS: Methotrexate (MTX)-naïve, erosive, RF- and/or ACPA-positive early RA patients (N = 509) were retrospectively evaluated. Baseline joint swelling was analyzed for large and small joints. Baseline erosions were analyzed for wrist, MCP1-5, IP1, PIP2-5 and MTP1-5. Remission rates were assessed after 6 months of treatment with abatacept (ABA) + MTX (N = 256) or MTX (N = 253). The following statistical tests were used: Chi-Square or Fisher's exact test (categorical variables); Student's t-test or Wilcoxon rank-sum test (continuous variables); continuity-corrected Chi-square test (efficacy remission endpoints). RESULTS: Baseline swelling was most frequent in wrist (91.9%) and MCP2 joint (89.1%), while baseline erosion was most frequent in MTP5 joint (43.5%). Swollen shoulder was significantly correlated (p < 0.0001) with swelling of almost all other large or medium joints. Baseline swelling in the knee, temporomandibular joint (TMJ), wrist and elbow was highly correlated (p < 0.001) with higher tender and swollen joint counts, higher DAS28(CRP) and higher SDAI and CDAI. Baseline swelling was not correlated with erosion per joint, except for MCP2. The largest difference in mean Boolean remission rates at 6 months was in patients with baseline swollen wrist favoring ABA + MTX (14.0% vs 4.4%; p < 0.001). CONCLUSIONS: Swelling in the large and medium joints (knee, TMJ, elbow and wrist) was highly correlated with severe disease activity while MCP2 swelling seemed to be correlated with joint damage. The correlation of joint locations at an early, treatment-naïve stage with poor prognostic factors, higher disease activity and joint damage, could establish a rapidly progressing anatomical pattern in early RA. TRIAL REGISTRATION: ClinicalTrials.gov NCT00122382, registered July 2005.

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 ß-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.

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

In type 1 diabetic patients insulin-producing pancreatic ß-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.

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.

Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice.

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.