Casein-based diet found to protect diabetic mice against autoimmunity

By Pooja Toshniwal PahariaFeb 10 2023Reviewed by Aimee Molineux

In a recent study published in Cell Host & Microbe, researchers studied diet-commensal microbial interactions in autoimmunity.

Commensal microbiota and diet can influence autoimmunity mechanisms in disorders such as type 1 diabetes (T1D). However, whether microbes mediate diet interventions is not clear and warrants further investigation. Identifying microbiome-independent diet interventions and exploring particular commensal-diet interactions that potentiate autoimmunity could aid in improving the management of autoimmune disorders.

About the study

In the present study, researchers explored the microbiome-regulated and microbiome-unregulated effects of dietary interventions in managing autoimmune disorders.

Non-obese diabetic (NOD).transglutaminase 2 (TGM2) knockout (KO) mice were selected for the experiments, and hydrolyzed casein (HC) was analyzed as the source of protein. To investigate whether the anti-diabetic effects of the casein-based diet depended on the microbiome, NOD mice were exposed, in germ-free (GF) and specific-pathogen-free (SPF) conditions, to formulations comprising intact casein protein (IC), or to chow (control dietary intervention).

To elucidate the mechanisms involved in protection against type 1 diabetes, the effects of casein on autoimmune T lymphocytes were evaluated, for which spleen cells from non-diabetic murine animals on casein-diet or chow diet were transferred into immunosuppressed NOD.TCRaKO or NOD.scid or mice. Subsequently, the number of regulatory T lymphocytes (Tregs) in the infiltrated pancreatic islet cells and pancreatic lymph nodes (PLNs) of the murine animals was determined.

To investigate whether casein influences insulin secretion/production, resulting in indirect autoimmunity attenuation, the intraperitoneal insulin tolerance test (IPITT) and intraperitoneal glucose tolerance test (IPGTT) were carried out. Gluten-digesting-bacterial microbes were detected, gluten digests were prepared, and the gluten digests stimulated peritoneal macrophages to investigate the contribution of gluten proteolysis to innate immunological immunity.

The culture supernatants were tested for short-term responses [tumor necrosis factor (TNF) expression] and delayed responses [interleukin-6 (IL-6) expression. The digests were treated with polymyxin B, and Limulus amebocyte lysate (LAL) assays were performed. Pancreatic islets of NOD.scid murine animals were subjected to single-cell ribonucleic acid (RNA) sequencing (SCS), T cell receptor (TCR) sequencing, and 16S ribosomal ribonucleic acid (rRNA) sequencing analyses, and the bacterial ribosomal RNA was amplified using polymerase chain reaction (PCR).

In addition, differential gene expression, multiparameter flow cytometry, enzyme-linked immunospot (ELISPOT), Western blot, and immunofluorescence analyses were performed. T lymphocyte proliferation was assessed in vitro and in vivo. CRISPR-Cas9 analysis was performed to investigate whether transglutaminase 2 gene activation promoted T1D. Further, the team investigated whether gnotobiotic mice colonization with E. faecalis would facilitate T1D development, and the role of lipopolysaccharide-driven signaling in T1D development was explored.

Results

The casein-based diet protected non-obese diabetic murine animals in traditional and GF conditions by physiologic improvements in insulin secretion to suppress the activation of autoimmune mechanisms independent of the microbiome composition. Gluten-activated autoimmune pathways, triggered by microbe-mediated gluten proteolysis. Cytokine expression was stimulated by lipopolysaccharide-dependent proteolytic gluten digestion.

Type 1 diabetes developed among germ-free animals colonized with E. faecalis containing gluten-digesting protease enzymes but not among animals colonized with bacteria lacking proteases. E. faecalis-mediated digestion of gluten-induced T lymphocyte-activating peptide molecules and enhanced innate immunological mechanisms by increasing the reactivity of macrophages to lipopolysaccharides. Gnotobiotic non-obese diabetic Toll4^- murine animals colonized with Enterococcus faecalis on casein+ gluten diets showed resistance to type 1 diabetes.

The HC diet lowered β lymphocyte stress and T1D in a microbiome-independent manner.  The promotion of gluten-mediated pancreatic islet cell inflammation depended on the gluten digestion ability of the commensal microbes. The findings indicated that altering the physiologic pathways of the target organ (such as the pancreas for insulin production) might inhibit or delay autoimmune activation, which is attainable irrespective of the commensal microbiome complexity.

Two critical biological functions of gut microbes associated with proteolytic gluten digestion were noted, i.e., the secretion of adaptive immune system-activating peptides, and enhancement of the LPS-regulated induction of innate immunological mechanisms. The digestion of gluten by microbial protease enzymes promoted type 1 diabetes development.

HC-regulated protection was not related to the active inhibition of immune effector pathways, but instead due to a decrease in the initial sensitization steps. The decrease in insulin production by casein did not depend on the adaptive immunological system-mediated injury, indicative of a direct impact on hormone-producing cells. Gluten potentiated autoimmunity by directly acting on the immunological system, and promoted diabetogenic pathways.

Overall, the study findings showed that casein-based diets could probably lower the incidence of autoimmune or type 1 diabetes by improving the insulin-secretion physiology, irrespective of the commensal microbiome composition. Protease-producing microbial organisms and gluten could reverse HC-regulated protection.