Immune recognition and response to the intestinal microbiome in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease resulting from T cell-mediated destruction of the insulin-secreting pancreatic beta cells. During the past 50 years T1D incidence has increased dramatically in many countries accompanied by an earlier age of onset especially in persons with lower genetic risk. These observations have prompted investigations of dynamic environmental factors that may contributor to risk for anti-pancreatic immunity. The gut and pancreas are anatomically and biochemically linked through the enteroinsular axis, a system in which gut-derived immune and metabolic signals have the potential to evoke effects in the pancreas. The gut microbiome (i.e. the 100 trillion symbiotic microorganisms which inhabit the mammalian gastrointestinal tract) influences numerous aspects of host metabolism, development and immunity. Here we examine recent evidence linking gut microbiome composition and function to pancreatic autoimmunity. Studies in children with genetic risk factors for T1D and analyses of the microbiome in rodent models have begun to associations between an altered microbiome composition potentially favoring a pro-inflammatory intestinal metabolic milieu and T1D. We discuss how environmental factors during critical developmental windows – gestation, birth, weaning and puberty may contribute to T1D risk. For example mode of delivery (vaginal or C-section) and exposure to antibiotics (pre- or post-natally) are two factors that modulate the maternal and/or offspring microbiome and can impact T1D development. Taken together, these emerging data underscore the requirement for longitudinal studies and mechanistic investigations in human subjects and rodent models to identify the basis for microbiome modulation of T1D and to identify biomarkers and therapeutics to improve the delayed onset and prevention of the disease.     

Clinical and experimental treatment of type 1 diabetes: Perspectives on immunopathology and Clinical and experimental treatment of type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of the insulin-producing pancreatic beta cells. Disease progression occurs along a trajectory from genetic risk, the development of islet autoantibodies, and autoreactive T cells ultimately progressing to clinical disease. Natural history studies and mechanistic studies linked to clinical trials have provided insight into the role of the immune system in disease pathogenesis. Here, we review our current understanding of the underlying etiology of T1D, focusing on the immune cell types that have been implicated in progression from pre-symptomatic T1D to clinical diagnosis and established disease. This knowledge has been foundational for the development of immunotherapies aimed at the prevention and treatment of T1D.     

The microbiome and regulation of mucosal immunity

The gastrointestinal tract is a mucosal surface constantly exposed to foreign antigens and microbes, and is protected by a vast array of immunologically active structures and cells. Epithelial cells directly participate in immunological surveillance and direction of host responses in the gut and can express numerous pattern recognition receptors, including Toll‐like receptor 5 (TLR5), TLR1, TLR2, TLR3, TLR9, and nucleotide oligomerization domain 2, as well as produce chemotactic factors for both myeloid and lymphoid cells following inflammatory stimulation. Within the epithelium and in the underlying lamina propria resides a population of innate lymphoid cells that, following stimulation, can become activated and produce effector cytokines and exert both protective and pathogenic roles during inflammation. Lamina propria dendritic cells play a large role in determining whether the response to a particular antigen will be inflammatory or anti‐inflammatory. It is becoming clear that the composition and metabolic activity of the intestinal microbiome, as a whole community, exerts a profound influence on mucosal immune regulation. The microbiome produces short‐chain fatty acids, polysaccharide A, α‐galactosylceramide and tryptophan metabolites, which can induce interleukin‐22, Reg3γ, IgA and interleukin‐17 responses. However, much of what is known about microbiome–host immune interactions has come from the study of single bacterial members of the gastrointestinal microbiome and their impact on intestinal mucosal immunity. Additionally, evidence continues to accumulate that alterations of the intestinal microbiome can impact not only gastrointestinal immunity but also immune regulation at distal mucosal sites.     

Respiratory microbiome and epithelial interactions shape immunity in the lungs

The airway epithelium represents a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli including microbes and allergens. However, lung epithelial cells are increasingly recognized as active effectors of microbial defence, contributing to both innate and adaptive immune function in the lower respiratory tract. These cells express an ample repertoire of pattern recognition receptors with specificity for conserved microbial and host motifs. Modern molecular techniques have uncovered the complexity of the lower respiratory tract microbiome. The interaction between the microbiota and the airway epithelium is key to understanding how stable immune homeostasis is maintained. Loss of epithelial integrity following exposure to infection can result in the onset of inflammation in susceptible individuals and may culminate in lung disease. Here we discuss the current knowledge regarding the molecular and cellular mechanisms by which the pulmonary epithelium interacts with the lung microbiome in shaping immunity in the lung. Specifically, we focus on the interactions between the lung microbiome and the cells of the conducting airways in modulating immune cell regulation, and how defects in barrier structure and function may culminate in lung disease. Understanding these interactions is fundamental in the search for more effective therapies for respiratory diseases.     

Application of zebrafish in the study of the gut microbiome

Zebrafish (Danio rerio) have attracted much attention over the past decade as a reliable model for gut microbiome research. Owing to their low cost, strong genetic and development coherence, efficient preparation of germ‐free (GF) larvae, availability in high‐throughput chemical screening, and fitness for intravital imaging in vivo, zebrafish have been extensively used to investigate microbiome‐host interactions and evaluate the toxicity of environmental pollutants. In this review, the advantages and disadvantages of zebrafish for studying the role of the gut microbiome compared with warm‐blooded animal models are first summarized. Then, the roles of zebrafish gut microbiome on host development, metabolic pathways, gut‐brain axis, and immune disorders and responses are addressed. Furthermore, their applications for the toxicological assessment of aquatic environmental pollutants and exploration of the molecular mechanism of pathogen infections are reviewed. We highlight the great potential of the zebrafish model for developing probiotics for xenobiotic detoxification, resistance against bacterial infection, and disease prevention and cure. Overall, the zebrafish model promises a brighter future for gut microbiome research.     

Immune mechanisms and the impact of the disrupted lung microbiome in chronic bacterial lung infection and bronchiectasis

Recent studies analysing immunogenetics and immune mechanisms controlling susceptibility to chronic bacterial infection in bronchiectasis implicate dysregulated immunity in conjunction with chronic bacterial infection. Bronchiectasis is a structural pathological end‐point with many causes and disease associations. In about half of cases it is termed idiopathic, because it is of unknown aetiology. Bronchiectasis is proposed to result from a ‘vicious cycle’ of chronic bacterial infection and dysregulated inflammation. Paradoxically, both immune deficiency and excess immunity, either in the form of autoimmunity or excessive inflammatory activation, can predispose to disease. It appears to be a part of the spectrum of inflammatory, autoimmune and atopic conditions that have increased in prevalence through the 20th century, attributed variously to the hygiene hypothesis or the ‘missing microbiota’. Immunogenetic studies showing a strong association with human leucocyte antigen (HLA)‐Cw*03 and HLA‐C group 1 homozygosity and combinational analysis of HLA‐C and killer immunoglobulin‐like receptors (KIR) genes suggests a shift towards activation of natural killer (NK) cells leading to lung damage. The association with HLA‐DR1, DQ5 implicates a role for CD4 T cells, possibly operating through influence on susceptibility to specific pathogens. We hypothesize that disruption of the lung microbial ecosystem, by infection, inflammation and/or antibiotic therapy, creates a disturbed, simplified, microbial community (‘disrupted microbiota’) with downstream consequences for immune function. These events, acting with excessive NK cell activation, create a highly inflammatory lung environment that, in turn, permits the further establishment and maintenance of chronic infection dominated by microbial pathogens. This review discusses the implication of these concepts for the development of therapeutic interventions.     

HLA genes associated with autoimmunity and progression to disease in type 1 diabetes

Insulin dependent diabetes mellitus (type I DM) is caused by an autoimmune process which culminates in destruction of pancreatic beta cells with resultant loss of insulin production. Preceding the clinical diagnosis of type I DM is a preclinical stage characterized by autoantibodies to insulin, glutamic acid decarboxylase (GAD) and a tyrosine phosphatase-like molecule (IA-2). We have studied both HLA class I and class 2 allele distributions in diabetic probands and autoantibody positive individuals in members of 452 families recruited for the Australian type I diabetes DNA repository. The results demonstrate that progression to autoimmunity as measured by the appearance of autoantibodies is strongly associated with the class 2 alleles DRB1*03 and DRB*04 and with DRB1*03/04 heterozygosity. In contrast, the progression to clinical disease appears associated with class I alleles A24, A30 and B18 while A1, A28, B14 and B56 appear negatively associated. The class 2 alleles appear to have a minimal role in the progression from autoantibody positivity to clinical disease. These results are consistent with the view that CD4+ T cells responding to peptides in the context of class 2 molecules are responsible for initiating autoantibody production, while the destruction of islet cells leading to clinical expression of the disease is the function of CD8+ T cells recognizing relevant peptides in the context of class I molecules.     

The role of the intestinal microbiota in type 1 diabetes

The digestive tract hosts trillions of bacteria that interact with the immune system and can influence the balance between pro-inflammatory and regulatory immune responses. Recent studies suggest that alterations in the composition of the intestinal microbiota may be linked with the development of type 1 diabetes (T1D). Data from the biobreeding diabetes prone (BBDP) and the LEW1.WR1 models of T1D support the hypothesis that intestinal bacteria may be involved in early disease mechanisms. The data indicate that cross-talk between the gut microbiota and the innate immune system may be involved in islet destruction. Whether a causal link between intestinal microbiota and T1D exists, the identity of the bacteria and the mechanism whereby they promote the disease remain to be examined. A better understanding of the interplay between microbes and innate immune pathways in early disease stages holds promise for the design of immune interventions and disease prevention in genetically susceptible individuals.     

Translational mini-review series on type 1 diabetes: Systematic analysis of T cell epitopes in autoimmune diabetes

T cell epitopes represent the molecular code words through which the adaptive immune system communicates. In the context of a T cell-mediated autoimmune disease such as type 1 diabetes, CD4 and CD8 T cell recognition of islet autoantigenic epitopes is a key step in the autoimmune cascade. Epitope recognition takes place during the generation of tolerance, during its loss as the disease process is initiated, and during epitope spreading as islet cell damage is perpetuated. Epitope recognition is also a potentially critical element in therapeutic interventions such as antigen-specific immunotherapy. T cell epitope discovery, therefore, is an important component of type 1 diabetes research, in both human and murine models. With this in mind, in this review we present a comprehensive guide to epitopes that have been identified as T cell targets in autoimmune diabetes. Targets of both CD4 and CD8 T cells are listed for human type 1 diabetes, for humanized [human leucocyte antigen (HLA)-transgenic] mouse models, and for the major spontaneous disease model, the non-obese diabetic (NOD) mouse. Importantly, for each epitope we provide an analysis of the relative stringency with which it has been identified, including whether recognition is spontaneous or induced and whether there is evidence that the epitope is generated from the native protein by natural antigen processing. This analysis provides an important resource for investigating diabetes pathogenesis, for developing antigen-specific therapies, and for developing strategies for T cell monitoring during disease development and therapeutic intervention.     

The role of liver-derived regulatory dendritic cells in prevention of type 1 diabetes

Development of type 1 diabetes has been attributed to T-cell-mediated autoimmunity, which is regulated by antigen-presenting cells. To study the role of liver-derived B220(+) regulatory dendritic cells (DCs) in the development of diabetes in non-obese diabetic (NOD) mice, we found that liver 220(+) DCs could easily be propagated from young NOD mice, but that such propagation was extremely difficult from mice older than 11 weeks, when insulitis began. This was not simply an age-related phenomenon, because liver B220(+) DCs were readily propagated from both young and old congenic non-obese diabetic-resistant (NOR) and normal BALB/c mice. It was therefore speculated that the development of diabetes might be associated with a lack of precursors of B220(+) DC in the liver in this animal model. Unfortunately, the specific marker for precursors of liver B220(+) DC has not been identified. An alternative approach to supplement liver B220(+) DCs by intravenous administration significantly inhibited the development of diabetes by inducing T-cell hyporesponsiveness via enhancement of their apoptotic death. Liver B220(+) DCs were capable of effectively presenting antigens but, unlike plasmacytoid DCs, did not express CD11c and were not interferon-alpha producers. These observations may throw new light on the aetiopathology of type 1 diabetes.     

Viral infections and molecular mimicry in type 1 diabetes

Type 1 diabetes (T1D) is a disease characterized by inflammation of pancreatic islets associated with autoimmunity against insulin‐producing beta cells, leading to their progressive destruction. The condition constitutes a significant and worldwide problem to human health, particularly because of its rapid, but thus far unexplained, increase in incidence. Environmental factors such as viral infections are thought to account for this trend. While there is no lack of reports associating viral infections toT1D, it has proven difficult to establish which immunological processes link viral infections to disease onset or progression. One of the commonly discussed pathways is molecular mimicry, a mechanism that encompasses cross‐reactive immunity against epitopes shared between viruses and beta cells. In this review, we will take a closer look at mechanistic evidence for a potential role of viruses in T1D, with a special focus on molecular mimicry.     

Viruses and type 1 diabetes: ignorance acquires a better vocabulary

The hypothesis that a virus might in some way be involved in the causation of type 1 diabetes has a long history, but decades of research have failed to resolve the issue beyond reasonable doubt. Viruses could potentially play a primary role in the pathogenesis of type 1 diabetes by initiating autoimmunity, a secondary role by promoting established immune responses, or a tertiary role by precipitating the onset of hyperglycaemia. There is currently little evidence to suggest that viruses play a primary role in the causation of type 1 diabetes, let alone a necessary or sufficient role. Secondary or tertiary roles remain possible, but have yet to be confirmed in prospective studies.     

Characterization of monocyte-derived dendritic cells in recent-onset diabetes mellitus type 1

Dendritic cells (DC) may play an important role in the immunopathogenesis of type 1 diabetes mellitus (DM-1). In this study, we have analyzed phenotypical changes during cytokine-driven maturation from CD14+ monocytes to mature DC and DC-dependent T-cell stimulation in recent-onset pediatric DM-1 patients and healthy controls. DC maturation was monitored by flow cytometric analyses for the expression of surface markers (HLA-DR, CD1a, CD40, CD80, CD86, CD83, CD14, CD32, mannose-receptor, and CD11c). Flow cytometric analysis of isolated peripheral blood monocytes did not reveal apparent differences between patients and controls. During DC maturation no obvious differences in the expression patterns of surface markers over time or evidence for maturation impairments in DM-1 patients could be appreciated. Solely, a marginal, but significant, transient down-regulation of CD1a on Day 3 (mean MDFI 3.82 vs 7.25; P = 0.021), which was accompanied by an increase of IL-6, could be observed. The comparison of mature DCs (Day 10) between patients and controls indicated no significant differences, except for CD83 (mean MDFI 1.7 vs 1.5; P = 0.042) and CD80 (mean MDFI 15.92 vs 12.73; P = 0.042). Moreover, no difference in T-cell stimulatory capacity was seen. In conclusion, our analysis of a cohort of recent-onset DM-1 patients and controls does not support a role for disease-related alterations in cytokine-driven maturation of monocyte-derived DC.     

NETosis‐associated serum biomarkers are reduced in type 1 diabetes in association with neutrophil count

As the immune pathways involved in the pathogenesis of type 1 diabetes (T1D) are not fully understood, biomarkers implicating novel mechanisms of disease are of great interest and call for independent evaluation. Recently, it was reported that individuals with T1D display dramatic elevations in circulating components of neutrophil extracellular traps (NETs), indicating a potential role for NETosis in T1D. Our aim was to evaluate further the potential of NET‐associated proteins as novel circulating biomarkers in T1D. We tested serum from subjects with T1D (n = 44) with a median age of 26·5 years and a median duration of 2·2 years, along with 38 age‐matched controls. T1D subjects did not show elevations in either neutrophil elastase (NE) or proteinase 3 (PR3), as reported previously. In fact, both NE and PR3 levels were reduced significantly in T1D subjects, particularly in subjects within 3 years of diagnosis, consistent with the known reduction in neutrophil counts in recent‐onset T1D. Indeed, levels of both NE and PR3 correlated with absolute neutrophil counts. Therefore, while not ruling out potential local or transient spikes in NETosis activity, the levels of these serum markers do not support a role for systemically elevated NETosis in the T1D population we studied. Rather, a modest reduction in these markers may reflect other important aspects of disease activity associated with reduced neutrophil numbers.     

Respiratory infections and type 1 diabetes: Potential roles in pathogenesis

Among the environmental factors associated with type 1 diabetes (T1D), viral infections of the gut and pancreas has been investigated most intensely, identifying enterovirus infections as the prime candidate trigger of islet autoimmunity (IA) and T1D development. However, the association between respiratory tract infections (RTI) and IA/T1D is comparatively less known. While there are significant amounts of epidemiological evidence supporting the role of respiratory infections in T1D, there remains a paucity of data characterising infectious agents at the molecular level. This gap in the literature precludes the identification of the specific infectious agents driving the association between RTI and T1D. Furthermore, the effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on the development of IA/T1D remains undeciphered. Here, we provide a comprehensive overview of the evidence to date, implicating RTIs (viral and non-viral) as potential risk factors for IA/T1D.     

Immune cell trafficking to the islets during type 1 diabetes

Inhibition of immune cell trafficking to the pancreatic islets during type 1 diabetes (T1D) has therapeutic potential, since targeting of T cell and B cell trafficking has been clinically effective in other autoimmune diseases. Trafficking to the islets is characterized by redundancy in adhesion molecule and chemokine usage, which has not enabled effective targeting to date. Additionally, cognate antigen is not consistently required for T cell entry into the islets throughout the progression of disease. However, myeloid cells are required to enable T cell and B cell entry into the islets, and may serve as a convergence point in the pathways controlling this process. In this review we describe current knowledge of the factors that mediate immune cell trafficking to pancreatic islets during T1D progression.     

Immunology in the clinic review series: focus on type 1 diabetes and viruses: the role of viruses in type 1 diabetes: a difficult dilemma

Convincing evidence now indicates that viruses are associated with type 1 diabetes (T1D) development and progression. Human enteroviruses (HEV) have emerged as prime suspects, based on detection frequencies around clinical onset in patients and their ability to rapidly hyperglycaemia trigger in the non-obese diabetic (NOD) mouse. Whether or not HEV can truly cause islet autoimmunity or, rather, act by accelerating ongoing insulitis remains a matter of debate. In view of the disease's globally rising incidence it is hypothesized that improved hygiene standards may reduce the immune system's ability to appropriately respond to viral infections. Arguments in favour of and against viral infections as major aetiological factors in T1D will be discussed in conjunction with potential pathological scenarios. More profound insights into the intricate relationship between viruses and their autoimmunity-prone host may lead ultimately to opportunities for early intervention through immune modulation or vaccination.     

16S rRNA gene sequencing analysis of gut microbiome in a mini-pig diabetes model

Background : Currently, increasing attention is being paid to the important role of in-testinal microbiome in diabetes. However, few studies have evaluated the characteris-tics of gut microbiome in diabetic miniature pigs, despite it being a good model animal for assessing diabetes. Methods : In this study, a mini- pig diabetes model (DM) was established by 9- month high- fat diet (HFD) combined with low- dose streptozotocin, while the animals fed standard chow diet constituted the control group. 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the characteristics of the intestinal microbiome in diabetic mini- pigs. Results : The results showed that microbial structure in diabetic mini- pigs was altered, reflected by increases in levels of Coprococcus_3 and Clostridium_sensu_stricto_1 , which were positively correlated with diabetes, and decreases in levels of the bac-teria Rikenellaceae , Clostridiales_vadinBB60_group , and Bacteroidales_RF16_group , which were inversely correlated with blood glucose and insulin resistance. Moreover, PICRUSt- predicted pathways related to the glycolysis and Entner- Doudoroff super-pathway, enterobactin biosynthesis, and the L - tryptophan biosynthesis were signifi-cantly elevated in the DM group. Conclusion : These results reveal the composition and predictive functions of the in-testinal microbiome in the mini- pig diabetes model, further verifying the relationship between HFD, gut microbiome, and diabetes, and providing novel insights into the application of the mini- pig diabetes model in gut microbiome research.