Type 1 diabetes (T1D) is an autoimmune disease characterized by the absence of insulin due to the specific destruction of the insulin-producing β cells of the pancreatic islets. This is a progressive process taking over 20 weeks in the NOD mouse and several years in human patients to be completed [1
]. The NOD animal model has been a valuable source of information for several aspects of disease pathogenesis [2
]. Genetic studies have contributed to portray the complexity of the disease and have established that multiple loci are carrying genes implicated in T1D in human [3
] and animal models [4
]. In human, more than 6 genes contribute to the disease [5
] while over 20 loci have been described in the NOD mouse [7
] but only few possible candidate genes have been unequivocally defined [8
] other than the H2g7 Idd1
Despite intensive research, the initial causal events remain elusive since the selection of individual mice at early stages, prior to the overt clinical signs, represents a challenge. Indeed, even though the NOD mice are inbred not all animals develop the disease, with an incidence of 40-90% in females, depending on the colony [2
]. Additional hindrances for selecting individual animals that will subsequently develop the disease with certainty are the low penetrance of the implicated genes and the influence of environmental factors. For these reasons, the exact mechanisms taking place prior to the onset of the pancreatic islet-damaging sustained inflammatory processes remain largely unknown.
The aim of our investigation was to evaluate the possibility of the existence of distinct gene expression profiles in order to eventually render possible the study of the early molecular changes, taking place before the onset of inflammation in autoimmune prone mice. One hypothesis is that during the post-weaning period in genetically autoimmune prone individuals (mouse or human), homeostatic changes prompt the immune system not to conform to physiological responses, but to instead trigger pathways that lead to the final autoimmune condition in later life. Exogenous factors including the more or less sustained presence of micro-organisms in otherwise microbe-free tissues may take place during early life stages [10
]. This may influence the immune regulatory processes put in place to prevent, attenuate and/or repair the acute or sustained inflammation. It has been reported that early immune stimulation prevents autoimmune diseases, while infections later in life might exacerbate their advent in genetically prone individuals [11
]. A single injection of BCG (the Mycobacterium bovis
vaccine strain) is highly protective against T1D in the NOD mice [12
] and BB rats [13
] when administered early but it has no beneficial effect given after disease onset; similar to what has been observed in human [14
]. These observations lead to the indication that the time "window" of opportunity to prevent disease in human may share similarities with the NOD mice and thus BCG or other immune-system signalling, safe biological preparation, should be given as therapy before the onset of inflammation of the pancreatic islets (insulitis). This early "window" coincides in the NOD mouse model with the early appearance of insulitis, occurring between 4 and 7 weeks [2
] and led to the hypothesis that early initiating mechanisms should be evaluated at this age i.e. over the weaning period. We have previously established that IAA are present early between 3 and 5 weeks of age and represent a diabetes sub-phenotype since their presence in NOD mouse sera, at this early period, correlated with the final disease phenotype [15
]. Thus Early-IAA (E-IAA) have been shown to mark the first measurable phenotypic checkpoint in T1D pathogenesis. Animals positive for E-IAA at 3-5 weeks after weaning, develop autoimmune diabetes earlier (between 16 and 20 weeks) than their E-IAA negative littermates [15
]. We have used this E-IAA sub-phenotype to select individual mice as diabetes prone and search for changes in gene expression patterns in the PLN, correlating with the presence of E-IAA in the NOD mouse sera.
Overt T1D is considered to require the dominant presence of islet-destructive pro-inflammatory T lymphocytes [16
]. The onset of T cell activation is known to deploy within organized secondary lymphoid tissues, draining the tissue that delivers the adequate signals for activation of T and B lymphocytes.
In the NOD mice, any early islet-disruptive remodelling process taking place, whether it implies β cell apoptosis or repair mechanisms [17
], might result in delivery of immune cell-signalling to the PLN. The endpoint of such signalling might include deletion of autoreactive regulatory T cells, and/or expansion of autoreactive pro-inflammatory T lymphocytes as well as T cell-dependent autoreactive B lymphocytes. The later further differentiate as insulin auto-antibody secreting plasma cells [18
]. Antigen specific T cells interact with dendritic cells (DC) within intact explanted lymph nodes (LN), and it has been observed that immunological synapse formation and prolonged interactions between these 2 cell types is followed by activation, dissociation and rapid migration of T cells away from the antigenic stimulus area [19
]. Even more strikingly, excision of PLNs at 3 weeks of age protects NOD mice against IAA, insulitis and diabetes development, almost completely, but has no effect when performed at 10 weeks of age [20
]. Therefore the time frame of 3 to 5 weeks seems to correspond to the initiation of the autoimmune process in the PLN and the related transcriptional profiling can be potentially sort out in this tissue.
We anticipated that at 5 weeks of age, differential gene expression correlated with the presence or absence of E-IAA, in the PLN of NOD mice, could be detected. This timing allowed for prior tests of E-IAA at 3 and 4 weeks. We used microarray analysis to determine gene expression patterns in the PLN. This approach has the potential i) to provide a global profiling of the modulated genes at this first measurable phenotypic checkpoint in T1D pathogenesis, ii) to allow novel hypothesis to be formulated concerning the initiation of the islet-destructive autoimmune process and eventually to further challenge these hypothesis. We identified ectopic high expression of insulin genes in the E-IAA positive PLN. This data confirm the possible cross talk between the pancreas and its adjacent lymph nodes via at least one pancreas specific autoantigen.
Functional annotations of up-regulated transcripts in the PLN of E-IAA positive NOD mice revealed a gene network otherwise found also to operate during inflammatory and tumour processes. Furthermore, we have identified several transcripts coding for extracellular proteins that have to be evaluated as potential disease pathogenesis associated markers and possibly therapeutic targets. This data represent a "freeze frame" of a first identifiable disease check point, associated with the presence of IAA and corresponding to the early steps of the autoimmune process.