I. General points on islet inflammation in diabetes

1. A brief overview of the immune response

Inflammation is traditionally characterized as an acute reaction leading to heat, pain, redness, and swelling—calor, dolor, rubor, and tumor—at the site of an inflammatory process, reflecting the acute effects of inflammatory mediators on blood vessels and the recruitment of plasma components and leukocytes during the first steps of inflammation.
Acute inflammatory responses are typically induced by local infections or tissue injuries. In addition, beyond acute inflammation, many pathological processes have been related to low-grade, chronic inflammation. Immune responses are complex processes that involve first-line defenses with low specificity for the causative event, but that progressively recruit and expand cells with high specificity for molecular determinants present at the inflammation site—lymphocytes—distinguishing innate and adaptive immunity, respectively.

Innate immunity refers to nonspecific defense mechanisms that come into play immediately or within hours of an antigen’s intrusion in the body, as common bacterial, viral, or fungal infections. It includes skin and mucosae as a first line of defense.
It relies on innate immune cells (among which macrophages, neutrophils, dendritic cells [DCs]) which express invariant receptors for invariant motifs, named pathogenassociated molecular patterns (PAMPs), shared by families of invading microbes.
There are several families of receptors (pattern-recognition receptors [PRRs]) in mammals, such as membrane-bound Toll-like receptors (TLRs) or cytoplasmic nucleotidebinding oligomerization domain receptors, in short, NOD-like receptors (NLRs). A number of PRRs do not remain associated with the cell that produces them. These secreted receptors bind to microbial cell surfaces, favoring phagocytosis by macrophages and neutrophils, and participating in the activation of the complement pathway. Innate immune cells also include natural killer (NK) cells, NK T cells (NKT), and newly characterized cells such as innate lymphoid cells that directly connect innate and adaptive immunity. Most PRRs, especially TLRs, drive the activation of nuclear factor-κB (NF-κB) and interferon-releasing factors (IRFs) that also allow a direct link with the activation of adaptive immune cells, such as T and B lymphocytes. This leads to the production of cytokines (such as interferons [IFNs] or interleukins [ILs]), chemokines, and expression of their receptors. Also, expression of costimulatory molecules for T-lymphocyte activation and key molecules for the presentation of antigens to T lymphocytes—class I and class II major histocompatibility complex (MHC) molecules—is enhanced. Interestingly, innate immune receptors can also sense nonphysiological sensors or stressors within and around cells or within the internal milieu to maintain their integrity.

Adaptive immunity refers to antigen-specific immune responses. The adaptive immune response is initiated by the recognition of an antigenic determinant by a lymphocyte that expresses a specific antigen receptor. It allows a considerable expansion of the antigen-specific lymphocytes. The diversity of antigen receptors that ensure the recognition of all possible antigenic motifs relies on random recombination of sets of genes that encode for the variable recognition domain of antigenic receptors. However, efficient adaptive immune responses are delayed in time and boosted by the innate immune response. Three types of cells are central to the adaptive immune response:
(i) Antigen-presenting cells (APCs) are commonly referred to as professional APCs.
Professional APCs express class I and class II MHC molecules and are able to activate T lymphocytes that have never encountered their specific antigen (naive T cells) or have previously been activated against this antigen and have escaped apoptosis following activation (memory T cells). More importantly, professional APCs can present phagocytosed, external antigens—microbes, but also cell debris—and express costimulation molecules that will interact with corresponding molecules on lymphocytes to allow their full activation. There are 3 main types of professional APCs: macrophages, DCs, and B lymphocytes. However, all nucleated cells within the organism, including pancreatic β-cells, can present antigens, but in a restricted manner. They express MHC class I molecules, but express neither class II nor costimulation molecules.
(ii) A variety of different types of T lymphocytes (also called “T cells”) exist, each with its own specialized function:
■ CD4+ cells (also called “T4 cells” or “helper T cells”) control the adaptive immune response, in particular, the activation of B lymphocytes and cytotoxic CD8+ T cells.
■ CD8+ cells (also called “T8 cells” or “cytotoxic T cells”) are able to kill cells presenting intracellular antigen on class I MHC molecules, eg, a peptide from a viral protein that has infected the presenting cell or an autoantigen.
■ Regulatory T cells (Tregs) are involved in regulating immune responses and ensuring immune homeostasis. They play a key role in the maintenance of immune tolerance to self-tissues.
T lymphocytes recognize intracellular proteins in the form of small fragments (peptides) complexed to MHC molecules. Class I MHC molecules are devoted to the presentation of antigens to CD8+ T cells. Class II MHC molecules are devoted to the presentation of antigens to CD4+ T cells.
(iii) Finally, B lymphocytes (also called “B cells”) are the actors in humoral immunity.
They recognize native motifs on extracellular antigens in their three-dimensional conformation, then they differentiate in cells that produce immunoglobulins (antibodies).
Their activation against T lymphocyte–dependent antigens requires the regulatory intervention of CD4+ helper T cells. B lymphocytes express class II MHC and costimulation molecules, and as such are efficient antigen-presenting cells.

Mechanism of adaptive immune response:
Whereas B cells are simply activated by recognition of an antigen by its cell surface receptors, an immunoglobulin-like structure, T cells must simultaneously recognize antigen on self-MHC molecules. Indeed, when a pathogen infects a tissue, it is engulfed and processed by an APC that becomes activated and inserts the processed antigen on the external domains of a MHC class II molecule to form a peptide/MHC complex on its cell surface. Along the same process, the APC migrates to a draining lymph node where it activates antigen-specific T cells through recognition by a T-cell receptor (TCR), thus amplifying the immune response through the activation of the adaptive immune pathway. Once the T cell recognizes the peptide/MHC complex, the APC sends out additional costimulatory signals, by the secretion of cytokines and the expression of membrane costimulatory molecules, to activate the T cell. Depending on the type of antigen, cytokines, and microenvironment, the activated T cell (CD4+ T
cell, Treg) will activate and expand other T-cell subsets and turn on specific signaling pathways and initiate the synthesis of transcription factors and cytokines leading to a second phase of recruitment of macrophages, NK cells, neutrophils, or B cells to eliminate the antigen.

Thus, the immune response is a complex process. Cells of the innate system play a crucial part in the initiation and subsequent direction of the adaptive immune response, participating in the removal of pathogens that have been targeted by an adaptive immune response, but can also sense altered self-structures. Activated APCs secrete cytokines that influence both innate and adaptive immune responses, making these cells essential gatekeepers that determine whether and how the immune system responds to the presence of infectious agents.1

2. Autoimmune type 1 diabetes

T1D is a chronic autoimmune disease in which pancreatic β-cells are inappropriately destroyed by the immune system.2 It is characterized by an exaggerated activation of the immune system which results in the generation of self-reactive lymphocytes and high-titer autoantibodies. It involves both innate and adaptive immunity. The initiating events leading to T1D pathogenesis still need to be elucidated. In the first steps of inflammation, it is possible that alterations of β-cells lead to a local inflammation and the release of β-cell debris and β-cell autoantigens, favoring the recruitment of innate immune cells such as neutrophils, macrophages, and NK cells in the pancreas, secretion of inflammatory cytokines (such as IL-1β, interferon γ [IFN-γ], and tumor necrosis factor α [TNF-α]), and other molecules (nitric oxide [NO], oxidative radicals, and chemokines) that further lead to functional damage and β-cell apoptosis.

Apoptotic β-cells make autoantigens available for uptake by DCs that in turn activate cytotoxic T cells, leading to β-cell destruction and progressive development of T1D.
The pathogenesis of T1D is believed to be a cell-mediated autoimmune disease because autoreactive T cells, but not autoantibodies, are necessary to transfer the disease in animal models and humans. The impairment of insulin-producing and secretory abilities make affected individuals dependent upon exogenous insulin for life and these individuals have a high risk of developing macrovascular and microvascular complications.

Several genetic as well as environmental factors are believed to contribute to the development of β-cell autoimmunity. Interestingly, epidemiologic studies have shown that obesity is a risk factor for both T2D and T1D, suggesting that autoimmune processes responsible for T1D may interact with risk factors associated with T2D.
A major task in T1D is the characterization in humans of T lymphocytes that are specific for major β-cell autoantigens, especially insulin, a major autoantigen in T1D. The characterization of the antigenic fragments—epitopes—recognized along the disease process is expected to open the door to new bioassays in the diagnosis of autoimmunity and to autoantigen-specific immunotherapy.

3. Autoinflammatory type 2 diabetes?

T2D is initiated by defective insulin secretion in an environment of increased resistance to the action of insulin on peripheral target tissues. In the physiological state, the decreased sensitivity to insulin is compensated by an increased secretory activity of β-cells and by expansion of the functional β-cell mass. However, with the advent of defective β-cells, mainly due to mitochondrial dysfunction, and oxidative and ER stresses within an external environment that drives glucolipotoxicity on a defavorable genetic background, adaptive mechanisms fail to compensate for the increased insulin needs, leading to insufficient hormone supply and hyperglycemia. Finally, the hyperstimulation of β-cells leads to β-cell failure and the progressive deterioration of insulin secretion accompanied by a loss of β-cell mass. Recent work has indicated that chronic inflammation is another important pathophysiological factor in the development of T2D that is favored by obesity, hyperglycemia, and insulin resistance. Indeed, the pancreas of T2D patients was shown to be infiltrated by immune cells, suggesting that immune-mediated islet damage may be a component of more than just classic T1D. In fact, in T2D patients, metabolic stresses and obesity can directly affect β-cells, leading them to launch a proinflammatory response that will impair insulin production and secretion. Chronic inflammation of the visceral adipose tissue is usually also induced3 and results in the production of proinflammatory mediators that affect both adipose cells and pancreatic islet cells through humoral and neuronal pathways. Such inflammation involves overexpression of proinflammatory proteins like C-reactive
protein (CRP), TNF-α, IL-6, and IL-1β, among others. These proinflammatory molecules activate the cells of innate immunity, as evidenced by the increased populations of residential macrophages and monocytes in the pancreatic islets. This causes damage to tissues in the vasculature, adipose tissue, and pancreas. Many studies have been conducted to determine whether adaptive immunity is also involved in T2D development.
Histological studies have concluded that neither T cells nor B cells are prominent in islets of human T2D or animal models of T2D. However, Tregs have been shown to play a fundamental role in the progression of the disease since they are involved in the regulation of body weight, adipocyte hypertrophy, glucose tolerance, and insulin resistance. It was also suggested that an imbalance in the adaptive immunity appears later in the course of T2D as a consequence of a chronic systemic inflammation state. Finally, Brooks-Worrell and collaborators found that T-cell reactivity to islet proteins in phenotypic T2D patients correlates more strongly with impaired β-cell function compared with autoantibody positivity, thus demonstrating not only the presence of islet autoimmune responses in T2D patients, but autoimmune disease4 (Barbara Brooks-Worrell, Lecture). But so far, they have not identified the antigens recognized by T lymphocytes along the inflammatory process.

“Islet Inflammation”
I. General points on islet inflammation in diabetes
II. What are the actors in inflammation in diabetes?
III. Conclusion
Lectures during IGIS meeting