Innate immunity and PPRs

Recognition by the mammalian immune system of invading micro-organisms depends on innate and adaptive components. The adaptive immune system recognizes antigens derived from micro-organisms by highly diverse T-cell surface receptors that are formed via somatic gene recombination. The innate immune system, on the other hand, recognizes microorganisms by pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I, MDA-5 and receptors of the nucleotide-binding oligomerization domain-like receptor (NLR) family (Box 1). Activation of these signaling molecules contributes to the development of various autoimmune diseases, such as systemic lupus erythematosis, rheumatoid arthritis and autoimmune hepatitis.⁴ Some components of the innate immunity response, including TLRs, contribute to the development of insulitis and T1DM in animal models.⁵

Ligands for PPRs in insulitis

Mouse and human pancreatic islets express TLR2, TLR3, TLR4 and TLR9; of which TLR3 and TLR4 are expressed at high levels.^{6, 7, 8} The expression of TLR3 is upregulated in cells by double-stranded RNA (dsRNA),^{7, 9} an intermediate nucleic acid which is generated during the life cycle of most viruses. In human islets that are infected with coxsackievirus B5 or exposed to interferon (IFN)- or IFN- and interleukin (IL)-1, increased expression of TLR3, RIG-I and MDA-5 have been observed.^{10, 11} Intracellular dsRNA and extracellular dsRNA, which is derived from damaged or dying cells, can both bind to TLR3 and trigger -cell apoptosis and cytokine and chemokine production, at least in part, through activation of the transcription factors NFB and IRF-3.^{9, 12, 13, 14} However, whereas the activation of these transcription factors by extracellular dsRNA is entirely dependent on TLR3, intracellular dsRNA uses alternative pathways, which might include RIG-I/MDA-5 activation¹⁴ (ML Colli, unpublished data).⁹

Internal dsRNA triggers a massive production of type I interferons. When prolonged or excessive, such interferon release can lead to dsRNA-induced -cell apoptosis, which is partly caused by endoplasmic-reticulum (ER) stress.¹⁴ Importantly, high levels of interferons are present in pancreatic tissue of patients with T1DM,¹⁵ and IFN- contributes to viral-induced experimental diabetes.^{16, 17} These observations suggest that activation of TLR3 and/or RIG-I and MDA-5 in cells leads to a complex molecular response that starts by activation of the key transcription factors NFB and IRF-3. This activation is followed by production of IFN- and IFN-, which leads to paracrine activation of the transcription factor STAT-1, overexpression of MHC class-I antigens, further production of IFN- and IFN- and the release of several chemokines (see below).^{9, 10, 12, 14, 18} The result of these combined factors is attraction of immune cells that release proinflammatory cytokines, such as IL-1, tumor necrosis factor (TNF) and IFN- (Figure 2). Local inflammation, coupled with triggering of intracellular and extracellular antiviral defenses, should in most cases eradicate the viral infection. In some genetically susceptible individuals, however, these cellular attempts to eradicate or neutralize the invading virus might go wrong: for example, an exaggerated inflammatory response and/or a defective triggering of intracellular anti-inflammatory or antiapoptotic responses could occur, such as expression of JunB,¹⁹ which induces progressive inflammation and prolonged -cell loss. How and why these pathological processes transpire is a critical question that remains to be answered.

Figure 2 | Interaction of cells and immune cells leads to induction and amplification of insulitis, and the transition from innate to adaptative immune response.

Recognition of endogenous and exogenous ligands by PRRs (TRL3/4, RIG-I, MDA5) leads to activation of key transcription factors, such as NFB, IRF3 and STATs. Activation of these transcription factors induces the release of chemokines and cytokines that recruit and activate immune cells; increase the cells' expression of MHC class-I antigens (that, when associated with ER stress, might lead to presentation of modified antigens to the immune cells), and activate proapoptotic signals that lead to -cell death. Signals from dying cells are presented by professional antigen-presenting cells that contribute to the activation of autoreactive T cells. During this local inflammation process, pro-inflammatory cytokines (for example, IL-1, TNF and IFN-//) are released by the immune cells and induce transcription factors (e.g STAT-1, NF-B and IRF-3) in cells that contribute to the maintenance and amplification of the network described above. This vicious circle can result in a progressive and selective destruction of pancreatic cells. Some transcription factors might function as negative regulators of the above signaling pathways, for instance, overexpression of JunB prevents cytokine-induced ER stress and -cell apoptosis. Abbreviations: ER, endoplasmic reticulum; IFN, interferon; IL, interleukin; PRR, pattern-recognition receptor; TLR, Toll-like receptor; TNF, tumor necrosis factor; +, stimulation; -, inhibition.

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The model described above assumes that binding of exogenous ligands (in this case, a virus) to PRRs triggers the innate immune response. This theory is supported by accumulating evidence that viral infections, especially enteroviruses, have a role in the etiology of T1DM.²⁰ Some enterovirus strains have specific -cell tropism, and, in a study where pancreatic tissue samples of organ donors were examined, such viruses were detected in the cells from three of six patients with recent-onset T1DM.²¹ Viruses might also be associated with T1DM by molecular mimicry: if viral antigens have epitopes which are highly similar to those of self-antigens, T-cell receptors sometimes fail to differentiate properly between these molecules. In these cases, T cells are initially activated by both viral antigens and self-antigens, which triggers an acute inflammatory response. After the resolution of the viral infection, the persistence of self antigens might lead to a chronic autoimmune response. This phenomenon has been elegantly demonstrated in the rat insulin promoter–lymphocytic choriomeningitis virus model of diabetes mellitus.²² For such an effect to occur, however, homology must be complete between the self epitope and the viral epitope, which is unlikely to occur in human disease. In fact, molecular mimicry is probably more relevant to the amplification of an ongoing autoimmune process than to initiate one.²³

Another possibility (which does not exclude a pathogenic role of viral infection in some individuals) is that endogenous ligands start the inflammatory process by binding to PRRs.⁴ In line with this theory, apoptotic mouse cells that are undergoing secondary necrosis trigger T-cell immunity through a TLR2-initiated signaling pathway. Importantly, autoimmune diabetes in two mouse models was markedly inhibited by TLR2 deletion through impaired activation of T cells by antigen-presenting cells following -cell injury.²⁴ This finding indicates that -cell death, and its detection by TLR2, is a putative trigger of the development of T1DM. Moreover, pancreatic islets isolated from TLR4-deficient mice (C57BL/10ScNJ background) are protected against allograft rejection when transplanted under the kidney capsule of BALB/c.²⁵

Although the above studies suggest that TLR2 and TLR4 have a central role in experimental T1DM, this hypothesis was challenged by a study that showed no change in the prevalence of diabetes in nonobese, diabetic (NOD) mice with genetic deletion of TLR2 and TLR4.²⁶ In this model, contribution of mouse protein MyD88—a key signaling molecule in TRL2 and TLR4 pathways—to the development of autoimmune diabetes depends on the constitution of the intestinal microflora, which suggests a complex interaction between the genetic background, enteric bacteria and the innate immune response.²⁶ From an immunological perspective, multiple pathways and diverse forms of human T1DM might exist.²⁷ Inbred NOD mice and BB-rats (a diabetes-prone strain of rat with abnormally low levels of lymphocytes) probably have defects in one of these pathways, which makes it particularly difficult to extrapolate findings on interactions between the environment and innate immune response from these models to the heterogeneous human disease.

Additional studies are now required to solve this issue, and to determine the mechanisms by which changes in gut microbiota regulate innate and adaptive immune responses of NOD mice. Polymorphisms of NLRs are associated with other autoimmune diseases and allergies, such as Crohn disease²⁸ and asthma,²⁹ but whether they contribute to T1DM remains unclear. These receptors detect intracytoplasmic bacterial molecules and trigger major inflammatory pathways by activation of NFB, MAP-kinases and caspase 1.³⁰ High amounts of NLRs are expressed in the intestine, and one of them, NOD1, has a key role in communication between intestinal bacteria and the innate and adaptive immune system.³¹ NLRs are thus interesting candidates for further study of the interaction between the gut microbiota and the immune response in NOD mice.

Dialog between cells and the immune system

Chemokines are signal molecules that direct leukocyte migration and activation during the early stages of an innate immune response and contribute to the transition to adaptive immunity.³² The specificity and complexity of the chemokine system derives from both the release of specific chemokines in various inflammatory reactions, and the regulated expression of their receptors in leukocytes, which varies in different immune responses.^{33, 34} Expression of chemokines and their receptors is altered in several autoimmune diseases, which seems also to be the case in T1DM. Increased levels of T-helper 1 (T_H1) cell-derived chemokines (CCL3, CCL4 and CXCL10) are present in the serum of patients who have recently been diagnosed as having T1DM,^{35, 36, 37} and a longitudinal study that included 256 such patients identified a negative correlation between the levels of CCL3 and C-peptide.³⁸ In this study, however, a major overlap was found between chemokine expression profiles in patients and in controls, which reflects the difficulties in understanding proinflammatory mechanisms in early T1DM from studies that have used traditional approaches to examine serum samples or peripheral cells.

Indeed, most of the pathological processes that occur in the early phases of T1DM take place in the islet microenvironment and pancreatic draining lymphnodes. These tissues are difficult to access in humans and, as the islets are just a small part of the pancreas (less than 2%), locally generated inflammatory signals are diluted in the general circulation. To circumvent these difficulties, a recent study has used microarray analysis of healthy, peripheral, mononuclear white blood cells exposed to sera from patients with recent-onset or long-standing T1DM, healthy controls, or siblings of patients with T1DM to identify the presence of proinflammatory factors in the serum.³⁹ Sera obtained from patients with recent-onset T1DM induced a specific expression pattern, including IL-1 cytokine family members and the chemokines CCL2 and CCL7 (which are both involved in the recruitment of monocytes and macrophages). This proinflammatory 'signature' or expression pattern was apparent years before the clinical onset of T1DM in three patients who were studied.³⁹ The results obtained with this elegant approach need now to be confirmed in large, prospective studies.

Studies in NOD mice have shown increased levels of CXCL10, CCL2, CCL20 and IL-15 mRNAs and/or proteins in pancreatic islets during the prediabetic stage.^{40, 41, 42, 43} During the course of diabetes, macrophages are the first cells to infiltrate the islets of NOD mice and BB rats, and depletion or inactivation of macrophages prevents the development of the disease.² CXCL10 and CCL2 attract macrophages, and their early expression in the islets of NOD mice described above contributes to macrophage recruitment during the early stages of insulitis. Transgenic expression, which leads to high production of CCL2 by cells, causes insulitis and autoimmune diabetes.⁴³ Of interest, high, basal CCL2 production by human islets correlates with a poor clinical outcome following islet transplantation in patients with T1DM.⁴⁴ In the adaptive immune response, diabetogenic T_H1 cells in NOD mice express the CCR5 receptor and its ligands (CCL3, CXC10), as well as XCL1, CCL2, CCL7 and CCL12.^{45, 46} Deletion of CCL3 in NOD mice ameliorates symptoms of insulitis and prevents autoimmune diabetes,⁴⁵ whereas deletion of CCR5 leads to a switch from a T_H1 response to a T_H2 response, which delays islet-allograft destruction in mice.⁴⁷

A relevant role for locally produced cytokines and chemokines was also observed in the mouse model of virus-induced autoimmune diabetes, as mentioned above. In these mice, the blockade of CXCL10, but not that of CCL5, prevented the development of autoimmune diabetes after infection with lymphocytic choriomeningitis virus.⁴⁸ Conversely, overexpression of CXCL10 accelerated the onset of T1DM.⁴⁸ The absence of CXCR3—the receptor for CXCL10, CXCL9 and CXCL11—delayed, but not prevented, the onset of insulitis and diabetes,⁴⁹ which suggests that these chemokines mainly have a role in the early stages of the disease.

An important source of chemokine production during insulitis can be the cells themselves. Isolated rat cells exposed to IL-1 and IFN- or to dsRNA have increased expression of mRNAs that encode several cytokines and chemokines, including CCL2, CXCL10, CCL20, CX3CL1 and IL-15.^{13, 40, 41, 50, 51} Human islets exposed to IL-1 and IFN- have increased mRNA expression of IL15, CXCL10, CCL2, CCL20 and CX3CL1, and secrete IL15, CCL2, CXCL10, CXCL9, CXCL11 and CCL20 into the culture medium.^{40, 41} -cell expression of chemokines is mostly regulated by the transcription factors NFB^{52, 53} and STAT-1.^{54, 55} These two transcription factors are also key mediators of cytokine-induced -cell death (see below).⁵⁶

The findings described above suggest the possibility of a dialog between immune cells and the target cells during the course of insulitis,^{34, 56} where activated macrophages, natural killer (NK) cells and T cells produce cytokines, such as IFN-, IL-1 and TNF, which induce cells to release chemokines and stimulatory cytokines (Figure 2). These molecules will attract more mononuclear cells that also release multiple chemokines.^{34, 42} If this vicious circle is not interrupted, it will evolve to progressive accumulation of activated macrophages and T cells around and inside the islets. The nature of the T cells that participate in this amplification phase remains an issue of debate, which is beyond the scope of the present article. Of interest is the recent discovery of T_H17 cells: T_H17 are potent inducers of tissue inflammation and autoimmunity,⁵⁷ and a study in NOD mice suggest that they might have a role in T1DM.⁵⁸ If confirmed, these observations should promote a re-evaluation of the role of T_H1 and regulatory T cells in T1DM.

Taking into account the points discussed above, chemokines and their receptors, or the transcription factors that regulate them, are interesting targets for therapeutic interventions to prevent T1DM.^{42, 56}

ER stress in cells and antigen presentation

-cell apoptosis is probably the main form of -cell death in patients with T1DM.^{2, 3} As discussed above, chemokine production and the triggering of -cell apoptosis seem to be regulated by similar intracellular signals, for example by the transcription factors NFB and STAT-1.⁵⁶ Thus, -cell death occurs parallel to intense inflammation in the islet microenvironment. Products of dying cells might be perceived as danger signals by the immune system, and antigens that are released from such cells—especially in the presence of inflammatory factors, such as TNF, interferons and chemokines—might be taken up by professional antigen-presenting cells in pancreatic lymph nodes and boost the autoimmune response.⁵⁹ In support of this scenario, knockout mice that lack caspase 3, the major downstream effector enzyme in the apoptotic pathway, are protected against diabetes that is induced by multiple, low-dose streptozotocin injections, which indicates that -cell apoptosis is a required step for T cell activation.⁶⁰ Furthermore, apoptotic cells that undergo secondary necrosis trigger TNF production by macrophages and activate autoreactive T cells (Figure 2).²⁴ This immune-response-enhancing role of dying cells is probably dependent on the physiological context. Low doses of streptozotocin injected in young NOD mice at the preinsulitis stage decrease the prevalence of diabetes, which suggests that the presence of low numbers of apoptotic cells in a noninflammatory environment might lead to tolerance against cell antigens.⁵⁹ These data, however, must be interpreted with caution, as streptozotocin might have effects on the immune system and/or on -cell gene expression, which also affects tolerance induction.

Exposure of cells to inflammatory cytokines or to dsRNA induces ER stress, which leads to accumulation of misfolded proteins in the ER and triggers the unfolded-protein response.^{14, 61, 62} The unfolded-protein response aims to alleviate stress on the ER and to restore homeostasis by decreasing the arrival of new proteins; increasing the amount of ER chaperones and increasing the extrusion and subsequent degradation of irreversibly misfolded proteins; when the steps described above fail, apoptosis is triggered.⁶² Dying cells can transfer immunologically relevant information to dendritic cells, which signals the nature of cell death and determines the immunological outcome of phagocytosis.⁶³ Peptides from within the ER of dying cells can be loaded onto MHC class-I molecules in dendritic cells without further cytosolic processing.⁶⁴ In this way, dying cells provide antigen-presenting cells with an accurate representation of what happened just before their death.⁶³

Although this signaling might be beneficial when viral infection has triggered apoptosis, it might have dire consequences for insulin-producing cells. Insulin production represents nearly 50% of the total production of protein by cells, and it accumulates in the ER during periods of increased stress. In this case, insulin accumulates (at least in part) in a misfolded configuration,⁶⁵ which might increase its antigenicity.⁶² High antigenicity results in increased presentation of proinsulin and insulin to antigen-presenting cells, especially in the presence of inflammation. Although this role of ER stress remains to be proved in T1DM, increased antigenicity due to misfolding of HLA-B27 has been suggested to occur in ankylosing spondylitis.⁶⁶ Of note, insulin is a key antigen for autoimmune diabetes in both humans and NOD mice.^{67, 68, 69, 70}