Immune cell crosstalk in type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune dis-ease, during which the pancreatic β-cells (which secrete insulin) are selectively destroyed. It is thought to be a T helper 1 (TH1) cell-mediated disease that involves CD8+ T cells and innate immune cells. Individuals with T1D develop hyperglycaemia and can develop diabetes-associated complications in several organ systems owing to a lack of insulin. This was a lethal disease until the 1920s when Banting and Best1 identified insulin as the hormone in the pancreas responsible for maintain-ing blood glucose homeostasis. The development of T1D is under polygenic control, with an additional role for environmental factors2. This role for environmental fac-tors is highlighted by the 40–60% concordance rate for diabetes onset in identical twins and also by the dramatic increase in the incidence of T1D in recent years3,4. As the development of T1D is increasing in the UK at a rate of 3% per year, which is faster than can be accounted for by genetic change, there has been a considerable effort to identify the environmental factors that predispose an individual to diabetes onset. There has been substantial focus on identifying agents, such as viruses, that might precipitate diabetes onset and, more recently, there has been a growing interest in establishing whether exposure to certain pathogens might have been the reason for a historically lower incidence of diabetes onset. Improved sanitation and living conditions, together with vaccina-tion strategies, have decreased our exposure to pathogens and development of infectious disease. It is therefore pos-sible that a reduced exposure to pathogens might be the environmental alteration over the last 60 years that has had a role in the increased incidence of T1D5.

Studies in animal models (BOX 1), particularly in non-obese diabetic (NOD) mice, have identified roles for several different immune cell types in β-cell destruc-tion. CD4+ and CD8+ T cells, as well as macrophages, have been shown to have a role in β-cell death. However, other cell types are present in the pancreatic infiltrate and in the pancreatic draining lymph node, where the initial presentation of islet antigen by dendritic cells (DCs) to islet antigen-specific T cells occurs6. These cells include B cells, natural killer (NK) cells and NKT cells, as well as DC subsets, and they could also contribute to β-cell death. Conversely, targeting or har-nessing the activities of many of these different immune cell types can also be effective in inhibiting β-cell destruction. This strongly suggests that there is substan-tial crosstalk between the immune cells that are involved in pathogenesis and those involved in immune regula-tion. The genetics of T1D or therapeutic approaches for disease prevention have been extensively reviewed elsewhere7,8 and therefore are not discussed here.

Epidemiological studies suggest that environ-mental factors influence the development of T1D in humans9,10. Pathogens, particularly viruses, could accelerate T1D onset through the induction of inflam-matory responses following direct infection of β-cells, as well as through other mechanisms (discussed below). In addition, other pathogens might inhibit T1D onset through effects on numerous cell types and the induction of mediators that suppress host pathol-ogy. These contrasting effects are mediated through interactions between cells of both the innate and adaptive immune system. In this Review, we discuss

*Institut National de la Santé et de la Recherche Médicale (INSERM) U986, Hôpital Saint Vincent de Paul, Bâtiment Petit, 82 Avenue Denfert-Rochereau, 75014 Paris, France.‡Department of Pathology, University of Cambridge, Tennis Court Rd, Cambridge CB21QP, UK.Correspondence to A.L. and A.C. e-mails: [email protected]; [email protected]:10.1038/nri2787

Non-obese diabetic (NOD) mice Mice that spontaneously develop type 1 diabetes as a result of islet antigen-specific T cell-mediated destruction of pancreatic β-cells.

Immune cell crosstalk in type 1 diabetesAgnès Lehuen*, Julien Diana*, Paola Zaccone‡ and Anne Cooke‡

Abstract | The development of type 1 diabetes involves a complex interaction between pancreatic β-cells and cells of both the innate and adaptive immune systems. Analyses of the interactions between natural killer (NK) cells, NKT cells, different dendritic cell populations and T cells have highlighted how these different cell populations can influence the onset of autoimmunity. There is evidence that infection can have either a potentiating or inhibitory role in the development of type 1 diabetes. Interactions between pathogens and cells of the innate immune system, and how this can influence whether T cell activation or tolerance occurs, have been under close scrutiny in recent years. This Review focuses on the nature of this crosstalk between the innate and the adaptive immune responses and how pathogens influence the process.

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X-linked agammaglobulinaemia A human immunodeficiency that is caused by mutations in the gene encoding Bruton’s tyrosine kinase (BTK) (which is located on the X chromosome).These mutations result in a block in B cell maturation and in poor antibody production. A naturally occurring mouse mutant of BTK, X-linked immune deficiency, is associated with less severe disease.

the role of innate immune cells in promoting or pre-venting diabetes onset and the potential for infection to influence the crosstalk between immune cells and pancreatic β-cells.

Lymphocytes and T1DThere is considerable evidence that T cells have an important role in the development and progression of T1D in both humans and animal models. The recur-rence of T1D in recipients of segmental pancreas grafts from HlA-identical donors showed a clear role for T cells — particularly CD8+ T cells — and monocytes, with little evidence for a humoral immune response, in β-cell destruction11. β-cell survival could only be achieved in these transplantation studies if the recipi-ent was immunosuppressed, indicating that T1D in humans was an autoimmune disease.

Studies in NoD mice have shown that T1D develop-ment depends on both CD4+ and CD8+ T cells: T1D can only be transferred to immunocompromised syngeneic recipients by a combination of splenic CD4+ and CD8+

T cells from donor NoD mice but not by either T cell subset alone12. The ability of a CD3-specific antibody, which induces T cell tolerance, to reverse T1D onset in NoD mice emphasized the key role of T cells in sus-tained β-cell destruction13. These studies led the way to the use of an aglycosyl CD3-specific monoclonal antibody in patients with recent-onset T1D, in which there was evidence that targeting T cells resulted in suppression of ongoing β-cell destruction14.

There are several ways in which T cell-mediated β-cell death might occur (FIG. 1). CD8+ T cells could kill pancreatic β-cells through mHC class I-mediated cytotoxicity, and both CD4+ and CD8+ T cells produce cytokines, such as interferon-γ (IFNγ), that induce expression of the death receptor FAS (also known as CD95) and chemokine production by β-cells. Activation of FAS by FAS ligand (FASl)-expressing activated T cells

could initiate β-cell apoptosis. Chemokine production by β-cells results in further recruitment of mononuclear cells to the site, thereby enhancing inflammation15. In addition, IFNγ can activate macrophages and induce increased pro-inflammatory cytokine production, including interleukin-1β (Il-1β) and tumour necrosis factor (TNF). β-cells express high levels of Il-1 recep-tor and seem to be more sensitive to Il-1β-induced apoptosis than other endocrine cells in the islet. This crosstalk between T cells and macrophages undoubtedly exacerbates the immune-mediated stress on β-cells and contributes to their destruction. IFNγ, Il-1β and TNF also induce the expression of reactive oxygen species (RoS) including nitric oxide by β-cells, and RoS have the potential to mediate apoptosis.

Although T cells have a pathological role in T1D onset, there is also evidence supporting a role for T cells in the prevention of β-cell destruction. Patients with IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome — who have muta-tions in forkhead box P3 (FoXP3; the regulatory T (TReg) cell-associated transcription factor) — can develop T1D16, which highlights the importance of TReg cells in controlling the onset of this autoimmune disease. Studies in NoD mice have shown the impor-tance of TReg cells in preventing T1D: CD28-deficient NoD mice, which lack TReg cells, develop accelerated disease17. In addition, strategies such as injection of Il-2 to increase TReg cell numbers are seen as a potential therapeutic approach18.

T cells are clearly pivotal for T1D development, but there are also data suggesting an involvement of other cell types such as B cells; however, there is little evidence for a role for antibody in the pathogenesis of T1D. B cell depletion in NoD mice, either through gene targeting or antibody treatment, impairs the develop-ment of T1D19,20. This has led to the treatment of newly diagnosed T1D patients with the B cell-depleting monoclonal antibody rituximab (Rituxan/mabthera; Genentech/Roche/Biogen Idec), resulting in improved β-cell function21. However, there is one description of a patient with X-linked agammaglobulinaemia who developed T1D22, which questions the role of B cells in disease pathogenesis. It may be that B cells have a role as antigen-presenting cells that maintain islet antigen-specific T cell activity19,23.

Innate immune cells in T1DAs islet antigen-specific T cells can differentiate into either pathogenic effector T cells (diabetogenic T cells) or protective TReg cells, many studies have investigated the role of innate immune cells in T1D, as these cells usu-ally determine the type of immune response that ensues. Innate cells producing pro-inflammatory or suppres-sive cytokines define the milieu in which islet antigen- specific T cells are activated and whether a deleterious or protective local immune response occurs in the pancreas (FIG. 2). we first discuss studies pertaining to the patho-genic role of innate immune cells, such as macrophages, NK cells and DCs, and then review the growing evidence for the protective effect of innate immune cells.

Box 1 | Animal models of type 1 diabetes

Animal models have contributed enormously to the understanding of processes leading to development of type 1 diabetes (T1D). T1D can be experimentally induced in laboratory animals with chemicals (such as streptozotocin and cyclophosphamide in genetically susceptible mouse strains) or by genetic manipulation (such as transgenic mice). In addition, two spontaneous murine models have been extensively used to understand the pathophysiology of T1D: the biobreeding (BB) rat and the non-obese diabetic (NOD) mouse models. In BB rats there is no sex difference in the incidence of T1D, but female NOD mice develop T1D at a higher incidence than male NOD mice. Besides this difference, both models resemble T1D in humans, including the presence of islet antigen-specific CD4+ and CD8+ T cells and genetic linkage to disease.

As in humans, specific MHC gene products are involved in T1D susceptibility in both models: RT1u/u in BB rats and I–Ag7 in NOD mice. In addition to the MHC locus, other loci contribute to disease development. No less than 12 loci were found to influence the diabetogenic process in BB rats, and more than 20 potential Idd loci have been identified in NOD mice, which include polymorphisms in the genes cytotoxic T lymphocyte antigen 4 (Ctla4), interleukin-2 (Il2) and protein tyrosine phosphatase, non-receptor type 22 (Ptpn22). In the past 20 years, numerous studies have shown that both genetic and environmental factors contribute to the pathogenesis of the disease in both models. In particular, infection studies have underlined the importance of co-evolution between pathogens and the mammalian immune system for both the induction and prevention of T1D.

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β-cell

TCR

β-cell antigen

Unknown ligand

NKp46

NKG2DRAE1

DiabetogenicCD8+ T cell

NK cell

DiabetogenicCD4+ T cell

PDL1PDL1 FAS

FASL

Microbial products

TLR2

TLR9

IFNγ TNFIL-12

IL-1β

TLR4

PD1

PD1

TCR

MHC class I

MHCclass II

Pro-insulinInsulin

WE14 peptide

Chromogranin A

β-cell damage

Lymphocyte and macrophage recruitmentby CXCL10, CCL2 and CCL20

Degranulation

Cytotoxicity

Apoptosis or induction of IDO

Toleranceor

apoptosis

TLR3

Macrophage

DC

Nature Reviews | Immunology

The pathogenic role of macrophages. Although numer-ous studies of both humans and NoD mice emphasize a predominant role for diabetogenic T cells in the patho-genesis of T1D, several studies support the involvement of macrophages in this disease24–28. Early experiments described the presence of macrophages in islet infil-trates of NoD mice, and showed that inhibition of the macrophage influx into the pancreas, by blocking an adhesion-promoting receptor on this cell, inhibited the development of T1D24. In vitro and in vivo studies in mice and rats showed that the deleterious effect of macrophages on β-cells can be mediated through the production of TNF and Il-1β29,30. Interestingly, pro-inflammatory macrophages can be detected in pancre-atic islets before T cell infiltration, as well as in NoD/scid (severe combined immunodeficient) mice, which lack functional B and T cells30.

macrophages have been shown to produce Il-12 (ReF. 26) and to promote efficient differentiation of diabetogenic CD8+ cytotoxic T lymphocytes (CTls) leading to T1D onset25. Genetic differences in Il-12 pro-duction have been observed in peritoneal macrophages from susceptible and resistant mouse strains: cells from NoD mice secrete more Il-12 than those from non-obese resistant (NOR) mice when stimulated with CD40 ligand or lipopolysaccharide (lPS)26. more recent data suggest that recruitment of macrophages to islets is mediated by the secretion of CC-chemokine ligand 1 (CCl1) and CCl2 by CD4+ T cells and pancreatic β-cells, respectively27,31.

macrophages recruited to the pancreas produce Il-1β, TNF and RoS that can cause β-cell death, revealing an additional role for macrophages in the destructive phase of T1D. Finally, TNF- and Il-1β-producing macrophages and DCs have been observed in pancreatic islet infiltrates from patients with recent-onset T1D32. Together, these studies support a pathogenic role for macrophages in both the initiation and destruction phases of T1D.

A role for NK cells. NK cells mediate early protection against viruses and are involved in the killing of infected cells and tumours. NK cells are both cytotoxic and pro-ducers of cytokines, particularly IFNγ. Thus, NK cells could contribute directly and indirectly to the destruction of β-cells. NK cells have been detected in the pancreas of patients with T1D and in many T1D mouse models33–37. moreover, several reports have described a correlation between the frequency and/or activation of NK cells with the destructiveness of the pancreatic infiltrate35,38.

NK cells isolated from the pancreas of NoD mice have a more activated phenotype than those isolated from the spleen and pancreatic lymph node, with higher expres-sion of CD25, CD69, programmed cell death 1 (PD1) and killer cell lectin-like receptor group G, member 1 (KlRG1)37. They also proliferate more and spontane-ously produce higher levels of IFNγ and express CD107a on their cell surface (a marker of granule exocytosis), reflecting their cytotoxic function39. Interestingly, NK cells were observed in the pancreas in NoD mice before T cell infiltration and in the pancreas of NoD–Rag mice (which lack mature B and T cells), suggesting that they could have a sentinel role in the pancreas.

Figure 1 | molecules expressed by pancreatic β-cells involved in their destruction or protection. Toll-like receptors (TLRs) are expressed by pancreatic β-cells to detect danger from microbial products and can trigger type 1 diabetes (T1D); for example, during a localized virus infection in the pancreas. Cytokines, such as interleukin-1β (IL-1β), IL-12, interferon-γ (IFNγ) and tumour necrosis factor (TNF), secreted by immune cells, such as macrophages, dendritic cells (DCs), CD4+ and CD8+ T cells and natural killer (NK) cells, could cause direct damage (such as apoptosis) of β-cells but could also induce self-defence mechanisms; for example, IFNγ induces indoleamine 2,3-dioxygenase (IDO) expression by β-cells. Chemokines such as CXC-chemokine ligand 10 (CXCL10), CC-chemokine ligand 2 (CCL2) and CCL20 secreted by β-cells recruit macrophages and other inflammatory cells to the islet area. Natural killer group 2, member D (NKG2D) expressed by NK cells binds to retinoic acid early transcript 1 (RAE1) expressed by β-cells in non-obese diabetic (NOD) mice, and is associated with β-cell damage. NKp46 engagement by an unknown ligand on β-cells causes degranulation of NK cells. Negative co-stimulatory molecules, such as programmed cell death ligand 1 (PDL1) expressed by β-cells can modulate diabetogenic T cell attack. β-cell antigens (such as peptides derived from insulin or pro-insulin) can be presented by MHC class I molecules and recognized by diabetogenic CD8+ T cells. MHC class II molecules expressed by antigen-presenting cells such as DCs can also present β-cell antigens (peptides such as WE14 from chromogranin A, insulin and pro-insulin) that can induce islet antigen-specific CD4+ T cell expansion. FASL-expressing T cells can mediate apoptosis through interaction with FAS expressed on β-cells. TCR, T cell receptor.

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β-cell antigen

β-cell damage: apoptosisand viral infection

cDC

cDC

ActivatedcDC

Tolerogenic cDCMHC

TCR

TReg cell

IL-12

Macrophage

Macrophage

Pancreas

Draining lymph node

Phase I: β-cell death and APC activation

Phase III: immune cell crosstalk and induction or prevention of β-cell death

Phase II: expansion of pathogenic or regulatory islet antigen-specific T cells

DiabetogenicT cell

Pathogenic islet antigen-specific T cell

Activation

IDO, IL-10,TGFβ and ICOSL

Cell death

Promote recruitment

IL-10 and TGFβ

IDO, IL-10, TGFβ and ICOSL

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IL-12

PDL1–PD1

PDL1–PD1

IFNγ, granzymesand perforin

IFNγ,granzymes,andperforin

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iNKT cellpDC

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pDC

β-cell

β-cell

TNF, IL-1β andnitric oxide

Non-obese resistant (NOR) miceNOR mice have the identical T cell developmental background as NOD mice, but they do not spontaneously develop type 1 diabetes.

BDC2.5 TCR-transgenic NOD miceT cells in these mice express a TCR specific for a pancreatic antigen but, interestingly, these mice have a decreased incidence of type 1 diabetes and develop a non-invasive insulitis. These mice are used as donors of islet antigen-specific CD4+ T cells and manipulation of these mice, such as injection of blocking CTLA4-specific antibody and infection with coxsackie virus B4, induces rapid type 1 diabetes.

A pathogenic role for NK cells has been suggested in various T1D mouse models such as NoD mice, SoCS1 (suppressor of cytokine signalling 1)-transgenic NoD mice infected with coxsackie virus B4, mice trans-genically expressing IFNβ in their β-cells, and CTlA4

(cytotoxic T lymphocyte antigen 4)-specific antibody treated BDC2.5 TCR-transgenic NOD mice34–36 (BOX 1). Depletion of NK cells using various antibodies against NK1.1 and asialo-Gm1 (ReFs 34–36) prevented T1D in all of these models. Natural killer group 2, member D

Figure 2 | Cellular and molecular mechanisms in the development or prevention of type 1 diabetes. The initiation phase of type 1 diabetes (T1D) takes place in the pancreas, where conventional dendritic cells (cDCs) capture and process β-cell antigens. β-cell damage can occur by ‘natural’ apoptosis or after viral infections. Invariant natural killer T (iNKT) cells and plasmacytoid DCs (pDCs) control viral replication, preventing subsequent inflammation and T1D (not shown). Activated cDCs prime pathogenic islet antigen-specific T cells after migration to the draining lymph node, and macrophages promote this activation through interleukin-12 (IL-12) secretion. B cells present β-cell antigen to diabetogenic T cells and secrete autoantibodies (not shown). The activation of islet antigen-specific T cells can be inhibited by cDCs through various mechanisms, such as engagement of programmed cell death ligand 1 (PDL1). iNKT cells can promote the recruitment of tolerogenic cDCs and pDCs that could expand regulatory T (T

Reg) cells

through the production of indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor-β (TGFβ) and inducible T cell co-stimulator ligand (ICOSL). In the pancreas, β-cells can be killed by diabetogenic T cells and NK cells through the release of interferon-γ (IFNγ), granzymes and perforin, as well as by macrophages through the production of tumour necrosis factor (TNF), IL-1β and nitric oxide. IL-12 produced by cDCs sustains the effector functions of activated diabetogenic T cells and NK cells (not shown). β-cell damage can be inhibited by T

Reg cells that inhibit

diabetogenic T cells and innate immune cells through IL-10 and TGFβ. Tolerogenic pDCs stimulated by iNKT cells could also control diabetogenic T cells through IDO production. Lastly, β-cells can inhibit diabetogenic T cells by expressing PDL1. This complex crosstalk between innate and adaptive immune cells results in the development or the prevention of T1D. APC, antigen-presenting cell; TCR, T cell receptor.

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RIP–LCMV transgenic model A transgenic mouse model of type 1 diabetes in which peptides derived from lymphocytic choriomeningitis (LCMV) are expressed in the pancreas under the control of the rat insulin promoter (RIP). Infection of the mice with LCMV leads to the development of type 1 diabetes as a result of infiltrating CD8+ effector T cells.

Perforin A component of cytolytic granules that participates in the permeabilization of plasma membranes, allowing granzymes and other cytotoxic components to enter target cells.

Peripheral tolerance The lack of self responsiveness of mature lymphocytes in the periphery to specific antigens. These mechanisms control potentially self-reactive lymphocytes that have escaped central tolerance. Peripheral tolerance is associated with the suppression of production of self-reactive antibodies by B cells and inhibition of self-reactive effector cells, such as cytotoxic T lymphocytes.

T cell anergyA state of T cell unresponsiveness to antigen-specific stimulation. It can be induced by stimulation with a large amount of specific antigen in the absence of the engagement of co-stimulatory molecules.

(NKG2D) and NKp46 are expressed by NK cells, and recent studies suggest that their ligands (retinoic acid early transcript 1 (RAE1) and NKp46 ligand, respec-tively) are expressed by β-cells from NoD mice40. moreover, NKG2D and NKp46 blockade could pre-vent T1D in NoD mice, suggesting that recognition of β-cells by NK cells may have a role in T1D39. However, although NKp46 expression is restricted to NK cells, NKG2D is also expressed by CTls, and NKG2D block-ade might affect the function of diabetogenic CTls that infiltrate the pancreas40. The deleterious role of NK cells in T1D has been further highlighted in a recent study38: TReg cell depletion of BDC2.5 TCR-transgenic NoD mice resulted in an increase in the percent-age of activated pancreatic NK cells that produced large amounts of effector molecules such as IFNγ, which promote the effector function of diabetogenic CD4+ T cells.

How do DCs promote T1D? Early studies described the presence of antigen-presenting cells (APCs) in transplanted pancreatic islets41 and showed that deple-tion of these cells facilitated graft survival in mice42. These data suggested that APCs could take up and process β-cell-derived proteins and subsequently ini-tiate the diabetogenic response. This hypothesis was tested using mice that express the lymphocytic chorio-meningitis virus (lCmv) glycoprotein under the con-trol of the rat insulin promoter (RIP) (the RIP–LCMV transgenic model)43. Repeated injection of conventional DCs (cDCs) constitutively expressing the immuno-dominant lCmv epitope into these transgenic mice resulted in severe destructive mononuclear infiltra-tion of the pancreatic islets and development of T1D through CTl activation. This development of CTl-mediated T1D was perforin independent, as it was also seen in perforin-deficient RIP–lCmv mice43. Further studies showed that self antigens released after β-cell death could be captured by cDCs in the pancreatic islets and presented to islet antigen-specific T cells in the pancreatic lymph node where the diabetogenic response is initiated6,44.

Initial β-cell death could occur physiologically in NoD mice at two weeks of age during tissue remodel-ling and at weaning owing to a metabolic change, or it could occur through injury mediated by viral infec-tions6,10. Such β-cell death could activate cDCs and initiate priming of islet antigen-specific CD4+ T cells, as revealed by the high frequency of cDCs present-ing β-cell-derived antigens in the pancreatic draining lymph node6. A role for Toll-like receptor 2 (TlR2) in this process has been suggested45, but a recent study showed similar T1D incidence in TlR2-deficient and wild-type NoD mice46. However, it is conceivable that other pattern recognition receptors (PRRs) may have a role in the absence of TlR2. Several reports have sug-gested that cDCs from NoD mice have increased abil-ity to activate T cells through higher Il-12 production and co-stimulatory molecule expression47,48. Together, these studies support a diabetogenic role for cDCs in the initiation of this disease.

Plasmacytoid DCs (pDCs) detect viral RNA or DNA through TlR7 and TlR9 and respond by secreting large amounts of type I IFNs, Il-12 and pro-inflam-matory chemokines. In contrast to cDCs, pDCs have a decreased capacity to take up, process and present solu-ble antigens49. A putative role for pDCs in the devel-opment of T1D is supported by observations in both human and murine models that type I IFNs, which are usually produced by this cell type, can in some situa-tions induce or enhance T1D50,51. Antibody-mediated blockade of type I IFNs identified a crucial role for these cytokines in the initiation of disease. However, these studies did not show that pDCs were the source of this type I IFN. mouse models have been used to examine the pDC populations in the pancreas and pancreatic lymph node at the early stage of T1D development. Two reports suggest a pathogenic role for pDCs in NoD mice. In one study, the initiation of T1D correlated with an increased production of type I IFNs by pDCs in the pancreatic lymph node52. FmS-like tyrosine kinase 3 ligand (FlT3l) treatment, which expands both cDC and pDC populations, accelerated T1D in 15-week-old NoD mice, an age when islet antigen-specific CTls were detectable in blood53.

However, there have been apparently discrepant data regarding the frequency of pDCs in the blood of patients with T1D, which may reflect differences in the time when the blood samples were taken54–56. Interestingly, pDCs from early-diagnosed patients were shown to present immune complexes to T cells more efficiently than cDCs, suggesting a possible detrimental role of pDCs in T1D onset.

Prevention of T1D by DCs. It is known that patients with a congenital DC deficiency develop autoimmune diseases57. This highlights a role for DCs in mediating peripheral tolerance and, indeed, antigen presentation by cDCs mediates tolerance by inducing T cell deletion, T cell anergy or the expansion of antigen-specific TReg cells58. TReg cells are key players in preventing T1D18, and various stud-ies have investigated whether modulation of cDCs could influence TReg cells and T1D development. Treatment of NoD mice with granulocyte colony-stimulating factor (G-CSF) to mobilize DCs prevented T1D development and increased the numbers of cDCs and pDCs in the spleen59. These cells mediated tolerance through effects on CD4+CD25+ TReg cells that suppress pathogenic T cells through the production of transforming growth factor-β (TGFβ)59. FlT3l injection protected NoD mice from T1D by enhancing CD4+CD25+ TReg cell frequency in the pancreatic lymph node and by modulating the balance of cDC subsets towards CD8+ cDCs, which have potentially tolerogenic functions60,61. of note, FlT3l treatment is pro-tective only when administered in the early stages of T1D development in NoD mice when islet antigen-specific T cells are still at a low frequency53. Thus, the opposing role of FlT3l depends on the time of injection relative to the disease progression in NoD mice. A recent study highlighted the in vivo peripheral feedback loop that exists between TReg cells and DC frequency involving FlT3l. Deletion of TReg cells induced FlT3l production, which

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Indoleamine 2,3-dioxygenase(IDO). An intracellular haem-containing enzyme that catalyses the oxidative catabolism of tryptophan. IDO suppresses T cell responses and promotes immune tolerance in mammalian pregnancy, tumour resistance, chronic infection, autoimmunity and allergic inflammation.

Invariant NKT (iNKT) cells Lymphocytes that express a particular variable gene segment, Vα14 (in mice) and Vα24 (in humans), precisely rearranged to a particular Jα (joining) gene segment to yield T cell receptor α-chains that have an invariant sequence. Typically, these cells co-express cell surface markers that are encoded by the NK locus, and they are activated by recognition of CD1d, particularly when α-galactosylceramide is bound in the groove of CD1d.

increased DC frequency and subsequently restored the normal TReg cell frequency required to prevent autoim-mune diseases62. Importantly, G-CSF and FlT3l could prevent T1D not only by increasing the numbers of cDCs but also the numbers of pDCs.

Several molecules expressed by pDCs might be involved in the induction of T cell tolerance, such as PDl1, inducible T cell co-stimulator ligand (ICoSl) and indoleamine 2,3-dioxygenase (IDo). A protective role for pDCs in T1D was first described by Saxena et al.63: by transferring naive BDC2.5 T cells to NoD-Scid mice, they showed that pDCs inhibited the CD4+ T cell diabe-togenic response, induced IDo in the pancreas and pre-vented T1D onset. IDo catalyses oxidative catabolism of tryptophan and, in this way, regulates T cell-mediated immune responses, as free tryptophan is an essential nutrient for T cells. Interestingly, a defect in IDo expres-sion has been described in young NoD mice64, and over-expression of IDo prolongs islet graft survival65. Using both virus-induced and spontaneous models of T1D, we recently observed that following viral infection, invariant NKT (iNKT) cell–pDC crosstalk inhibits diabetogenic CTl responses in the pancreas66. In this infectious context, pDCs induced the conversion of CD4+ T cells to FoXP3+ TReg cells, which are crucial for preventing T1D (j.D. and A.l., unpublished observations). Together, these studies support a protective role of pDCs in T1D and strengthen their potential use in new therapeutic strategies.

Protective role of NK cells in T1D. Despite their poten-tial pathogenic role in T1D, several studies suggest a protective function of NK cells. NK cells from the blood of patients with T1D and lymphoid tissues from NoD mice exhibit impaired function67–69. As this defect was observed in patients with long-term T1D it could be a consequence of the disease69. However, in NoD mice, the NK cell defect is present before disease onset, suggesting that impaired NK cell function could contribute to dis-ease development67,68. Consistent with this hypothesis, injection of young NoD mice with complete Freund’s adjuvant (CFA) induces IFNγ production by NK cells and prevents T1D70. The protective role of NK cells in this setting was shown by their ability to inhibit T1D in transfer experiments. The beneficial role of NK cells in islet allograft tolerance has been shown to involve killing of DCs by NK cells in a perforin-dependent manner71. NK cells seem to have a protective role following CFA immunostimulation of young NoD mice and in islet allografts, whereas they have a deleterious effect in older NoD mice that have massive islet infiltrates or in more aggressive mouse models of T1D.

iNKT cells. iNKT cells are innate-like T cells that express an invariant TCR α-chain (vα14–jα18 in mice and vα24–jα18 in humans). Following activation, iNKT cells promptly produce large amounts of various cytokines and chemokines, thereby providing signals to other immune cells, including DCs, NK cells and lymphocytes. Although iNKT cells can promote immunity to pathogens, many studies have shown their role in preventing auto-immune diseases, particularly T1D (reviewed in ReF. 72).

Increasing the frequency of iNKT cells, either by adop-tive transfer or through the introduction of a vα14–jα18 transgene, substantially reduces the incidence of T1D in NoD mice73,74. A similar protective effect was observed after specific iNKT cell stimulation with the exogenous ligand α-galactosylceramide or its analogues75–78. Early reports suggested that iNKT cell-mediated protection was associated with the induction of TH2 cell responses to islet autoantigens75,76,79. However, studies using the transfer of islet antigen-specific CD4+ and CD8+ T cells showed that iNKT cells inhibit the differentiation of these T cells into effector T cells during their priming in the pancreatic lymph node80,81. Interestingly, these islet antigen-specific CD4+ T cells became anergic and remained in the pancreatic lymph node and pancreas80 where they could have a regulatory role. The abortive priming of islet antigen-specific T cells in the pancreatic lymph node could be explained by their ability to recruit tolerogenic DCs81. Interestingly, type 2 NKT cells, which express variable TCRs, can also prevent T1D in NoD mice, although the precise mechanism is still under investigation82. Although the number and function of iNKT cells from NoD mice are defective, which could contribute to T1D susceptibility67,83, there is no consen-sus on whether iNKT cell defects occur in humans with T1D84,85. However, manipulations of iNKT cells prevent and even cure T1D in various mouse models, encour-aging the development of new therapeutic strategies to target iNKT cells.

The dual role of DCs and NK cells in the development of T1D could depend on different parameters: first, the type of cell subsets involved (for example CD8– cDCs, CD8+ cDCs and/or pDCs); second, different pathways of activation through surface and cytoplasmic recep-tors (such as TlR-mediated recognition of endogenous and exogenous ligands); third, a pro-inflammatory or regulatory cytokine milieu; and fourth, genetic defects such as increased Il-12p40 production by DCs and impaired NK cell function in NoD mice. The integra-tion of all of these parameters could result in prevention or exacerbation of T1D development by DCs and NK cells. Interestingly, based on studies of mouse models, these innate cells often seem to have a protective role in the early phases of the disease, whereas increasing their frequency or activating them at later stages, when diabetogenic T cell frequency have reached a certain threshold, may precipitate disease.

Infection and T1DPotentiating disease. The previous section describes the innate cells involved in the regulation of T1D, and DCs have a pivotal role both in inducing and preventing dis-ease, depending on various factors. As these cells express several pathogen recognition receptors (PRRs) to sense micro organisms, it is not surprising that infections can modulate DCs, and other innate immune cells, and influ-ence islet antigen-specific T cell responses and the devel-opment of T1D (FIG. 3). TlR3 and the cytoplasmic proteins retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (mDA5; also known as IFIH1) have been identified as crucial intracellular

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http://www.uniprot.org/uniprot/Q9BYX4

Viral or self antigen


Macrophage

CytoplasmicPRR

Viral molecule

cDC

Inflammatorymolecules

Cell death

Recruitment

Cytotoxicity

OX40–OX40L

TGFβIL-10

Molecularmimicry

TReg cell induction by tolerogenic DC

Stimulation of antiviral innate immunity andviral clearance

Tolerogenic cDC

TReg cell

Islet antigen-specific T cell

β-cell antigen

β-cell

NK cell

iNKT cell

MHC

Virus

Viral antigenTCR

MHCTCR

pDC

IFNα

NKG2D

RAE1

Molecular mimicry Resemblance between epitopes contained in microbial and host proteins, leading to cross- reactivity of T cells in the host.

Insulitis An infiltration of lymphocytes into pancreatic islets during the progression of type 1 diabetes. Insulitis can be innocuous or destructive.

receptors for host responses to viral dsRNA86. Several studies have suggested a genetic association between mDA5 allelic variants and susceptibility to T1D87. Different viruses can induce or promote T1D through various mechanisms, including molecular mimicry, induc-tion of bystander damage of β-cells, release of sequestered antigens and changes in the balance between the num-bers of TReg cells and effector T cells (TABLe 1). There is some evidence that viruses can directly destroy pancreatic β-cells but in many cases there seems to be a requirement for immune cells in the β-cell destruction process.

Picornavirus encephalomyocarditis virus (EmCv) induces T1D in wild-type mice. Infection with a high dose of the D strain of EmCv results in acute onset of T1D in more than 90% of infected animals within 4 days of infection88. T1D onset in EmCv-infected mice seemed to depend on macrophages but not T cells, as shown by blocking and depleting antibody treatments89. The mechanism of β-cell destruction by macrophages could be mediated through the release of cytokines (TNF) and RoS89,90. Interestingly, fulminant T1D in humans is associated with a macrophage-dominant, rather than T cell-dominant, infiltrate91. These data show the role of innate immune cell activation following virus infection in promoting T1D. In most examples in which viruses induce T1D, infections lead to a cascade of innate and adaptive immune cell activation with a clear role for diabetogenic T cells (as described below).

Activation of autoimmune T cells through molecular mimicry. Cross-reactive immune responses arising through regions of sequence similarity between viral- and self-antigen epitopes could theoretically lead to diabetogenic T cell activation and tissue destruction following viral infection. Numerous mimics between β-cell antigens and viral epitopes have been identified, including coxsackie virus B4 (ReF. 92), rotavirus93, rubella virus94 and Cmv95. The RIP–lCmv mouse model, which is based on transgenic expression of virus anti-gens in β-cells, has been extensively used to investigate mechanisms that could be involved in virus-induced T1D and how self tolerance could be broken96,97. These mice seemed to be tolerant of viral antigen, as no sponta-neous T1D developed. However, following lCmv infec-tion all of the transgenic mice became diabetic within a few weeks owing to the destruction of pancreatic β-cells by virus-specific CTls. These virus-induced T1D mod-els have help to reveal the key role for cytokines and chemo kines, produced by immune cells and even the β-cells themselves, in the development of T1D. Blocking or depletion of IFNγ, TNF or CXC-chemokine ligand 10 (CXCl10) reduced T1D incidence in these mice follow-ing infection, revealing the crucial role for the inflam-matory milieu in the development of virus-induced T1D98–100. These studies show that viral infections could induce T1D possibly by the activation of diabetogenic T cells that are cross-reactive for both viral and self anti-gen, but more importantly they show that viruses can induce T1D by creating an inflammatory environment favourable for diabetogenic T cell activation.

Activation of diabetogenic T cells through bystander damage. The bystander damage resulting from virus infection could involve nonspecific activation of auto-immune cells by a pro-inflammatory milieu and/or T cell activation through exposure to released islet antigens. The association of bystander damage with T1D onset was first revealed after coxsackie virus B4 infection of BDC2.5 TCR-transgenic NoD mice, which induced rapid T1D101. T cells in these mice express a TCR specific for a pancreatic antigen but interestingly, these mice have a lower incidence of T1D and develop

Figure 3 | Effects of viral infection on the regulation of type 1 diabetes. Infection of the pancreas by viruses triggers the activation and recruitment of innate immune cells such as macrophages, dendritic cells (DCs) and natural killer (NK) cells, which produce pro-inflammatory cytokines through cytoplasmic pattern recognition receptor (PRR) activation. In parallel, viruses can enhance the release of β-cell antigens through direct or indirect β-cell damage. Activated antigen-presenting cells can present either self antigen or viral antigens (in the case of molecular mimicry) to islet antigen-specific T cells. The pro-inflammatory milieu created by the innate immune cells promotes the recruitment and activation of islet antigen-specific T cells in the pancreas. Viral infection can be controlled by invariant NKT (iNKT) cell–plasmacytoid DC (pDC) crosstalk leading to a rapid elimination of the virus from the pancreas. In a second step, activated tolerogenic conventional DCs (cDCs) and pDCs loaded with viral and/or self antigens can induce regulatory T (T

Reg) cells, which mediate bystander suppression, through

transforming growth factor-β (TGFβ) and interleukin-10 (IL-10). The complex interplay between viruses and immune cells reflects the dual role, beneficial or detrimental, of infections in the development of type 1 diabetes. IFN, interferon; NKG2D, natural killer group 2, member D; RAE1, retinoic acid early transcript 1; TCR, T cell receptor.

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Table 1 | Infections associated with the regulation of type 1 diabetes

Pathogen Effect on type 1 diabetes

mechanisms of action Refs

Viral infections

Coxsackie B3 and B4 viruses Acceleration Bystander damage, release of sequestered antigens and molecular mimicry 92,101, 104–107,

110,111,132Prevention Upregulation of PDL1 expression and TReg

cell-mediated control of diabetogenic CTL expansion

Cytomegalovirus Acceleration Molecular mimicry and congenital infections 95,133

Encephalomyocarditis virus Acceleration Direct destruction 88–90

Mumps virus Acceleration Unknown 9

Parvovirus Acceleration Change in TReg

cell and effector T cell balance 133,134

Rhesus monkey rotavirus Acceleration Acceleration by induction of pro-inflammatory cytokines 108,109

Prevention Prevention mechanism unknown

Rubella virus Acceleration Molecular mimicry and congenital infections 94

Lymphocytic choriomeningitis virus Prevention Upregulation of PDL1 expression and TReg

cell-mediated control of diabetogenic CTL expansion

111,135

Lactate dehydrogenase virus Prevention Unknown (possible role for antigen-presenting macrophages) 136

Mouse hepatitis virus Prevention Unknown (only observational) 137

Murine gammaherpes virus-68 Delay Alteration of self-antigen presentation by DCs 138

Viral antigens

CpG DNA Prevention Decrease in T cell proliferative responses and possible shift to TH2 cell responses 116

CpG DNA No effect Not applicable 115

PolyI:C Prevention Expression of type I IFNs and induction of suppressor T cells 114

Bacterial infections

Salmonella enterica subsp. enterica serovar Typhimurium

Prevention TH1 cell- and IFNγ-mediated inhibition of type 1 diabetes and phenotypic

changes in DCs and IDO production123,124*

Mycobacterium avium Prevention Induction of suppressor T cells 139

Bacterial antigens

Mycobacterium bovis bacille Calmette–Guérin

Prevention Unknown (only observational) 120,121

Complete Freund’s adjuvant (Mycobacteria)

Prevention Expansion of natural suppressor cells and NK cells 70,122

Streptococcal OK-432 Prevention Unknown (only observational) 140

Klebsiella pneumoniae glycoprotein extract and Escherichia coli LPS

Prevention Expansion of natural suppressor cells 126

OM-85 (complex mix of bacterial extracts)

Prevention Induction of TGFβ, TReg

cells and NKT cells 141

Fungal antigens

Zymosan Prevention Activation of innate cells, TReg

cells 127

Helminth infections

Schistosoma mansoni Prevention Shift to TH2-type response 130

Trichinella spiralis Prevention Shift to TH2-type response 142

Heligmosomoides polygyrus Prevention Shift to TH2-type response 142

Litomosoides sigmodontis Prevention Shift to TH2-type response and T

Reg cell increase 143

Helminth antigens

L. sigmodontis antigen Prevention Shift to TH2-type response and T

Reg cell increase 143

Dirofilaria immitis IgE-inducing antigen Prevention Shift to TH2-type response 144

S. mansoni soluble worm antigen or soluble egg antigen

Prevention Expansion of alternatively activated macrophages, tolerogenic DCs and iNKT cells, T

H2 cells and T

Reg cells

128,129, 131

S. mansoni eggs Prevention Induction of TH2 cells 130

APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; IDO, indoleamine 2,3-dioxygenase; LPS, lipopolysaccharide; NK, natural killer; PDL1, programmed cell death ligand 1; polyI:C, polyinosinic–polycytidylic acid; TGFβ, transforming-growth factor-β; T

H, T helper; T

Reg, regulatory T.

*Also observed by S. Newland and A.C., unpublished observations.

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non-invasive insulitis102,103. Coxsackie virus B4 infec-tion of NoD mice also led to the acceleration of T1D development but only in mice at least 8 weeks of age with a pancreatic infiltrate of islet antigen-specific T cells; younger mice were resistant to the disease104. In the NoD mouse model, an accumulation of a crucial number of islet antigen-specific T cells was required for the induction of T1D by coxsackie virus B4 and other coxsackie virus B strains105. Treatment with the TlR3 ligand polyinosinic–polycytidylic acid (polyI:C), a syn-thetic double-stranded RNA that induces the secretion of large amounts of IFNα by DCs and macrophages, is not sufficient to induce T1D in BDC2.5 TCR-transgenic NoD mice106. These results suggest that nonspecific acti-vation of T cells mediated by virus-induced inflammatory cytokines is not sufficient to induce disease.

Coxsackie virus B4-specific innate immune responses result in pancreatic tissue damage and the capture of released self antigens by APCs. Coxsackie virus B4 did not induce direct β-cells death after infec-tion; it triggered the upregulation of cell surface mark-ers on β-cells associated with cellular stress, particularly death receptor 4, and subsequent engulfment of infected islets by macrophages107. These macrophages induced T1D following transfer into uninfected BDC2.5 TCR-transgenic NoD mice, showing their involvement in the induction of T1D following viral infection. Therefore, viral infection could have a role in T1D development by potentiating ongoing autoimmune disease through the induction of inflammatory cytokines and the stimula-tion of self-antigen-presenting APCs.

Inhibiting disease. Although some viruses have been shown to promote T1D pathogenesis, infections have also been shown to have a protective role in T1D (TABLe 1). This dual role of viruses in T1D depends on the different effects that the virus might have on β-cells or in the modulation of molecules and cells involved in controlling T1D.

An example of the dual role of a virus in T1D is the rhesus monkey rotavirus (RRv), which provides protec-tion when inoculated into young NoD mice but exac-erbates disease if administered when insulitis is already established108,109. Similar effects on T1D development are observed with coxsackie virus infection of NoD mice. In this case, the virus precipitates disease after a crucial level of islet antigen-specific immune cells has accumulated in the pancreas, but can also prevent T1D in pre-diabetic NoD mice110. Coxsackie viruses can control the expan-sion of islet antigen-specific CTls by upregulating PDl1 expression on lymphocytes. As the interaction between PD1 and PDl1 is known to control not only T cell immunity to viral infection but also autoimmune T cell responses, the hypothesis that viral infection can potentiate regulatory mechanisms is consistent with the protective effects observed on T1D development. Increased numbers of CD4+CD25+ TReg cells are also observed in coxsackie virus infection111. TReg cells from infected NoD mice express high levels of TGFβ, CTlA4 and glucocorticoid-induced TNF-receptor-related protein (GITR), and have increased ability to regulate the onset of T1D during the pre-diabetic phase, when

compared with naive TReg cells from uninfected mice. Positive correlations have been observed between lCmv infection and PDl1 upregulation, enhanced TReg cell number and tolerogenic function following infection111. It would be interesting to determine whether viral infections upregulate PDl1 expression on β-cells, which is already expressed in uninfected NoD mice and participates in the control of diabetogenic T cell attack112.

The use of RIP–lCmv model has enabled the identification of a new immune cell crosstalk that con-trols pancreatic viral load and thereby T1D onset66,113. Following infection with lCmv, iNKT cells promote pDCs recruitment into the pancreas and their produc-tion of type I IFNs. This local iNKT–pDC crosstalk is dependent on the oX40–oX40l pathway. iNKT cells in the pancreas, but not in lymphoid tissues, express oX40, and experiments with blocking antibodies, as well as the transfer of wild-type iNKT cells to oX40-deficient mice, have shown the crucial role of oX40–oX40l in the iNKT cell–pDC interaction. As a result, in the pan-creas (but not in the spleen), iNKT cells enhance IFNα production by pDCs and inhibit local viral replication. The low viral burden in the pancreas results in a weak local adaptive immune response, thereby preventing tis-sue damage and T1D development. These results reveal that the diabetogenic effect of lCmv can be controlled by efficient innate immune cell crosstalk, limiting the activation of diabetogenic T cells.

viral antigens in the absence of infection also prevent diabetes in NoD mice. For example, polyI:C can pro-tect NoD mice from developing T1D114 and, although controversial115, vaccination using CpG-containing oligodeoxynucleotides (which are TlR9 ligands) also inhibited T1D development in NoD mice116. Recent data from the streptozotocin model of T1D suggest that TlR9 agonists might inhibit diabetes onset through the induction of IDo expression by DCs117. TlR3 and TlR9 are expressed not only by cells of the immune system but also by β-cells118, raising the possibility that these TlR ligands function at several levels.

There are many studies showing reduced insulitis and inhibition of T1D development following expo-sure of NoD mice to mycobacterial species or anti-gens119–122 and Salmonella enterica subsp. enterica serovar Typhimurium123. Given that T1D is a TH1 cell-mediated disease, this is intriguing as both infections are known to activate macrophages and DCs that induce a typical TH1 cell-mediated immune response. S. Typhimurium infection prevents T1D in NoD mice with an already established infiltrate in the pancreas123. The transfer of DC-enriched populations from S. Typhimurium-infected mice to NoD mice injected with the chemotherapeutic drug cyclophosphamide to accelerate T1D prevented dis-ease development124. In this model, the immunomodula-tory effect was due to the ability of the transferred DCs to prevent the trafficking of islet antigen-specific T cells into the pancreas. The paradoxical protective effect of TH1 cell-inducing infections (and their antigens (TABLe 1)) on a TH1 cell-mediated autoimmune disease indicates that a classic pro-inflammatory immune response is not suf-ficient to exacerbate disease per se. Intracellular infections

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such as S. Typhimurium can induce the expression of IFNγ, resulting in induction of anti-inflammatory mol-ecules such as IDo, not just in lymphoid tissues but also in the pancreas (S. Newland and A.C., unpublished observations). IDo has been shown to be produced by human pancreatic β-cells in response to IFNγ and its role has been proposed as a self-defence mechanism against inflammation125. Functional evidence of a tolerogenic role of islet IDo in vivo has been shown by prolonged survival of IDo-expressing islets following transplan-tation65. Furthermore, an inverse correlation has been observed between IDo and apoptosis, suggesting that IDo may have an additional protective role by reducing oxidation and mHC class I expression.

PRR ligands of either bacterial126 or fungal origin127 can also prevent diabetes in NoD mice. The fungal cell wall component zymosan is recognized by innate cells through an interaction with TlR2 and the C-type lectin receptor dectin 1 (also known as ClEC7A). Zymosan induces phenotypic changes in DCs and macrophages, which results in the secretion of both pro-inflammatory cytokines (Il-6 and TNF) and anti-inflammatory cytokines (Il-10 and TGFβ)127. Zymosan also upregulated the expression of Il-10 and TGFβ by CD4+ T cells127. These changes seem to be involved in the suppression of insulitis.

The expression of TlR2 and TlR4 by β-cells adds another site at which these agents can potentially func-tion118. Interestingly, it has been shown that gut microbi-ota may have a crucial role in T1D development46. Studies in myeloid differentiation primary-response protein 88 (myD88)-deficient NoD mice kept in germ-free con-ditions have revealed normal disease development in these mice, whereas a decreased incidence was observed following gut colonization with commensal microorgan-isms. This suggests that some organisms can prevent T1D development in a myD88-independent manner but does not exclude the involvement of other PRRs.

Perhaps less surprising, but also complex, is the effect of parasitic infections on T1D. Parasitic worms (and their products) can rapidly skew the immune response of the host towards an anti-inflammatory response, in order to secure their own survival. The common immune response observed during helminth infection (or immunization with parasite products) is character-ized by the induction of an immature phenotype in DCs and the alternative activation of macrophages128, as well as TH2 and TReg cell129 development. Schistosoma mansoni infection or injection of S. mansoni antigens dramatically reduces T1D incidence in NoD mice130,131. After exposure to S. mansoni soluble egg antigen (SEA), for example, the composition of the cellular infiltrate in the pancreas of NoD mice is markedly changed. In particular, there is increased expression of FoXP3, Il-4, Il-10 and TGFβ by CD4+ T cells128,129. The number of iNKT cells is also increased in NoD mice exposed to SEA131. Thus in the context of a live infection with S. mansoni, several different cell types of both the innate and the adaptive immune responses are affected with the potential to influence each other and reinforce their ability to inhibit autoimmune pathology.

Pathogens such as parasites and bacteria are usu-ally protective against T1D, whereas viruses have a dual role. The ability of viruses to induce or promote T1D can depend on a susceptible genetic background, the tro-pism of the virus for islet cells and its ability to induce a strong pro-inflammatory response10. Conversely, virus infection in the presence of a limited number or absence of diabetogenic T cells is usually beneficial against T1D. The local pancreatic viral load is a crucial parameter and, therefore, the efficiency of immune cell crosstalk con-trolling viral replication, subsequent β-cell damage and pro-inflammatory cytokine secretion will determine the outcome of the infection on T1D.

Concluding remarks and future directionsStudies of the pathogenesis of T1D have largely focused on the analysis of diabetogenic T cells and their control by TReg cells. However, there is increasing evidence that innate immune cells have crucial roles in T1D patho-genesis. Innate cells such as macrophages, DCs and NK cells are required for the development of T1D in vari-ous mouse models and these cells have been detected in the pancreas of patients with T1D. Innate cells such as DCs, NK and iNKT cells have also been shown to be involved in protection against this disease. This Review highlights the potential for the ambivalent function of innate immune cells in T1D. many observations sup-port a protective role for these cells following their activation, by specific agonist or following microbial infection, in younger mice harbouring limited β-cell destruction and diabetogenic T cell frequency. one of the key questions that remain to be addressed is why these innate immune cells are inefficient in preventing T1D in the absence of exogenous triggering but instead participate in the development of the disease. one hypothesis is that T1D could be associated with some immune deficiency of innate immune cells render-ing them unable to induce tolerance to islet antigens, whereas chronic low activation of these cells through continued β-cell death and/or persistent virus activates their pathogenic functions.

The knowledge gained from the analysis of the pre-ventative mechanisms induced by some infections sug-gests two main pathways for the development of new therapeutic approaches for T1D. The first could be based on parasite or bacterial components that are pro-tective in mouse models of T1D or in other immune-mediated pathologies in humans such as inflammatory bowel disease and asthma. The second could focus on specific triggering of innate cells, such as pDCs and NKT cells, that preferentially exert a protective role against T1D. However, as pDCs can also have delete-rious effects, similar to cDCs, their triggering by PRR ligands should be restricted to individuals at risk but still devoid of any sign of islet-specific autoimmunity, as indicated by the presence of specific autoantibodies and islet antigen-specific T cells. Concerning the exog-enous activation of iNKT cells, many investigations are currently underway to generate and screen new agonists to obtain molecules that preferentially direct NKT cells towards regulatory pathways.

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AcknowledgementsWe apologize to all the authors whose work we could not cite owing to space constrictions. The Cooke laboratory is sup-ported by The Wellcome Trust, Medical Research Council (MRC) and Diabetes UK. P.Z. is supported by the MRC. The Lehuen laboratory is supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Agence Nationale de la Recherche (ANR-GENOPAT) and the European Foundation for the Study of Diabetes (EFSD).

Competing interests statementThe authors declare no competing financial interests.

DATABASESUniProtKB: http://www.uniprot.orgdectin 1 | FLT3L | ICOSL | IDO | IFNγ | IL-1β | KLRG1 | MDA5 | NKG2D | PD1 | PDL1 | RIG-I | TLR2 | TLR3 | TLR7 | TLR9 | TNF

FURTHER INFORMATIONAnne Cooke’s homepage: http://www.path.cam.ac.uk/research/investigators/cooke/

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