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TRPV1
+
Sensory Neurons Control
b
Cell Stress and Islet Inflammationin Autoimmune Diabetes
Rozita Razavi,
1,2,4,7
 Yin Chan,
1,2,4,7
F. Nikoo Afifiyan,
1,2,4,7
 Xue Jun Liu,
1,3,7
 Xiang Wan,
1,3
Jason Yantha,
1,2,4
HubertTsui,
1,2,4
LanTang,
1,2,4
SueTsai,
5
PereSantamaria,
5
JohnP.Driver,
5
DavidSerreze,
6
MichaelW.Salter,
1,3
and H.-Michael Dosch
1,2,4,
*
1
Neurosciences and Mental Health Program
2
Department of Pediatrics
3
Department of Physiology
4
Department of ImmunologyThe Hospital for Sick Children, Research Institute, University of Toronto, Toronto, ON, Canada, M5G 1X8
5
Julia McFarlane Diabetes Research Centre and Department of Microbiology and Infectious Diseases, Faculty of Medicine,University of Calgary, Calgary, AB, Canada T2N 4N1
6
The Jackson Laboratory, Bar Harbor, ME 04609, USA 
7
These authors contributed equally to the work.*Contact:hmdosch@sickkids.caDOI 10.1016/j.cell.2006.10.038
SUMMARY 
In type 1 diabetes, T cell-mediated death of pancreatic
b
cells produces insulin deficiency.However, what attracts or restricts broadlyautoreactive lymphocyte pools to the pancreasremainsunclear.WereportthatTRPV1
+
pancre-atic sensory neurons control islet inflammationand insulin resistance. Eliminating these neu-rons in diabetes-prone NOD mice preventsinsulitis and diabetes, despite systemic persis-tence of pathogenic T cell pools. Insulin resis-tance and
b
cell stress of prediabetic NODmice are prevented when TRPV1
+
neurons areeliminated. TRPV1
NOD
, localized to the
Idd4.1
diabetes-risk locus, is a hypofunctional mutant,mediating depressed neurogenic inflammation.Delivering the neuropeptide substance P byintra-arterial injection into the NOD pancreasreverses abnormal insulin resistance, insulitis,and diabetes for weeks. Concordantly, insulinsensitivityisenhancedin
trpv1
À
 / 
À
mice,whereasinsulitis/diabetes-resistant NODxB6
Idd4
-con-genic mice, carrying wild-type TRPV1, show re-stored TRPV1 function and insulin sensitivity.Ourdatauncoverafundamentalroleforinsulin-responsive TRPV1
+
sensory neurons in
b
cellfunction and diabetes pathoetiology.
INTRODUCTION
Type1diabetes(T1D)isanautoimmunediseasegovernedby multiple genetic and environmental risk factors. Overtdiabetes reflects glucose intolerance due to insulin defi-ciency.Itistheendresultofprediabetes,withprogressivelymphoid infiltration around and then inside pancreaticislets of Langerhans and subsequent destruction of insu-lin-producing
b
-cells by autoreactive T lymphocytes(  Anderson and Bluestone, 2005 ). T1D is characterized byapermissiveimmunesystemthatfailstoimposetolerancetoarraysofself-antigens.Althoughtheinitiatingeventsarenotfullyunderstood,
b
cellstressanddeathinthecourseof early islet restructuring are thought to provide sensitizingautoantigens, which expand autoreactive T cell pools inpancreatic lymph nodes ( Mathis et al., 2001; Rosmalenet al., 2002; Trudeau et al., 2000; Zhang et al., 2002 ).Self-antigens targeted in T1D are expressed in
b
cellsand, in most cases, elsewhere in the body. They promi-nently include neuronal antigens, recognized by T cellswith pathogenic potential ( Salomon et al., 2001; Wineret al., 2001 ). It is unclear why, in T1D, T cells infiltrateonly islets and their associated glia ( Winer et al., 2003 ). Itis also unclear whether autoimmunity and islet inflamma-tion are related to hyperinsulinism and insulin resistancetypical for even young NOD mice (  Amrani et al., 1998;Chaparro et al., 2006 ).There is evidence for functional interactions betweennervous and immune systems (e.g.,Wang et al. [2003] ),but connections between islet autoimmunity and thenervous system remain ill defined ( Carrillo et al., 2005 ).The interface between nervous system and external andtissue environments is the primary sensory afferent neu-ron. Primary afferents also have efferent function throughlocal release of mediators such as neuropeptides (e.g.,substance P [sP], CGRP). Islets may be innervated byprimary sensory neurons, but their local function is uncer-tain(  Ahren,2000 ).Aprominentsubsetof sensoryneuronsexpresses the transient receptor potential vanilloid-1(TRPV1) protein, a nonspecific cation channel that was
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127 
, 1123–1135, December 15, 2006
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2006 Elsevier Inc.
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first identified as the receptor for capsaicin ( Caterinaet al., 2000; Prescott and Julius, 2003 ). TRPV1
+
neuronsare of known importance in proinflammatory reactions( O’Connor et al., 2004 ), islet infiltrating lymphocytesexpress receptors for neuropeptides ( Persson-Sjogrenet al., 2005 ), and we asked if these sensory neurons mayhave a role in T1D.
RESULTS
TRPV1
+
Sensory Afferents Control Onset of IsletInflammation and Diabetes
Using immunofluorescence, we determined that murineislets are associated with meshworks of TRPV1
+
fibers( Figure 1 A). TRPV1 was undetectable in endocrine isletcells by immunofluorescence ( Figures 1 A and 1B) andbyRT-PCRofpurifiedNODislets( FigureS1intheSupple- mental Dataavailable with this article online). Based onthis evidence of islet innervation by TRPV1
+
primary affer-ent sensory neurons,we investigated their possible role inT1D pathogenesis, using neonatal treatment of diabetes-prone NOD mice with capsaicin to permanently removethese neurons ( Caterina and Julius, 2001; Jancso et al.,1977 ). Two-day-old NOD mice received capsaicin (50mg/kg, s.c.) or vehicle (NOD
caps
, NOD
ctrl
 ). As expectedfrom the voluminous literature, capsaicin-treated micewere viable, fertile, and without abnormalities in growthor gross tissue structure, including pancreas. We con-firmed the lack of TRPV1 expression in NOD
caps
mice,using immunofluorescence ( Figures 1 A and 1B, TRPV1green), western blots (8, 12, and 20 week), and standardhot-plate testing in randomly selected 5- to 6-week-oldmice or in diabetic animals ( Figure 1C). Consistent withloss of neuropeptide secreting TRPV1
+
neurons, NOD
caps
mice showed no sP staining ( Figure S2 ).Islet infiltration by hematopoietic inflammatory cells be-gins by 4–5 weeks of age, accumulating at the peri-isletSchwann cell (pSC) border; the autoimmune destructionof the pSC mantle is extensive by 8 weeks of age, 8–12weeks before the onset of overt diabetes ( Winer et al.,2003 ). InNOD
caps
mice, isletinfiltrationswere significantlyreduced, compared with NOD
ctrl
( Figure 1D, p < 0.0001).More than 70% of NOD
caps
islets were free of lympho-cytesbytheageof20weeks,whereaseveninnondiabeticNOD
ctrl
mice of that age, very few islets were lymphocytefree ( Figure 1D). In one-third of NOD
caps
mice, infiltratedislets wereentirelyabsent,asdemonstrated byextensive,serial sectioning. In the remaining two-thirds of NOD
caps
mice, most islets remained free of lymphocytes, withsome degree of inflammation in rare islets, but strikingly,there was little of the typical insulitis progression overtime ( Figure 1E). Concordantly, capsaicin treatment de-layed the onset of diabetes (p = 0.0002) and reduced itsincidence ( Figure 1F, p < 0.0001, 35 weeks, n = 52 mice/ group, life table analysis). No further NOD
caps
mice devel-oped disease over the next 12 weeks, with about 80%reduction in final diabetes incidence (p < 0.0001, Fisher’sexact test).NOD mice spontaneously develop a Sjo ¨ gren-like siali-tis/lacrimitis, which is under genetic controls separatefrom diabetes ( Boulard et al., 2002; Cha et al., 2002 ).NOD
caps
miceexhibitedthesamesubmandibularlympho-cyte infiltrates as NOD
ctrl
( Figure S3 ). Capsaicin treatmentthus causes a dramatic reduction in pancreatic isletinflammation and development of diabetes without ageneralized effect on autoimmune infiltrations elsewherein the NOD mouse.Capsaicin treatment effects on islet infiltration anddisease development could reflect a failure to generateislet autoreactive T cell pools, a block of their tissueaccumulation, or changes in immunoregulatory mecha-nisms. Capsaicin was reported to affect some immunefunctions in other animal models ( Chancellor-Freelandetal.,1995;Helmeetal.,1987;Nilssonetal.,1991;Santonietal.,1996 ),andwecompareddevelopmentandfunctionsofsystemic( Figures2 A–2E)andpancreaticTcells( Figures 2F–2I) in NOD
caps
and NOD
ctrl
mice. Systemic T cell poolsautoreactive to islet- (Insulin, GAD65), pSC- (GFAP,S100
b
 ), and other disease-associated antigens (HSP60,BSA)( Wineretal.,2003 )wereindistinguishableinNOD
caps
andNOD
ctrl
spleencells( Figure2 A).Toprobethedevelop-ment of diabetogenic T cell pools in NOD
caps
mice, wemeasured the peripheral frequency of pathogenic CD8
+
T cells that recognize residues 206–214 of the glucose-6-phosphatase catalytic subunit related protein (IGRP)and its homologous, high-avidity mimotope, NRP-V7(  Amrani et al., 2000, 2001; Anderson et al., 1999; Lieber-man et al., 2003; Verdaguer et al., 1996, 1997 ). The sizeof the circulating NRP-V7-reactive CD8
+
T cell pool wassimilar in NOD
caps
and NOD
ctrl
spleens (0.25%
±
0.1%,p = 0.69) ( Figure 2D). Lymphoid organ cellularities and Tcellsubsetdistributions werealsonotdifferentinNOD
caps
andNOD
ctrl
,comparingsplenocytes,axillarylymphnodes,and thymus ( Figure S4 ). Delayed-type hypersensitivityreactions developed normally in NOD
caps
mice ( Fig-ure S5 ), suggesting intact antigen presentation andeffector cell generation ( Cua et al., 1995; Morikawa et al.,1993 ).Incontrast,pancreaticNOD
caps
lymphnodetissuecon-tained significantly reduced proportions and absolutenumbers of CD8
+
and of activated CD8
+
CD69
+
effector Tlymphocytes critical for islet destruction ( DiLorenzoetal.,1998 )( Figures2Fand2G).Asahallmarkofprediabe- tes progression, prediabetic NOD mice selectively loseCD4
+
CD25
+
and Foxp3
+
regulatory T cell subsets inpancreatic lymph node tissue ( Bluestone and Tang,2005; Pop et al., 2005 ). However, NOD
caps
mice main-tained their regulatory T cell compartment in pancreaticlymph nodes beyond 12–16 weeks of age ( Figures 2Hand 2I). Thus, there are significant differences in the pan-creatic, local immune system of NOD
caps
and NOD
ctrl
mice, consistent with the absence of chronic progressiveislet inflammation in these animals.Conceivably, undetected abnormalities in the NOD
caps
immune system might have influenced T cell pathogenic-ity. NOD
caps
animals that did develop disease had severe
1124
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127 
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ª
2006 Elsevier Inc.
 
insulitis, and spleen cells from these animals transferredT1D with normal kinetics to lymphocyte-free NOD.
 scid 
recipients not treated with capsaicin ( Figure S6 ). We alsocompared the ability of splenocytes from randomlyselected NOD
caps
and NOD
ctrl
to initiate insulitis in suchNOD.
 scid 
mice.NOD
caps
andNOD
ctrl
splenocytesinitiatedinsulitis equally ( Figure 2C). Moreover, we analyzedBDC2.5 T cell receptor transgenic NOD mice treated withcapsaicin(BDC2.5
caps
 )( Jietal.,1999 ).BDC2.5
caps
spleno-cytes transferred T1D to NOD
ctrl
mice (p > 0.1). Low-dosecyclophosphamide accelerates NOD diabetes by multiplemechanisms ( Hadaya et al., 2005 ). Low-dose cyclophos-phamide accelerated diabetes development in bothNOD
caps
and NOD
ctrl
(p = ns,Figure 2E) and was associ-ated with reversal of the regulatory T cell accumulationpresent previously in NOD
caps
versus NOD
ctrl
pancreaticlymph nodes ( Figure S7 ). Thus, NOD
caps
mice retain theprincipal ability to generate diabetogenic T cell pools.Collectively, our observations separate loss of self-tol-erance from target tissue invasion as distinct elementsof T1D pathogenesis, and they demonstrate that theNOD
caps
immune system retains pathogenic potential.TRPV1
+
sensory neurons thus appear critical for theimmune-cell accumulation in the pancreas.
NOD
trpv1
Is Polymorphic
The above findings identify an important role of TRPV1
+
primary afferent neurons in the initiation and progressionof islet inflammation. TRPV1 maps to the
Idd4.1
NODdiabetes-risk sublocus (mouse chromosome 11) into an
$
0.3 cM interval downstream of D11Ndsl ( Figure 3 A)( Grattan et al., 2002; Ivakine et al., 2005; McAleer et al.,1995 ). Congenic replacement of the NOD
Idd4
locuswith the homologous B6 genomic interval protects frominsulitis and, consequently, diabetes, although spleno-cytes from these congenic animals transfer both insulitisand diabetes to NOD.
 scid 
mice ( Grattan et al., 2002 ).The NOD
Idd4
risk locus differs from the homologousgenomic region in the insulitis- and diabetes-resistantNORstrain,whichcarriesnearly90%oftheNODgenome,including most other T1D risk loci ( Ivakine et al., 2005;Serreze et al., 1994 ).WeclonedandsequencedTRPV1cDNAfromNODandNOR mouse dorsal root ganglia (DRG) and confirmedselected sequence regions in NOD and NOR genomicDNA. The NOR
trpv1
was identical to the publishedwild-type (B6, DBA) sequence, but the NOD sequencehas two in-frame base exchanges, leading to predictedP
322
/
 A 
322
and D
734
/
E
734
amino acid replacements
Figure 1. Removal of TRPV1
+
NeuronsReduces Islet Infiltration and DiabetesProgression
Immunohistochemistry of TRPV1
+
neuronsin pancreas of 8-week-old NOD
ctrl
(A) andNOD
caps
(B). Insulin, blue; GFAP, red; andTRPV1, green; insert, dual-color stain forTRPV1 and GFAP in an adjacent, serial section(A). Western blot analysis of TRPV1 expressionin spinal cordproteinextracts from NOD
ctrl
andNOD
caps
mice at 12 (first lane) and 20 weeks of age (second lane) (C). Insulitis was scored in atleast 300 islets from NOD
ctrl
and from NOD
caps
mice, 20 weeks of age (n = 5/group) (D). Kinet-ics of insulitis (E) and diabetes development inNOD
ctrl
and NOD
caps
mice (F).
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