Autoimmunità e Vitamina D: quali evidenze?

Laura Andreoli U.O. Reumatologia e Immunologia Clinica

Spedali Civili di Brescia

Dipartimento di Scienze Cliniche e Sperimentali

Università degli Studi di Brescia

Vitamin D: the need for sunlight

Cholecalciferol

Calcidiol

Calcitriol Image modified from Holick, NEJM 2007

80%

20%

Serum levels of Vitamin D:

>30 ng/ml: sufficiency 10-30 ng/ml: insufficiency <10 ng/ml: deficiency

DOSED IN CLINICAL PRACTICE

BIOLOGICALLY ACTIVE FORM

Macrophages, dendritic cells, T cells & B cells express:

- Vit D receptor (VDR)

- key vitamin metabolizing enzymes:

• CYP27A1: 25-hydroxylase

• CYP27B1: 1--hydroxylase

1,25 (OH)2D3 binds to the

VDR in the cytoplasm. The

1,25 (OH)2D3 -VDR complex

enters the nucleus where it

binds to VITAMIN D

RESPONSIVE ELEMENTS

(VDRE) located on DNA

Vitamin D: from the Skeleton……

…….to the Immune System

Mora JR, et al. Nat Rev Immunol 2008; 8: 685-98

VDR POLYMORPHISMS

Vitamin D immunomodulatory properties

Image modified from Shoenfeld, Ann Rheum Dis 2007

Th1 and Th17 Response

Th2 Response

T regulatory

Differentiation

Proliferation

Antibody production

B cells

Tolerogenic properties of DCs

IFNalfa signature

Aranow C. J Invest Med 2011; 59:881-6. Ben-Zvi I et al, PLoS One 2010; 5:e9193

Ritterhouse LL et al, Ann Rheum Dis 2011; 70:1569-74.

Mathieu C et al, J Steroid Biochem Mol Biol 2004; 89-90:449-52.

Adorini L, Cellular Immunology 2005

Adorini & Penna, Nat Clin Pract Rheumatol 2008

Animal models of autoimmune diseases

Beneficial effects of VDR agonists treatment

EXPERIMENTAL

MODELS

MAIN EFFECT REFERENCES

ARTHRITIS Decreased incidence and severity of collagen-

induced or Lyme arthritis, also when given at

disease onset

Larsson 1998

Cantorna 1998

AUTOIMMUNE

DIABETES

Inhibition of insulitis and reduction of diabetes, even

when given after islet infiltration

Mathieu 1994&1995,

Casteels 1998,

Inaba 1992

EXPERIMENTAL

ALLERGIC

ENCEPHALOMYELITIS

Prevention and treatment of disease, inhibition of

relapses

Mattner 2000,

Lemire 1991,

Branisteanu 1995,

Nashold 2001

INFLAMMATORY

BOWEL DISEASE

Significant amelioration of symptoms, block of

disease progression

Cantorna 2000

PSORIASIS Inhibition of leukocyte activation and amelioration of

histological and clinical signs of disease in human

psoriatic skin grafts transplanted to SCID mice

Dam 1999

SYSTEMIC LUPUS

ERYTHEMATOSUS

Inhibition of proteinuria, prevention of skin lesions Koizumi 1995

Lemire 1992

274 162

99 57 229 199 151

935 European patients

Orbach H, et al., Ann.N.Y.Acad.Sci. 1109, 385-400; 2007

Vit D in autoimmune diseases

Antico A, Tozzoli R, Giavarina D, Tonutti E & Bizzaro N, Clin Rev Allergy Immunol 2012

Vit. D in the general population

Distribution of 25-OH vit.D

concentration

in 36,384 subjects

of the Northeast Italian

population

Mean vit.D level 21.7 ng/ml

Upper reference limits

9.4 – 43.7 ng/ml

Epidemiological studies on vit.D

in systemic and organ-specific autoimmune diseases

Agmon-Levin N et al., Clinic Rev Allerg Immunol 2012

AUTOIMMUNE DISEASE LOWER VIT.D

LEVELS vs.

HEALTHY

SUBJECTS

CORRELATION WITH

CLINICAL

MANIFESTATIONS OR

DISEASE ACTIVITY

VIT.D RECEPTORS

POLYMORPHISM

SYSTEMIC LUPUS ERYTHEMATOSUS + + +

ANTIPHOSPHOLIPID SYNDROME + + NR

RHEUMATOID ARTHRITIS + + +

SYSTEMIC SCLEROSIS + + NR

SJOGREN DISEASE Normal levels + NR

UNDIFFERENTIATED CONNECTIVE

TISSUE DISEASE + +/- NR

MIXED CONNECTIVE TISSUE DISEASE + NR NR

INFLAMMATORY MYOPATHIES Normal levels - NR

MULTIPLE SCLEROSIS + + +

AUTOIMMUNE THYROID DISEASE + + +

CELIAC DISEASE Normal levels - NR

DIABETES MELLITUS + + +

CROHN’S DISEASE + + +

PRIMARY BILIARY CIRRHOSIS + + +

AUTOIMMUNE SKIN DISEASES + NR NR

ALOPECIA AREATA + NR +

Autoimmunity Reviews 2012; 12:127-136

Autoimmunity Reviews 2012; 12:127-136

Low vit.D levels are

widespread in most

autoimmune diseases

Severe vit.D deficiency

(<10 ng/ml) is associated

with a more severe

disease course

Mechanism: loss of

immune tolerance in

subjects with predisposing

VDR polymorphisms

Observational

design,

uncontrolled

studies

Different type of

compound

Different dose

Different duration

of supplementation

Need for RCTs

No association

with the risk of

developing

Rheumatoid

Arthritis

Lower risk of

Multiple Sclerosis

in young white

subjects

Autoimmunity Reviews 2012; 12:127-136

Vitamin D intake & risk of developing autoimmune diseases:

Type 1 Diabetes Mellitus

INFANCY INTAKE MATERNAL INTAKE

The risk of T1DM was reduced in infants who were supplemented with vitamin D (pooled OR 0.71; 95% CI 0.60 to 0.84)

Dose-response effect Timing of supplementation: lower chances of developing T1DM if between 7

and 12 months of age rather than between 0 and 6 months

Vitamin D & Autoimmunity

Sjögren’s Syndrome

Neonatal Lupus (Congenital Heart Block)

Antiphospholipid Syndrome

Systemic Lupus Erythematosus

Novel aspects of Sjögren’s Syndrome in 2012

Vitamin-D levels were determined in 176 primary SS patients and 163 matched

healthy volunteers utilizing the LIAISON chemiluminescent immunoassays

(DiaSorin-Italy). Mean vitamin-D levels were comparable between SS

patients and controls (21.2±9.4ng/ml and 22.4±10ng/ml)

PHERIPHERAL NEUROPATHY

was diagnosed in 23% of SS

patients and associated with

lower vitamin-D levels

(18.6±5.5ng/ml vs. 22.6±8ng/ml

(p=0.04)).

LYMPHOMA was diagnosed in

4.3% of SS patients, who had

lower levels of vitamin-D,

13.2±6.25 ng/ml, compared to

SS patients without lymphoma

(22±8 ng/ml; p=0.03).

Agmon-Levin N, Kivity S, Tzioufas AG, López Hoyos M, Rozman B, Efes I, Shapira Y, Shamis A, Amital H, Youinou P, Shoenfeld Y:

Low levels of vitamin-D are associated with neuropathy and lymphoma among patients with Sjögren's syndrome. J

Autoimmun 2012, 39(3):234-9.

Fibroblast polarization in

myofibroblast and

collagen production

Myocardial

fibrosis

Proposed pathogenic mechanism of cardiac damage in Neonatal Lupus Buyon JP et al, Arthritis Rheum 2004; 50:173-82; Figure from Tincani A et al, Lupus 2006, 15: 1-7

Macrophages bind

opsonized cardiocytes

A greater proportion of children with CHB

were born during the summer.

This means that the gestational period of

enhanced CHB susceptibility (18-24 weeks

of gestation) occurred during January-

March, which was the time of the year in

which vitamin D levels were at their lowest.

The seasonal timing of the

pregnancy may be critical

to the onset of CHB and

vitamin D is possibly the

mediator of such seasonal

variations.

CHB and Vitamin D

Di Santo G, BMC Medicine 2012; 10:69

APRIL

OCTOBER

Vitamin D levels in APS

Agmon-Levin N, et al., Ann Rheum Dis. 2011; 70(1):145-50 EUROPEAN MULTICENTER STUDY

APS is close to SLE from a clinical, serological and

genetic point of view, but:

•patients are generally not advised to avoid sun

exposure and to use sunscreen;

•patients do not usually take corticosteroids or

other immunosuppressive drugs and therefore

Vitamin D supplementation is rare.

September December April

Se

rum

25

-OH

vit

. D

(n

g/m

l)

p<<0,01 p=0,03 p=ns

NORMAL SUBJECTS

(median value)

PAPS PATIENTS

(median value)

115 PAPS PATIENTS vs 128 NORMAL SUBJECTS

Rheumatology and Clinical

Immunology, Brescia – Italy

Lupus 2012; 21:736-40

Grouped

manifestations

Vit-D <15

ng/ml

Vit-D ≥15

ng/ml

p

Thrombotic 58% 42% 0.04

Pulmonary 42% 24% 0.02

Livedo reticularis

Skin ulcers

42%

26%

22.5%

8.5%

0.01

0.006

Neurologic 70% 54% 0.05

Ophthalmic 26% 10% 0.01

Obstetric 32.4% 41% NS

Arthritis, arthralgia 44% 45% NS

Hematologic 40% 37% NS

Renal 15% 11% NS

Cardiac/valvular 19.5% 15% NS

Thrombosis: the main clinical manifestation of APS

Any relationship with Vitamin D levels?

Agmon-Levin N, et al., Ann Rheum Dis. 2011; 70:145-50

SEPTEMBER

THROMBOSIS

(n=74) OBSTETRIC

DISEASE

(n=40)

P=0,01

se

rum

25

-OH

vit

. D

(n

g/m

l)

Rheumatology and Clinical Immunology, Brescia - Italy

A = anti- 2GPI (APS Patient 1)

B = anti- 2GPI (APS Patient 1) + 1,25 OH vitamin D 10 nM

C = anti- 2GPI (APS Patient 2)

D = anti- 2GPI (APS Patient 2) + 1,25 OH vitamin D 10 nM

E = control IgG

F = control IgG + vitamin D 10 nM (including ethanol)

A B C D E F

Tissue Factor expression

GAPDH expression

Vitamin-D inhibits anti- b2GPI mediated tissue factor expression

Agmon-Levin N, et al., Ann Rheum Dis. 2011; 70(1):145-50

1. Eritema malare

2. Rash discoide

3. Fotosensibilità

4. Ulcere orali

5. Artrite non erosiva

6. Sierosite (pleurite e/o pericardite)

7. Alterazioni renali (proteinuria o cilindruria)

8. Coinvolgimento neurologico (epilessia o psicosi)

9. Alterazioni ematologiche (anemia emolitica; leucopenia; linfopenia; piastrinopenia)

10. Alterazioni immunologiche: positività per anticorpi Anti-DNA nativo, Anti-Sm, Anti-fosfolipidi (Anti-cardiolipina, LAC)

11. Positività per anticorpi Anti-Nucleo (ANA).

Criteri classificativi Lupus Eritematoso Sistemico (LES): American College of Rheumatology (ACR) 1997

Almeno 4 di questi criteri

per la diagnosi di LES

Radiazioni UV

Cute

Chemochine Cellule Apoptotiche Cellule Necrotiche

Apoptosi

Antigene LE

IFN α IFN γ

Cellule T

pDC

Immagine modificata da Meller et al. Arthritis Rheum 2005; 52-1504

I pazienti con LES

vengono invitati a non

esporsi al sole e ad

applicare creme solari a

protezione totale.

IL LES e la mancata esposizione solare

SLE & Vit. D in human studies: contrasting results

POSITIVE RESULTS NEGATIVE RESULTS

CROSS-SECTIONAL STUDIES

Amital H, 2010

Wu PW, 2009

Wright TB, 2009

Borba VZC, 2009

Cutolo M, 2008

Thudy A, 2008

Kamen D, 2006

Birmingham DJ, 2012

Brescia cohort (in press)

Kim HA, 2011

Toloza SMA, 2010

Ruitz-Irastorza G, 2010

Chen S, 2007

Muller K, 1995

DISEASE ACTIVITY

CARDIOVASCULAR DISEASE

WINTER FLARES

Wu PW, 2009 (CV risk)

Przybelski RJ, 2007 (CV risk)

Reynolds, 2012 (vascular stiffness)

Moc CC, 2012

(subclinical atherosclerosis)

Winter flares are associated with lower vit.D levels

Winter FLARE

Winter REMISSION

Summer REMISSION

Rheumatology and Clinical Immunology, Brescia - Italy

SLE & Vit. D in PROSPECTIVE studies: contrasting results

POSITIVE RESULTS NEGATIVE RESULTS

Studio Terrier B. Aranow C. Abou-Raya A. Petri M.

Anno 2012 2012 2013 2013

N. di pazienti 24 57 267 1006

Metodo di supplementazione

Per 2 mesi 100.000 UI/sett D3, poi per 6 mesi 100.000 UI/mese D3

Per 3 mesi: • Gruppo 1: 2.000 UI/die D3 • Gruppo 2: 4.000 UI/die D3

Per 12 mesi: 2.000 UI/die D3

Per 32 mesi: 50.000 UI/sett D2 + 200 UI/2 volte die di calcio e D3

SLEDAI a T0 2

(media) <5

4,8 (media)

NR

Modificazioni in relazione alla supplementazione

Anti-dsDNA Riduzione

NR ma non diminuita l’espressione

genica dell’IFN

Riduzione Nessuna variazione

Complemento Nessuna

variazione Aumento Aumento

SLEDAI Nessuna

variazione Riduzione Riduzione

Ipercalcemia Nessun caso Nessun caso 2% 0,002 % (2 casi)

STUDIO PROSPETTICO di supplementazione in donne premenopausali con LES

CARDIOVASCULAR ASSESSMENT

(at baseline, 12 and 24 months)

• transtoracic echocardiography (assessment

of left ventricular and valvular function, in

addition to indexes of myocardial tissue

characterization)

• carotid ultrasound (with evaluation of carotid

intima-media thickness and local

distensibility)

• pulse wave velocity and analysis (carotid-

femoral) for large-artery stiffness

• ABPM

EVERY 3 MONTHS:

• 25-OH vitamin D, Intact PTH

• lupus activity (SLEDAI), including serology (anti-DNA,

C3, C4, CH50)

• serum calcium and phosphorus

EVERY 6 MONTHS:

• T regs (phenotype study): CD3+CD4+ (CD25highCD127low)

• central memory T cells (CM) CCR7+CD45Ra-

• effector memory T cells (EM) CCR7-CD45Ra-

• terminal differentiated effector memory (TDEM) CCR7-CD45Ra+

• CD28- T cells, an effector T subtype able to resist to apoptosis

(senescent cells) and to bypass regulation mediated by Treg

In collaborazione con Spedali Civili e Università degli Studi di Brescia

Laboratorio di Diagnostica Cardiovascolare – 2^Divisione di Medicina

Laboratorio di Immunologia e Patologia Generale

BASELINE 12 MONTHS 24 MONTHS

STANDARD REGIMEN

STANDARD REGIMEN

INTENSIVE REGIMEN

INTENSIVE REGIMEN

STANDARD REGIMEN: CHOLECALCIPHEROL 25.000 UI once a month (300.000 UI annually) INTENSIVE REGIMEN: CHOLECALCIPHEROL 300.000 UI starting bolus, 50.000 UI once a month (850.000 UI annually)

Efficacia della supplementazione

T12

STANDARD

28% sufficienti

INTENSIVO

75% sufficienti

p = 0,02 Test del Chi Quadrato

NESSUN CASO DI SUPERAMENTO DELLA SOGLIA DI TOSSICITÀ

Test di Mann-Whitney

34 donne con LES in età premenopausale

31,7

24,8 23,8 25,7 24,8

31,9

40,6

31,1 32,5 32,0

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

T1 T2 T3 T4 T5 T6 T7

25-O

H v

it. D

(n

g/m

l)

Tempo (mesi)

Livelli di 25-OH vit.D

STANDARD

INTENSIVO

T0 T3 T6 T9 T12

p = 0,38 p = 0,002

p = 0,0047

p = 0,0075 p = 0,047

Sicurezza della supplementazione

• Nessun caso di ipercalcemia • Nessun caso di soppressione del PTH • 3 casi di ipercalciuria transitoria, non sintomatica

T0 vs. T12

GRUPPO INTENSIVO

T0 vs. T3

PTH (pg/ml)

0

10

20

30

40

50

60

70

T0 T12 T0 T12

STD INT

PTH (pg/ml)

10

15

20

25

30

35

40

T0 T3

8,4

8,6

8,8

9

9,2

9,4

9,6

9,8

10

Ca sierico (mg/dl)

T0 T3

Ca urinario (mg/24h)

0

50

100

150

200

250

300

350

T0 T0 T12 T12

STD INT

Ca sierico (mg/dl)

7

7,5

8

8,5

9

9,5

10

10,5

T0 T12 T0 T12

STD INT

Effetti sui parametri sierologici e sull’attività di malattia

• T0 vs. T12: nessuna variazione significativa • Gruppo intensivo T0 vs. T3: nessuna variazione significativa • Standard vs. Intensivo (C3, C4 e CH50): nessuna differenza significativa • SLEDAI stabile nei 12 mesi di controllo

Anti ds-DNA (UI/ml)

0

10

20

30

40

50

60

70

T0 T12 T0 T12

STD INT

C3 (mg/dl)

50

60

70

80

90

100

110

120

130

T0 T0 T12 T12

STD INT

C4 (mg/dl)

0

5

10

15

20

25

30

T0 T0 T12 T12

STD INT

CH50 (%)

0

20

40

60

80

100

120

140

160

180

T0 T0 T12 T12

STD INT

PAZIENTI CLINICAMENTE SILENTI, SIEROLOGICAMENTE ATTIVE

Steiman A. J. et coll. Arthritis Care & Research 2012

IMMUNOMODULATORY PROPERTIES THAT REDUCE

AUTOIMMUNITY

ANIMAL MODELS

IN VITRO STUDIES

1, 25-OH Vitamin D (CALCITRIOL)

25-OH Vitamin D (COLECALCIPHEROL)

HUMAN STUDIES

VDR POLYMORPHISMS

NON-HYPERCALCEMIC

VDR AGONISTS (PARICALCITOL and

others) PREVENTION OR

TREATMENT OF THE DISEASE

DISCORDANT RESULTS ON THE EFFECTS ON DISEASE ACTIVITY AND SEVERITY

REPARTO e LABORATORIO

U.O. Reumatologia e Immunologia Clinica

Direttore: Prof.ssa Angela Tincani

Laboratorio di Diagnostica

Cardiovascolare –

2^Divisione di Medicina Generale

Laboratorio di Immunologia –

Dip. Medicina Molecolare e

Traslazionale

DiaSorin S.p.A.

Abiogen S.p.A.