Photo Augusto Domínguez Barros

ORAL DRYNESS IN SJÖGREN’S SYNDROME PATIENTS: NOT JUST A QUESTION OF WATER

María Julieta González Burgos

Simposio de Autoinmunidad en Reumatología Septiembre 9 , 2016, Bogotá, Colombia

The standard aetiopathogenic model employed to describe SS disease suggests that alterations in the immune system trigger a pathological response, which eventually destroys the exocrine glands. Apoptotic model

Some antecedents related with dryness mouth

Dawson et al proposed an alternative model of glandular dysfunction, referred to as the non-apoptotic model, in which mechanisms reducing or inhibiting water transport are thought to cause reduced salivary flow.

Perez et al. (2009) performed gene expression profiling of fractions enriched in epithelial cells from LSGs SS-patients and controls using cDNA microarrays. 528 genes were differentially expressed in SS-patients relative to controls. Among the up-regulated genes, 28 were pro-apoptotic and 15 were anti-apoptotic . These findings suggest that the balance between death and survival signals is altered on a large scale in LSG from SS-patients.

The severity of secretory dysfunction does not correlate with the degree of lymphocytic infiltration and glandular destruction in SS-patients (Fox RI).

In salivary glands of SS-patients, altered cholinergic signaling, calcium tunneling, among other factors, have been detected that may result in reduced water release. However, mouth dryness correlates only poorly with the USF.

Non-apoptotic model of glandular hypofunction

1. Cytokines induce inhibition of neurotransmitters

2. Degradation of acetylcholine by increase of cholinesterase

3. Anti-M3 antibodies block Muscarinic receptors

4. Impaired production of ON

5. Disturbance in the release of calcium induced calcium(CICR) by changes of cADPr

8. Altered expression and distribution of AQP5

Adapted of L. Dawson y cols., Rheumatology 2006.45:792–798

Autoimmunity Diseases: High Complexity and Difficult to Understand

Intercambiador Na+/H+

Intercambiador Cl-/HCO3-

Canal K+

Canal Cl-

Na+/K+ ATPasa

Intercambiador K+/H+

Na+/K+/2Cl-

AQP5

Canal de Na+

Receptor muscarínico

AcetilcolinaReceptores α- y β- adrenérgicosAdrenalina o noradrenalina

α β

NAACh

Dibujado por María José Barrera, PhD en Ciencias Biomédicas, 2015

Exocrine Epithelium Complexity Water and electrolytes

ACINUSIsotonic Saliva

DUCTHypotonic Saliva

IONIC COMPONENTSOF SALIVA

-140 mEq/L NaCl

25 mEq/L NaCl

Increased value(1.5 – 4) in SS patients

H2O

H2O

H2O

Components and functions of Saliva

Adapted of A. van Nieuw Amerongen y cols., Caries Res 2004.38:247–253

99% waterelectrolytes

(Na+, K+, Cl-, HCO3-)

proteins (immunoglobulins,

antibacterial products and antifungals,

enzymes)Glycoproteins/mucins

Non-stimulated salivary flow normal ≥

1,5ml/15minutes

SSTPGTAHTLTVLTTTATTPTATGSTATP

TTAAPPTPSATTPAPPSSSAPPE

GSTAPPAHGVTSAPDTRPAP

MUC1/TM

MUC5B

MUC7

GSTAPPAHGVTSAPDTRPAP

MUC1/SEC

Dominio citoplasmático

SEA

Secuencia de transmembrana

Secuencia VSIGLSFPMLP del C-terminal de MUC1/SEC

Sitio de clivaje

EGF

Dominio rico en Cisteína

Dominio D (D4)

Sitio de clivaje autocatalíticoSecuencia única

Secuencia señal

Dominio D (D1-D3)

Dominio rico en Cisteína

Repeticiones en tandem

Modificada de M. Hollingsworth y B. Swanson, Nat Rev Cancer, 2004. 4(1): p. 45-60

Características estructurales de las mucinas salivales

90-300 kDa

200-250 kDa

1 MDa

220 kDa

H2O

H2O

H2OH2O

H2OH2O

H2OH2O

H2OH2O

H2O

H2OH2O

H2O H2O

Mucinas poliméricas secretadasMucinas de membrana

C

N

Y

Y

YY Y Y YYY YN CC N

GalNAcGlcNAcGal

Ácido siálicoFucosaÉster sulfatoSO3

_

Y N-glicosilaciónDominio VNTRDominio citoplasmático Dominio Transmembrana

SO3

_SO3

_SO3

_

SO3

_SO3

_

SO3

_ SO3

_

SO3

_SO3

_ SO3

_

SO3

_

Mucinas salivales

Efecto higroscópico

Mucinas asociadas a membrana

Secuestro de factores solubles

Poder estequiométrico

Transducción de señales

Efecto de filtraciónEfecto de intercambio

iónico

Mucinas solubles

Célula

Modificada de M. Hollingsworth y B. Swanson, Nat Rev Cancer, 2004. 4(1): p. 45-60

Biosíntesis de mucinas

Modificada de D. Thornton y cols. Annu Rev Physiol, 2008. 70: p. 459-86.

RER

Dominio N-terminal (no plegado)Dominio N-terminal (plegado)Dominio C-terminal (no plegado)Dominio C-terminal (plegado)Puentes disúlfuro intermolecularesN-acetilgalactosaminaN-acetilglucosaminaGalactosaFucosaÁcido siálicoDominio de mucina glicosilado

Trans-Golgi

Cis-MedialGolgi

Altered secretion of salivary mucins in Sjögren's syndrome patients

Controls Patients

C1 C2 C3 C4 C5 P1 P2 P3 P4 P5 P6

193 kd

b-actin

stacking gelMUC5B

A B

p = 0.33

Rela

tive

leve

ls of

pro

tein

M

UC5

B/b-

actin

0

5

10

15

20

Controls Patients

Expression of MUC5B in LSG

A

E

B

m sm

s

C

d

ms

D

f

ms

f

Ann Rheum Dis 2008;67:1480–1487

Localization of MUC5B

MUC5B

Sulfo-Lewisa:SO3Galβ1-3[Fuc1-4]GlcNAc

SO3SO

3SO3 SO

3

Levels of Sulfo Lewisa -MUC5B

p < 0.001

Controls Patients

0

5

10

15

20

Rela

tive

leve

ls of

Sul

fo-M

UC

5B

BA

Controls Patients

193 kd

b-actin

Stacking gel

C1 C2 C3 C4 C5 P1 P2 P3 P4 P5 P6

SulfoMUC5B

Localization of Sulfo-Lewisa -MUC5B

m

A

m s

m

C

m

mf

B

a

B

a

C

Abso

rban

ce (A

U)

SS PatientsControl0.0

0.1

0.2

0.3 A p=0.04

Microdensitometric analysis of total sulfate groups and correlation with the basal lamina organization

Ann Rheum Dis 2008;67:1480–1487

Comparison between unstimulated salivary flow and the percentage of mucous acini Sulfo-Lewisa antigen from SS patients and controls

Ann Rheum Dis 2008;67:1480–1487

Disorganization of the basal lamina observed in patients with Sjögren syndrome may lead to dedifferentiation of acinar mucous cells and, as a consequence, alter sulfation of MUC5B. These changes are suggested to represent a novel mechanism that may explain xerostomia in these patients.

S

O

-O

O

ppGalNAcT

β1-3

Core 1

Esqueleto proteico

C1GalT

β1-3

2-3 β1-3

ST3GalTGal3ST

Serina o treonina del esqueleto proteicoGalNAc Gal Neu5Ac

Ser/Thr

Ser/Thr

Ser/Thr

Ser/Thr

Ser/Thr

Ser/Thr

C2GnT

β1-3

Ser/Thr

GlcNAcS

O

-O

OEster sulfato

β1-3

Ser/Thr

β3/4GalT

Core 2

C3GnT

β1-3

Ser/Thr Core 3

β1-3

Ser/Thr Core 4

C2GnT2

β1-3

Ser/Thr

β3/4GalT

β1-6 β1-6

β1-6 β1-6

β1-3/4 β1-3/4

Biosíntesis de oligosacáridos en GSL de pacientes SS

I. Castro y cols., Rheumatology. 2012. 51:482-490

p=0,92 p=0,085

Controles Pacientes SS0.0

0.5

1.0

1.5

2.0

Nive

les re

lativo

sGa

l3ST-

2/b-

actin

a

Controles Pacientes SS0

1

2

3

4

5

Nive

les re

lativo

sGa

l3ST-

4/b-

actin

a

I. Castro y cols., Rheumatology. 2012. 51:482-490Controls SS-Patients

0

5

10

15*

pmol

/h/m

gGa

l3ST

acti

vity

DrynessUSF TNF- Focus

Scoreeye mouth

Gal3ST activity -0.8660* -0.4880* 0.2205 -0.7204* -0.8271*

Correlation coefficients between Gal3ST activity and clinical parameters of SS-patients

Correlación actividad Gal3-O-sulfotransferasa y niveles de TNF-

The Gal3ST activity levels showed an inverse and significant correlation with the symptoms of oral and ocular dryness, with glandular levels of TNF-α and the degree of lymphocyte infiltration in the GS from SS patients.

These results suggest that in GS of SS patients there is an alteration in some of the machinery components involved in the sulfation of MUC5B and this alteration is correlated with the degree of inflammation presented glands.

Citoquinas pro-inflamatorias

CONTROL PACIENTES SS

?

Estos antecedentes sugieren que en GS de pacientes SS, el ambiente inflamatorio podría afectar la localización y sulfatación de mucinas.

H2O H2O

S/T S/T S/T S/T

SO3- SO3

- SO3-

SO3-

H2OH2O

S/T S/T S/T S/T

F. Wang y cols., International Immunopharmacology. 2015. 28:764–772

T. Kondo y cols., Modern Pathology. 2005. 18: 1199–1210

Células HeLa

Glándula endocervical normal teñida con Alcian Blue pH 1.0

Células HeLa estimuladas con TNF-

DMSO

TNF-

Detección de MUC5B y Sulfo-Lewisa en células HeLa estimuladas con TNF- o IFN- durante 48 horas.

0 1 10 1 10 ng/ml, 48hTNF-

SulfoLewisa

b -actina

kDa

IFN-

MUC5B

250 >

50 >

250 >

,

,

,

,

,

,

,

,

,

,

Estos resultados demuestran que TNF- e IFN- reducen la presencia de oligosacáridos sulfatados asociados a MUC5B y sugieren que en GSL de pacientes SS, los altos niveles de citoquinas pro-inflamatorias podrían explicar la hiposulfatación de esta mucina.

Citoquinas pro-inflamatoriasCONTROL PACIENTES SS

H2O H2O

S/T S/T S/T S/T

SO3- SO3

- SO3-

SO3-

H2OH2O

S/T S/T S/T S/T

Gracias

Colaborators

NationalSergio AguileraClaudio MolinaSergio GonzálezCecilia AlliendeMarcela HermosoUlises UrzúaAndrew QuestLisette Leyton Cecilia Leyton

InternacionalJuan Manuel Anaya Enno VeermanUlla MandelBruce BaumHenrik ClausenInka Brockhausen

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