8. Julieta Gonzalez. Bogota epithelial cells in lsg from ss patients

ARE THE SALIVARY GLAND EPITHELIAL CELLS A TARGET OR AN ACTOR IN THE SJÖGREN'S SYNDROME PATHOGENESIS?

Simposio de Autoimunidad en Reumatología: Septiembre 9, 2016 | Bogotá, Colombia

María Julieta González Burgos

Salivary Glands of control subjects and Sjögren’s syndrome patients are highly complex system

Reflection

Our physiology is a large puzzle; we know little about each one of the different systems

How can we get specific answers that help us to understand highly complex diseases such as autoimmune diseases?

So, an intriguing question is:

For me it's a mystery

Yes

No

a dream

……but step by step the answers have slowly been emerging ¡¡¡¡

General aspects of Sjögren's syndrome patients

Sjögren's syndrome is a chronic autoimmune disease that mainly affects the salivary and lachrymal glands producing severe disorders of mouth and eye dryness.

This disease has the second highest prevalence after rheumatoid arthritis and with a ratio of 9:1 exhibiting one of the highest female-to-male ratios among autoimmune diseases. It is diagnosed around the fourth and fifth decade and its progress is insidious.

The symptoms are unclear and difficult to diagnose, causing much frustration for the patient who becomes a conflictive person for their family, friends and colleagues.

Like all autoimmune diseases, treatments are palliative and the action of drugs is highly variable. As life expectancy has increased, the autoimmune diseases have increased considerably and the scientists who study them are still very scarce.

The National Institute of Health estimated that autoimmune disorders affected 24 million Americans (17 in 100).

¿A que nivel nos ubicamos para hacer una observación?

Salvador Felipe Jacinto Dalí

What level do we stand to make an observation?

ControlsA B C

D E F

Bars: 100m

SS-patientsA B C

D E F

The role of intrinsic epithelial activation in the pathogenesis of Sjögren’s syndrome

Adapted of: Sjögren’s SyndromeNovel Insights in Pathogenic, Clinical and Therapeutic AspectsChapter | 12 page 195, 2016

Glandular epithelial cells are leading actors in the development and maintenance of Sjögren’s syndrome autoimmune responses.

Salivary gland epithelial cells (SGECs) are suitably equipped to mediate the recruitment activation and/or differentiation of T and B lymphocytes, macrophages (MΦs), and dendritic cells (DCs), as well as the organization of infiltrates in the minor salivary glands (MSGs) of SS patients through the production of cytokines/chemokines. Chapter | 12 page 198, 2016

Journal of Autoimmunity 34 (2010) 400-407J Immunol 2009; 182:3540-3547;

Loss of Acinar Cell Polarity

Normal Acinus Altered Acinus

LL

Control SS-patients

It is necessary to keep in mind

When we started studying this disease our first finding was to observe acinar cells that had lost their cell polarity.

So the obvious question was “why did the acinar cells lose their attachment to the basal lamina?

The acinus function depends on a polarized architecture that is based on continuous dialogue between:

Acinar cells

Cells and Basal lamina

Acinar and myoepithelial cells

ACINUS

BASAL

APICAL

Cell polarity is maintained by master regulators

PAR6aPKCCDC42

CrbPATJPALS

PAR3aPKCPTEN

ScribLglDlg

vect

oria

lity

Autoimmunity Reviews 10:175–179,2011

CYTOPLASM

BASAL LAMINA

STROMA

Hemidesmosome Components

Basal Pole

Apical Pole

PLASMA MEMBRANE

Matrix Biology 22: 49–54, 2003Autoimmunity Reviews 10:175–179,2011

ARTHRITIS & RHEUMATISM 43:2807–2817, 2000.

ARTHRITIS & RHEUMATISM 48: 2573–2584, 2003

ARTHRITIS & RHEUMATISM 52: 2751–2760, 2005

Ann Rheum Dis 2006;65;178-183

Salivary glands from SS-patients showed an increased MMP-3 and MMP-9 expression, as well as MMP-9 catalytic activity. These enzymes were synthesized by exocrine epithelial cells.

An altered balance between MMPs/TIMPs was detected

Salivary glandular extracts of SS-patients degraded purified ECM proteins, such as laminins, fibronectin, type 1, III and IV collagens, among others.

Basal lamina becomes disorganized as a result of degradation of at least two components, laminin 1 and type IV collagen.

Ann Rheum Dis 2009;68:991–996.

ARTHRITIS & RHEUMATISM 54: 3465–3475,2006.

ARTHRITIS & RHEUMATISM 63: 1106–1115,2011

Lateral redistribution of α6β4 integrin and the formation of new cell–cell interactions was observed

Significant increase in BP230 protein levels and the formation of new hemidesmosomes was found.

Significant increase in both mRNA and protein levels of laminin α1, α4, and 2 chains showing that the basal lamina of SG from SS-patients is undergoing active remodeling.

Together these results showed that the salivary glands from SS-patients display a molecular potential to disorganize their ECM thus inducing detachment of acinar cells. However, acinar cells also have activated mechanisms for remodeling of basal lamina and the renewal of new cell-cell and cell-basal interactions, which help maintain acinar organization and promote cell survival.

Adapted of Am J Physiol Gastrointest Liver Physiol 279: G250–G254, 2000.

AF6

Rab 3BRab 13

PKC

7H6

Simplekin

ASIP

ZO-2

ZO-3

BAP-1

Sec 6/8

ZAKZONAB

Miosin

Occludin

Claudins

Microfilaments of actin

Simplekin

VAP3

3

JAM

CAR

ZO1ZO1

Plasma membrane

CAR

Coxsackievirus and Adenovirus Receptor

Cell 1 Cell 2

Pole Apical

Tight Junctions are Multiproteic Complexes

ProteinRelative Protein Levels (Mean ± SD)

Controls Patients p RatioP/C

Claudin-1 0.2 ± 0.2 0.4 ± 0.2 0.02 2.0

Claudin-3 1.5 ± 0.4 1.2 ± 0.5 0.66 0.8

Claudin-4 0.13 ± 0.1 0.34 ± 0.4 0.04 2.6

Occludin 1.1 ± 1.20.5 ± 0.7 0.089 0.45

a1.32 ± 0.8 0.38 1.2b0.13 ± 0.15 0.004 0.12

ZO-1 0.5± 0.6 0.1 ± 0.1 0.002 0.2

Tight Junction Relative Protein Levels from LSG of SS Patients

In SS patients, Claudin 1 and 4 were up-regulated, while Occludin and ZO1 were down-regulated. However, Claudin 3 showed no change ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

Claudin-3 showed re-distribution from apical to basal in Acini and Ducts from MSG of SS patients

Acini Ducts

Control

Patient

A A´ B´

C´ D´

B

C D

`LL

LLLL

L

L

L

L

ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

Ultrastructure of Tight Junctions of Acinar

Cells

TJ ultrastructure in the acinar cells of SS patients showed an electron-dense material extending from the apical to basolateral plasma membrane.

This distribution was similar to the Claudin 3 distribution.

Cytoplasmic vesicles containing a similar material were also observed. Vesicles like exosomes were observed in the lumen

L

L

B

LA

d

d

L

Control

Patient


IC

L

L

D

Patient

L

L

IC

Patient

C

LL

Control

A

L

Patient

BL

Claudin 3 relocation non-dependent on the proximity of inflammatory cells


What factors could be participating in this redistribution?

In other epithelia it has been reported that cytokines may disorganize TJ. In addition, salivary glands from SS patients present high levels of these cytokines.

So, we incubated control acini with TNF- alone and with both TNF- and INF- and then occludin was identified in these samples.

Control Patient

A B

IC

IC

da

aa

Patient

C

d

a

IC

Patient

D

TNF- immunolocalization in acini, ducts and inflammatory cell infiltrates of control and SS-patients


C

L

B

L

A Control

TNF- 10ng/ml

TNF- 30ng/ml

L

D

L

TNF- + IFN- 20 ng/ml

Effect of TNF- and/or IFN- on acinus and localization of occludin

In all experimental conditions we observed re-distribution of occludin


IFN- IFN- and TNF-

(Sci STKE.2006 (316):pe1 Review)

APICAL

What cytokine induced mechanisms cause TJ Disorganization?

STX3

VAMP8

SNAP-23

STX3VAMP8

STX4

STX4 and SNAP-23

VAMP8

VAMP8

STX4 and SNAP-23

VAMP8

Munc18

Rab3D

Synaptotagmin

GTP

Syntaxin 4SNAP-23

Ca2+

Ca2+

Ca2+

NSF

α-SNAP

Mature secretory granule

Plasma membrane

1. Rab GTPases 2. SNARE proteins 3. NSF/αSNAP 4. Modulating proteins

SNARE proteins in control acinar cells

SNARE proteins are receptors for membrane fusion

How do changes in cell polarity affect the localization of proteins involved in regulated exocytosis?

In control acini, VAMP8 was localized in the apical secretory granules and the nuclei were localized in the basal region of acinar cells.

In SS-patients, VAMP8 was observed all over the cytoplasm and the nuclei had lost their polarity.

apical

C

Patient

VAMP8/nuclei

apical

D

apical

B

apical

A

Control

Re-distribution of VAMP8 and loss of nuclear polarity in acinar cells from MSG of SS-patients


STX3, in control acini is located in the apical cytoplasm, while in SS-patients it is observed in all over the cytoplasm.

STX4, in control acini, is mainly localized next to the basolateral plasma membrane and to a lesser extent to the apical plasma membrane. In SS-patients STX-4 is mainly localized in the basal plasma membrane.

SNAP-23, in control acini, is mainly localized in the plasma membrane, while in SS-patients, it has lower immunoreactivity, particularly at the apical and lateral plasma membranes.

Subcellular localization of STX3, STX4, and SNAP-23 in SS-patients

Journal of Autoimmunity 39: 83-92, 2012

PatientControl

SNAP-23E

apical

A STX3

apical

B

F

DC STX4

C

Patient

apical

basal

BA

apical

basal

Control Control

apical

basalB

D

basal

Patient

apical

Considering the re-distribution of SNARE proteins we were interested in determining if SNARE complexes localized ectopically were formed

1. In acinar cells of SS patients there was a significant increase in the levels of SNARE complexes for VAMP8 and STX4.

2. In controls, VAMP8 localized to the apical cytoplasm of acinar cells, whereas in SS-patients VAMP8 was observed all over the cytoplasm.

Formation of SNARE-complexes in vivo

3. In controls, STX4 localized in all the plasma membrane, in SS-patients STX4 co-localized with VAMP8, mainly in basolateral plasma membrane


Control

32

100

Monomeric STX4

P 25

ºC

C 25

ºC

100

18

kDa

Monomeric VAMP-8

P 10

0ºC

C 10

0ºC

P 25

ºC

C 25

ºC

kDa

STX4 SNARE Complex

VAMP8 SNARE Complex

C 10

0ºC

P 10

0ºC

Patient

SNARE COMPLEXES ARE STABLE AT 25ºC

Could these ectopic SNARE complexes participate in the basal exocytosis of mucins?

Patient

Control

D

basal

m

S

ECM

S

m

basalS

A

MUC7/LAMININB

DICC

MUC7/LAMININ/DIC

E F

Presence of secreted MUC7 in the extracellular matrix of MSG from SS-patients

In controls, MUC7 localized in the cytoplasm of acinar cells. In SS patients MUC7 maintained its localization in the acinar cells, but was also detected atypically in the ECM. The Laminin a component of the basal lamina marked the boundary of the acinus.


Aberrant exocytosis of MUC5B and MUC7

Controls SS-Patients

BASAL

APICAL

LATERAL

VAMP8

STX3

SNAP-23

STX4

Rab3D

Occludin

Claudin-4

Claudin-3

ZO-1

TJ

BasalLamina

Summary

Nat Rev Rheumatol 21:186, 2012

J Autoimmunity 42: 7-18,2013

So, we wanted to determine whether exogenous mucins were able to induce the expression of pro-inflammatory cytokines in cultured human salivary cells (HSG).

These results allowed us to conclude that the loss of acinar cell polarity leads to ectopic exocytosis, which could explain the oral dryness in these patients.

Golgi/Nucleus

3D acini from HSG cells

Relative mRNA levels of pro-inflammatory cytokines in HSG cells stimulated with mucins

A significant increase (*) in relative mRNA levels of pro-inflammatory cytokines was observed. Differences found for BAFF were not significant.

……..So we wondered………..

00.5

11.5

22.5

33.5

44.5

5

CXCL-8 TNF-α IFN-α IFN-β IL-6 IL-1β BAFF

Cyto

kine

/h18

S (R

elati

ve e

xpre

ssio

n ra

tio)

0µM0.001 µM0.01 µM0.1 µM0.2µM0.4 µM

*

**

*

*

*

*

**

*

BSM

Barrera et al, under revision

Are the mucin’s sugar residues involved in the induction of these cytokines?

Effect of purified MUC5B and Sulfo-Lewis residues in the relative expression levels of cytokines in HSG cells

Sulfo-Lewis showed a comparable response to purified MUC5B. These results allowed us to postulate that the mucins might be recognized by their sugar residues.

Sulfo Lewis : SO3-Galβ1-3GlcNAc is a sulfated disaccharide presents in MUC5B

MUC5B Sulfo-Le BSM

*

*

*

*

*

*

*

*

*

*

*

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

0 0.005 0.015 21.6 54 0.4 µM

Cyto

kine

/h18

S (R

elati

ve e

xpre

ssio

n ra

tio)

IL-6

TNF-α

IL-1

Barrera et al, manuscript under review

Ligands containing sugar residues can bind Toll like receptors, such as TLR4. We used the lipopolysaccharide (LPS), a ligand of TLR4, to induce expression of cytokines. Then, we compared the LPS-induced cytokine profile with the mucin-induced profile.

Comparison between relative mRNA levels of pro-inflammatory cytokines in HSG cells stimulated with LPS or mucins

Stimulation of HSG cells with TLR4 ligand induced the expression of IL-6 and TNF-α in a similar manner to stimulation with mucins.

LPS BSM

* *

*

*

*

*

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.01 0.1 0.2 0.4 µM

Cyto

kine

/h18

S (R

elati

ve e

xpre

ssio

n ra

tio)

IL-6TNF-α


The role of TLR4 in cytokine induction was evaluated using TBX2, a peptide that specifically inhibits TIRAP binding to TLR4. The TNF- and IL-6 expression were determined.

TBX2: GKMADWFRQTLLKKPKKRPNSPESTLQLRDATPGGAIVS

TBX2

Modified of Cytokine 42 (2008) 145–151Modified of JBiotechnology 129 (2007) 555–564

Dominant-negative domain

TIRAPTBX2

TLR4

MyD88

Molecular modeling and prediction of protein–protein interaction. TLR4, TIRAP, MyD88 and TBX2 are shown in ribbon representation.

Relative mRNA levels of TNF-α and IL-6 in HSG cells treated with a TIRAP inhibitory peptide of TLR4

TBX2 selectively inhibited the production of pro-inflammatory cytokines under the induction of mucins or LPS. This inhibition might have been mediated by the disruption of the recruitment of downstream effector molecules. Mucins induced the expression of pro-inflammatory cytokines via TLR4.

TNF-α#

*

#*

0

1

2

3

4

5

6

UT TBX2 LPS TBX2/LPS BSM TBX2/BSM

TNF-

α/h1

8S (R

elati

ve e

xpre

ssio

n ra

tio) IL-6

#*

#*

00.5

11.5

22.5

3

3.54

4.5

UT TBX2 LPS TBX2/LPS BSM TBX2/BSM

IL-6/

h18S

(Rel

ative

exp

ress

ion

ratio

)


anti-hTLR4-IgG

Modified of Cytokine 42 (2008) 145–151

To confirm the involvement of TLR4, we used blocking antibodies against TLR4 and measured TNF- and IL-6 expression in LPS-treated or mucin-treated cells.

Relative IL-6 mRNA levels in HSG cells treated with a TLR4-blocking antibody

The specific antibody against TLR4 inhibited the expression of cytokines. These results demonstrated that mucins induced the expression of pro-inflammatory cytokines via a TLR4 mediated mechanism.

*

*#*

*

#

0

0.5

1

1.5

2

2.5

3

3.5

4

UT

anti-TLR

4IgG

1BSM

anti-TLR

4/BSM

IgG1/B

SM LPS

anti-TLR

4/LPS

IgG1/LP

S

IL-6/

h18S

(Rel

ative

exp

ress

ion

ratio

)


In HSG cells, exogenous MUC5B and Sulfo-Lewis can induce, via TLR4, the mRNA expression of pro-inflammatory cytokines. However, we aren’t discarding the possibility that galectins and TIM family receptors might also play a role.

A similar response might be observed in the salivary gland extracellular matrix of SS patients, where the ectopically secreted mucins may promote the infiltration of inflammatory cells, a hallmark in the pathogenesis of the disease.

CONCLUSIONS

Concluding RemarksCellular interactions are essential for the maintenance of morphological

and functional homeostasis of salivary epithelia.

An important element involved in the development of autoimmunity is the loss of the protective function of mucosal barriers by disruption of tight junctions.

The potentially pathogenic ectopic secretion of mucins and other factors into the ECM “may be the initial step in the genesis of the disease”.

Colaborators

NationalSergio AguileraClaudio MolinaSergio GonzálezCecilia AlliendeMarcela HermosoUlises UrzúaAndrew QuestLisette Leyton Cecilia Leyton

InternacionalJuan Manuel Anaya Enno VeermanUlla MandelBruce BaumHenrik ClausenInka BrockhausenFONDECYT

Our Students

Paola PérezEduardo GoicovichYoon-Jeoung KwonPatricia EwertMarianela SánchezMaría José BarreraRodrigo PintoMónica BritoJuan CortésIsabel CastroVerónica BahamondesHsiao Hsin SungCamilo TapiaDenisse SepúlvedaHery UrraJosé VelozoSebastián IndoKatherine Bravo Camilo TapiaSebastián PugaCarolina Lagos`Patricia CarvajalNicolás Albornoz

A heartfelt gratitude to our Patients

Model of the changes in assembly of type I-HD in LSG from SS patients

González S et al ARTHRITIS & RHEUMATISM 63, 2011, 1106–1115

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8. Julieta Gonzalez. Bogota epithelial cells in lsg from ss patients

Health & Medicine