.

1. 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

2. 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

3. 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 ¡¡¡¡

4. 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).

5. ¿A que nivel nos ubicamos para hacer una observación?
Salvador Felipe Jacinto Dalí
What level do we stand to make an observation?

6. Controls
A B C
D E F
Bars: 100m
SS-patients
A B C
D E F

7. The role of intrinsic epithelial activation in the pathogenesis of Sjögren’s syndrome
Adapted of:
Sjögren’s Syndrome
Novel Insights in Pathogenic,
Clinical and Therapeutic
Aspects
Chapter | 12 page 195, 2016

8. 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

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

10. Loss of Acinar Cell Polarity
Normal Acinus Altered Acinus
L
L
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?

11. 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

12. BASAL
APICAL
Cell polarity is maintained by master regulators
PAR6
aPKC
CDC42
Crb
PATJ
PALS
PAR3
aPKC
PTEN
Scrib
Lgl
Dlg
vectoriality
Autoimmunity Reviews 10:175–179,2011

13. CYTOPLASM
BASAL LAMINA
STROMA
Hemidesmosome Components
Basal Pole
Apical Pole
PLASMA MEMBRANE
Matrix Biology 22: 49–54, 2003Autoimmunity Reviews 10:175–179,2011

14. 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.

15. 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.

16. Adapted of Am J Physiol Gastrointest Liver Physiol 279: G250–G254, 2000.
AF6
Rab 3B
Rab 13
PKC
7H6
Simplekin
ASIP
BAP-1
Sec 6/8
ZAK
ZONAB
Occludin
Claudins
Microfilaments of actin
Simplekin
VAP33
JAM
CAR
ZO1
ZO1
Plasma
membrane
CAR
Coxsackievirus and Adenovirus Receptor
Cell 1 Cell 2
Pole Apical
Tight Junctions are Multiproteic Complexes

17. Protein
Relative Protein Levels (Mean ± SD)
Controls Patients p
Ratio
P/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 ± 1.2
0.5 ± 0.7 0.089 0.45
a1.32 ± 0.8 0.38 1.2
b0.13 ± 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

18. 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
`L
L
LLL
L
L
L
L
L
ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

19. 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
L
A
d
d
L
Control
Patient
ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

20. IC
L
L
D
Patient
L
L
IC
Patient
C
L
L
Control
A
L
Patient
B
L
Claudin 3 relocation non-dependent on the proximity of
inflammatory cells
ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

21. 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.

22. Control Patient
A B
IC
IC
d
a
a
a
Patient
C
d
a
IC
Patient
D
TNF- immunolocalization in acini, ducts and inflammatory
cell infiltrates of control and SS-patients
ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

23. 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
ARTHRITIS & RHEUMATISM 62:1280–1289, 2010

24. IFN- IFN- and TNF-
(Sci STKE.2006 (316):pe1 Review)
APICAL
What cytokine induced mechanisms cause TJ Disorganization?

25. STX3
VAMP8
SNAP-23
STX3
VAMP8
STX4
STX4 and
SNAP-23
VAMP8
VAMP8
STX4 and SNAP-23
VAMP8
Munc18
Rab3D
Synaptotagmin
GTP
Syntaxin 4
SNAP-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?

26. 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
ARTHRITIS & RHEUMATISM 63:3126–3135, 2011

27. 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

28. C
Patient
apical
BA
apical
Control Control
apical
B
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
Journal of Autoimmunity 39: 83-92, 2012
Control
32
100
Monomeric STX4
P25ºC
C25ºC
100
18
kDa
Monomeric VAMP-8
P100ºC
C100ºC
P25ºC
C25ºC
kDa
STX4 SNARE Complex
VAMP8 SNARE Complex
C100ºC
P100ºC
Patient
SNARE COMPLEXES ARE STABLE AT 25ºC

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

30. Patient
Control
D
m
S
ECM
S
m
S
A
MUC7/LAMININ
B
DIC
C
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.
Journal of Autoimmunity 39: 83-92, 2012

31. 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
Basal
Lamina
Summary
Nat Rev Rheumatol 21:186, 2012
J Autoimmunity 42: 7-18,2013

32. 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.

33. Golgi/Nucleus
3D acini from HSG cells

34. 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………..
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
CXCL-8 TNF-α IFN-α IFN-β IL-6 IL-1β BAFF
Cytokine/h18S(Relativeexpressionratio) 0µM
0.001 µM
0.01 µM
0.1 µM
0.2µM
0.4 µM
*
*
*
*
*
*
*
**
*
BSM
Barrera et al, under revision

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

36. 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
Cytokine/h18S(Relativeexpressionratio)
IL-6
TNF-α
IL-1
Barrera et al, manuscript under review

37. 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.

38. 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
Cytokine/h18S(Relativeexpressionratio)
IL-6
TNF-α
Barrera et al, manuscript under review

39. 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.

40. 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/h18S(Relativeexpressionratio)
IL-6
#
*
#
*
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
UT TBX2 LPS TBX2/LPS BSM TBX2/BSM
IL-6/h18S(Relativeexpressionratio)
Barrera et al, manuscript under review

41. 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.

42. 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
IL-6/h18S(Relativeexpressionratio)
Barrera et al, manuscript under review

43. 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

44. Concluding Remarks
Cellular 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”.

45. Colaborators
National
Sergio Aguilera
Claudio Molina
Sergio González
Cecilia Alliende
Marcela Hermoso
Ulises Urzúa
Andrew Quest
Lisette Leyton
Cecilia Leyton
Internacional
Juan Manuel Anaya
Enno Veerman
Ulla Mandel
Bruce Baum
Henrik Clausen
Inka Brockhausen
FONDECYT
Our Students
Paola Pérez
Eduardo Goicovich
Yoon-Jeoung Kwon
Patricia Ewert
Marianela Sánchez
María José Barrera
Rodrigo Pinto
Mónica Brito
Juan Cortés
Isabel Castro
Verónica Bahamondes
Hsiao Hsin Sung
Camilo Tapia
Denisse Sepúlveda
Hery Urra
José Velozo
Sebastián Indo
Katherine Bravo
Camilo Tapia
Sebastián Puga
Carolina Lagos`
Patricia Carvajal
Nicolás Albornoz
A heartfelt gratitude to our Patients

46. 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