University of Khartoum Institute of Endemic Diseases

Department of Molecular Pathology and Immunology

Vitiligo as organ specific Autoimmune disease

Prepared by : Omer Yahiaomerflagella@yahoo.com

M.sc. Program of molecular medicine

Human skinEpidermis:• keratinized epithelial cells • Langerhans cells• Intraepithelial lymphocytes. Dermis: is mainly composed of connective tissue produced by dermal fibroblasts , and• Dermal DCs .• Mast cells .• Cutaneous lymphocyte antigen (CLA)-positive memory T cells. Dermal post-capillary venules: constitutively express low levels of • E-selectin.• CC-chemokine ligand 17 (CCL17) •ICAM1.These support the margination and baseline emigration of CLA+ memory T cells into non-inflamed skin.

Innate immune mechanisms in the skin Epithelial-cell injury or pathogen invasion leads to the release of primary cytokines and the activation of:skin cells (keratinocytes and fibroblasts).resident innate immune cells(Langerhans cells (LCs), dermal dendritic cells (DCs) and mast cells).which stimulating downstream activation cascades.

Activated Langerhans cells and dermal DCs are stimulated to mature and emigrate from the tissue to the draining lymph node, carrying antigen for presentation to naive and memory T cells.

The cytokines and chemokines act on the local endothelia through nuclear factor-κB

NF-kB (NF-κB)-mediated pathways to upregulate the expression of •Adhesion molecules(including E-selectin, P-selectin and intercellular adhesion molecule 1 (ICAM1).•Direct the recruitment of additional innate immune components according to the specific signals that are generated (for example, neutrophils, eosinophils and natural killer (NK) cells. CCL17, CC-chemokine ligand 17).

Adaptive immune responses in the skin•Cytokines released by keratinocytes, fibroblasts and resident antigen-presenting cells stimulate the upregulation of expression of E-selectin and intercellular adhesion molecule 1 (ICAM1) through nuclear factor-κB (NF-κB)-mediated activation pathways. •Production and presentation of T-cell-specific chemokines, such as CC-chemokine ligand 17 (CCL17), CCL22 and CCL27, on the local endothelium results in the recruitment of CLA+ T cells in an antigen non-specific manner.

• Circulating cutaneous lymphocyte antigen (CLA)-positive T cells represent a library of memory T cells with T-cell receptors (TCRs) specific for antigens previously encountered in the skin.

• T cells entering the tissue that encounter their specific antigen presented by local macrophages or dendritic cells will be activated to proliferate and carry out their specific functions. Those that do not encounter their cognate antigen, which might be most of the cells that are recruited, will enter the lymphatics and return to the general circulation.

• In the skin there are CLA+,CD45O+,CCR4+ skin resident memory T cells

• they are 20 times more than that present in blood.

• 5% of these cells are resident memory (CD4+,CD25+,FOXP3+, CD127+) regulatory T cells.

• They are antigen specific and expanded by Langerhans dendritic cells which occur side by side with skin resident CD45O+ effector memory cells

Tolerance

Tolerance • Type of negative regulators:• Regulatory T cells CD4+,CD25+,FOXP3+ ( natural nTreg. and induced iTreg) (Nrp-1: It is a peptide of a role in nreg. T cells differentiation and is not

expressed on induced T reg cells). Marine CD4CD25 Treg cells constitutively express CTLA-4 , which is known

as a negative regulator of T cell activation, and expression of this molecule is required for these cells to suppress immune responses.

Regulatory T cells inhibition of autoreactivity, explaining why autoreactive T cells are present in the circulation in the absence of autoimmune symptoms

• CD3+CD8+CD161 −CD56+ regulatory CD8 T cells were discovered in PBMC (regulatory CD8 T cells occurred at a frequency of 3.2% of total CD8 T cells,

they do not express CD45RO or CCR7, could lyse autologous and allogeneic activated CD4 T cell targets)

Sequential immunohistochemical study of depigmenting and

repigmenting minigrafts in vitiligo• Marwa ABDALLAH*1• Mohamed B. ABDEL-NASER*1• Manal H. MOUSSA2• Chalid ASSAF3• Constantin E. ORFANOS3

Sequential melanocytes disappearance in vitiligo patient

HMB-45 staining of minigrafts of non-responders showing a sequential decrease of melanocytes

from day 14 (a), 17(b) and 21 (c), with a complete disappearance on

day 28 (d). Meanwhile, keratinocytes show striking cytoplasmic vacuolation.

Day 14

Day 17

Day 21

Day 28

Local Immune Response in Skin of Generalized Vitiligo

PatientsRené van den Wijngaard1, Anna Wankowicz-Kalinska1,2, Caroline Le Poole4, Bert Tigges1, Wiete Westerhof2,3 and

Pranab Das1,2

Melanocytes disappearance

(A) Immunohistochemistry with the melanocyte-specific antibody NKI-beteb shows a partial absence of melanocytes from perilesional (PL) epidermis.

(B) CD3-immunoreactive T cells in PL skin.

CD1a-reactive Langerhans

• Basal localization of CD1a-reactive Langerhans cells in the lesional (L) epidermis of a vitiligo donor.

ICAM-1 reactivity in PL skin

• ICAM-1 reactivity in PL skin of vitiligo donor. Note: the focal epidermal expression (arrow).

Immunoreactivity (A)NKI-beteb (blue)/HLA-DR (red) double-label immunoreactivity showing HLA-

DR immunoreactivity of one remaining melanocyte (arrow) in PL skin. (B) NKI-beteb (red)/CD68 (blue) double-label immunoreactivity of normal

control (NC) skin showing CD68+ melanocytes (arrows). C to F, Serial sections of PL vitiligo skin.(C), NKI-beteb (blue)/CD8 (red) double-label immunoreactivity; long arrow,

CD8+ T cell apposed to melanocyte; short arrows, CD8+ T cells apposed to melanocytic remnants.

(D) CD3 (red)/CLA (blue) double-label immunoreactivity showing a cluster of double-immunoreactive T cells (arrows) at the epidermal/dermal junction and CD3 single-immunoreactive cells in the dermal compartment (asterisks).

(E) Granzyme-B (F) perforin immunoreactivity in cells at the PL epidermal/dermal junction.

macrophage infiltrates

(A) CD68 immunoenzymatic reactivity of NL skin.(B) PL skin. (C)Epidermal CD68 expression pattern in PL skin

using high antibody concentration.

Immunohistochemical Findings in Active Vitiligo Including DepigmentingLesions and

Non-Lesional SkinFlavia M.N.P. Aslanian*,1, Rosangela A.M. Noé1, Daniela P. Antelo1, Rogerio

E. Farias2,Pranab K. Das3, Ibrahim Galadari4, Tullia Cuzzi1 and Absalom L. Filgueira1

(A)Significantly decreased number of CD1a positive cells (inbrown) in the non-lesional skin of active vitiligo patients.

(B) In contrast, there are abundant marked CD1a positive cells in the control skin.

(A)Presence of CD8+ cells infiltrate in the dermis of nonlesional skin in active vitiligo.

(B)Such infiltrate was not observed in any control skin.

Th17 Cells and Activated Dendritic Cells Are Increased

in Vitiligo LesionsClaire Q. F. Wang,#1 Andres E. Cruz-Inigo,#2 Judilyn Fuentes-Duculan,1 Dariush

Moussai,2 Nicholas Gulati,1 Mary Sullivan-Whalen,1 Patricia Gilleaudeau,1 Jules A. Cohen,1 and James G. Krueger1*

Characterization of Langerhans cells, dermal dendritic cells subpopulations in matched

non-lesional, leading edge and depigmented lesional skin

NALP-1 positive Langerhans cells are found in lesional vitiligo skin

T cell infiltration and loss of melanocytes in lesional and leading edge vitiligo skin.

Review

Apoptosis of melanocytes in vitiligo results from antibody

penetrationAlejandro Ruiz-Argüellesa, , , Gustavo Jiménez Britob, Paola Reyes-Izquierdoa, Beatriz Pérez-Romanoa, Sergio

Sánchez-Sosac

• Note : this is the only paper that

show B cells in the perilesional area of vitiligo patient.

• Lymphocyte infiltrate (solid oval) in juxtaposition to a depigmented area of the skin (dotted oval)

• The arrow indicates the reactivity of MoAb HMB-45 in an adjacent area where abundant melanocytes are still present.

Note :the specimen showed lymphocyte infiltrates that, most strikingly, were located in juxtaposition to areas of overt depigmentation while not in those where residual melanocytes were present. Staining for Bcl-2 revealed that infiltrates consisted mainly of B lymphocytes

Role of T.reg in the autoimmune disease

• Several T cell subsets with immunoregulatory functions have been described, and their crucial roles in certain animal autoimmune disease models have been shown.(Maloy, K. J., and F. Powrie. 2001.)

• One such T cell subset was initially identified in mice, as CD4T cells that constitutively express CD25, the IL 2R -chain (regulatory T (Treg) 4 cells).( Asano.1996 ;Takahashi. 1998; Thornton. 1998.)

• This population is characterized by its ability to inhibit the development of autoimmune gastritis after neonatal thymectomy and constitutes 10% of peripheral CD4 cells.( Asano.1996)

• Murine Treg cells are anergic when stimulated in vitro with anti-CD3 mAbs, but proliferate upon addition of exogenous IL-2. (Takahashi. 1998; Thornton. 1998.) and show remarkable suppressive capacity both in vitro and in vivo .(Suri-Payer 1998; Salomon. 2000 ; Read. 2000; Sakaguchi. 1995; Itoh. 1999)

• After TCR-mediated stimulation, CD4CD25 Treg cells suppress the activation and proliferation of other CD4 and CD8 T cells in an Ag-nonspecific manner (Takahashi. 1998;

Thornton. 2000 )via a mechanism that requires cell-cell contact and in most systems is independent of the production of immunosuppressive cytokines. (Takahashi. 1998; Thornton. 1998.)

• Murine CD4CD25 Treg cells constitutively express CTLA-4 (Salomon. 2000), which is known as a negative regulator of T cell activation, and expression of this molecule is required for these cells to suppress immune responses in vivo. (Read. 2000; Takahashi. 2000).

• Regulatory T cells inhibition of autoreactivity, explaining why autoreactive T cells are present in the circulation in the absence of autoimmune symptoms (Baecher-Allen and Hafler, 2006).

• While the mechanism of action for Treg is still not fully understood, TGF-b and IL-10 contribute to Treg mediated immunosuppression.

• TGF-b is important for imposing a regulatory phenotype to the Treg subset and regulatory activity is dependent on cell-cell contact (Bala and Moudgil, 2006;

Joetham et al., 2007; Zhu and Paul, 2008).

• Markers expressed by Treg include FoxP3, GITR, CTLA-4 and CD25, yet only FoxP3 expression is relatively unique to regulatory T cells (De Boer et al.,

2007). This transcription factor affects the expression of many genes (Zheng and Rudensky, 2007)

• Mutations in FoxP3 can cause severe autoimmune disease as in IPEX (human) and scurfy mice, supporting the importance of Treg to keep autoreactive T cells in check (Lahl et al., 2007).

• Among circulating Treg, a large proportion is set to home to the skin (Hirahara et al., 2006).

• Chemokine CCL1 and its receptor CCR8 are involved in the chemoattractive process that guides Treg to the skin, as are the combinations of CCL5 and CCR5 and of CCL22, CCL17 and CCR4 (Colantonio et al., 2002; Hirahara et al., 2006).

• The cutaneous lymphocyte antigen (CLA) is a ligand for selectin like molecules PCAM and ECAM on the endothelial cell surface in a process that determines skin homing of lymphocytes including Treg (Iellem et al., 2003).

• CLA is abundantly expressed by CD4+ and CD8+ T cells that infiltrate vitiligo skin (Van den Wijngaard et al., 2000).

What is new?

• Progressive depigmentation in vitiligo involves a CTL mediated autoimmune response to melanocytes.

• Although circulating CTL targeting melanosomal antigens are observed in healthy individuals, their autoimmune impact is limited by the presence of regulatory T cells.

• In vitiligo patients however, CD8+ T cells are cytotoxic towards melanocytes and Treg apparently fail to keep autoimmunity in check.

• Studies presented here demonstrate a significant reduction in the abundance of Treg associated with reduced CCL22 expression in patient skin, whereas functional Treg are abundant in the circulation.

• These studies represent an important step forward in understanding how depigmentation can progress in vitiligo patients.

Paucity of Treg in vitiligo skin

• Treg abundance quantified. (A)Immunostaining of all T cells (blue) and Tregs (red) was light microscopically evaluated by two investigators.

Paucity of Treg in vitiligo skin

• (B) The percent Treg among T cells was calculated for 4–8 samples in each group, looking for significant differences to the % Treg found in adult normal skin by Student’s T test, with **P < 0.01, and *P < 0.05.

Paucity of Treg in vitiligo skin

Abundance of circulating Treg quantified by FACS analysis

(A) Representative FACS plots for a vitiligo and a control blood sample, with CD4+CD25+CD127low and FoxP3+ used to identify Treg.

Abundance of circulating Treg quantified by FACS analysis

(B) The average percentage of Treg among lymphocytes was quantified and compared among control and vitiligo samples. According to these data, the number of Treg circulating in patients does not support a systemic defect in Treg in vitiligo.Control Vitiligo.

Skin homing marker expression among circulating Treg analyzed by FACS.

(A) Representative control subject plots show sequential gating strategies for identifying Treg, as well as CD4+FoxP3) and CD4)FoxP3) subsets among CD3+ enriched lymphocytes. These subsets were analyzed for expression of CCR4, CCR5, CCR8 and CLA.

Skin homing marker expression among circulating Treg analyzed by FACS.

• (B) show a similar percentage of skin homing marker expression among patient and control Treg, and CD4)FoxP3) populations whereas CCR8 expression among non-Treg T cells was significantly higher in vitiligo subjects (not shown). The MFI representing CCR4, CCR5, CCR8 and CLA expression was similar among patient and control Treg

Skin homing chemokine CCL22 is markedly reduced in vitligo patient skin

Representative images are given for A. control and non-lesional vitiligo B. frozen skin immunostained for CCL22.C. The data reveal a significantly reduced abundance of CCL22

expressing non-lesional and intra-lesional vitiligo skin (P < 0.05).

D. Treg migration in response to CCL22 was also determined in control and vitiligo samples .

Treg abundance among migrated CD4+ lymphocytes was measured in the absence (spontaneous migration) or presence of CCR4 ligand CCL22. Treg migration data were summarized showing no difference in the % Treg responding to CCL22 among control and vitiligo CD4+ lymphocytes.

• Type 1 diabetes is characterized by destruction of insulin-producing β cells in the pancreatic islets by effector T cells.

• Tregs, defined by the markers CD4 and FoxP3, regulate immune responses by suppressing effector T cells and are recruited to sites of action by the chemokine CCL22.

• production of CCL22 in islets after intrapancreatic duct injection of double-stranded adeno-associated virus encoding CCL22 recruits endogenous Tregs to the islets and confers long-term protection from autoimmune diabetes in NOD mice.

• In addition, adenoviral expression of CCL22 in syngeneic islet transplants in diabetic NOD recipients prevented β cell destruction by autoreactive T cells and thereby delayed recurrence of diabetes.

• CCL22 expression increased the frequency of Tregs, produced higher levels of TGF-β in the CD4+ T cell population near islets, and decreased the frequency of circulating autoreactive CD8+ T cells and CD8+ IFN-γ–producing T cells.

• The protective effect of CCL22 was abrogated by depletion of Tregs with a CD25-specific antibody.

• Results indicate that islet expression of CCL22 recruits Tregs and attenuates autoimmune destruction of β cells.

• CCL22-mediated recruitment of Tregs to islets may be a novel therapeutic strategy for type 1 diabetes.

CCL22 expression in islets protects from diabetes development

(A) NOD females were injected at 8 weeks of age with 1.5 × 1011 vg of dsAAV8-RIP/CCL22, dsAAV8-RIP/GFP, or PBS (as control) via the pancreatic duct. Kaplan-Meier survival curves were derived from blood glucose data and compared using the log-rank test (P < 0.001 CCL22 versus PBS control; P < 0.01 versus GFP; P = 0.001 versus untreated).

(B) i.p. glucose tolerance tests 2 and 4 months after injection of dsAAV8-RIP/CCL22 in NOD mice and untreated NOD/SCID mice as control (n = 4 in each group). No significant difference in glucose tolerance was observed after intraductal injection and CCL22 expression. Results are expressed as mean ± SEM.

(C) Immunostaining for insulin-positive β cells (red) and islet-infiltrating leukocytes by CD45 staining (green) of PBS- and CCL22-NOD mice at indicated time points.

Note: the presence of intact islets containing strong insulin staining in CCL22-NOD mice compared with islets of PBS-NOD mice. Scale bar: 100 μm.

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Discussion

• I suggest that Local elevation of CCL22 will be a mechanism of therapy in T reg. pausing associated Autoimmune diseases.

• This peptide is constitutively expressed by macrophages and B cells but only expressed under activation by langerhans skin dendritic cells , so I think the expression by langerhans cell is the committed step in triggering of the mechanism of pathogenesis .

• This CCL22 is expressed in response to the activation effect of the transcription factor NF-kappa (a key link between the innate and adaptive immune systems) which has multiple hits .

• about the mechanisms I think they may selectively affect the expression of CCL22 as follow:

o Epigenetic mechanism: as there are 3 regions in the promoter of CCL22 gene regulate its expression and affected by methylation.

o Micro RNA silencing mechanism: this need verification.

o Post translational modification defect. this need verification.

o Protein - m RNA interactions . this need verification.

• The recent mechanisms revealed for some immunosuppressive therapies highlight the role of langerhance cells :

o Glucocorticosteroids Modify Langerhans Cells To ProduceTGF-b and Expand Regulatory T Cells 2010. (but no talk about CCL22).

o Effects of vitamin D3 on expression of tumor necrosis factor-alpha and chemokines by monocytes 2010 (talked about elevation in CCL22 )

• Mechanisms of UV-induced immunosuppression “review”2005

(talked about depletion of langerhans cells by UVA and in particular UVB “inhibits antigen presentation, induces the release of immunosuppressive cytokines and causes apoptosis of leukocytes, however, does not cause general immunosuppression but rather inhibits immune reactions in an antigen-specific fashion”) .

THANKS