**4. T cells differentiated from bone marrow derived hematopoietic cells of psoriatic patients are functionally different from normal T cells**

Since T cells are derived from bone marrow hematopoietic cells, it is suggested that hematopoietic cells are partly relevant to the dysfunction of T cells in psoriasis. To demonstrate whether T cells are produced inherently dysfunctional from the immune system, Zhang et al. cultured bone marrow CD34+ cells from psoriatic patients and induced them to differentiate into T cells and CD4+CD25+ regulatory T cells *in vitro*. A further functional study revealed abnormal characters of these cells compared to normal bone marrow derived ones.

The main hallmark of CD4+CD25+ T cells is their immune regulatory function by interacting with effector T cells. Several studies have reported that the CD4+CD25+ T-lymphocyte subpopulation in peripheral blood and lesional skin demonstrates a less inhibitory effect on effector T cells, leading to accelerated proliferation of pathogenic/effector T-cells in autoimmune diseases, especially in psoriasis. Although the proportion of CD4+CD25+ T cells and FOXP3 gene expression are comparable in both psoriatic and healthy samples, proliferation of psoriatic bone marrow derived CD4+CD25+ T cells is significantly attenuated and secretion of cytokines IL-2 and IL-10 is decreased compared to normal controls in response to streptococcal superantigen (Strep-A). In particular, CD4+CD25+ T cells differentiated from psoriatic CD34+ cells are functionally insufficient to restrain proliferation of activated effector T-cells. That is to say, the function of CD4+CD25+ T cells derived from psoriatic bone marrow CD34+ cells *in vitro* is similar to that of peripheral CD4+CD25+ Tlymphocytes of psoriatic patient *in vivo.*

In another study, bone marrow CD34+ hematopoietic cells from psoriatic patients with family history were induced into effector T cells and their functions such as in vitro proliferation ability, secretion of cytokines IL-4, IL-8 and IFN–γ, and their ability to induce human keratinocytes producing C-myc, Bcl-xL, and Ki67 proteins were compared with their counterpart from healthy objects. The differentiated T cells from CD34+ cells of psoriatic patients showed higher proliferative activity and stronger capacity to secret Th1 cytokines in response to streptococcal superantigen and could induce expression of C-myc and Ki67, but not Bcl-XL in keratinocytes co-cultured with psoriatic differentiated T cells.

These studies show that regulatory as well as effector T cells differentiated from CD34+ cells of psoriatic patients, but not normal controls, are functionally similar to those psoriatic circulating T cells and suggest that dysfunctional activity of T cells in psoriatic patients can be traced back to the early development of hematopoietic cells.

Psoriasis and Stem Cells 7

Sabat R, Philipp S, Hoflich C, et al. Immunopathogenesis of psoriasis. Exp Dermatol 2007,

Abrams J R, Kelley S L, Hayes E, et al. Blockade of T lymphocyte costimulation with

Nickoloff BJ, Schrohder JM, von den Driesch P, et al. Is psoriasis a T- cell disease? [J]. Exp

Li X, Fan X, Zhang K, Yin G, Liu Y. Influence of psoriatic peripheral blood CD4+T and

Woods A C, Mant M J. Amelioration of severe psoriasis with psoriatic arthritis for 20 years

Snowden J A, Heaton D C. Development of psoriasis after syngeneic bone marrow

Altmeyer P, Munz DL, Chilf G, et al. Morphological and functional findings of fixed

Zhang K, Zhang R, Li X, et al. The mRNA expression and promoter methylation status of

K. Zhang, R. Hou, X. Niu, *et al*. Decreased colony formation of high proliferative potential

Yin G, Li J, Wan Y, et al. Abnormality of RUNX1 signal transduction in psoriatic CD34+bone

Helms C, Cao L, Krueger J G, et al. A putative RUNX1 binding site variant between

Zhang K, Zhang R, Li X, et al. Promoter methylation status of p15 and p21 genes in HPP-

Zhang K, Li X, Yin G, et al. Functional characterization of CD4+CD25+ regulatory T cells

phagocytes in psoriatics. Arch Dermatol Res 1983, 275: 95-9.

psoriasis. British Journal of Dermatology 2010, 163, 93–101

with psoriasis. Clin Exp Dermatol, 2007, 32: 702.

marrow cells. Br J Dermatol. 2011, 164, 1043-1052.

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cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (CTLA4Ig) reverses the cellular pathology of psoriatic plaques, including the activation of keratinocytes, dendritic cells, and endothelial cells. J Exp Med 2000, 192: 681–694. Gordon K B, Papp K A, Hamilton T K, et al. Efalizumab for patients with moderate to severe plaque psoriasis: a randomized controlled trial. JAMA 2003, 290: 3073–3080. Boyman O, Hefti H P, Conrad C, et al. Spontaneous development of psoriasis in a new

animal model shows an essential role for resident T cells and tumor necrosis factor-

CD8+T lymphocytes on C-myc, Bcl-xL and Ki67 gene expression in keratinocytes.

after allogeneic haematopoietic stem cell transplantation. Ann Rheum Dis 2006, 65:

transplant from psoriatic donor: further evidence for adoptive autoimmunity. Br J

the p16 gene in colony-forming cells with high proliferative potential in patients

colony-forming cells and granulocyte-macrophage colony-forming units and increased Hes-1 expression in bone marrow mononuclear cells from patients with

SLC9A3R1 and NAT9 is associated with susceptibility to psoriasis. Nat Genet 2003:

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bone marrow-derived CD34+ cells of psoriatic patients with family history.

differentiated in vitro from bone marrow-derived haematopoietic cells of psoriasis patients with a family history of the disorder. Br J Dermatol, 2008; 158 (2): 298-305.

**6. References** 

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Dermatol 1997, 137: 130–132.

#### **5. The bionomics of psoriatic bone marrow mesenchymal stem cells**

Mesenchymal stem cells, also referred as marrow stromal cells, are another important type of stem cells in bone marrow. Cytokines secreted by bone marrow mesenchymal stem cells (BMSCs) along with extracellular matrix compose the hematopoietic microenvironment and influence hematopoiesis. More than 30 hematopoietic cytokines and growth factors including TNF-a, IL-1, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ and IL-18 are reportedly secreted by BMSCs and many of them could influence immune reaction of peripheral blood. Secretion of SCF, granulocyte colony-stimulating factor (G-CSF) and IL-6 is increased in *in vitro* cultured BMSCs from psoriatic patients, while that of IL-1a, IL-1b, IL-3, IL-8, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), TNF-a, leukaemia inhibitory factor (LIF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) is decreased and the levels of GM-CSF, IL-11 or IL-7 is not altered. *Pearson* correlation analysis demonstrates that those cytokine levels are not correlated with PASI scores, indicating that abnormal secretion of cytokines is due to anomaly of BMMSCs themselves rather than systemic inflammatory response.

On the other hand, BMSCs are also characterized by their ability to differentiate into multiple mesenchymal lineages, including osteocytes, chondrocytes, adipocytes, endothelial cells and skeletal muscle cells under controlled *in vitro* conditions. Studies have found that BMSCs from psoriatic patients have lower proliferative and passage ability and are more prone to differentiate into vascular endothelial cells (VEC) compared with those from healthy subjects under the same induction conditions. Moreover, this differentiation ability is paralleled with the disease severity. In addition, specimens from a patient whose parents also have psoriasis could spontaneously differentiate into VECs. Further studies on gene expression using RNA sequencing showed a total of 475 genes mostly enriched in prostaglandin (PG) and prostanoid metabolic process (unpublished data) are differentially expressed in this patient.

Studies on differential gene expression of BMSCs from 4 psoriatic patients and 3 healthy subjects found a total of 1617 genes were differently expressed by more than 2-fold between the two groups, among which 324 genes were upregulated and 1293 genes were downregulated in psoriatic patients. GO analysis revealed the first five gene-enriched GO terms were immune response, inflammatory response, antigen processing and presentation of peptide, chemotaxis, and cell adhesion. While the first five highly enriched factor terms were positive regulation of CD4+CD25+ alpha-beta regulatory T cell differentiation, lipoprotein particle clearance, antigen processing and presentation of peptide, negative regulation of peptidase activity, and positive regulation of cholesterol storage (unpublished data). These terms have been confirmed to participate in the onset and development of psoriasis.

Taken together, these studies suggest that BMSCs of psoriatic patients are abnormal in proliferation, differentiation, passage ability, secretion of multiple cytokines and gene expression, and may partly participate in the occurrence and development of psoriasis. In other words, psoriasis is a multi-system disease that involves not only the epidermis, but also the hematopoietic system, immune system, neuroendocrine system, and so on. With continued research, various stem cells may be confirmed to be involved in the generation and development of psoriasis.

#### **6. References**

6 Psoriasis

Mesenchymal stem cells, also referred as marrow stromal cells, are another important type of stem cells in bone marrow. Cytokines secreted by bone marrow mesenchymal stem cells (BMSCs) along with extracellular matrix compose the hematopoietic microenvironment and influence hematopoiesis. More than 30 hematopoietic cytokines and growth factors including TNF-a, IL-1, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-γ and IL-18 are reportedly secreted by BMSCs and many of them could influence immune reaction of peripheral blood. Secretion of SCF, granulocyte colony-stimulating factor (G-CSF) and IL-6 is increased in *in vitro* cultured BMSCs from psoriatic patients, while that of IL-1a, IL-1b, IL-3, IL-8, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), TNF-a, leukaemia inhibitory factor (LIF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) is decreased and the levels of GM-CSF, IL-11 or IL-7 is not altered. *Pearson* correlation analysis demonstrates that those cytokine levels are not correlated with PASI scores, indicating that abnormal secretion of cytokines is due to anomaly of BMMSCs

On the other hand, BMSCs are also characterized by their ability to differentiate into multiple mesenchymal lineages, including osteocytes, chondrocytes, adipocytes, endothelial cells and skeletal muscle cells under controlled *in vitro* conditions. Studies have found that BMSCs from psoriatic patients have lower proliferative and passage ability and are more prone to differentiate into vascular endothelial cells (VEC) compared with those from healthy subjects under the same induction conditions. Moreover, this differentiation ability is paralleled with the disease severity. In addition, specimens from a patient whose parents also have psoriasis could spontaneously differentiate into VECs. Further studies on gene expression using RNA sequencing showed a total of 475 genes mostly enriched in prostaglandin (PG) and prostanoid metabolic process (unpublished data) are differentially

Studies on differential gene expression of BMSCs from 4 psoriatic patients and 3 healthy subjects found a total of 1617 genes were differently expressed by more than 2-fold between the two groups, among which 324 genes were upregulated and 1293 genes were downregulated in psoriatic patients. GO analysis revealed the first five gene-enriched GO terms were immune response, inflammatory response, antigen processing and presentation of peptide, chemotaxis, and cell adhesion. While the first five highly enriched factor terms were positive regulation of CD4+CD25+ alpha-beta regulatory T cell differentiation, lipoprotein particle clearance, antigen processing and presentation of peptide, negative regulation of peptidase activity, and positive regulation of cholesterol storage (unpublished data). These terms have been confirmed to participate in the onset and development of

Taken together, these studies suggest that BMSCs of psoriatic patients are abnormal in proliferation, differentiation, passage ability, secretion of multiple cytokines and gene expression, and may partly participate in the occurrence and development of psoriasis. In other words, psoriasis is a multi-system disease that involves not only the epidermis, but also the hematopoietic system, immune system, neuroendocrine system, and so on. With continued research, various stem cells may be confirmed to be involved in the generation

**5. The bionomics of psoriatic bone marrow mesenchymal stem cells** 

themselves rather than systemic inflammatory response.

expressed in this patient.

and development of psoriasis.

psoriasis.


**1. Introduction** 

**2. Psoriasis pathogenesis** 

**2** 

*Italy* 

**by Keratinocytes** 

Anna Balato, Nicola Balato, Matteo Megna,

Maria Schiattarella, Serena Lembo and Fabio Ayala *Department of Dermatology – University of Naples Federico II* 

**Pathogenesis of Psoriasis: The Role of Pro-Inflammatory Cytokines Produced** 

Psoriasis is a chronic, inflammatory skin disease affecting 2 to 3% of the white population (Gudjonsson & Elder, 2007). It is a multifactorial disease since its development depends on a complex interplay of genetic and environmental factors. As no pathogen has been consistently identified within psoriatic plaques (indeed skin infections are rare in lesions because of antimicrobial peptides) (Nomura et al., 2003), an autoimmune basis for the chronic inflammation is the dogma for this complex disorder. Psoriasis is characterized by macroscopic (clinical) and corresponding microscopic (histological) skin alterations and leads to considerable impairment of the quality of life of the affected patients. Special forms of psoriasis (e.g. arthropathic form) can be accompanied by severe extra-cutaneous changes.

Psoriasis is usually identified by erythematous, raised, scaly skin lesions. These clinical features are explained by impressive growth and dilation of superficial blood vessels (elongated/hyperplastic capillaries in the papillary dermal region) and equally impressive hyperplasia of the epidermis. Epidermal growth occurs in a pattern termed ''psoriasiform'' hyperplasia, which describes both elongated rete pegs, thickening (acanthosis), and differentiation changes (Krueger & Bowcock, 2005). In psoriatic epidermis, keratinocytes proliferate and mature rapidly so that terminal differentiation, normally occurring in granular keratinocytes and then squamous corneocytes, is incomplete. Hence, squamous keratinocytes aberrantly retain intact nuclei (parakeratosis) and release few extracellular lipids that normally cement adhesions of corneocytes. The failure of psoriatic corneocytes to stack normally and to secrete extracellular lipids cause scaling and a break in the protective barrier whereas marked dilation of blood vessels in the dermis causes the visible redness of psoriatic skin lesions. The extensive infiltration of mononuclear immune cells in the dermis and epidermis (T cells and dendritic cells in the dermis and polymorphonuclear leucocytes such as neutrophils within small foci in the stratum corneum) is another defining feature of psoriasis histopathology and a key point of its pathogenesis. The pathogenesis of psoriasis is considered to be an immunologically mediated process that takes place upon a favourable

Zhang K, Liu R, Yin G, et al. Differential cytokine secretion of cultured bone marrow stromal cells from patients with psoriasis and healthy volunteers. Eur J Dermatol 2010; 20 (1): 1-5.
