Impact of Adalimumab Treatment on Interleukin-17 and Interleukin-17 Receptor Expression in Skin and Synovium of Psoriatic Arthritis Patients with Mild Psoriasis.

Interleukin (IL)-17 and tumor necrosis factor-alpha (TNF)-α are key players in psoriatic arthritis (PsA) pathogenesis. While both cytokines can be therapeutically targeted with beneficial clinical outcome, it is unclear whether inhibiting one cytokine will affect the other at sites of inflammation. If both act independently, this might provide a rationale for dual or combined inhibition of both cytokines. Here, we evaluated the effect of TNF blockade in PsA patients on IL-17 levels in both skin and synovial tissue biopsies. PsA patients with mild psoriatic skin lesions were randomized to receive either adalimumab or placebo for four weeks. Synovial and skin biopsies were obtained at weeks zero and four. Skin from healthy donors (HDs) was used for comparison. Expression of IL-17A, IL-17F, IL-17RA and IL-17RC was assessed by immunohistochemistry and analyzed with digital image analysis. We found relatively low levels of IL-17 and its receptors in the skin of PsA patients compared to HD, and only IL-17F in the dermis of lesional psoriatic skin was significantly higher compared to HD skin (p = 0.0002). Histologically IL-17A, IL-17F, IL-17RA and IL-17RC in skin and synovial tissue were not downregulated by adalimumab treatment. Thus, in this cohort of PsA patients with mild psoriasis, TNF blockade did not affect the protein levels of IL-17 cytokines and its receptors in skin and synovium, despite reduced cellular inflammation and improved clinical outcome for joint involvement.

Labeling and tracking of immune cells in ex vivo human skin.

Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.

c-Kit-positive ILC2s exhibit an ILC3-like signature that may contribute to IL-17-mediated pathologies.

Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44- ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1ß and IL-23. We also report a role for transforming growth factor-ß in promoting the conversion of c-Kit- ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis.

Tissue patrol by resident memory CD8+ T cells in human skin.

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.

Research Techniques Made Simple: High-Throughput Sequencing of the T-Cell Receptor.

High-throughput sequencing (HTS) of the T-cell receptor (TCR) is a rapidly advancing technique that allows sensitive and accurate identification and quantification of every distinct T-cell clone present within any biological sample. The relative frequency of each individual clone within the full T-cell repertoire can also be studied. HTS is essential to expand our knowledge on the diversity of the TCR repertoire in homeostasis or under pathologic conditions, as well as to understand the kinetics of antigen-specific T-cell responses that lead to protective immunity (i.e., vaccination) or immune-related disorders (i.e., autoimmunity and cancer). HTS can be tailored for personalized medicine, having the potential to monitor individual responses to therapeutic interventions and show prognostic and diagnostic biomarkers. In this article, we briefly review the methodology, advances, and limitations of HTS of the TCR and describe emerging applications of this technique in the field of investigative dermatology. We highlight studying the pathogenesis of T cells in allergic dermatitis and the application of HTS of the TCR in diagnosing, detecting recurrence early, and monitoring responses to therapy in cutaneous T-cell lymphoma.

Reduced CLEC9A expression in synovial tissue of psoriatic arthritis patients after adalimumab therapy.

OBJECTIVES: We aimed to investigate the early changes in expression of C-type lectin domain family 9, member A (CLEC9A), a C-type lectin that is specifically expressed by the CD141(+) dendritic cell subset that is involved in cross-presentation to CD8(+) T cells, by evaluating gene and/or protein expression in three different compartments [skin, synovial tissue (ST) and serum] after short-term adalimumab treatment in PsA patients compared with placebo. METHODS: Patients with active PsA and psoriasis were randomized to receive adalimumab or placebo for 4 weeks. Synovial and skin biopsies were obtained before and after 4 weeks of treatment and serum samples 4 weeks, 12 weeks and 1 year after treatment. Skin and serum from healthy donors were used as control. CLEC9A expression was assessed by immunohistochemistry, double immunofluorescence using terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick-end labelling (TUNEL), quantitative PCR and ELISA. RESULTS: CLEC9A expression was significantly higher in psoriatic skin compared with healthy donor. In psoriatic skin and PsA ST, CLEC9A(+) cells were in close proximity to TUNEL(+) cells. SF CLEC9A levels were significantly lower compared with paired PsA serum. Adalimumab treatment did not affect CLEC9A serum level and skin expression. However, ST CLEC9A protein expression was significantly decreased after adalimumab treatment compared with the placebo group while CLEC9A gene expression remained unchanged. There was a positive correlation between T cell numbers and ST CLEC9A protein expression. CD141(+) cell numbers and chemokine (C motif) receptor 1 expression were not affected with adalimumab treatment. CONCLUSION: Altogether, the present study suggests that the downregulation of synovial CLEC9A might be associated with a novel mechanism by which anti-TNF therapy might reduce CD8-mediated inflammation in PsA patients.

Composition of innate lymphoid cell subsets in the human skin: enrichment of NCR(+) ILC3 in lesional skin and blood of psoriasis patients.

Innate lymphoid cells (ILCs) are increasingly appreciated as important regulators of tissue homeostasis and inflammation. However, their role in human skin remains obscure. We found that healthy peripheral blood CD117(+) ILC3, lacking the natural cytotoxicity receptor (NCR) NKp44 (NCR(-) ILC3), CD117(-)NCR(-)CRTH2(-)CD161(+) ILC1, and CRTH2(+) ILC2, express the skin-homing receptor cutaneous lymphocyte antigen (CLA). NCR(+) ILC3 were scarce in peripheral blood. Consistently, we identified in normal skin ILC2 and NCR(-) ILC3, a small proportion of CD161(+) ILC1, and hardly any NCR(+) ILC3, whereas NCR(+) ILC3 were present in cultured dermal explants. The skin ILC2 and NCR(+) ILC3 subsets produced IL-13 and IL-22, respectively, upon cytokine stimulation. Remarkably, dermal NCR(-) ILC3 converted to NCR(+) ILC3 upon culture in IL-1ß plus IL-23, cytokines known to be involved in psoriatic inflammation. In line with this observation, significantly increased proportions of NCR(+) ILC3 were present in lesional skin and peripheral blood of psoriasis patients as compared with skin and blood of healthy individuals, respectively, whereas the proportions of ILC2 and CD161(+) ILC1 remained unchanged. NCR(+) ILC3 from skin and blood of psoriasis patients produced IL-22, which is regarded as a key driver of epidermal thickening, suggesting that NCR(+) ILC3 may participate in psoriasis pathology.

Cutting edge: virus selectively primes human langerhans cells for CD70 expression promoting CD8+ T cell responses.

The two outermost compartments of skin are populated by different Ag-presenting dendritic cell types. Epidermal Langerhans cells (LCs) are evolutionarily adapted to the continuous presence of harmless skin commensals by the selective lack of cell surface TLRs that sense bacteria. In this article, we analyze the ability of LCs and dermal dendritic cells (DDCs) to respond to virus infection. Live virus and intracellular TLR3-agonist dsRNA commit LCs more effectively than DDCs to stimulate naive CD8(+) T cell expansion and their differentiation into effector cells. This potent CD8(+) T cell-promoting capacity of LCs is causally related to high levels of virus-induced CD70 expression but not to IL-12 production. These data suggest a remarkable specialization of LCs in the induction of pathogen class-specific adaptive immunity. Whereas LCs ignore bacteria, they are superior to DDCs to initiate effective CD70-mediated CD8(+) T cells in response to virus stimulation.

Overrepresentation of IL-17A and IL-22 producing CD8 T cells in lesional skin suggests their involvement in the pathogenesis of psoriasis.

BACKGROUND: Although recent studies indicate a crucial role for IL-17A and IL-22 producing T cells in the pathogenesis of psoriasis, limited information is available on their frequency and heterogeneity and their distribution in skin in situ. METHODOLOGY/PRINCIPAL FINDINGS: By spectral imaging analysis of double-stained skin sections we demonstrated that IL-17 was mainly expressed by mast cells and neutrophils and IL-22 by macrophages and dendritic cells. Only an occasional IL-17(pos), but no IL-22(pos) T cell could be detected in psoriatic skin, whereas neither of these cytokines was expressed by T cells in normal skin. However, examination of in vitro-activated T cells by flow cytometry revealed that substantial percentages of skin-derived CD4 and CD8 T cells were able to produce IL-17A alone or together with IL-22 (i.e. Th17 and Tc17, respectively) or to produce IL-22 in absence of IL-17A and IFN-γ (i.e. Th22 and Tc22, respectively). Remarkably, a significant proportional rise in Tc17 and Tc22 cells, but not in Th17 and Th22 cells, was found in T cells isolated from psoriatic versus normal skin. Interestingly, we found IL-22 single-producers in many skin-derived IL-17A(pos) CD4 and CD8 T cell clones, suggesting that in vivo IL-22 single-producers may arise from IL-17A(pos) T cells as well. CONCLUSIONS/SIGNIFICANCE: The increased presence of Tc17 and Tc22 cells in lesional psoriatic skin suggests that these types of CD8 T cells play a significant role in the pathogenesis of psoriasis. As part of the skin-derived IL-17A(pos) CD4 and CD8 T clones developed into IL-22 single-producers, this demonstrates plasticity in their cytokine production profile and suggests a developmental relationship between Th17 and Th22 cells and between Tc17 and Tc22 cells.

Reduction of different inflammatory cell types of the innate immune system in psoriatic skin during etanercept treatment.

To investigate whether specific markers for innate immunity would diminish with successful treatment in psoriasis, we analyzed lesional and non-lesional skin biopsies taken from patients with moderate to severe psoriasis during 12 weeks of treatment with etanercept in correlation with the clinical response. In the clinical responders (PASI reduction >50%), all markers (CD3, CD68, CD161, elastase, BDCA-2, TNF-alpha) showed a decline during treatment, indicating a pivotal role for innate immunity in the pathogenesis of psoriasis.

Immunohistochemical analysis of regulatory T cell markers FOXP3 and GITR on CD4+CD25+ T cells in normal skin and inflammatory dermatoses.

Regulatory T cells (Treg) are a subset of T lymphocytes that play a central role in immunologic tolerance and in the termination of immune responses. The identification of these cells in normal and inflammatory conditions may contribute to a better understanding of underlying pathology. We investigated the expression of FOXP3 and GITR in normal skin and in a panel of different inflammatory dermatoses. Immunohistochemical double stainings in skin tissue sections revealed that FOXP3 and GITR were almost exclusively present on T cells that express both CD4 and CD25. Further, immunohistochemical double staining, as well as fluorescence-activated cell sorter analysis, on peripheral blood T cells showed that most FOXP3(+) cells expressed GITR and vice versa, whereas a minority were single-positive for these markers. The mean frequency of FOXP3(+) T cells in spongiotic dermatitis, psoriasis, and lichen planus was in the same range (25-29%), but the frequency of these cells in leishmaniasis appeared to be lower (approximately 15%), although this was not statistically significant. The mean frequency of GITR(+) T cells was fairly similar in all conditions studied (14-20%). Normal human skin also contained FOXP3(+) and GITR(+) cells in the same frequency range as in diseased skin, but the absolute numbers were, of course, much lower. In conclusion, frequencies of FOXP3(+) and GITR(+) T cells were similar in all inflammatory skin diseases studied and normal skin, despite the well-known differences among the inflammatory conditions under investigation.

Lichen planus remission is associated with a decrease of human herpes virus type 7 protein expression in plasmacytoid dendritic cells.

The cause of lichen planus is still unknown. Previously we showed human herpes virus 7 (HHV-7) DNA and proteins in lesional lichen planus skin, and significantly less in non-lesional lichen planus, psoriasis or healthy skin. Remarkably, lesional lichen planus skin was infiltrated with plasmacytoid dendritic cells. If HHV-7 is associated with lichen planus, then HHV-7 replication would reduce upon lichen planus remission. HHV-7 DNA detection was performed by nested PCR and HHV-7 protein by immunohistochemistry on lesional skin biopsies from lichen planus patients before treatment and after remission. Biopsies were obtained from lichen planus lesions before treatment (n = 18 patients) and after remission (n = 13). Before treatment 61% biopsies contained HHV-7 DNA versus 8% after remission (P = 0.01). HHV-7-protein positive cell numbers diminished significantly after remission in both dermis and epidermis. Expression of HHV-7 was mainly detected in BDCA-2 positive plasmacytoid dendritic cells rather than CD-3 positive lymphocytes. HHV-7 replicates in plasmacytoid dendritic cells in lesional lichen planus skin and diminishes after remission. This study further supports our hypothesis that HHV-7 is associated with lichen planus pathogenesis.

Deactivation of endothelium and reduction in angiogenesis in psoriatic skin and synovium by low dose infliximab therapy in combination with stable methotrexate therapy: a prospective single-centre study.

Psoriasis and psoriatic arthritis are inflammatory diseases that respond well to anti-tumour necrosis factor-alpha therapy. To evaluate the effects of anti-tumour necrosis factor-alpha treatment on expression of adhesion molecules and angiogenesis in psoriatic lesional skin and synovial tissue, we performed a prospective single-centre study with infliximab therapy combined with stable methotrexate therapy. Eleven patients with both active psoriasis and psoriatic arthritis received infusions of infliximab (3 mg/kg) at baseline, and at weeks 2, 6, 14 and 22 in an open-label study. In addition, patients continued to receive stable methotrexate therapy in dosages ranging from 5 to 20 mg/week. Clinical assessments, including Psoriasis Area and Severity Index (PASI) and Disease Activity Score (DAS), were performed at baseline and every 2 weeks afterward. In addition, skin biopsies from a target psoriatic plaque and synovial tissue biopsies from a target joint were taken before treatment and at week 4. Immunohistochemical analysis was performed to detect the number of blood vessels, the expression of adhesion molecules and the presence of vascular growth factors. Stained sections were evaluated by digital image analysis. At week 16, the mean PASI was reduced from 12.3 +/- 2.4 at baseline to 1.8 +/- 0.4 (P

T cells in psoriatic lesional skin that survive conventional therapy with NB-UVB radiation display reduced IFN-gamma expression.

The type 1 T cell-derived cytokine interferon gamma (IFN-gamma) is overexpressed in psoriatic lesional skin. Recently, we have shown that a single high erythemal dose of broad-band ultraviolet B (UVB) irradiation reduces type 1 and favors type 2, i.e. interleukin-4 (IL-4), cytokine expression in normal and psoriatic skin. In this study, we wanted to see whether conventional narrow-band UVB (NB-UVB) therapy (i.e. repeated exposure to nonerythemal doses) also affects type 1/type 2 cytokine expression of T cells present in chronic plaque type psoriatic lesions. Staining of cryostat sections showed decreased expression of both IFN-gamma and IL-4 in situ after NB-UVB therapy. CD4(+) dermal T cell lines, derived from psoriatic lesional skin, displayed significantly decreased intracellular IFN-gamma expression during and after NB-UVB therapy as compared to pretreatment values. Intracellular IL-4 expression was increased in most patients after therapy. Analysis of the supernatants of these stimulated dermal T cells revealed that IFN-gamma production decreased significantly following NB-UVB therapy, whereas IL-4 expression increased in the T cell supernatants from most patients, confirming the intracellular determinations. In addition, IL-10 and transforming growth factor-beta levels in the supernatants appeared to be increased in the majority of patients following UVB therapy. Apart from the well-known killing effect of UVB on T cells, our results show that the improvement in psoriatic skin following NB-UVB therapy is also due to a reduced capacity of the surviving dermal T cells to express the proinflammatory cytokine IFN-gamma.

IL-4 expression by neutrophils in psoriasis lesional skin upon high-dose UVB exposure.

BACKGROUND: Upon a single high dose of UVB irradiation of psoriatic lesional skin, IFN-gamma expression is decreased, whereas IL-4 expression is enhanced. A similar type 1 to type 2 shift was found in dermal T cells derived from irradiated lesional skin as compared to unexposed lesional psoriatic skin. We have found recently that the IL-4 protein detected in situ upon UVB exposure of normal skin was not associated with T cells but with infiltrating neutrophils. OBJECTIVE: To determine which cell types express IL-4 in psoriatic skin after UVB irradiation. METHODS: Skin biopsies were obtained from healthy controls and psoriasis patients before and after local UVB exposure. Double immunohistochemical stainings were performed to determine the identity of IL-4-expressing cells. RESULTS: In the irradiated skin of both healthy controls and patients, IL-4-positive cells coexpressed elastase and CD15, but not CD3. CONCLUSION: IL-4-expressing cells found in psoriatic skin after a single high-dose UVB exposure appeared to be neutrophils.

Ultraviolet-B irradiation decreases IFN-gamma and increases IL-4 expression in psoriatic lesional skin in situ and in cultured dermal T cells derived from these lesions.

Type 1 cytokine producing T cells play an important role in the pathogenesis of psoriasis. Ultraviolet-B (UVB) irradiation is effective in the treatment of this disease. In normal skin, UVB causes a change in dermal microenvironment, leading to a decrease of IFN-gamma expressing type 1 T cells and a concurrent increase of IL-4 expressing type 2 T cells. The aim of this study was to show whether UVB irradiation causes a like-wise shift of type 1 and type 2 responses in psoriatic skin. For this purpose, biopsies were obtained from the lesional skin of psoriatic patients before, 2 days and 14 days after a single exposure to 4 MED UVB. Sections from these biopsies were immunostained (CD3, IFN-gamma and IL-4) or RNA was extracted and analyzed for the expressions of IFN-gamma and IL-4 by PCR. In addition, primary cultures of T cells from dermal cell suspensions were stained intracellularly for IFN-gamma and IL-4 expression and CD4+ and CD8+ T subsets were analyzed by flow cytometry. IFN-gamma was abundantly expressed in situ before irradiation and decreased in all patients after UVB irradiation, whereas IL-4 expression was variably expressed before irradiation and increased in different degrees after irradiation. Cytokine mRNA expressions determined by PCR showed a clear decrease of IFN-gamma and increase of IL-4 following UVB irradiation. Both CD4+ and CD8+ dermal T cells were found to produce less IFN-gamma and more IL-4 following UVB irradiation as determined by flow cytometry. Decrease in IFN-gamma expression and increase in IL-4 expression of dermal T cells in psoriatic lesions after UVB irradiation may lead to decrease in local immunoreactivity. These changes could be part of the therapeutic effects of UVB on psoriasis.

Ultraviolet B radiation induces a transient appearance of IL-4+ neutrophils, which support the development of Th2 responses.

UVB irradiation can cause considerable changes in the composition of cells in the skin and in cutaneous cytokine levels. We found that a single exposure of normal human skin to UVB induced an infiltration of numerous IL-4(+) cells. This recruitment was detectable in the papillary dermis already 5 h after irradiation, reaching a peak at 24 h and declining gradually thereafter. The IL-4(+) cells appeared in the epidermis at 24 h postradiation and reached a plateau at days 2 and 3. The number of IL-4(+) cells was markedly decreased in both dermis and epidermis at day 4, and at later time points, the IL-4 expression was absent. The IL-4(+) cells did not coexpress CD3 (T cells), tryptase (mast cells), CD56 (NK cells), and CD36 (macrophages). They did coexpress CD15 and CD11b, showed a clear association with elastase, and had a multilobed nucleus, indicating that UVB-induced infiltrating IL-4(+) cells are neutrophils. Blister fluid from irradiated skin, but not from control skin, contained IL-4 protein as well as increased levels of IL-6, IL-8, and TNF-alpha. In contrast to control cultures derived from nonirradiated skin, a predominant type 2 T cell response was detected in T cells present in primary dermal cell cultures derived from UVB-exposed skin. This type 2 shift was abolished when CD15(+) cells (i.e., neutrophils) were depleted from the dermal cell suspension before culturing, suggesting that neutrophils favor type 2 T cell responses in UVB-exposed skin.