IL-17 Expression Negatively Correlates with γδ T-Cell Accumulation in Human Psoriasis Lesions: A Novel Implication for Disease Pathogenesis

doi:10.21203/rs.3.rs-4713172/v1

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IL-17 Expression Negatively Correlates with γδ T-Cell Accumulation in Human Psoriasis Lesions: A Novel Implication for Disease Pathogenesis

https://doi.org/10.21203/rs.3.rs-4713172/v1

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Psoriasis, an inflammatory autoimmune disease, arises from intricate interactions between the immune system and epithelium. Recent reports have suggested new roles for gamma delta (γδ) T-cells in addition to immune surveillance, however, it remains to be determined whether the mechanisms identified in psoriasis murine models have a similar role in humans. The present study aimed to examine the relationship between IL-17 mRNA level and the γδ T-cell proliferation and to clarify their function in the psoriatic human samples. The study involved 20 patients diagnosed with psoriasis and 16 control subjects. Expression of the IL-17 gene was measured in formalin-fixed paraffin-embedded (FFPE) tissues by qRT-PCR method. TCRγδ⁺ immunofluorescence staining was performed to measure the proliferation and distribution of γδ T-cells in the same samples. In psoriatic lesion biopsies, TCRγδ⁺ T-cell percentage was found higher than the control samples. Additionally, psoriasis patients exhibited elevated levels of IL-17 gene expression. In addition, this study showed a weak negative correlation between γδ T-cell proliferation and IL-17 gene expression in psoriatic skin samples, highlighting the novel effector functions of these cells in psoriasis pathogenesis.

Autoimmune Disease

Gamma Delta T Cell

IL-17

Inflammation

Psoriasis

Psoriasis is an autoimmune disease that results from cross-talk between the epithelium and the immune system. Its prevalence ranges from 0.09–11.43%. Also, it has been linked to several comorbidities and other autoimmune diseases, such as inflammatory bowel disease, arthritis, cardiovascular disease, obesity, and diabetes mellitus (Qi et al. 2021).

Gamma-delta (γδ) T cells are a distinct subgroup of T lymphocytes (Ribot et al. 2021). Interleukin-17 (IL-17) is a pro-inflammatory cytokine that is linked to inflammatory and autoimmune diseases including psoriasis (Martin et al. 2013). In psoriasis, the immune response adapts and associates with pro-inflammatory subtypes of Th1 (T-helper), Th17, and Th22 cells. Th17 cells are widely recognized as the primary IL-17 producers. However, according to recent data, innate immune cells are responsible for producing a significant amount of the IL-17 released during inflammation (Keijsers et al. 2014).

Psoriasis does not occur naturally as a disease in mice. However, various xenotransplantation models and immunological reconstitution approaches have elucidated the psoriasis immunopathogenesis (Keijsers et al. 2014). In addition to IL-17-producing CD4⁺ T cells (Th17) in inflammatory diseases, γδ T-cells are the primary sources of IL-17 in mouse models (Akitsu and Iwakura 2018). The available evidence demonstrated that γδ T-cells that are abnormally activated can regulate the pathogenesis of psoriasis (Qi et al. 2021). However, the local immune response, cellular changes, and tissue-specific gene expression directly within the skin have not been fully elucidated (Qi et al. 2021). This lack of understanding hinders the development of more effective treatments for psoriasis. Additionally, human γδ T-cells display notable flexibility throughout their lifespan, with the capacity to generate various cytokines when exposed to specific triggers which contributes further to the difficulty in translating observations from murine models to humans (Qu et al. 2022).

While murine and human γδ T cells are similar in psoriasis pathogenesis, there are notable differences. Mice mainly use Vγ5Vδ1 TCR for psoriasis-like inflammation, while humans have more diverse TCR expressions like Vδ1, Vδ2, and Vδ3. The tissue distribution of γδ T cell subsets varies between mice and humans, possibly affecting their role in psoriatic inflammation. Human γδ T cells have diverse cytokine production profiles, including IL-17A and IFN-γ, unlike murine γδ T cells that are known for robust IL-17A production. Also, some γδ T-cell subsets in humans recognize stress-induced self-ligands on keratinocytes, contributing to psoriasis, while murine γδ T cells may respond to different antigens in the skin (Hu et al. 2023). Therefore, translating these findings to humans remains a challenge. Also, most studies on gamma-γδ T cells and IL-17 have been conducted using peripheral blood, cell cultures, or animal models (Moens et al. 2011; Sandrock et al. 2018; Agerholm et al. 2019). Nevertheless, the in-depth understanding of γδ T-cell function in the local immune response, cellular alterations, and gene expression in human psoriatic skin samples is not well understood.

Therefore, this study aimed to investigate the function of γδ-T cells in the pathophysiology of psoriasis and to gain new insights into its management by examining the relationship between IL-17 gene expression and γδ T-cell proliferation in human psoriatic tissue samples.

Participants

The sample size of the study was determined with a power analysis using G*Power software version 3.1.9.4. The statistical error rate for type I was set at 0.05, with a power of 0.80. Based on the analysis, a significant intergroup difference required a minimum of 14 individuals per group.

Each participant was chosen from the Goztepe Prof. Dr. Suleyman Yalcın City Hospital's Pathology Department. After receiving approval from the Ethics Committee at Goztepe Prof. Dr. Suleyman Yalcin City Hospital (School of Medicine, Istanbul Medeniyet University), the control group comprised 16 healthy volunteers while the patient group comprised 20 volunteers diagnosed with psoriasis. Following the Helsinki Declaration, each participant gave their informed permission. The skin samples of the participants were obtained from skin biopsies that were directly impacted by psoriasis, whereas the skin samples of the control group were collected from healthy skin tissue confirmed by the H&E-stained sections.

Histopathology

Histopathological assessment was conducted on formalin-fixed paraffin-embedded (FFPE) tissue samples of psoriasis and the control group. The histo-morphology of the samples was evaluated using the hematoxylin and eosin (H&E) staining method.

Immunofluorescence

Immunofluorescence staining was performed on FFPE tissue samples of psoriasis lesioned biopsies. After the deparaffinization and dehydration, tissue antigens were retrieved on the slides with 1X Citrate Buffer (pH:6) in the decloaking chamber. After the retrieval, samples were incubated in 0.3% H₂O₂ solution to prevent endogenous peroxidase activity. The sections were incubated with superblock solution and then anti-TCRγδ primary antibody solution (GTX15594, Irvine, CA, USA) at a dilution of 1:200 at + 4°C. After the secondary antibody incubation for 2 hours at room temperature (Alexa 488, ab150113, Cambridge, UK), Hoechst 33342 (Thermo Scientific, Massachusetts, USA) was used in a 1:1 glycerol mounting medium for counterstaining. Sections that underwent the same procedure without the primary antibody incubation were used as negative controls. After that, the specimens were examined and studied with a fluorescence microscope (DMi8-S; Leica). After counting all inflammatory cells, the percentage of TCRγδ⁺ T cells in total inflammatory cells was analyzed.

RT-PCR

IL-17 mRNA expression in each sample was examined. Trizol reagent (tripleXtractor; GRiSP Research Solutions, Porto, Portugal) was used to extract Total RNA samples from FFPE tissues. UV absorbance ratio at 260 and 280 nm (DeNovix Inc., Wilmington, USA) was used to assess the purity of the RNA. For RNA, a ratio of 1.8 to 2.0 was regarded as pure. Complementary DNA (cDNA) synthesis was performed with 500 ng of the total RNA by using the SCRIPT cDNA Synthesis Kit (Jena Bioscience, Germany). For the PCR procedures, 40 ng of cDNA was used with Taq PCR Master Mix (EurX, Poland). The Light Cycler® 480 System II (Roche Applied Science, Switzerland) was used for RT-qPCR analysis. The IL-17 mRNA expression was normalized using 18S rRNA as a housekeeping gene. The experiment was performed three times. The level of IL-17 expression was determined using the 2^−ΔΔCT calculation method.

Statistical Analysis

GraphPad Prism 8.4.2 was used for statistical calculation and analysis of the data. The data was analyzed with the Mann-Whitney U-test. The Spearman correlation coefficient was then calculated to measure the correlation between the γδ T-cell proliferation and IL-17 mRNA level in psoriasis patients. Significance was attributed to p-values below 0.05. Statistical significance was marked as *: p < 0.05, ***: p < 0.001.

Participants' clinical characteristics

Twenty psoriasis patients were included in the research; nine (45%) were male and eleven (55%) were female. The patient's average age was 49.05 ± 19.95 years. In the control group (n = 16), 2 participants were male (12.5%) and 14 participants were female (87.5%). The mean age distribution of the participants was 38.81 ± 6.48 years.

Histopathological assessment

The diagnosis of psoriasis was made based on the observation of the following symptoms: perivascular regions of parakeratosis in the epidermal keratinocytes in the stratum corneum, accompanied by neutrophil plaques; regular acanthosis with elongated rete; and irregular areas of hypergranulosis and hypogranulosis in the epidermis. The dermis showed signs of mostly lymphocytic infiltration, with neutrophil aggregates in the layer of stratum spinosum and dilated arteries in the dermal papillae (Fig. 1a). Control skin dermis showed no signs of inflammation (Fig. 1b).

TCRγδ⁺ T-cells increased with psoriasis

Antibodies directed against TCRγδ were used to stain TCRγδ⁺ T cells in both control and psoriasis tissue samples (Fig. 1c). The total number of inflammatory cells and TCRγδ⁺ T cells in the reticular and papillary dermis were analyzed in each biopsy with a psoriasis lesion. In the psoriasis group, the percentage of TCRγδ⁺ T cells was substantially higher in comparison to the control group (p < 0.0001) (Fig. 2a).

mRNA level of IL-17 in psoriasis patients

RT-PCR was performed to evaluate the level of IL-17 mRNA expression in biopsies taken from patient lesions with psoriasis and control biopsies. Compared to the control samples, the psoriasis group's IL-17 mRNA expression was shown to be up-regulated (p = 0.03) (Fig. 2b). A weak negative correlation was determined in psoriatic skin samples between the γδ T-cell proliferation and IL-17 mRNA level (p = 0.021).

Recent reports have indicated the new roles of γδ T-cells in tissue physiology in humans and mice, in addition to immune surveillance. However, it remains to be determined whether the mechanisms identified in murine models can be applied to human γδ T-cells (Qu et al. 2022). This study re-examines the involvement of γδ T-cells in the skin of psoriatic and healthy individuals. We demonstrated that although there was an increase in the number of γδ T-cells and the level of IL-17 mRNA, a weak negative correlation was evident in psoriatic human skin in vivo.

Psoriasis is a well-known inflammatory skin condition that has been used as a model for studying inflammatory diseases. Upon calculating the percentage of γδ T cells in psoriatic skin, we found a significant γδ T-cell population in psoriatic lesions. In addition to tissue-resident cells, the increase in the number of γδ T-cell populations demonstrated the accumulation of these cells at the inflammation site. The enhanced γδ T-cell infiltration in the dermis suggests that these cells may migrate from the periphery into the skin dermis via chemotaxis or expand locally (Cai et al. 2011). In skin colonization, γδ T-cells are attracted by keratinocyte-produced chemokines such as CCL20 and CCL27 that act as chemoattractant for γδ T-cells with specific receptors of CCR6 and CCR10. The interaction between chemokines and receptors may prompt γδ-T cells to migrate toward the epidermal layer for self-renewal and residency (Hu et al. 2023).

Since γδ⁺ T-cells are relatively infrequent in human skin compared to murine skin (Cai et al. 2011), it is important to determine if they have a similar role in immune control as their murine homologs. Although cells expressing the pan-γδ TCR have been identified in psoriasis (Seung et al. 2007), we demonstrated for the first time the negative correlation between IL-17 and γδ T-cell expansion, suggesting complex immunological interactions in the disease microenvironment. The qRT-PCR evaluation revealed an upregulation of IL-17 gene expression in the psoriasis biopsies. This finding is consistent with previous research (Cai et al. 2011). Our current study's findings depicted that IL-17-producing dermal γδ T-cells have a major function in psoriasis pathogenesis. The mechanism behind this phenomenon is considered to be the increase in the number of γδ T-cells at the inflammation site in the dermis.

Given that γδ T cells are the primary infiltrating immune cells that secrete IL-17 in psoriasis, an increase in IL-17 mRNA level was not unexpected in this study (Akitsu and Iwakura 2018). However, γδ T-cells represent a heterogeneous group with cytotoxic or regulatory functions depending on the local environment and the specific signals they encounter (Hu et al. 2023). Recent studies have revealed profound tissue-specific transcriptional signatures for human and mouse γδ T-cells, indicating that tissue residency and the impact of the microenvironment on γδ T-cell function are reflected in their transcriptional profiles. It has been demonstrated that γδ T-cells are shaped by the microenvironment and exert tissue-specific functions depending on the signals they receive (Qu et al. 2022). In the context of psoriasis, the higher prevalence of γδ T-cells does not necessarily imply an elevated production of IL-17. In models of psoriasis and dermatitis, IL-17 is produced by dermal Vγ4⁺ and Vγ6⁺ γδ T cells. Paradoxically, another study demonstrated that IL-17-producing γδ T-cells are also essential for maintaining skin homeostasis in steady-state skin physiology (Qu et al. 2022). Alternatively, these cells may be engaged in a different function, such as regulating other immune cells or participating in tissue repair reflecting the complexity of their roles in immune responses, including psoriasis. (Bonneville et al. 2010; Vantourout and Hayday 2013). Therefore, our investigation into IL-17 expression and γδ T-cell distribution in psoriasis patients underscores the heterogeneity of immune responses across rodents and humans.

The clinical significance of our findings lies in the weak association observed between IL-17 expression and γδ T-cell proliferation in skin samples from psoriasis patients, offering insights into immune dysregulation in this chronic skin condition. This study also highlights the importance of considering species-specific factors in the development of therapeutic strategies targeting γδ T-cell-associated cytokines. Given that our research involved direct examination of human biopsy tissues, the findings are likely to have a greater impact on addressing different characteristics of γδ T-cells in humans.

In conclusion, our findings suggest a negative association between IL-17 production and γδ T-cell accumulation in the dermis. However, further understanding of the factors that regulate the plasticity of γδ T-cell effector functions and the molecular cues that drive their differentiation into specific functional subsets in human tissue samples is essential for the development of more effective therapeutic applications.

Conflicts of interest/competing interests:

The authors have no financial or proprietary interests in any material discussed in this article.

Ethics approval:

This study was approved by the Ethics Committee of Goztepe City Hospital, Faculty of Medicine, Medeniyet University, Turkey on November 2, 2022, with the number 2022/0634.

Consent

Informed consent was obtained from all participants.

Funding:

We would like to express our gratitude to the Turkish Academy of Sciences (TUBA) for their invaluable support in conducting this study.

Author Contribution

All authors conceived, designed, analyzed, interpreted the data, and drafted or critically revised the article for important intellectual content.

Acknowledgement

The authors would like to acknowledge the support provided by the Turkish Academy of Sciences.

Data Availability

The data is available upon request.

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You are reading this latest preprint version

IL-17 Expression Negatively Correlates with γδ T-Cell Accumulation in Human Psoriasis Lesions: A Novel Implication for Disease Pathogenesis

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Participants

Histopathology

Immunofluorescence

RT-PCR

Statistical Analysis

Results

Participants' clinical characteristics

Histopathological assessment

TCRγδ⁺ T-cells increased with psoriasis

mRNA level of IL-17 in psoriasis patients

Discussion

Declarations

Conflicts of interest/competing interests:

Funding:

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1

IL-17 Expression Negatively Correlates with γδ T-Cell Accumulation in Human Psoriasis Lesions: A Novel Implication for Disease Pathogenesis

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Participants

Histopathology

Immunofluorescence

RT-PCR

Statistical Analysis

Results

Participants' clinical characteristics

Histopathological assessment

TCRγδ+ T-cells increased with psoriasis

mRNA level of IL-17 in psoriasis patients

Discussion

Declarations

Conflicts of interest/competing interests:

Funding:

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Status:

Version 1

TCRγδ⁺ T-cells increased with psoriasis