In a recent study published in the journal Frontiers in Immunology, researchers use a multi-omics approach combining immune cell-enriched single-cell transcriptomics (scRNA-seq), microarray analysis, and immunohistochemistry to elucidate the translational role of anti-interleukin-17A (IL-17A) in suppressing the autoimmune functions of type 17 T-cells (T17), thereby effectively treating human psoriasis.
Their results show that systemic IL-17A blockade depleted almost all IL17A+ and IL17F+ T-cells and caused significant downregulations in interleukin-23A (IL23A) gene expression, causing long-term reduction in psoriasis symptoms, even after medication has been halted. The IL-17A blockade was also found to upregulate CD1C and CD14 gene expression. Together, these results highlight the benefits of biologics blocking the IL-23/Type 17 T-cell autoimmune axis, with the potential for novel monoclonal antibodies that might finally present a cure for psoriasis, a hitherto uncurable condition.
Psoriasis is a non-communicable, autoimmune skin condition characterized by scaly and itchy rashes that most commonly present on affected patients' scalp, trunk, elbows, and knees. It is generally a chronic, life-long disease, and while topical treatments have been proven effective in symptomatically treating mild- to moderate disease manifestations, a lasting cure for the disease remains elusive.
Psoriasis is one of the most common autoimmune disorders in humans, with over 3.2% of all people estimated to be afflicted. While not directly lethal, psoriasis presents comorbidities, including metabolic abnormalities, psoriatic arthritis, overt vascular inflammation, diabetes, and cardiovascular disease that result in severe reductions in patients' health and have together been indicated to reduce patient's lifespan by 3-5 years or more.
Recent research has identified the benefits of biologics blocking interleukin-23 (IL-23) in suppressing Type 17 T-cells (T17), the cells responsible for autoimmune response, with studies suggesting that the blockades can cause durable psoriasis improvement in some patients even after treatment withdrawal. However, the transcriptomic mechanisms accompanying the systematic blockade of the IL-23/T17 axis and the resulting immune tolerance remain poorly understood.
Given the prevalence of the condition and the potentially life-threatening implications of the diseases' comorbidities, understanding the role of the IL-23/T17 axis will allow for the development of novel drugs and therapeutics that may present the first cure for the disease.
About the study
In the present study, researchers aimed to reveal translational evidence that blockades of a related interleukin, interleukin-17A (IL-17A), may modify the regulatory transcriptome of skin immune cells, thereby reversing the effects of human psoriasis. Their research combined immune cell-enriched single-cell RNA sequencing (scRNA-seq), microarray analysis, and immunohistochemistry to evaluate the impact of IL-17A blockades on human psoriasis lesions before and after treatment.
The study cohort comprised 23 adult psoriasis patients who were administered secukinumab, an anti-IL-17A monoclonal antibody. With a phase II clinical trial methodology, study participants received drug injections for at least 12 weeks in the following dosage regime – 300 mg of secukinumab injections at weeks one, two, three, four, and then every four weeks. Psoriasis skin biopsy samples (6 mm diameter) were obtained during this clinical trial along with anthropometric information, including age, sex, psoriasis vs. internal control, and biopsy timepoint data. Biopsy samples were bisected for analyses. Half of the biopsy was grown in a culture medium for scRNA-seq experiments, while the other half was snap-frozen before being embedded in an Optimal Cutting Temperature (OCT) compound for immunohistochemistry and microarray analyses.
Reverse transcription was carried out using cultured cells at a concentration of 10,000 cells per sample. Single-cell suspensions, gel beads, and oils were used in tandem with a 10X Genomics single-cell chip for single-cell capture and copy DNA (cDNA) preparation. Amplified DNA thus obtained was aligned to a human genome reference sequence (GRCh38) and quality-controlled.
"Genes expressed in <3 cells, and cells with <100 or >5,000 genes and a mitochondrial gene percentage of >25% were filtered out to eliminate partial cells and doublets. Ubiquitously expressed ribosomal protein-coding (RPS and RPL) and MALAT noncoding RNA, miRNA, and snoRNA genes were excluded from the analysis as we described in our prior single-cell analysis publication with the publicly available R scripts."
Graph-based clustering and principal component analysis (PCA) were used to estimate the average gene expression between psoriasis positive (+) and internal control gene expressions. Clusters expressing gene signatures from more than two immune cell subsets were excluded from downstream analyses to account for the confounding effects of doublets. Adjacent clusters of common immune cell subsets were merged to evaluate differences between psoriasis + and control immune cells.
Snap-frozen biopsy samples were mechanically disaggregated and subject to RNA extraction for microarray analysis. The GCRMA algorithm, an expression algorithm that uses probe sequence information to estimate probe affinity to non-specific binding (NSB), was used to compute differential microarray expression between psoriasis + and control immune cells. Immunostaining of flash-frozen vertical biopsy lesions embedded in OCT compound (6 μm [+/- 1 μm]), following which x10 magnification images were obtained for immunohistochemistry analysis.
Finally, statistical analyses were employed under a general framework using the Wilcoxon rank sum test to estimate the differential expression of scRNA-seq data between case and control cells. Cells were categorized into T-cells, dendritic cells (DC), and keratinocytes (KC) and analyzed separately. Moran's I, a spatial autocorrelation analysis, was used to further investigate differentially expressed genes within cohorts.
Immunohistochemistry staining revealed that 12-week-long secukinumab treatment resulted in reduced T-cell and DC signatures, and evidence of KC hyperproliferation. This IL-17A systematic blockade was further found to increase the expression of genes that regulate molecular expression in excised lesions following treatment. These results were consistent with single-cell cluster analysis that depicted reduced T17 cell signatures.
Analyses of DC cell signatures at the single-cell cluster level revealed that IL23A expression in both semi-mature and mature DCs decreases after secukinumab treatment. Immunohistochemistry results showed that the absolute population number of both mature and semi-mature DC also reduced, but found an upregulation of regulatory transcriptome expression in semi-mature DCs. Specifically, expression levels of CD1C and CD14, genes responsible for suppressing antigen-specific T-cell responses, significantly increased following patient treatment.
"IL-17A blockade reduces IL-17-driven inflammatory mediator expression in suprabasal keratinocytes and increases keratinocyte stem cell marker expression in basal keratinocytes in psoriasis lesional skin at single-cell cluster levels"
In the present study, researchers used cutting-edge multi-omics approaches to elucidate the differential gene expressions and potential molecular mechanisms underpinning the effectiveness of anti-IL treatments in treating the hitherto untreatable psoriasis. Their before-and-after phase II trial of secukinumab, an anti-IL17A biologic, on 23 adult psoriasis patients revealed that after 12 weeks of treatment, significant reductions in autoimmune responses mediated by T-cells, DCs, and KCs could be noted, highlighting the efficacy of the therapeutic in controlling and even reversing the effects of the disease.
These results persisted even after treatment termination, thereby showing promise in future drugs that might finally cure this common and chronic condition. While extensive phase II and phase III clinical trials need to be carried out before drugs with this mode of action are released into the market, this research highlights a promising first step in the war against this potentially life-threatening disease.