Effect of baricitinib in regulating programmed death 1 and ligand programmed cell death ligand 1 through JAK/STAT pathway in psoriasis

Abstract

Objectives: Psoriasis is a chronic infectious skin disease triggered by an autoimmune process involving T-cell-mediated hyper-proliferation of keratinocytes. The objective of this study is to assess the modulation of programmed death 1 (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) through JAK/STAT pathway during the development of a psoriasis-like disease by both in vitro and in vivo model. Baricitinib, a known inhibitor of JAK1 and JAK2, was used to study the impact on PD-1 and PD-L1.

Materials and methods: Human peripheral blood mononuclear cells (PBMC) were stimulated with either anti-CD3/CD28 or PMA/Ionomycin, to modulate level of PD-1 and PD-L1 under psoriasis-like condition. Interferon-gamma (IFNγ) was used to treat HaCaT cells to mimic the diseased keratinocytes found in Psoriatic patients. Psoriasis was induced with Imiquimod (IMQ) in animal model to study the cross-talk between different cell types and pathways.

Results: Expression levels of PD-1 and PD-L1 in PBMC, and secretion of cytokines, namely tumor necrosis factor-α (TNFα), IFNγ, interleukin (IL)-6, and IL-1 β, were down-regulated on treatment with baricitinib. Further, in IFNγ-treated HaCaT cells (keratinocytes) mRNA levels of KRT-17 and PD-L1 were up-regulated.). Interestingly, in IFNγ-treated HaCat cells baricitinib decreased the levels of inflammatory cytokines such as IL-1 β, IL-6, and TNFα along with KRT-17 and PD-L1. On IFNγ-treatment. Data from both PBMC and HaCaT suggest an anti-inflammatory role for this compound. Accordingly, baricitinib was able to alleviate disease symptom in IMQ induce mice model of psoriasis. As a consequence of baricitinib treatment down-regulation of p-STAT3, PD- and PD-L1 expression levels were observed.

Conclusion: This study demonstrates a crosstalk between JAK/STAT and PD-1/PD-L1 pathways. It also demonstrates that cytokines such as IFNγ and IL-17 are down-regulated by baricitinib. We believe decreased expressions of PD-1 and PD-L1 may be a consequence of baricitinib-induced down-regulation of IFNγ and IL-17. More importantly, our data from the acute model of psoriasis indicates that PD-L1 behaves as a T-cell-associated T-cell-associated surrogate activation marker rather than immunosuppressive marker in early phase of psoriasis. Therefore it does not exhibit a causal relationship to disease.

Keywords: Baricitinib; HaCaT; imiquimod; programmed death 1; psoriasis.

Figure 1

Effect of co-stimulation with anti-CD3/anti-CD28 and PMA/Ionomycin: PBMC were incubated with and without stimulation for 72 h. Gene expression levels were analyzed with RT-qPCR. Relative gene expression was assessed for each marker by comparing DCt value and normalized with housekeeping gene, (a) Gene markers upregulated upon stimulation with anti-CD3/anti-CD28 in PBMC, (b) Gene markers upregulated upon stimulation with PMA/Ionomycin in PBMC. Cells were stained with fluorescently conjugated monoclonal antibodies directed against PD-1 and PDL-1. Samples were acquired with a FACSCalibur system (Becton Dickinson), and the resulting data were analyzed using FlowJo software, (c) shows MFI of PD-1, (d) shows MFI of PDL-1 with and without stimulation, (e) Release of cytokines TNF alpha, IFN gamma, IL-6, and IL-1 beta upon stimulation with anti-CD3/anti-CD28 and PMA/Ionomycin in PBMC. *P < 0.05, **P < 0.01, ***P < 0.001. PD-1: Programmed death 1, PD-L1: Programmed cell death ligand 1, IFNγ: Interferon gamma, TNFα: Tumour necrosis factor-α, IL: Interleukin, IMQ: Imiquimod

Figure 2

Effect of Baricitinib on proliferation rate and release of IFNγ. (a) Shows significant decrease in proliferation rate in baricitinib-treated group in comparison to group stimulated with anti-CD3/anti-CD28. (b) Shows decrease in release of IFNγ cytokine upon baricitinib treatment in comparison to group stimulated with anti-CD3/anti-CD28. ^###P < 0.001, ***P < 0.001. IFNγ: Interferon gamma

Figure 3

Effect of IFNγ on HaCaT cells: Cells were treated with 100 ng/ml of IFNγ for 24 h; (a) Cells were stained with fluorescently conjugated monoclonal antibodies directed against PDL-1. Samples were acquired with a FACSCalibur system (Becton Dickinson), and the resulting data were analyzed using FlowJo software. (b) Gene expression levels of PD-L-1, KRT-17, ICAM, IL-6 were analyzed with RT-qPCR. Relative gene expression was assessed for each marker by comparing DCt value and normalized with housekeeping gene. *P < 0.05, **P < 0.01, ***P < 0.001. PD-1: Programmed death 1, PD-L1: Programmed cell death ligand 1, IFNγ: Interferon gamma, TNFα: Tumour necrosis factor-α, IL: Interleukin, IMQ: Imiquimod

Figure 4

Baricitinib downregulates IFNγ-induced psoriatic condition in HaCaT cell; (a) Baricitinib treatment downregulated gene expression of PD-L1, KRT-17, and ICAM in IFNγ-treated HaCaT cells. (b) Baricitinib downregulated release of cytokines such as TNFα, IL-6, and IL-1 β in IFN gamma-treated HaCaT cells. ^###P < 0.001, *P < 0.05, **P < 0.01, ***P < 0.001. PD-1: Programmed death 1, PD-L1: Programmed cell death ligand 1, IFNγ: Interferon gamma, TNFα: Tumor necrosis factor-α, IL: Interleukin, IMQ: Imiquimod

Figure 5

Baricitinib treatment ameliorated IMQ induced psoriasis: (a) Topical application of baricitinib at 300 μg/20 μl/animal in mice experienced significant reduction in ear thickness when compared to IMQ treated control. (b) The spleen weight and weight of ear biopsy punch of the IMQ + Baricitinib group was significantly lower than those of the IMQ. (c) Representative images: Morphological analysis of inflamed ear and skin treated with IMQ alone and combination of IMQ and baricitinib compared with control. (d) Hematoxylin and eosin (H and E) staining of mouse ear from IMQ alone and combination of IMQ and baricitinib compared with control, reflecting the hallmarks of psoriasis with abnormally thickened epidermis, thickened keratinized upper layer (hyperkeratosis (double-headed arrow) and increased immune cell influx. Scale bars = 100 μm (e) Gene expression analysis of the ear tissue was performed, Cytokine genes IL-22, IFN γ, IP-10, IL-6, TNF α, IL-1 and KRT-17 were elevated along with PD-1 and PD-L1 in IMQ alone group, Baricitinib treatment significantly reduced the pro-inflammatory gene and expression profile of immune checkpoints as compared to IMQ treated group. Compared with control: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 and when compared with IMQ group: *P < 0.05, **P < 0.01, ***P < 0.001. PD-1: Programmed death 1, PD-L1: Programmed cell death ligand 1, IFNγ: Interferon gamma, TNFα: Tumour necrosis factor-α, IL: Interleukin, IMQ: Imiquimod

Figure 6

Effect of Baricitinib treatment on protein expression and cytokine production: (a) Protein expression analysis by western blotting shows Baricitinib downregulates elevated levels of PD-1, PD-L1, KRT-17, pSTAT3 and STAT3 in comparison with IMQ treated groups. (b) Densitometry analysis of the blots shows significant downregulation of PD-1, PD-L1, KRT-17 and p-STAT3 when compared to IMQ-treated group. (c) Cytokine analysis from the ear homogenate showed elevated levels of IL-17, IL-6, TNF and IL-1 beta in the IMQ treated group, the same were downregulated in Baricitinib-treated group. Compared with control: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 and when compared with IMQ group: *P < 0.05, **P < 0.01, ***P < 0.001. PD-1: Programmed death 1, PD-L1: Programmed cell death ligand 1, IMQ: Imiquimod