The effect of immunomodulatory celecoxsib on the gene expression of inhibitory receptors in dendritic cells generated from monocyte cells

Abstract

Autoimmune diseases are characterized by irregular immune responses that disrupt self-tolerance. This research explores the effects of the immunomodulatory drug celecoxib on the expression of immune checkpoint receptors in monocyte-derived dendritic cells (DCs). Key receptors, including CTLA-4, VISTA, BTLA, PDL-1, B7H7, and LAG3, play critical roles in initiating and regulating immune responses and maintaining self-tolerance. Previous studies have highlighted the significance of immune checkpoints in preventing autoimmune conditions, with animal research supporting their effectiveness in immunotherapy. Our findings demonstrate that the upregulation of immune checkpoint receptors can enhance the inhibitory functions of DCs, thereby promoting self-tolerance. As a result, tolerogenic DCs present a promising therapeutic avenue for treating autoimmune diseases. Although these results are promising, further trials are required to validate this approach before it can be applied clinically. This study underscores the potential of targeting immune checkpoint receptors as a therapeutic strategy for autoimmune disorders.

Introduction

Autoimmunity disorders are more than 70 diseases that mostly have a common immuno-pathogenesis [1], Such as lupus, multiple sclerosis, psoriasis, rheumatoid arthritis, and Hashimoto thyroiditis. Failure of immunologic-tolerance can cause the activation of immune responses against self-antigens resulting in autoimmune disorders [2]. Previous studies showed that Dendritic cells (DCs) are one of the critical factors involved in the induction of pro- and anti-inflammatory immune responses [3] Immunogenic DCs (imDCs) can induce immune responses, while DCs named as tolerogenic DCs (tolDCs) are inhibitory cells and suppress immune responses by the induction of T cell apoptosis, generation of regulatory or FOPX3 + T (Treg) cells, expression of various inhibitory-immune checkpoint receptors, such as PD-L1, hemooxygenase1, CD95L, HLA-G, galectin-1, C-type lectin (DC-SIGN), and production of various anti-inflammatory factors (IL-10, IDO1, TGFβ, IL-27, and nitric oxide (NO)) [4,5,6]. Dendritic cells with regulatory properties have taken significant attention due to their crucial role in keeping immunity homeostasis [7, 8]. Based on recent studies, tolDCs have shown an immunotherapeutic potential in autoimmune diseases, such as MS, because of inhibiting immune responses and changing of T-cell reactions to allogeneic against specific antigens in vitro. Transfection of tolDCs to humans is safe without any serious adverse effects [9, 10].

Induction of immune checkpoints can effectively prevent the immune responses and protect tissues from destroying activated immune cells against them. Tumor cells can wisely express the immune checkpoints and reduce the ability of immune cells specially T lymphocytes in recognition and destruction of tumor cells [11]. CTLA-4, LAG3, TIM3, BTLA, and VISTA are the most important immune checkpoint molecules that have been identified in recent years. These molecules signal using ITIM and ITSM and send inhibitory signals to cells [12]. After binding of CTLA-4 molecules [13] to CD80 /CD86 ligands, the T lymphocytes-mediated immune responses will suppress and T-reg cells will differentiate [14, 15]. Given that the excessive expansion of the immune responses leads to the induction and progression of the autoimmune diseases, induction of the inhibition of immune system could be effective in the treatment of this type diseases. Celecoxib is the first specific COX-2 and MTOR pathway inhibitor confirmed via the US FDA for the treatment of rheumatoid arthritis, osteoarthritis, and pain managing. Celecoxib is more effective than other common NSAIDs, such as diclofenac, and naproxen [16, 17]. In current study, the therapeutic effects of celecoxib on the expression of inhibitory molecules on human DC cells generated from monocytes investigated.

Materials and methods

Materials

The Medium consisted of RPMI − 1640 in the presence of 15% FBS, penicillin, streptomycin and, l-glutamine purchased from Gibco (USA). Recombinant human interleukin (rh IL)-4 and human recombinant granulocyte–macrophage colony-stimulating factor (rh GM-CSF) were purchased from BioLegend (San Diego, United States). Phenotyping antibodies including anti-CD86-PE and anti-HLA-DRAPC were purchased from BioLegend (San Diego, United States) and Anti-CD11c- Fluorescein isothiocyanate (FITC) were obtained from Immunostep (Salamanca, Spain). LipoPolySaccharide (LPS), Ficoll were provided from Sigma (Germany), and celecoxib was purchased from Cayman Chemical (United States). Human CD 14 monocyte isolation kit was obtained from BioLegend Company (USA), and apoptosis detection kit was provided from Immunostep (Spain).

Separation of human monocyte cells from PBMCs by Ficoll

Peripheral human blood cells (hPBMCs) from healthy people were gathered in germ-free falcons containing heparin. Based on density gradients, hPBMCs were separated by Ficoll, and then magnetic activated cell sorting (MACS) through a positive selection was applied for the isolation of monocytes from hPBMCs by streptavidin-nanobead and biotinylated Anti-CD14+. In brief, the MACS buffer was added to hPBMC, and incubated with biotin- CD14 Ab (10 µL/10⁷ cells) for 15 min, then incubated with streptavidin (10 µL/10⁷ cells). Separated cells washed and resuspended in MACS buffer. Using MACS separator’s magnetic field monocytes purified and their purification was confirmed through the MACSQuant cytometer (Miltenyi Biotec) and their viability was assessed by the trypan-blue Test (Fig. 1).

Fig. 1

Purification of monocytes and verification of high purified monocytes by flow cytometry and CD14-FITC antibody

Apoptosis analysis for defining the ideal dosage of celecoxib drug

Based on recent studies, DCs were treated with three different doses of Celecoxib (1, 5, 10 μm) for 24 h at 37 ◦C, while control group did not receive celecoxib. The optimal dose of Celecoxib was assessed by the FITC AnnexinV Apoptosis assay and Flow Cytometry method. Flow cytometry data were analyzed by FlowJo software v10.5.3. The maximum dose of celecoxib with the minimum rate of cell death (5 µM) was considered the optimal dose.

Differentiation of monocyte cells to DC cells

Collected monocytes were cultured in RPMI-1640 with 10% FBS, 50 µM 2ME, 40 ng/mL rh GM-CSF, and IL-4 (25 ng/mL) for five days. After 48 h, the media were replaced with a fresh culture medium containing rh-IL-4 and rh-GM-CSF leading to the production of DCs.

Morphological and phenotypic characterization of dendritic cells

An Inverted light microscope from Optika, (Italy) was used to examination of DCs morphology. DCs phenotyping was assessed by cell surface markers staining, including anti-HLADR-APC, anti-CD86PE, anti-CD14FITC, and anti-CD11c FITC were used. In brief, cells stained with mentioned Abs for 30 min at 4 °C and evaluated with a MACSQuant ^® analyzer from Miltenyi Biotec Company, (USA) and FlowJo (v10.5.3) software.

RNA isolation and quantitative PCR system

Total RNA of DCs was extracted using the TRIzol reagent (Roche Company, Germany) according to the manufacturer’s instructions. The concentration of extracted total RNA was analyzed by Spectrophotometer. Then, cDNA (complementary DNA) was synthesized using AddScript cDNA Synthesis Kit. The expression level of immune checkpoints was done by real-time PCR (Biosystems Company USA.). The normalization of target genes was provided by the evaluation of 18s gene expression level as an internal control. The primer sequences of genes were summarized in Table 1.

Table 1 The Forward/reverse primer sequences of inhibitory- checkpoint receptors gene

Statistical analysis

Statistical Analysis was done by GraphPad- Software Prism v8.0.2 (San Diego, USA). The T-test was used to compare results between two different groups. Each test was evaluated in triplicate. data were represented as mean ± standard deviation (SD) and P < 0.05 was considered statistically significant (*P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001, and ns: not significant).

Results

Optimum dose of celecoxib for the induction of TolDCs in vitro

The optimal dose of celecoxib in the induction of tolDCs was determined by flow cytometry. Cells were treated with different dose of celecoxib, 1µM, 5µM, and 10µM. As shown in the Fig. 2, different celecoxib doses did not cause remarkable death of treated cells. Therefore, 10µM was selected as the optimum dose of celecoxib in evaluating its effects on dendritic cells.

Fig. 2

Apoptosis assay; Determination the optimum dose of celecoxib for inducting tolDCs with Annexin V and PI

Induction of tolerogenic-related markers on DCs by celecoxib in vitro

Monocytes in vitro were cultured and differentiated to DCs. As shown in Fig. 3a, morphological alterations of differentiated DCs were assessed by microscopic examination. The low expression level of CD14 molecule on cells verified the differentiation of monocytes to DCs (Fig. 3b).

Fig. 3

(A) Morphological analysis of monocytes and DCs in vitro by the inverted light microscope. (B) Evaluation of phenotypic characterization and surface markers expression (CD11c, HLA-DR, CD86, and CD14) of mDCs and celecoxib-treated mDCs by flow cytometry. DC: dendritic cell, HLA-DR: Human leukocyte antigen-DR isotype, mDC: Mature dendritic cell

Celecoxib-treated mDCs showed increased surface expression level of maturation- and Ag presentation-related markers such as CD11c, HLA-DR, and CD86 (Fig. 3b). Mean fluorescence intensity (MFI) was also used to evaluate expression levels of CD11c, HLA-DR, and CD86. As shown in Fig. 4, celecoxib treated DCs showed high expression level of CD11c and HLA-DR, while there was no significant expression level of CD86 molecule on treated cells.

Fig. 4

The expression levels of CD11c, HLA-DR, and CD86 based on MFI in mDC and celecoxib-treated DCs (ns, not significant, **P ≤ 0.01 and ***P ≤ 0.001)

Inhibitory function of celecoxib on DCs was approved by expression level of immune check points

Celecoxib-treated DCs were evaluated for immune checkpoint molecules, including CTLA-4, PD-L1, LAG3, VISTA, and BTLA. qRT-PCR analysis showed that celecoxib could significantly increase the expression level of CTLA-4, PD-L1, and LAG3 expression level. The high expression level of VISTA and BTLA have also shown following treatment with celecoxib, but the differences were not statistically significant (Fig. 5).

Fig. 5

mRNA expression level of immune checkpoints in mDCs and celecoxib-treated mDCs (ns, not significant, **P < 0.01, and ****P ≤ 0.0001)

Expression level of B7H7 molecule on DCs was also increased after treatment with celecoxib, which it was statistically significant.

Discussion

Dendritic cells (DCs) are heterogens, which in mice, they are classified into four types: Langerhans cells, plasmacytoid dendritic cells (pDCs), conventional DCs (cDCs), and monocyte-derived DCs (MoDCs). Besides that, classification, DCs are divided into immature DCs (iDCs) and mature DCs (mDCs). mDCs are mainly responsible for the induction of immune responses, whereas iDCs are involved in the induction of immune tolerance. Therefore, because of this function, DCs could be immunogenic (imDCs) or tolerogenic (tolDCs).

imDCs are the most important antigen presenting cells (APCs) that regulate T-related immune responses, while tolDCs are involved in the induction of central and peripheral immune tolerance. imDCs express molecules on their surface for T cell inducing, such as CD80/CD86 co-stimulatory molecules and peptide-MHC complexes. CD28 molecule expressed on T cells interacts with CD80/CD86 molecules resulting in the activation of T cells and production of IL-2. imDCs from cancer patients show lower expression level of co-stimulatory molecules than healthy subjects. Besides CD80/CD86, CD40-CD40L and CD30-CD30L interactions are also required for fully activation of T cells [18]. Therefore, blockade of CD80/CD86/CD28, CD30-CD30L, and CD40-CD40L interactions can modulate developing and severity of autoimmune diseases, such as Experimental Autoimmune Encephalitis (EAE).

Although cytotoxic T lymphocyte associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) immune check point molecules are mainly expressed in (regulatory T) Treg cells, they express also in tolDCs. CTLA-4 binds to CD80/CD86 ligands and suppresses CD28 interaction resulting in the inhibition of T cell responses. PD-1 mainly is involved in suppression of CD8 + T cells proliferation and function. PD-1 can also induce the expression of FOXP3, which lead to the development of Treg cells involved in immune tolerance. Lymphocyte activation gene-3 (LAG-3) is another negative regulator of immune responses that controls excessive lymphocyte activation and induces T cell exhaustion.

Given that tolDCs are essential in homeostatic maintenance, can be considered as a therapeutic agent for the treatment of various autoimmune diseases, such as Type I diabetes, inflammatory bowel disease, systemic lupus erythematosus, and rheumatoid arthritis [19]. In vitro or ex vivo generation of tolDCs can be considered as safe and effective treatment options for various autoimmune diseases. However, there is some challenges for developing and applying the effective tolDCs therapy. One of these limitations is that we could not consider it to all types of autoimmune diseases because of lacking associated Ags, specifically migration of DCs to the target organ or stability of tolDCs in proinflammatory environments [20]. Therefore, sometimes tolDCs therapy needs more manipulation or other therapeutic strategy besides it such as CAR-T cells. In vitro generation of tolDCs could be a first step for safely treating autoimmune diseases and introducing them to the clinic.

The immunomodulatory effects of celecoxib have not been studied. Based on our results, increased expression level of markers on the cell surface of celecoxib-treated mDCs, including CD11c, HLA-DR, and CD86, is because of DCs maturation in vitro. The high gene expression level of various immune checkpoints is also resulting in immune modulatory effects of celecoxib. Celecoxib-treated DCs showed high mRNA expression level of CTLA-4, PD-L1, and LAG3. Previous studies suggested that generation of tolDCs can lead to the inhibition of autoreactive-induced T cell responses and induction of Treg and Th2 differentiation. After induction of tolDCs, there was also decreased inflammatory cytokines production.

In summary, Immunologic tolerance is essential in modulating excess immune responses leading to autoimmune diseases. Modulation of DCs and generation of tolDCs could be considered as a possible target in treating autoimmune diseases because of their role in the suppression inflammatory responses. However, we need to do more research in preclinical and clinical studies to confidently introduce celecoxib for the treatment of patients with autoimmune diseases.