Influence of adalimumab on interleukin 12/23 signalling pathways in human keratinocytes treated with lipopolysaccharide A

Introduction

The skin consists of 3 layers, with the outermost being the epidermis, followed by the dermis and finally subcutaneous tissue [1]. The epidermis is built up by cells, called keratinocytes, undergoing a continuous process of proliferation and differentiation [2–4]. The mesenchymal cells of the dermis proper, as well as elements of the immune system supplied with blood to the various layers of the epidermis, are responsible for regulating the aforementioned processes [2–5]. Other signals regulating the process of proliferation and differentiation of keratinocytes are antigens of microorganisms and mechanical factors causing damage to the outer layers of the skin [2–4]. In addition, induced epidermal cells secrete growth factors and numerous cytokines, as well as adhesion molecules and antibodies [2–4].

In psoriasis, we observe an excessive, approximately 8-fold accelerated proliferation of keratinocytes [6]. The pathomorphological picture of a section of psoriasis-affected skin is characterized by thickening of the epidermis, elongation of epidermal icicles, parakeratosis of the stratum corneum, atrophy of the stratum granulosum, hypertrophy of the stratum spinosum, dilatation of dermal papillary capillaries, and focal accumulation of neutrophil granulocytes (Munro’s microabscesses) [7, 8].

Psoriasis is classified as an autoimmune disease in which there is an abnormal functioning of the immune system and abnormal secretion of cytokines, chemokines, and growth factors, which translates into disturbances in molecular signalling pathways [9, 10]. Activation of the IL-12/Th1/IFN-g axis and Th17/IL-23 plays a key role [11]. In the group of cytokines, proteins called interleukins should be distinguished. They are signalling molecules in hematopoietic reactions and immune system responses. Currently, cytokines are divided into 7 groups. Class I is divided sequentially according to the receptor subunit. Thus, the second group of class I cytokines is the IL-12 family, which includes IL-12, IL-23, IL-27, IL-35, and IL-39, which form soluble heterodimers composed of an α subunit and a b subunit linked by a disulfide bond. Human interleukin 12 is formed by IL-12p35 and IL-12p40, while interleukin 23 is formed by IL-23p19 and IL-12p40. The function of IL-12 is to stimulate NK cells, monocytes, and macrophages, as well as to stimulate interferon production, while the function of IL-23 is to regulate the cellular response mechanisms that determine the inflammatory response [12, 13].

In in vitro models, bacterial lipopolysaccharide (LPS) [14], a building block of the outer cell membrane of the bacterial envelope, is used to induce inflammation. It is found in cyanobacteria and Gram-negative bacteria, constituting an essential element of their life processes, which determines their pathogenicity. Thanks to the recognition of LPS by macrophages, an inflammatory reaction is induced in the infected organism. This is mediated by the cell membrane protein CD14 and the TLR4 receptor, which, when activated by lipopolysaccharide, activates signalling pathways responsible for the production of, among other things, pro-inflammatory cytokines [15, 16].

Patients with confirmed moderate-to-severe plaque psoriasis (PASI > 10, BSA > 10, DLQI > 10) who are characterized by poor tolerance or ineffectiveness of previously used systemic treatment at the maximum recommended doses, for the maximum recommended time, are eligible for biologic therapy [17, 18]. A biologic drug used successfully in the treatment of inflammatory and autoimmune diseases is adalimumab [17, 18]. It is a recombinant human monoclonal antibody that, by blocking the p55 and p75 receptors for tumor necrosis factor (TNF-a) on the cell surface, inhibits its activity. In addition, it affects leukocyte migration [19, 20].

Aim

Therefore, the current study aimed to determine the expression pattern of mRNAs and miRNAs related to the IL-12/23 signalling pathways in human keratinocyte culture exposed to liposaccharide A (LPS) and then adalimumab in comparison with the untreated cells.

Material and methods

Results

Microarray profile of IL-12/23-related genes in LPS-stimulated HaCaT cells treated with adalimumab

Among the 22,283 mRNAs present on HGU-133A_2 microarrays, we selected 149 mRNAs associated with IL-12/23 signalling pathways. As a first step, we performed an ANOVA with Benjamini-Hochberg correction. Based on this test, we observed that of the 149 mRNAs, 23 mRNAs differentiated LPS-exposed cultures compared to control cultures, and 65 mRNAs were differentially expressed in HaCaT cultures treated with LPS and adalimumab compared to controls. In the next step, a Tukey post-hoc assay was performed. We observed that as the incubation time of keratinocytes on LPS and drug increased, the number of differentiating mRNAs decreased (H_2 vs. C = 49 mRNAs; H_8 vs. C = 21 mRNAs; H_24 vs. C = 14 mRNAs). In contrast, genes STAT1, STAT3, STAT5, IL-6, IL-6R, SOCS3, and JAK3 differentiated the HaCaT culture with the drug from the control regardless of the time the cells were exposed to the drug (Figure 1). Table 2 shows the differential gene expression changes of keratinocyte cultures incubated with and/or without LPS and/or adalimumab regardless of drug exposure time compared to the control culture.

Figure 1

Venn diagram

STAT1 – signal transducer and activator of transcription 1, STAT3 – signal transducer and activator of transcription 3, STAT5 – signal transducer and activator of transcription 5, IL-6 – interleukin 6, IL-6R – interleukin 6 receptor, SOCS3 – suppressor of cytokine signalling 3, JAK3 – Janus kinase 3.

Table 2

Changes in the expression profiles of genes differentiating keratinocyte cultures exposed to LPS and adalimumab compared to controls (p < 0.05)

mRNA	HaCaT with LPS	HaCaT with LPS and adalimumab
	H_8 vs. C	H_2 vs. C	H_8 vs. C	H_24 vs. C
STAT1	(–)2.87 ±0.44	(+)3.88 ±0.41	(+)3.98 ±.081	(+)2.11 ±0.34
STAT3	(–)3.45 ±0.23	(+)6.18 ±0.11	(+)5.12 ±0.76	(+)2.01 ±0.54
STAT5	(–)2.98 ±0.14	(+)3.01 ±0.58	(+)3.87 ±0.12	(+)2.14 ±0.11
IL-6	(+)3.87 ±0.45	(–)2.98 ±0.12	(–)4.91 ±0.11	(–)1.76 ±0.34
IL-6R	(–)2.34 ±0.12	(+)2.11 ±0.31	(+)2.98 ±0.12	(+)2.01 ±0.47
SOCS3	(+)3.98 ±0.98	(–)2.38 ±0.31	(–)2.14 ±0.15	(–)2.77 ±0.67
JAK3	(+)4.56 ±0.34	(+)2.98 ±0.45	(+)2.71 ±0.17	(+)2.22 ±0.65

[i] (+) – overexpression in comparison to the control, (–) – downregulated in comparison to the control, STAT1 – signal transducer and activator of transcription 1, STAT3 – signal transducer and activator of transcription 3, STAT5 – signal transducer and activator of transcription 5, IL-6 – interleukin 6, IL-6R – interleukin 6 receptor, SOCS3 – suppressor of cytokine signalling 3, JAK3 – Janus kinase 3, H_2, H_8, H_24 – time of exposure of keratinocytes to LPS or adalimumab.

RTqPCR analysis of IL-12/23-related genes in LPS-stimulated HaCaT cells treated with adalimumab

In the next step, we conducted a validation of the microarray experiment. Based on the results of changes in the expression patterns of selected genes determined by RTqPCR, the same direction of expression changes as in the microarray experiment was noted. Exposure of keratinocytes to LPS resulted in decreases in the transcriptional activity of STAT1, STAT3, STAT5, and IL-6R and increases in the expression of IL-6, SOCS3, and JAK3 compared to controls. In contrast, the addition of adalimumab to a culture previously exposed to LPS resulted in the silencing of SOCS3 and IL-6 expression compared to the control, while for the other transcripts they were found to be overexpressed compared to the control culture (Figure 2).

Figure 2

Expression profiles of genes differentiating keratinocyte cultures with LPS and adalimumab independently of the time of exposure of cells to the drug determined by the RTqPCR technique

STAT1 – signal transducer and activator of transcription 1, STAT3 – signal transducer and activator of transcription 3, STAT5 – signal transducer and activator of transcription 5, IL-6 – interleukin 6, IL-6R – interleukin 6 receptor, SOCS3 – suppressor of cytokine signalling 3, JAK3 – Janus kinase 3.

Discussion

The rationale for the study presented in this paper was the loss of adequate response to molecularly targeted therapy, including adalimumab, prompting the search for new complementary molecular markers of early drug resistance [25, 26]. The inclusion of molecular systems in diagnostics is important because molecular changes precede phenotypic changes [25, 26]. In our previous paper, we noted that adalimumab significantly altered the gene expression profiles of IL-12/23 signalling pathways in skin fibroblasts and whole blood obtained from psoriasis patients treated with the drug [27].

The determined microarray expression profile of genes related to IL-12/23 signalling pathways indicated that induction of inflammation and addition of adalimumab to HaCaT cultures significantly altered the expression of genes related to the JAK/STAT cascade, as well as IL-6. We noted that mRNA STAT1, STAT3, STAT5, JAK3, and IL-6R were overexpressed in the cell culture with the drug, and the expression levels of IL-6 and SOCS3 were reduced when adalimumab was added to the culture.

IL-6 is categorized as a pro-inflammatory cytokine that, through interaction with the receptor complex, which consists of the non-signalling α subunit of IL-6R and the b subunit of gp130, causes activation of JAK1/JAK2 and TYK2, and through this exerts a modulating effect on the JAK/STAT and MAPK cascades [28]. Ongoing observations confirm the involvement of IL-6 in the pathogenesis of psoriasis, with elevated IL-6 levels in the serum of patients with psoriasis indicating the severity of the disease, as well as its being a sensitive marker of response to treatment with TNF-a inhibitors [29]. IL-6 inhibitors are a promising group of biologic drugs used in the treatment of psoriatic arthritis [30]. Mease et al. evaluated the efficacy of clazakizumab (a monoclonal antibody against IL-6) in a group of 165 patients with psoriasis vulgaris, noting a modest relief of joint symptoms, with a concomitant lack of effect or exacerbation of skin symptoms [31]. According to Fritz et al., this unfavourable aspect of anti-IL-6 therapy for psoriasis vulgaris is the result of excessive, uncontrolled synthesis of other pro-inflammatory cytokines in response to the inhibition of transmission along the IL-6 pathway [32]. This may partly explain the overexpression of genes STAT1, STAT3, STAT5, and JAK3 in the cell culture under adalimumab exposure [32] and confirms the “vicious circle” hypothesis of self-perpetuating inflammation [33], although at the protein level, STAT5 and JAK3 levels were significantly lower in the drug-exposed culture compared to the control culture. This appears to be due to the regulatory effect of miRNAs on the expression profile of the aforementioned genes [27].

Nevertheless, Biesemann et al. demonstrated in an in vitro model of rheumatoid arthritis that IL-6 and TNF-a have synergistic effects, and that the simultaneous use of anti-IL-6 and anti-TNF-a inhibitors results in a longer quieting of the inflammatory process than when these inhibitors are used separately [34]. Concurrent silencing of IL-6 expression is accompanied by overexpression of IL-6R. Simondurairaj et al. reported an increase in IL-6R expression in gastric cancer, indicating that IL-6R may be both a therapeutic target and a prognostic marker of gastric cancer [35]. Overexpression of both IL-6 and IL-6R has been observed in patients with psoriasis [36, 37]. It is therefore possible that blocking the action of TNF-a by adalimumab affects only the expression of IL-6, not its receptor IL-6R.

Walker et al. determined changes in the expression profiles of JAK3, STAT1, and STAT4 in synoviocytes from patients with rheumatoid arthritis, inflammatory spondyloarthropathy, and arthrosis, noting overexpression of all 3 of these proteins compared to synoviocytes from healthy volunteers (control) [38]. Ivashkiv et al. emphasized that genes encoding proteins of the IL-12/23-dependent JAK/STAT signalling pathway play an anti- or pro-inflammatory role depending on the biological context, cell type, and microenvironment [39]. Thus, the overexpression of STAT1, STAT3, STAT5, and JAK3 that we observed may be due to the choice of the study model as well as to the decrease in IL-6 expression [32]. Also, Aerts et al. found overexpression of STAT4 and STAT6 in adalimumab-treated patients during 12 weeks of follow-up. They suggested that the noted expression pattern is due to the response of immune cells to anti-TNF-a immunosuppressive therapy [40]. Therefore, the overexpression of selected STAT family proteins we observed in cultured keratinocytes with induced inflammation after the addition of adalimumab to the culture indicates that the IL-12/23-dependent JAK/STAT signalling pathway is also stimulated in keratinocytes and activates adaptive mechanisms in response to the drug.

In the next stage of our study, we made a predictive assessment of the effect of miRNA molecules on the expression of the selected mRNA transcripts based on the target score value. The analysis showed that 2 miRNA molecules, i.e. hsa-miR-373-5p and hsa-miR-1827, were most likely to affect the expression profiles of JAK3, SOCS3, and STAT5. Tan et al. in their study confirmed the involvement of hsa-miR-373-5p as an important modulator of the expression of the gene encoding beta-lactamase-like (LACTB) [41], whose changes in expression pattern may be a complementary molecular marker in predicting hepatocellular carcinoma progression [42]. Further, Jing et al. showed that reduced transcriptional activity of hsa-miR-373-5p in glioma patients was an unfavourable prognostic marker of metastasis [43].

Interestingly, overexpression of hsa-miR-373-5p has been noted in tongue and esophageal squamous cell carcinoma, and the biological effect of the mentioned miRNA molecule was to promote the growth of tumour cells [44, 45]. This indicates that the duality of action of the hsa-miR-373-5p molecule depends on the biological context [43–46]. In contrast, in our study, we noted an increase in hsa-miR-373-5p expression under conditions of induced inflammation, as well as after adalimumab was added to keratinocyte cultures, with hsa-miR-373-5p expression higher in the drug-exposed than in the LPS-exposed culture. Thus, the pattern of hsa-miR-373-5p expression found may be due to the immunosuppressive effect of the TNF-a inhibitor and indicates a higher risk of cancerous lesions in patients during adalimumab therapy [21, 47–49]. Also, the evaluation of the expression pattern of the second miRNA, i.e. hsa-miR-1827, differentiating drug-exposed cultures from control cultures is widely discussed in the context of the tumorigenesis process [50, 51].

The final step of our study was to determine changes in the profiles of JAK3, SOCS3, and STAT5 protein concentrations in HaCaT cultures exposed to LPS, and adalimumab, and in control cultures. We observed reductions in JAK3, SOCS3, and STAT5 protein levels both under induced inflammatory conditions and after the cells were exposed to the anti-TNF-a drug. Thus, the opposing pattern of JAK3, SOCS3, and STAT5 expression at the mRNA and protein levels may be the result of post-transcriptional regulation of gene expression by miRNAs molecules. It should be kept in mind that there is no full complementarity between the selected mRNAs and miRNAs, resulting in decreased expression at the protein level, rather than complete degradation of the mRNA transcript, which would prevent the formation of a protein product.

Conclusions

Thus, our study indicates that adalimumab exerts the strongest modulating effect on mRNA and miRNA expression of JAK/STAT and IL-6-dependent IL-12/23 pathways. Under the influence of adalimumab, we noted overexpression of STAT1, STAT3, STAT5, JAK3, and IL-6 at the mRNA level, with a concomitant decrease in SOCS3 and IL-6R expression levels. The assessment indicated the strongest connections between JAK3 and hsa-miR-373-5p (target score 96); SOCS3, STAT5, and hsa-miR-1827 (target score 96).