Inhibition of CEACAM1 expression in cytokine-activated neutrophils using JAK inhibitors

doi:10.21203/rs.3.rs-3215559/v1

Download PDF

Research Article

Inhibition of CEACAM1 expression in cytokine-activated neutrophils using JAK inhibitors

https://doi.org/10.21203/rs.3.rs-3215559/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 01 Oct, 2024

Read the published version in BMC Immunology →

You are reading this latest preprint version

Objectives: Carcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is an adhesion molecule that acts as a coinhibitory receptor in the immune system. We previously demonstrated that CEACAM1 is predominantly expressed on peripheral blood neutrophils in patients with RA. The aim of the present study was to investigate the effects of Janus kinase inhibitors (JAKi) on cytokine-activated human neutrophils and CEACAM1 expression.

Methods: Peripheral blood neutrophils were obtained from healthy subjects. Isolated neutrophils were stimulated with tumor necrosis factor-alpha (TNF-α) or granulocyte–macrophage colony-stimulating factor (GM-CSF) in the presence or absence of JAKi. The expression of CEACAM1 in peripheral blood neutrophils was analyzed by flow cytometry. Protein phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 was assessed by Western blot using phospho-specific antibodies.

Results: We found that TNF-α-induced CEACAM1 expression was marginally suppressed after pretreatment with pan-JAK inhibitor, tofacitinib. Moreover, TNF-α induced STAT1 and STAT3 phosphorylation at the late stimulation phase (4 to 16 h). The expressions of CEACAM1 on neutrophils were markedly up-regulated by GM-CSF not by interleukin (IL)-6 stimulation. All JAKi inhibited GM-CSF-induced CEACAM1 expressions on neutrophils, however, the inhibitory effects of baricitinib were larger compared to those of tofacitinib or filgotinib. Moreover, CEACAM1 was marginally upregulated in IFN-γ stimulated neutrophils. Similarly, JAKi inhibited interferon (IFN)-γ-induced CEACAM1 expressions on neutrophils.

Conclusions: We demonstrated that JAKi prevent GM-CSF-induced CEACAM1 expression in neutrophils, and JAKi-induced inhibition depends on their selectivity against JAK isoforms. These findings suggest that JAKi can modulate the expression of CEACAM1 in cytokine-activated neutrophils, thereby limiting their activation.

Carcinoembryonic-antigen-related cell-adhesion molecule 1

Janus kinase inhibitors

granulocyte-macrophage colony-stimulating factor neutrophils

tumor necrosis factor-alpha

rheumatoid arthritis

Neutrophils play a pivotal role in innate immunity, especially in host defense against pathogenic infections [1]. Neutrophils are also involved in the progression of a broad range of disorders, including autoimmune and inflammatory diseases [2]. Rheumatoid arthritis (RA) is a complex systemic autoimmune disease mediated by the abnormal activation of various immune cells, and neutrophils have been linked to the onset and progression of RA [3]. In the rheumatoid synovium, neutrophils infiltrating synovial membranes or fluids play an important role in the pathogenesis of RA [4]. We previously reported that CEACAM1 is selectively expressed in neutrophils not in T cells or monocytes, upregulated in peripheral blood neutrophils isolated from patients with active RA [5]. This observation suggests that inflammatory stimuli induce upregulation of the CEACAM1 gene on neutrophils in patients with active RA. CEACAM1, also known as cluster of differentiation (CD)66a, is a carcinoembryonic antigen involved in intercellular adhesion, cellular growth, differentiation, and immune function [6]. CEACAM1 also plays a role in immune tolerance, acting with the T cell immunoglobulin and mucin domain-3 (TIM-3) regulatory molecule to inhibit immune responses [7]. Although CEACAM1 is a well-known cell surface protein expressed by activated myeloid cells, including neutrophils [8], its expression mechanism in inflammatory processes has not yet been characterized. Herein, we explored whether and how CEACAM1 expression is regulated in response to rheumatoid inflammation. Further, we explored molecular mechanisms by which inflammatory cytokines alter CEACAM1 expression in human neutrophils and the interplay between these mechanisms and the Janus kinase (JAK) / signal transducer and activator of transcription (STAT) signaling pathway.

Effects of JAK inhibitors (JAKi) on TNF-α-induced CEACAM1 expression

Although TNF-α stimulation should not activate the JAK/STAT pathway, we assessed the effect of a pan-JAKi, tofacitinib, on CEACAM1 expression in TNF-α-activated neutrophils. We pretreated neutrophils with a pan-JKA inhibitor, tofacitinib, and then stimulated with TNF-α (20 ng/ml) for 24 hr. We observed that the level of CEACAM1 increased in response to stimulation with TNF-α, however, tofacitinib pretreatment marginally suppressed the TNF-α-induced CEACAM1 expression (Fig. 1).

TNF-α induces STAT1 and STAT3 phosphorylation

To determine whether TNF-α induces the activation of the JAK/STAT pathway in neutrophils, we assessed the phosphorylation status of JAK/STAT proteins in TNF-α-activated neutrophil cell lysates (Fig. 2A). Western blot analysis revealed that TNF-α did not promote JAK1/2/3 phosphorylation at any experimental time points (data not shown). However, STAT1 and STAT3 phosphorylation was detected in TNF-α-stimulated neutrophils after 4 to 16 h from the start of stimulation (Fig. 2A). These results may suggest the possibility that TNF-α induces other inflammatory cytokine that activates JAK/STAT pathways.

GM-CSF induces CEACAM1 expression

We hypothesized that JAKi suppress TNF-α-induced CEACAM1 expression by affecting other inflammatory cytokines. Therefore, we examined the effects of inflammatory cytokines which are involved in RA pathogenesis by activating the JAK/STAT pathway. Notably, inflammatory cytokines such as GM-CSF or IL-6 have been reported to be involved with the progression of RA [10]. We explored whether proinflammatory cytokines, GM-CSF and IL-6, modulate CEACAM1 expression in neutrophils. Interestingly, CECAM1 levels were markedly upregulated by GM-CSF stimulation after 24 h, whereas CECAM1 expression did not significantly change in IL-6-treated neutrophils (Fig. 2B). Moreover, GM-CSF stimulation induced CEACAM1 expression on neutrophils in a dose-dependent manner (Fig. 3A).

Effects of JAKi on GM-CSF-induced CEACAM1 expression

To investigate the effects of tofacitinib, baricitinib, and upadacitinib on the level of CEACAM1, we pretreated isolated neutrophils with each JAKi at different concentrations for 1 h and then incubated them with GM-CSF for the next 24 h. The expression of CEACAM1 in neutrophils was assessed by flow cytometry. The pretreatment with JAKis at low concentrations (40 nM) had only marginal effects on the level of CEACAM1, whereas high concentrations (200–1000 nM) significantly inhibited GM-CSF-induced CEACAM1 expression in neutrophils (Fig. 3B). JAKi showed variable inhibitory effects, and baricitinib caused the most potent suppression of CEACAM1 among JAKi used in this experiment (Fig. 3B).

IFN-γ induced CEACAM1 expression

IFN-γ stimulation induces CEACAM1 expressions in human neutrophils, however, their induction levels were lower compared to those in GM-CSF-stimulated neutrophils (Fig. 4A). Similarly, IFN-γ induced CEACAM1 expressions were inhibited by JAKi pretreatment and baricitinib most efficiently inhibited IFN-γ induced CECAM1 expressions (Fig. 4B).

Neutrophil’s function is modulated by cytokines during the inflammatory processes [11]. Activated neutrophils express various adhesion molecules and produce inflammatory cytokines and chemokines associated with immune responses [12]. Inflammatory cytokines such as TNF-α and GM-CSF induce neutrophil activation and survival at inflammatory sites [13]. In rheumatoid synovium, these cytokines suppress apoptosis and promote neutrophil activation [14]. Moreover, CEACAM1 is a key signaling molecule that regulates multiple cellular processes, including cell proliferation, tumor growth, apoptosis, angiogenesis, and granulocyte activation [15].

CEACAM1 has been shown to be upregulated in inflammatory responses [16]. Indeed, we previously reported the increased expression levels of CEACAM1 in neutrophils isolated from patients with active RA [5]. In rheumatoid neutrophils, CEACAM1 expression could be induced by inflammatory cytokines, including TNF-α, an essential cytokine in the pathogenesis of RA [17]. Other cytokines that activate the JAK/STAT pathway (e.g., IL-6 and GM-CSF) have been reported to be produced in the rheumatoid synovium [18]. In the present study, we investigated the effects of JAKi on CEACAM1 expression in neutrophils activated by TNF-α, which may not involve in the activation of the JAK/STAT pathway. Unexpectedly, we observed that JAKi marginally downregulate the expression of CEACAM1. Although TNF-α may not directly activate JAK/STAT signaling [19], TNF-α stimulation resulted in the delayed phosphorylation of STAT1 and STAT3 in neutrophils. Therefore, we investigated the mechanisms by which JAKi inhibit cytokine-induced CEACAM1 expression. Among inflammatory cytokines, we focused on GM-CSF because CEACAM1 expression is strongly induced in GM-CSF-treated but not in IL-6-treated neutrophils. Untreated neutrophils barely express CEACAM1 on their cell surface, whereas CEACAM1 is highly upregulated after GM-CSF stimulation.

We determined the effect of JAK inhibition using JAKi (tofacitinib, baricitinib and filgotinib) on CEACAM1 expressions in GM-CSF-activated neutrophils. The GM-CSF receptor is a homodimer complex containing two JAK2 subunits [20]; therefore, it can be efficiently blocked by baricitinib. Indeed, the inhibitory effects of JAKi on CEACAM1 expression were significantly higher in neutrophils pretreated with baricitinib compared to those with tofacitinib or filgotinib. These findings suggest that each JAKi inhibite cytokine-induced CEACAM1 expressions with some variations and the selectivity against JAK2 contribute to the inhibitory properties JAKi on CEACAM1 expressions. Our data demonstrate that JAKi suppress the inflammatory functions by affecting GM-CSF-induced CEACAM1, one of the key molecules during the neutrophil’s activation processes. These findings suggest that the suppressive effects against neutrophils may contribute to the therapeutic effect of JAKi in RA.

GM-CSF plays a pivotal role in the rheumatoid cytokine network, and JAK2 is a protein kinase that regulates GM-CSF-induced signal transduction [21]. Baricitinib is a potent and selective inhibitor of JAK1 and JAK2 [22]. Although tofacitinib was developed as a selective JAK3 inhibitor, subsequent studies demonstrated that it also inhibits JAK1 [23]. Moreover, filgotinib was reported as a selective JAK1 inhibitor [24]. In accord to these findings, we observed that GM-CSF induces CEACAM1 expression in neutrophils, and JAK1/2 inhibitor, baricitinib, most efficiently suppress CEACAM1 expression in neutrophils. It is expected that inhibitory effects depend on the selectivity of JAKi against different JAK isoforms [25]. We hypothesize that JAKi-mediated STAT inactivation decreases CEACAM1 expression in neutrophils. Our results show that the JAK/STAT pathway is associated with cytokine-induced CEACAM1 expression. We first demonstrated that JAKi inhibit CEACAM1 expression in cytokine-stimulated neutrophils, thereby supporting the interplay between CEACAM1 and the JAK/STAT pathway. Further studies are required to dissect the regulatory mechanism of CEACAM1 and its association with the JAK/STAT pathway, as well as to elucidate the role of this protein in RA pathogenesis.

Nonetheless, our study has limitations. Several transcription factors, which are identified to bind the promoter region of the CEACAM1 gene (e.g., interferon regulatory factor 1) [26], were not investigated because of the limited number of neutrophils available for study. These CEACAM1-binding factors may act the downstream of JAK/STAT signaling pathway, thus interfering with our judgment of the results. Both GM-CSF and IL-6 induce JAK/STAT pathways after ligation of their receptors, however, IL-6 did not upregulate CEACAM1 expression in neutrophil. It is presumed that distinct signaling pathways located in the downstream of JAK-STAT also contribute the gene expression of CEACAM1. However, these pathways were not investigated in this study.

In conclusion, we demonstrated that a pan-JAKi, tofacitinib, marginally inhibits CEACAM1 expression in TNF-α-stimulated neutrophils. Moreover, GM-CSF markedly upregulates CEACAM1 and baricitinib efficiently inhibits GM-CSF-induced CEACAM1 expression in neutrophils. We hypothesize that the inhibition of CEACAM1 in neutrophils is one of the mechanisms through which JAKi exert their anti-inflammatory effects in inflammatory disorders.

Reagents

Recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and interferon (IFN)-γ were purchased from Peprotech (Rocky Hills, NJ). Anti-β-actin antibody was purchased from Santa Cruz Biotechnology Inc. (Dallas, USA). Phospho-specific antibodies against JAK-1 (Tyr1022/1023), JAK-2 (Tyr1007/1008), JAK-3 (Tyr980/981), STAT-1 (Tyr701), STAT-5 (Tyr701) and STAT-3 (Tyr705) were purchased from Cell Signaling Technology (Beverly, MA). Tofacitinib, barictinib and filgotinib were purchased from Selleck Chemicals (Huston TX, USA).

Neutrophil’s isolation

Venous peripheral blood was collected from healthy volunteers. Written informed consent for blood donation was obtained from each individual. The blood was layered on a Polymorphprep TM (Axis-Shield, Oslo, Norway) cushion and cells were isolated according to the manufacturer’s protocol. Briefly, neutrophils were isolated on the basis of density, washed once in 0.5 N RPMI-1640 to restore osmolality, and then washed once more in RPMI-1640. The cells were subsequently diluted in complete medium consisting of RPMI-1640. To investigate the effects of JAKi on TNF-α-mediated signaling, freshly isolated neutrophils were pretreated with JAKi for 1 h then stimulated with TNF-α and protein extracts were analyzed by Western blotting.

Flow cytometry

Cultured neutrophils were used for flow cytometric analysis of the surface expression of CEACAM1, CD14, and CD16 using antihuman CEACAM1 (PE; mouse. No. 283340, diluent 2:100; R&D Systems, Minneapolis, MN, USA), antihuman CD14 (FITC; mouse. No.301804, diluent 2:100; Biolegend), and anti-human CD16 antibodies (PerCP; mouse. No. 302029, diluent 2:100; Biolegend), respectively. Dead cells were excluded using LIVE/DEAD™ Fixable Dead Cell Stain Kits. CEACAM1 expressions on the surface of CD16(+) CD14(–) neutrophil populations were analyzed (Fig. 5). Anti–CEACAM1–specific mouse monoclonal antibody (clone number 283340) was shown to be non-cross-reactive with other CEACAM families as described previously [9]. Flow cytometry was performed on a FACS–Canto II (BD Biosciences, Franklin Lakes, NJ, USA) and data were analyzed using FlowJo software (BD Biosciences).

Cell lysis and Western blotting

Neutrophils were stimulated with TNF-α or GM-CSF for the indicated times in the figure legends and the cells were washed by ice-cold PBS and lysed with RIPA Buffer (Sigma-Aldrich) supplemented with 1.0 mM sodium orthovanadate, 10 µg/mL aprotinin and 10 µg/mL leupeptin) for 20 min at 4°C. After 5 min on ice, the cell lysates were centrifuged at 10,000 g for 10 min at 4°C. After centrifugation, cellular lysates (30 µg) were also subjected to 12% SDS-PAGE, followed by western blot with antibodies against phospho-specific STATs (1:1000 dilution) and β-actin (1:2000 dilution). Western blots were visualized using the enhanced chemiluminescence (ECL) system (Amersham, Little Chalfont, UK). Densitometry was done using the automated digitizing software (Image J, NIH, Bethesda, USA). All phosphorylation levels were normalized to the protein expression of β-actin.

Statistical analysis

Between-group differences were examined for statistical significance using Student’s t-test. P-values of < 0.05 were considered significant. Bonferroni correction was per-formed for multiple comparisons.

CD, cluster of differentiation; CEACAM1, carcinoembryonic-antigen-related cell-adhesion molecule 1; FSC, forward scatter; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; JAKi, Janus kinase inhibitors; MFI, mean fluorescence intensity; RA; rheumatoid arthritis; SD, standard deviation; SSC, side scatter; STAT, signal transducer and activator of transcription; TIM-3; T cell immunoglobulin and mucin domain-3; TNF-α, tumor necrosis factor-alpha.

Ethical Approval and Consent to participate

This study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Fukushima Medical University (protocol code No. 2021-158). Written informed consent for blood donation was obtained from each individual.

Consent for publication

Not applicable.

Availability of supporting data

All data generated or analysed during this study are included in this published article.

Competing interests

K.M. has received research grants from Chugai, Pfizer, AbbVie, and Eli Lilly. The rest of the authors declare that they have no competing interests.

Funding

This study was supported by the Japan Grant-in-Aid for Scientific Research (JP20K08777) to KM.

Author contributions

HM, RS, YF, KS, YS, SY, JT, NM, TA, SS, ES, KM carried out the molecular biochemical studies, participated in the sequence alignment and drafted the manuscript. HM, KM participated in the sequence alignment and drafted the manuscript. HM participated in the design of the study, performed the statistical analysis. MO, TM contributed as supervisors in this study. All authors discussed the results and commented on the manuscript.

Acknowledgments

We are grateful to Ms Sachiyo Kanno for her technical assistance in this study.

Kobayashi SD, Voyich JM, Burlak C, et al. Neutrophils in the innate immune response.Arch Immunol Ther Exp (Warsz). 53 (2005) 505-517, https://www.ncbi.nlm.nih.gov/pubmed/16407783.
Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation.Nat Rev Immunol. 13 (2013) 159-175, https://www.ncbi.nlm.nih.gov/pubmed/23435331.
Wright HL, Moots RJ, Edwards SW. The multifactorial role of neutrophils in rheumatoid arthritis. Nat Rev Rheumatol. 2014;10:593-601.
Cecchi I, Arias de la Rosa I, Menegatti E, Roccatello D, Collantes-Estevez E, Lopez-Pedrera C, et al. Neutrophils: Novel key players in Rheumatoid Arthritis. Current and future therapeutic targets. Autoimmun Rev. 2018;17(11):1138-1149.
Matsumoto H, Fujita Y, Onizawa M, Saito K, Sumichika Y, Yoshida S, et al. Increased CEACAM1 expression on peripheral blood neutrophils in patients with rheumatoid arthritis. Front Immunol. 2022;13:978435.
Kim WM, Huang YH, Gandhi A, Blumberg RS. CEACAM1 structure and function in immunity and its therapeutic implications. Semin Immunol. 2019;42:101296.
Huang YH, Zhu C, Kondo Y, Anderson AC, Gandhi A, Russell A, et al. CEACAM1 regulates TIM-3-mediated tolerance and exhaustion. Nature. 2015;517:386-390.
Gray-Owen SD, Blumberg RS. CEACAM1: contact-dependent control of immunity. Nat Rev Immunol. 2006;6:433-446.
Saiz-Gonzalo G, Hanrahan N, Rossini V, Singh R, Ahern M, Kelleher M, et al. Regulation of ceacam family members by ibd-associated triggers in intestinal epithelial cells, their correlation to inflammation and relevance to ibd pathogenesis. Front Immunol. 2021;12:655960.
Burmester GR, Feist E, Dörner T. Emerging cell and cytokine targets in rheumatoid arthritis. Nat Rev Rheumatol. 2014;10:77-88.
Ley K. Integration of inflammatory signals by rolling neutrophils. Immunol Rev. 2002;186:8-18.
Futosi K, Fodor S, Mócsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol. 2013;17:638-650.
Hampson P, Hazeldine J, Lord JM. Neutrophil apoptosis and its induction as a potential treatment for chronic inflammatory disease. Curr Opin Hematol. 2013;20:10-15.
Wright HL, Lyon M, Chapman EA, Moots RJ, Edwards SW. Rheumatoid Arthritis Synovial Fluid Neutrophils Drive Inflammation Through Production of Chemokines, Reactive Oxygen Species, and Neutrophil Extracellular Traps. Front Immunol. 2021;11:584116.
Pan H, Shively JE. Carcinoembryonic antigen-related cell adhesion molecule-1 regulates granulopoiesis by inhibition of granulocyte colony-stimulating factor receptor. Immunity. 2010;33:620-631.
Gray-Owen SD, Blumberg RS. CEACAM1: contact-dependent control of immunity. Nat Rev Immunol. 2006;6:433-446.
Feldmann M, Maini SR. Role of cytokines in rheumatoid arthritis: an education in pathophysiology and therapeutics. Immunol Rev. 2008;223:7-19.
Simon LS, Taylor PC, Choy EH, Sebba A, Quebe A, Knopp KL, et al. The Jak/STAT pathway: A focus on pain in rheumatoid arthritis. Semin Arthritis Rheum. 2021;51(1):278-284.
Ihnatko R, Kubes M. TNF signaling: early events and phosphorylation. Gen Physiol Biophys. 2007;26:159-167.
Watanabe S, Itoh T, Arai K. Roles of JAK kinases in human GM-CSF receptor signal transduction. J Allergy Clin Immunol. 1996;98:S183-191.
Khwaja A, Linch DC. Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on neutrophil formation and function. Curr Opin Hematol. 1994;1(3):216-220.
Choy EHS, Miceli-Richard C, González-Gay MA, Sinigaglia L, Schlichting DE, Meszaros G, et al. The effect of JAK1/JAK2 inhibition in rheumatoid arthritis: efficacy and safety of baricitinib. Clin Exp Rheumatol. 2019;37(4):694-704.
Vijayakrishnan L, Venkataramanan R, Gulati P. Treating inflammation with the Janus kinase inhibitor CP-690,550. Trends Pharmacol Sci. 2011;32:25-34.
Raimondo MG, Biggioggero M, Coletto LA, Ramming A, Caporali R, Favalli EG. Clinical pharmacology of filgotinib in the treatment of rheumatoid arthritis: current insights. Expert Rev Clin Pharmacol. 2021;14(6):661-670.
Choy EH. Clinical significance of Janus Kinase inhibitor selectivity. Rheumatology (Oxford). 2019;58:953-962.
Dery KJ, Kujawski M, Grunert D, Wu X, Ngyuen T, Cheung C, et al. IRF-1 regulates alternative mRNA splicing of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) in breast epithelial cells generating an immunoreceptor tyrosine-based inhibition motif (ITIM) containing isoform. Mol Cancer. 2014;13:64.

Competing interest reported. KM has received research grants from Chugai, Pfizer, AbbVie, and Eli Lilly. The rest of the authors declare that they have no competing interests.

Download PDF

Journal Publication

published 01 Oct, 2024

Read the published version in BMC Immunology →

Editorial decision: Revision requested
24 Nov, 2023
Reviews received at journal
18 Nov, 2023
Reviewers agreed at journal
13 Nov, 2023
Reviews received at journal
17 Oct, 2023
Reviewers agreed at journal
06 Oct, 2023
Reviewers invited by journal
29 Aug, 2023
Editor assigned by journal
08 Aug, 2023
Submission checks completed at journal
08 Aug, 2023
First submitted to journal
29 Jul, 2023

You are reading this latest preprint version

Inhibition of CEACAM1 expression in cytokine-activated neutrophils using JAK inhibitors

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Effects of JAK inhibitors (JAKi) on TNF-α-induced CEACAM1 expression

TNF-α induces STAT1 and STAT3 phosphorylation

GM-CSF induces CEACAM1 expression

Effects of JAKi on GM-CSF-induced CEACAM1 expression

IFN-γ induced CEACAM1 expression

Discussion

Conclusions

Materials and Methods

Reagents

Neutrophil’s isolation

Flow cytometry

Cell lysis and Western blotting

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1