ANOTHER NOTCH IN THE BELT OF RHEUMATOID ARTHRITIS

Abstract

Notch ligands and receptors, including JAG1/2, DLL1/4, and Notch1/3, are enriched on macrophages (MΦs), fibroblast-like synoviocytes (FLS), and/or endothelial cells in rheumatoid arthritis (RA) compared to normal synovial tissues (ST). Power Doppler ultrasound-guided ST studies reveal that the Notch family is highly involved in early active RA, especially during neovascularization. In contrast, the Notch family is not implicated during the erosive stage, evidenced by their lack of correlation with radiographic damage in RA STs. TLRs and TNF are the common inducers of Notch expression in RA MΦs, FLS, and endothelial cells. Among Notch ligands, JAG1 and/or DLL4 are most inducible by inflammatory responses in RA MΦs or endothelial cells and trans-activate their receptors on RA FLS. TNF plays a central role on Notch ligands, as anti-TNF good responders display JAG1/2 and DLL1/4 transcriptional downregulation in RA ST myeloid cells. In in vitro studies, TNF increases Notch3 expression in MΦs, which is further amplified by RA FLS addition. Specific disease-modifying anti-rheumatic drugs (DMARDs) reduced JAG1 and Notch3 expression in MΦ and RA FLS cocultures. Organoids containing FLS and endothelial cells have increased expression of JAG1 and Notch3. Nonetheless, Methotrexate, IL-6R antibodies, and B cell blockers are mostly ineffective at decreasing Notch family expression. NF-κB, MAPK, and AKT pathways are involved in Notch signaling, whereas JAK/STATs are not. Although Notch blockade has been effective in RA preclinical studies, its small molecule inhibitors have failed in phase I and II studies, suggesting that alternative strategies may be required to intercept their function.

Graphical Abstract

INTRODUCTION

Notch signaling in mammals is mediated by four Notch receptors (Notch1–4) and five ligands (JAG1, JAG2, DLL1, DLL3, and DLL4). The ligands are both membrane-bound and have soluble forms. Notch ligand binding to the receptor expressed on neighboring cells results in transactivation, while ligation to the receptor within the same cell prevents their signaling.^{1 2} In the canonical pathway, Notch ligand binding to its receptor results in conformational changes facilitated via two cleavages.³ Initially, Notch receptors are cleaved by the ADAM family and later via γ-secretase, which releases the Notch intracellular domain (NICD) into the nucleus, where it modulates the expression of genes important for immune cell remodeling and activation. Interaction of NICD with DNA binding transcription factor RBP-Jκ is associated with recruitment of histone acetyltransferases (HAc) and Mastermind-like (MAML), leading to transcription of target genes (including HES1 and HEY1).³ However, without NICD interaction, RBP-Jκ interacts with a corepressor, resulting in transcriptional inhibition. Additionally, the ligand-independent noncanonical regulation of Notch via NICD signals through PI3K/AKT, NF-κB, MAPK, mTORC, WNT, HIPPO, or TGFβ pathways.³

The Notch pathway is a conserved intracellular cascade that directs a broad spectrum of cellular processes involved in normal development, including proliferation, differentiation, tissue/organ development, and homeostasis.⁴ However, recent data indicate that Notch and its ligands can be linked to cancer metastasis and autoimmune inflammatory diseases by manipulating innate and adaptive responses.³ Notch receptor signaling in breast, ovarian, colorectal, lung adenocarcinoma, and hepatocellular cancer has been described as oncogenic.³ Nevertheless, the observed malignancies linked to Notch activation are complex, as the effect was shown to be tissue- and ligand-dependent.

More recently, Notch ligand and receptor abnormalities have been connected to autoimmune inflammatory diseases, including rheumatoid arthritis (RA).^{5 6} Early preclinical studies showed that blockade of Notch signaling via γ-secretase inhibitor, DAPT, shifts Th1/Th17 cell remodeling to regulatory T cells (Tregs),⁷ while in TNF transgenic arthritic mice, the Notch1 inhibitor shifts the inflammatory macrophage (MΦ) reprogramming to a pro-repair profile.⁸ Intriguingly, human synovial tissue JAG1, JAG2, DLL1, and Notch3 expression are linked to active RA vascularization evaluated by ultrasound Power Doppler imaging.⁹ However, this pathway is less critical in established RA during osteoclast remodeling.¹⁰ Recent studies revealed that Toll-like receptor (TLR) activation and TNFα are the common denominators that enhance Notch protein levels in RA MΦs, FLS, or endothelial cells.¹¹ TNFα is implicated in Notch-mediated MΦ and RA FLS trans-interaction via JAG1 and Notch3.¹¹ Notch ligands, particularly JAG1 and DLL4, are highly expressed in endothelial cells and may also be involved in RA FLS trans-activation and differentiation.¹¹

RA patients with a good response to anti-TNF therapy (evaluated using ACR/EULAR DAS28 with good response to therapy defined as ΔDAS [DAS28 at baseline – DAS28 at 12 weeks after treatment] > 1.2 with DAS28 at 12 weeks ≤ 3.2) display lower JAG1, JAG2, DLL1, and DLL4 transcription in RA synovial tissue myeloid but not fibroid pathotypes.¹¹ Yet, in good responders to anti-IL-6R antibody therapy, only synovial tissue DLL4 was responsive.¹¹ Methotrexate and B cell depletion were ineffective on Notch family members.¹¹ Here, we will review recent findings that indicate Notch family members are responsible for active disease facilitated by MΦ and endothelial cell trans-activation by RA FLS.

NOTCH LIGANDS AND RECEPTORS

The four functional Notch ligands in mammals are single-pass transmembrane proteins: members of the Delta family, DLL1 and DLL4, and members of the Serrate family, JAG1 and JAG2. In contrast, as DLL3-deficient animals display a phenotype consistent with Notch activation and gain-of-function, it appears that the DLL3 gene encodes a decoy receptor that serves as an inhibitor.¹² Expression patterns of Notch ligands are less well-defined than those of receptors. However, knockout mouse models and some congenital human disorders have suggested specialized functions and preferred ligand-receptor pairs.

Mammals express four different Notch receptors, Notch 1–4. Notch receptors are also single-pass transmembrane proteins composed of a series of domains. The extracellular region of Notch receptors encompasses a series of N-terminal EGF repeats followed by a juxtamembrane negative regulatory region (NRR) consisting of 3 Lin12/Notch repeats (LNRs) and a heterodimerization domain.¹³ The intracellular region of Notch receptors contains a protein-binding RAM region, an ankyrin repeat domain (ANK), a C-terminal domain rich in the amino acids proline, glutamine, serine, and threonine (PEST), and a transcriptional activation domain.¹³ Notch1 and Notch2 receptors are widely distributed in various tissues and organs, and their knockout animal models are embryonic lethal.¹³ Notch3 is predominantly expressed by vascular smooth muscle cells, pericytes, synovial FLS, and developing T cells, while Notch4 is expressed on endothelial cells and Tregs.¹³ Knockout models of either Notch3 or Notch4 are viable and exhibit modest abnormalities confined to blood vessels.¹³

CANONICAL NOTCH SIGNALING PATHWAY

Notch receptors are first synthesized in the endoplasmic reticulum (ER), where they undergo glycosylation at the EGF-like repeats (Figure 1, Step 1). Afterward, Notch receptors undergo activation throughout a sequence of cleavages, the first of which is carried out by a furin-like protease at site S1 in the Golgi system (Figure 1, Step 2).³ This creates a non-covalently associated heterodimer composed of a Notch extracellular subunit and a transmembrane Notch subunit.³ The resulting receptor is held in the “inactive state” by the juxtamembrane Notch negative regulatory region (NRR), which consists of three Lin12/Notch repeats (LNRs) and the heterodimerization domain. ³

Figure 1. Overview of the Notch signaling pathway.

Step 1) Notch receptors are first synthesized in the ER, where they undergo glycosylation at the EGF-like repeats by ER enzymes (ex POFUT). Step 2) In the Golgi apparatus, Notch receptors are cleaved at the S1 site by a furin-like protease, which generates a mature heterodimeric form that is transported to the cell membrane. Also, fringe family glycosyl transferases extend O-fucose residues by adding N-acetylglucosamine. Step 3) Notch receptors are activated by binding to a ligand found on a neighboring cell (trans-activation). Step 4) Endocytosis of Notch ligands produces an essential mechanical pulling force impacting the bound Notch receptor by exposing the S2 site for subsequent cleavage by ADAM metalloproteases. Steps 5 and 6) S2 cleavage by ADAM10, ADAM17, or ADAMTS1 produces the NEXT fragment, which is further cleaved by the γ-secretase complex, releasing the NICD. In the absence of NICD, RBP-J binds corepressors, inhibiting target gene transcription. Step 7A) Once NICD translocates into the nucleus, it binds to RBP-J, facilitating the release of corepressors (Co-R) and the binding of transcriptional co-activators (Co-A), MAMLs and histone acetyltransferase p300 (P300) to activate transcription of target genes. Step 7B) NICD can also stay in the cytoplasm to crosstalk with other signaling pathways.

Notch receptor binding to its ligand expressed on a neighboring cell releases the autoinhibition imposed by the NRR (Figure 1, Step 3). Furthermore, endocytosis of Notch ligands produces an essential mechanical pulling force affecting the bound Notch receptor (Figure 1, Step 4).¹³ This exposes the S2 site for subsequent cleavage by ADAM metalloproteases (Figure 1, Step 5). This second rate-limiting cleavage relies on ligand binding to the receptor, and then the signal-sending cell delivers mechanical force to the receptor.¹³ The disintegrin and metalloproteinase enzymes ADAM10, ADAM17, and ADAMTS1 have been implicated in S2 cleavage, with recent data suggesting a predominant role for ADAM10 in physiological systems.^13–15 S2 cleavage creates a short-lived form of Notch consisting of the transmembrane and intracellular domains, called NOTCH extracellular truncation (NEXT).³ NEXT is rapidly cleaved within its transmembrane segment at the S3 site, releasing the Notch intracellular domain (NICD) (Figure 1, Step 6).¹³ S3 cleavage is performed by γ-secretase, which contains many catalytic subunits, including presenilin1 or presenilin2 (PS1 or PS2), nicastrin (NCT), PEN-2, and APH-1.³ Notably, one Notch receptor can only release one NICD after ligand-receptor interaction.¹²

Once freed from the membrane, NICD translocates to the nucleus and forms a Notch transcription complex (NTC) consisting of NICD, the DNA binding factor RBP-J (also known as CSL), and co-activators of the Mastermind-like (MAML) family (Figure 1, Step 7A). ³ The binding of the NTC to Notch regulatory elements (NREs) is followed by the recruitment of transcriptional coregulators that initiate transcription of Notch target genes. The primary canonical Notch target gene families are Hairy/Enhancer of Split (HES) and Hairy/Enhancer of Split related to YRPW motif (HEY) families. ³

NONCANONICAL NOTCH SIGNALING PATHWAY

Noncanonical Notch signaling is defined broadly as any form of signaling carried out by Notch independent of interactions with RBP-J (CSL) proteins. Such forms of signaling do not necessarily cause modifications in RBP-J-dependent target gene expression.³ Various noncanonical models have been described, yet such pathways have not been fully characterized, making this topic an active research area. One described noncanonical signaling model is the ligand-independent activation of endocytosed Notch receptors, which can be activated in endosomes that contain ADAM and γ-secretase.³ Another described example of ligand-independent activation is T cell receptor (TCR)-mediated self-amplification.³

Independent of RBP-J, NICD was found to possess the ability to interact with several pathways, including the NF-κB, PTEN, AKT, WNT, Hippo, mTOR, and TGFβ pathways at the cytoplasmic and/or nuclear level to modify the transcription of target genes (Figure 1, Step 7B). ³ This interaction between NICD and NF-κB plays a role in various malignancies, including cervical cancer and small-cell lung cancer. ³

In the canonical pathway, S3 cleavage is required for Notch receptors to release NICD, thus regulating target gene transcription. However, the membrane-associated form of Notch before S3 cleavage may activate the PI3K/AKT pathway, promoting the transcription of IL-10 and IL-12, providing another example of noncanonical signaling and its interplay with other signaling pathways.³

NICD REGULATION AND POSTTRANSLATIONAL MODIFICATIONS

Notch signaling is regulated at multiple levels. Such regulation impacts both the strength and orientation of downstream signaling. The first layer encompasses Notch receptor modifications, which can modulate the ligand-receptor affinity, impacting signal strength. A remarkable example is the addition of fucosylation sites by POFUT1, which increases the capacity of Notch receptors to bind its ligands (Figure 1, Step 1).¹³ Also, adding N-acetylglucosamine to O-linked fucose residues by fringe family glycosyl transferases improves the responsiveness to Delta family ligands (Figure 1, Step 2).¹³ Furthermore, Notch ligands and receptors are subjected to continuous endocytosis, recycling, and degradation, which impacts the strength of Notch signaling.³

Another aspect of Notch regulation at the ligand-receptor interaction level is the distinct effects of trans and cis binding within the canonical model. Trans-interactions encompass ligand-receptor interactions between neighboring cells. Meanwhile, cis-interactions involve an interface between ligands and receptors on the same cell. Trans-interaction is the typical activator of canonical signaling, while cis-interaction inhibits ligand and receptor signaling.¹ The traditional trans-activation and cis-inhibition determine the balance between stimulatory or inhibitory Notch signaling.²

Uniquely, Notch signaling does not contain a signaling intermediate for amplification, unlike other pathways that involve kinase-mediated signaling.¹² Notably, the scale and duration of the transcription process is sensitive to the amount of NICD present in the nucleus, as RBP-J-NICD binding triggers NICD ubiquitinylation and subsequent proteosome-mediated degradation. ¹³ This “off-switch” is crucial, as sustained Notch activation due to PEST domain mutation has been demonstrated to lead to abnormally increased cellular proliferation, driving the development of chronic lymphocytic leukemia and B-cell lymphoma. ¹³

ROLE OF THE NOTCH FAMILY IN INFLAMMATORY DISEASES AND RA

The significance of Notch as an oncogene is well characterized in solid tumors, including breast, lung, ovarian, and colorectal cancers. ³ Nevertheless, our understanding of the Notch family function is evolving in the context of inflammatory diseases. Notch signaling dysregulation is linked to several inflammatory diseases, including RA, atherosclerosis, systemic lupus erythematosus (SLE), sepsis, and systemic sclerosis.^{6 11 16 17} Growing evidence demonstrates that Notch family members protect the heart endothelium from damage caused by inflammatory cytokines and NF-κB activation, preventing atherosclerosis.¹⁸ Conversely, Notch and its downstream signaling pathway have a pathogenic outcome in RA, SLE, sepsis, and systemic sclerosis.^{6 11 16 17} Intriguingly, the importance of the Notch pathway in inflammatory diseases is partly due to monokines, especially TNFα and TLR ligands, regulating the expression of Notch ligands and receptors.^{19 20} TNFα and TLRs play a crucial role in Notch signaling as they are instrumental for NF-κB and MAPK activation in addition to chromatin modulation of the Notch target gene, HES1.²¹ This review will present the functional importance of the Notch family and focus on the significance of Notch expression and signaling in RA MΦs, T cells, FLS, endothelial cells, and osteoclasts.

NOTCH IN EARLY AND ESTABLISHED RA

Transcriptome data analysis from RA synovial tissue (ST) displays that JAG1/2, DLL1/4 and Notch1/3 are expressed at a higher level in early rather than established disease (Figure 2A–F,2K).¹⁰ In line with this idea, Power Doppler ultrasound imaging of RA ST reveals that JAG1/2, DLL1, and Notch3 are involved in joint neovascularization during early active RA (Figures 2G–J).⁹ In contrast, RA ST from patients with or without radiographic erosion show no difference in the expression of Notch ligands (Figures 3A, C, E, G) or receptors (Figure 3I, K), suggesting that Notch ligand/receptor expression does not correlate with disease burden.²² Consistently, RA ST thickness evaluated by ultrasound did not correlate with JAG1/2 (Figure 3B, D), DLL1/4 (Figure 3F, H), and Notch1/3 (Figure 3J, L) transcription levels.²³

Figure 2. Notch ligands and receptors are involved in early active but not established RA.

A-F) Relative expression of A) JAG1, B) JAG2, C) DLL1, D) DLL4, E) Notch1, and F) Notch3 was determined by RNA-seq in early and established RA ST biopsies.¹⁰ G-J) Linear regression of delta biopsy joint power Doppler ultrasonographic measures across 12 representative joints (delta Ultrasound PD 12) following treatment against G) JAG1, H) JAG2, I) DLL1, and J) Notch3 expression.⁹ K) Summary of Notch ligand and receptor expression in early active vs. established RA. p values were calculated using linear regression models or the Mann-Whitney test.

Figure 3. Notch ligands and receptors are not involved in the erosive stage of RA.

Relative expression of A) JAG1, C) JAG2, E) DLL1, G) DLL4, I) Notch1, and K) Notch3 was determined by RNA-seq of RA ST from patients with or without radiographic erosion.²² Linear regression of biopsy joint synovial thickness measures across 12 representative joints (delta Ultrasound ST 12) against B) JAG1, D) JAG2, F) DLL1, H) DLL4, J) Notch1, and L) Notch3 expression.²³ p values were calculated using linear regression models or the Mann-Whitney test.

IMPACT OF DIFFERENT CELL TYPES IN NOTCH SIGNALING RELEVANT TO RA PATHOPHYSIOLOGY

NOTCH SIGNALING IN MACROPHAGES

Macrophages are critical in host defense and inflammatory responses to endogenous and exogenous signals in RA patients. Nevertheless, MΦ involvement in Notch signaling is poorly defined. Morphological studies have revealed that JAG1, JAG2, DLL1, DLL4, and Notch1/3 are enriched in RA compared to normal ST CD68⁺CD14⁺ MΦs (Figure 4A).¹¹ Extending these findings, others exhibit that human and murine MΦs expressing Notch ligands and receptors can be receivers or senders of Notch signaling.^{24 25}

Figure 4. Notch ligands and receptors are differentially expressed in RA MΦs, FLS, and endothelial cells and are upregulated by RA inflammatory mediators.

A) DLL4 is ubiquitously highly expressed across all three cell types. Other Notch ligands are predominantly expressed on RA MΦ and FLS. Notch receptors are highly expressed on RA MΦs but also displayed at intermediate levels by RA FLS and endothelial cells. B) Inflammatory mediators found in the RA joint tend to upregulate several Notch ligands and, in a select few cases, Notch receptors in RA MΦs. In RA FLS, LPS/IFNγ and TNFα upregulate JAG2 and DLL1, but LPS/IFNγ and IL-6 upregulate Notch receptors. In endothelial cells, several inflammatory mediators upregulate Notch ligands, but only flagellin and IL-6 upregulate Notch receptors. lo, low; int, intermediate; hi, high. C) Heatmap displaying log(normalized counts) for Notch signaling components, target genes, and receptor modifiers in M-CSF differentiated CD14⁺ monocytes treated with LPS, IFNγ or primed with IFNγ then activated with LPS compared to control.²⁶ D) Heatmap displaying log(normalized counts) for Notch signaling components, target genes, and receptor modifiers in TNF-treated RA FLS compared to untreated cells.⁵³ p values were generated using the Wald test statistic and adjusted for multiple testing. *p < 0.05, ** p < 0.01, *** p < 0.001.

JAG1 and DLL4 transcription is mutually elevated in RA MΦs reprogrammed by LPS/IFNγ, flagellin, or TNFα exposure (Figure 4B).¹¹ However, Syntenin1 activation increases JAG1, DLL4, and Notch1 expression in RA MΦs (Figure 4B).¹¹ Consistently, microarray data show that TLR4-activated monocytes display increased levels of Notch transcription factors, HES1 and HEY1 accompanied by histone phosphorylation.¹⁹ These investigators uncovered that the crosstalk between the Notch component, RBP-J, and TLR pathways leads to escalated TNFα, IL-6, and IL-12 levels in MΦs.¹⁹ Additionally, IFNγ inhibits TLR-induced HES1 and HEY1 transcription, by impairing Notch2 signaling, and reducing TNF and IL-6 expression.¹⁹ This theme was further supported by an RNA-seq analysis of Notch signaling components in M-CSF differentiated CD14⁺ monocytes treated with LPS, IFNγ, or primed with IFNγ then activated with LPS (Figure 4C).²⁶ Interestingly, in LPS-stimulated myeloid cells, Notch signaling components and target genes, including receptor modifiers (POFUT1 and LFNG), ADAM10, ADAM17, MAML2, HES1, HES4, HEY1, and MYC expression levels were elevated.²⁶ In comparison, myeloid cells exposed to IFNγ exhibited higher expression of Notch target genes, MAML2 and HES4 (Figure 4C). Myeloid cells primed with IFNγ before LPS stimulation demonstrated upregulation of fewer Notch target genes than LPS treatment alone (Figure 4C).²⁶ These data highlight the role of TLR4 in activating downstream Notch signaling and underline the suppressive effect of IFNγ on this process.

Notch1 and Notch2 are implicated in TLR bidirectional regulation in monocytes.^{19 27} DLL4 ligation to Notch1 is involved in NF-κB transcription and its interaction with TNFα and iNOS promoters.²⁷ In the meantime, DLL1 activates IL-6 secretion through STAT3 signaling in monocytes.²⁸ Conversely, using a Notch small molecule inhibitor or knockout mice shifts proinflammatory MΦs to a pro-repair profile.^{3 27} IRF8 facilitates Notch1/3-mediated MΦ differentiation along with NF-κB and MAPK signaling.¹⁹ Distinctly, DLL4 impairs IL-4-induced pro-repair MΦ remodeling via IRF5 and STAT1 transcriptional upregulation.²⁹ Macrophages reprogrammed by Notch are oxidatively stressed and express high levels of mitochondrial reactive oxygen species (mtROS) and HIF1α. ³⁰

Additionally, TNFα plays a pivotal role in JAG1, JAG2, DLL4, and Notch3 expression, while IL-6 exclusively modulates DLL4 transcription in RA MΦs.¹¹ It is postulated that TLR-activated MΦs are the primary source of TNFα and IL-6 secretion.³¹ Corroborating these findings, RA ST MΦs are the main producers of TNFα, whereas both RA synovial MΦs and FLS participate in IL-6 regulation.³¹ Others have shown that in the TNF transgenic model, joint inflammation was mitigated by Notch1 inhibitor through expansion of pro-repair MΦs⁸, suggesting that TNFα has a central role in Notch-mediated signaling in myeloid cells.

In agreement with these observations, the JAG1/JAG2/DLL1/DLL4 transcriptome was repressed in anti-TNF good responders with myeloid pathotype but was unaffected in the fibroid pathotype except for DLL4.¹¹ Notch family members modulated by TNFα and TLRs depend on NF-κB and MAPK signaling to influence transcription factors, target genes, and chromatin modification.¹⁹

TNFα amplification of JAG1, Notch3, and HEY1 in MΦs is further advanced by RA FLS coculture (Figure 5A).³² Naproxen, Dexamethasone, A77/leflunomide, Hydroxychloroquine, or triple DMARD therapy can diminish JAG1, Notch3, and/or HEY1 transcription in MΦ and RA FLS cocultures (Figure 5B).³² On the contrary, treatment with Methotrexate, sulfasalazine, auranofin, or Tofacitinib is ineffective on Notch family members in MΦ and FLS cocultures (Figure 5B).³²

Figure 5. Impact of RA medications on Notch ligands, receptors, and target genes.

A) Heatmap displaying average log(normalized counts) for Notch ligands, receptors, and target genes in MΦs alone, TNF-activated MΦs, or TNF-activated MΦs exposed to RA synovial fibroblasts.³² B) Heatmap displaying average log(normalized counts) for Notch ligands, receptors, and target genes for TNF-activated MΦs exposed to RA FLS treated with RA medications compared to TNF-activated MΦs + RA FLS in the absence of treatment.³² C) Heatmap displaying the scaled mean expression for Notch ligands, receptors, and target genes for a fibroblast plus endothelial cell organoid compared to a fibroblast only organoid.⁶ D) Summary table showing the impact of Methotrexate, anti-TNF, anti-IL-6R Ab, and B cell blocker therapies on Notch ligands, receptors, and target genes.^{9 11} p values were calculated using linear regression models. *p < 0.05, ** p < 0.01. Nap, Naproxen; Dex, Dexamethasone; Meth, Methotrexate; Sulfa, sulfasalazine; A77, leflunomide; Hyd, Hydroxychloroquine; Tri, triple DMARD (Hydroxychloroquine + sulfasalazine + Methotrexate); Aur, auranofin; Tofa, Tofacitinib.

Overall, these findings indicate that although Notch ligands (JAG1/2 and DLL1/4) and receptors (Notch1/3) are overexpressed on RA ST MΦs, Notch ligands are more susceptible to increased expression by inflammatory stimuli.

NOTCH SIGNALING IN T CELLS

The Notch family promotes lymphocyte development, T-cell differentiation, and function. ³ Notch ligand and receptor interaction between antigen-presenting cells (APCs) and T cells can facilitate T cell survival and differentiation.³ NICD signaling through AKT results in mitochondrial dynamic remodeling by Mitofusins (MFN)1/2 to rescue cell survival and mitochondrial function and repair nuclear damage.³³ Others have found that DLL4 fosters CD4⁺ T cell differentiation through PI3K signaling.³⁴ Consistently, myeloid cell depletion of DLL4 impairs CD4⁺ T cell differentiation,³⁴ suggesting that APCs serve as the source of Notch ligands for T-cell polarization.

The Th1-associated genes, TBX21 and IFNγ, were directly activated by Notch signaling independent of IL-12.³⁵ Nonetheless, inhibition of Notch signaling with γ-secretase inhibitors impaired Th1 cell differentiation by IL-12 in in vitro studies.³⁵ On the other hand, in vivo studies using Notch knockout mice resulted in inconsistent results related to Th1 cell responses.³⁶ In Leishmania-infected mice, Th1 cell differentiation was diminished in Notch1/2 knockout mice. Similarly, Th1 cell polarization was independent of RBP-Jκ as these cells were detected in RBP-Jκ conditional T-cell deficient mice.³⁷ The use of DLL4 neutralizing antibodies demonstrated that DLL ligands promote Th1 reprogramming. While JAG/Notch involvement via NICD1/2 reprograms naïve CD4⁺ T cells into Th2 cells.³⁸ Confirming these findings, DLL4 or JAG1 overexpression in APCs reconfigured naive CD4⁺ T cells into Th1 or Th2 cells in vitro, respectively.³⁹ It has been postulated that Notch ligands’ differential ability to support specific polarizing conditions can influence Th1 or Th2 cell reprogramming.

Notch family members have also been connected to Th17 cell remodeling. Th17 cells secrete IL-17A, IL-17F, IL-21, and IL-22, which are associated with various inflammatory conditions and autoimmune diseases.⁴⁰ Earlier studies reported that Notch1 triggers Th17 cell reconfiguration through RORγT and IL-23R activation in part via RBP-J. ⁴¹ Corroborating these findings, Notch deactivation impairs IL-6 and TGFβ-induced RORC/Th17 cell polarization in vitro.^{42 43} Similarly, using γ-secretase inhibitors to block Notch signaling or antibodies against DLL4 impairs Th17 cell differentiation.⁴³ Two independent studies revealed that CIA was ameliorated by γ-secretase inhibitor or Notch anti-sense therapy by reducing neutrophil migration via NF-κB and ICAM1 downregulation or by abrogating joint Th1 or Th17 cell differentiation.⁷

IL-2, IL-2R, and glucose transporters are crucial in Notch-mediated T-cell reprogramming.^{34 44} T-cell glucose metabolism is controlled by the mammalian target of rapamycin (mTOR) via complex I and II (TORC1 and TORC2).⁴⁵ Notably, TORC1 advances Tbet or HIF1α, contributing to Th1 or Th17 cell reprogramming.⁴⁵ In the absence of Notch ligands, NICD-dependent responses are responsible for mTOR and NF-κB signaling in Th-cell differentiation through TCR and CD28.^{46 47} At the same time, Notch ligands are accountable for RBP-Jκ-induced Th1/Th17 cell expansion via APCs.^{34 42} Comparatively, in collagen-induced arthritis (CIA), DLL1 ligation to Notch3 escalates spleen Th1 and Th17 polarization without affecting Tregs.⁴⁸ Previous reports highlight the impact of DLL ligands on Th1/Th17 cells while emphasizing the influence of JAG ligands in Th2 and Treg rewiring.⁴⁹ Supporting this notion, DLL ligands participate in TLR-amplified Th1/Th17 cell differentiation.^{39 50} Furthermore, fringe family glycosyl transferases can adjust recognition to potentiate affinity for DLL over JAG ligands.⁵¹ Altogether, previous studies underscore the significance of surrounding cues, such as cytokines and microenvironment, in guiding Notch-driven T-cell reprogramming.

NOTCH SIGNALING IN RA FLS

Recent studies show that JAG1 and Notch3 are widely expressed in RA FLS subtypes, while the expression of DLL1 and Notch1 is modest in these cells (Figure 4A).^{6 11} The modest levels of DLL1 and JAG2 in RA FLS are upregulated by TLR4 activation and TNFα stimulation (Figure 4B).¹¹ Others have found that RA FLS exposed to TNFα exhibit JAG2, Notch1, and Notch4 transcriptional upregulation.⁵² Interestingly, RA FLS activated by TNFα displayed elevated Notch-related cleavage enzymes (ADAM10 and ADAM17), transcription factors (MAML2), and target genes (MYC and HES4), suggesting the importance of this inflammatory factor in Notch regulation (Figure 4D).⁵³ Extending these findings, the blockade of Notch signaling suppressed TNF-mediated IL-6 secretion in RA FLS, which was rescued by DLL1 recombinant protein.⁵⁴ Furthermore, in RA FLS, γ-secretase inhibitor therapy reduced HEY1 while increasing transcription of TGFβ and IL-10. ⁵⁵ Sublining CD90⁺ RA FLS were reconfigured via Notch3 trans-activation by JAG1/DLL4 expressed on arterial endothelial cells.⁶ Notch3 signaling affects the transcriptional and spatial gradients in vascular endothelial cells in connection with RA FLS.⁶ In K/BxN arthritic mice, Notch3 is expressed on mural cells and perivascular FLS, and its deletion or blockade attenuates joint inflammation.⁶ Additionally, blockade of DLL1 in K/BxN mice attenuates arthritis by interfering with myeloid DLL1 binding to Notch2 expressed on FLS, impairing IL-6 and MMP3 production.^{56 57}

Hypoxia is crucial for HIF1α stabilization as its level is orchestrated by oxygen availability. HIF1α is escalated in RA synovial tissues and is associated with FLS angiogenesis.⁵⁸ Hypoxia strongly initiates FLS and endothelial cross-regulation, proliferation, and survival. In RA FLS, hypoxic cellular responses link HIF1α and Notch pathways.⁵ IL-17 and TNFα collaboratively promote HIF1α-induced invasiveness in RA FLS.⁵⁹ Low oxygen levels advance IL-17, IL-1β, and TNFα angiogenic effects in RA FLS via HIF1α and NF-κB signaling.^{60 61}

Notch1 and Notch3 intracellular domains (N1ICD, N3ICD) are enriched in RA FLS by hypoxia. Knockdown of HIF1α downregulates Notch1, Notch3, N1ICD, and N3ICD levels in RA FLS exposed to hypoxia.⁵ Moreover, while Notch1 influences RA FLS migration during hypoxia, Notch3 impacts RA FLS survival.⁵ HIF1α-induced NF-κB activation plays a pivotal role in RA FLS invasiveness via IL-17, as NF-κB inhibition impairs HIF1α-induced angiogenesis and joint inflammation in CIA.⁶⁰ Consistent with this notion, intermittent hypobaric hypoxia pretreatment alleviated CIA by HIF1α, TNFα, and IL-17 dysregulation.⁶² It was also suggested that the N1ICD/N3ICD inhibitor mitigates CIA by blocking the connection between HIF1α and Notch1/3 signaling in FLS.⁵ However, during normoxia, TNFα stimulation or RA FLS exposure potentiates myeloid cell expression of JAG1, Notch3, and HEY1 (Figure 5A).

ADAM10 and ADAM17, Notch cleavage enzymes, are overexpressed on RA ST and RA FLS.^{63 64} Intercellular Adhesion Molecule (ICAM) and TNFα upregulate ADAM17 in RA FLS, which promotes monocyte adhesion.⁶⁴ Meanwhile, VEGF produced from ADAM10-activated RA FLS cultivates endothelial tube formation and angiogenesis.⁶³ In short, Notch family members expressed on RA FLS are sensitive to hypoxia and inflammatory mediators in the milieu, which can impact endothelial cell and/or MΦ function.

ROLE OF ENDOTHELIAL CELLS IN NOTCH SIGNALING

Notch signaling orchestrates endothelial cell proliferation, differentiation, migration, and sprouting.³ Endothelial cells express high levels of DLL4 and intermediate levels of Notch1/3 (Figure 4A).¹¹ JAG1, DLL1/4, and Notch1/3 expressed on RA synovial tissue endothelial cells are upregulated by TLR4 or TLR5 stimulation of these cells (Figure 4B).¹¹ Uniquely, IL-6 increased DLL4 and Notch1 expression levels, while JAG1 transcription was elevated by TNFα and IL-1β exposure in endothelial cells.¹¹ VEGF enhances DLL4 expression on sprouting endothelial tip cells.⁶⁵ Moreover, elevated VEGF, MAPK, and WNT component levels potentiate DLL4 expression.⁶⁶ DLL4 binding to Notch1 downregulates VEGFR2 and its responsiveness to VEGF, which prevents excessive angiogenesis.^{65 67} DLL4/Notch1 signaling in endothelial cells interferes with VEGF-induced vessel structure and function, hence rebalancing the ratio of stalk-to-tip cells for proper sprouting.^{65 68} Bone morphogenic proteins (BMPs) are also involved in VEGF and Notch cross-signaling and vascular expansion. Differential expression of BMP, VEGF, and Notch family members results in new vessel sprouting, whereas synchronized signaling results in vessel enlargement.⁶⁹

Notch signaling was implicated in VEGF/Ang2-induced endothelial cell invasion, angiogenesis, and migration, which was suppressed by Notch1 knockdown.⁷⁰ In RA explants, Notch blockade abrogated VEGF/Ang2-induced IL-6, IL-8, MMP2, and MMP9 levels.⁷⁰ Exposure of endothelial cells to hypoxia elevates HIF1α, DLL4, hairy-related transcription factor (HRT)1, and HRT2 expression. Endothelial Notch1 knockdown nullified hypoxia-induced endothelial cell migration, invasion, and HIF1α expression, supporting the crosstalk between Notch/HIF1α pathways.¹⁶

The counterbalance between DLL4 and JAG1 is manifested by JAG1’s negative impact on DLL4/Notch activity.⁷¹ In JAG1 transgenic mice, angiogenesis is amplified, whereas DLL4-induced sprouting is obstructed.⁷² The glycosyltransferase fringe directs the antagonistic interaction between DLL4 and JAG1 in endothelial cells, which regulates Notch posttranslational modifications. Fringe family glycosyltransferases sensitize Notch ligation to DLL4, simultaneously reducing JAG1 responsiveness.⁷³ Changes in RA ST metabolism shift mitochondrial oxidative phosphorylation to glycolysis.⁷⁴ The combination of hypoxia and glycolytic metabolites, such as lactate, can activate HIF1α and NF-κB to replenish oxygen and nutrients.⁷⁴ The use of glucose uptake inhibitor in endothelial cells disrupts inflammatory factors (IL-6, IL-8, CCL5) as well as HIF1α, STAT3, and Notch1 signaling.⁷⁴ Concurrently, in anti-TNF good responders, GLUT1 staining was markedly diminished in RA ST.⁷⁴

Increased deacetylating enzymes such as sirtuins (SIRT)1 can redirect metabolism from glycolysis to oxidative phosphorylation. SIRT1 inhibits Notch signaling in part by deacetylation of NICD.⁷⁵ SIRT1 can also eliminate Notch-activated metabolic reprogramming, angiogenesis, and angiogenic factors.⁷⁵

Recent single-cell RNA-Seq clustering has identified 4 RA FLS subpopulations that display distinct transcriptomic profiles.⁶ The CD90⁺HLA-DRA⁺ RA FLS cluster exhibits overlapping transcription and unique signatures with Notch3 family members associated with endothelial cells.⁶ In RA synovial organoids, JAG1 expressed on endothelial cells transactivates Notch3 on RA FLS (Figure 5C).⁶ This suggests that CD90⁺ FLS reprogramming may be fostered by the endothelial link mediated via Notch family members responsive to oxidative stress. Taken together, the inflammatory or hypoxic microenvironment in RA ST triggers FLS and endothelial cell crosstalk through the Notch family.

IMPACT OF NOTCH IN OSTEOCLASTS

Circulating monocytes home into RA ST and differentiate into MΦs that remodel into osteoclasts during inflammation. Monokines, particularly TNFα, IL-1β, and IL-6, also transform precursor osteoclasts into bone-eroding cells.⁷⁶

Earlier studies reveal that JAG1 ligation to Notch1 suppresses osteoclast precursor cells’ differentiation into mature counterparts.⁵⁶ On the contrary, DLL1 promotes osteoclast formation via Notch2, and the blockade of DLL1 mitigates K/BxN arthritis and osteoclastogenesis.⁵⁶ However, osteoclast precursor cells were unaffected by DLL4.⁵⁶. Distinctly, in Notch1 deficient mice, osteoclast formation was amplified by JAG1-induced RANKL activation.⁵⁶ Others have shown that in murine and RA cells, TNFα upregulates RELB binding to NICD, resulting in HES transcription.⁷⁷ Further, Notch signaling disrupted osteoblast differentiation in TNF-Tg mice via the noncanonical NF-κB pathway.⁷⁷

The interpretation of the role of the Notch pathway is complicated by the dual negative and positive effects RBP-J has on the transcription of the same genes. Indeed, RBP-J deletion increases RANKL-induced osteoclastogenesis and enables osteoclast maturation and pathological bone loss by TNF in the absence of RANK, partly by removing a requirement for ITAM receptor-mediated co-stimulation and regulating IRF8 and microRNAs.^{20 78–80}

Our newly generated data shows that RA ST JAG1/2, DLL1/4, and Notch1/3 are similarly expressed in patients with erosive and non-erosive RA as indicated by x-ray (Figure 3).²² Substantiating these findings, ultrasound-guided studies of synovial thickness could not detect any connection between Notch family members and disease activity (Figure 3).²³ Altogether, the role of Notch in osteoclastogenesis is ligand-dependent. Furthermore, radiographic erosion or RA synovial thickness assessed by ultrasound does not corroborate Notch’s involvement in later disease stages, suggesting that Notch signaling is important in early disease.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Despite the efficiency of Notch blockade in preclinical arthritis, employing Notch small molecule inhibitors (OMP-52M51, OMP-59R5, PF-06650808) in clinical cancer studies was either ineffective or terminated due to side effects.³ Hence, this review focuses on how current RA standard-of-care therapies impact Notch family members (Figure 6). Interestingly, treatment with Methotrexate or B cell depletion (Rituximab) did not affect the Notch family transcriptome in RA ST, irrespective of response (Figure 5D).^{9 11} Conversely, JAG1/2, DLL1/4, and HEY1 transcription was markedly reduced in anti-TNF good responders in the RA ST myeloid pathotype (Figure 5D). ^{11 22} The effectiveness of anti-TNF therapy may be due to the ability of TNFα to modulate JAG1, JAG2, and DLL4 expression in RA MΦs.¹¹ Additionally, the influence of JAK/STAT and IL-6 signaling is minimal on the Notch family, as evidenced by the inefficacy of Tofacitinib in MΦs + RA FLS and the limited effect of IL-6R antibody on only DLL4 transcription (Figure 5B, D).

Figure 6. Summary depicting the impact of current RA therapies on Notch ligands, receptors, and target genes.

Naproxen impacts Notch3 and HEY1 expression. Dexamethasone (a steroid) reduces JAG1 and Notch3 expression. DMARD therapies do not impact Notch ligands but reduce Notch3 and HEY1 expression. Anti-TNF therapy broadly impacts expression of all Notch ligands and the downstream target HEY1. Anti-IL-6R Ab therapy only downregulates DLL4 expression. A77, leflunomide; Triple DMARD (Hydroxychloroquine + sulfasalazine + Methotrexate).

Given that Notch small molecule inhibitors were unsuccessful in phase 2 studies (NCT01859741), novel strategies are required to intercept Notch signaling in autoimmune diseases. In MΦs, RA FLS, and endothelial cells, Notch signaling promotes NF-κB, AKT/mTOR, and MAPK activity; therefore, inhibitors against these signaling intermediates may block Notch function.

AVAILABILITY OF DATA AND MATERIAL:

All findings are exhibited in the paper, and the material and data are available for transparency.