Transcriptional Regulation of Mouse Mast Cell Differentiation and the Role of Human Lung Mast Cells in Airway Inflammation

Summary

Mast cells (MCs) play a critical role in allergic inflammation, anaphylaxis, and chronic inflammatory diseases such as asthma, COPD, and osteoarthritis. Dysregulated MC activation can lead to MC activation syndrome (MACS), which is observed in patients with long COVID. MCs express the high-affinity receptor for IgE and, upon activation, release mediators and cytokines that trigger anaphylactic shock and promote allergic inflammation. They also interact with epithelial and nerve cells, crucial in forming a complex network of cell-cell and gene-gene interactions driving chronic inflammation that can confer resistance to treatment. In this review, in the context of the literature, we focus on experiments conducted in our laboratory investigating how transcription factors and enhancers regulate genes critical in mouse MC differentiation and function related to human lung inflammation.

Introduction

Mast cells (MCs) play a crucial role in developing allergic inflammation, anaphylaxis and many chronic inflammatory diseases, such as asthma^1,2, COPD^3,4, osteoarthritis⁵ and MC activation syndrome (MACS). MCs express the high-affinity receptor for IgE and, upon activation, release pre-made mediators and induce de novo cytokine synthesis that promotes allergic inflammation. By interacting with epithelial and nerve cells, MCs provide a foundation for forming a complex network of cell-cell and gene-gene interactions driving chronic inflammation that can confer resistance to treatment. This review focuses on our work to further understand how transcription factors and enhancers regulate the MC genes that play critical roles in MC differentiation and fuction. We also discuss the role of human MCs in lung inflammation.

Mast cell biology

MCs are essential for allergic inflammation and anaphylaxis. Mice that are either deficient in MCs or have had their MCs depleted can't develop IgE-mediated anaphylactic shock by treatment with anti-stem cell factor antibodies^6,7. The effectiveness of the anti-IgE monoclonal antibody TNX-901 in ameliorating peanut allergy underscores the critical role of IgE/MCs in mediating anaphylactic shock in humans⁸. MCs express FcεRI, the high-affinity receptor for IgE, which is composed of one IgE-binding α subunit (FcεRIα), one signaling β subunit (FcεRIβ), and a signaling γ subunit dimer (FcεRIγ)^9,10. MCs can be activated through the crosslinking of FcεRI-bound antigen (Ag)-specific IgE, which is produced by B cells as part of the adaptive immune response¹¹. The activation of MCs by Ag crosslinking of the IgE/FcεRI complex involves the release of pre-made histamine stored in MC granules and the triggering of Src family tyrosine kinases, including Lyn, Fyn, and Syk¹². Fyn is required to activate the PI3K pathway, while Syk is essential for activating the mitogen-activated protein kinase (MAPK) pathway. PI3K pathway signaling promotes MC degranulation and survival, while the MAPK pathway regulates MC gene expression^12,13. While cytokines released by MCs such as IL-4, IL-5, IL-9 and IL-33 induce allergic inflammation¹⁴, histamine has a significant role in causing IgE-mediated anaphylactic shock^6,7,15.

MCs are strategically located at the interface of tissues and environments. These cells interact with epithelial cells and nerve cells. MCs express high levels of the IL-33 receptor (ST2, encoded by the IL1RL1 gene)^16-18, enabling them to interact with epithelial cells via the IL-33/ST2 pathway. IL-33, produced by epithelial cells, drives the differentiation of MC progenitor cells into mature MCs that produce Th2 cytokines IL-5 and IL-13¹⁸. Upon IL-33 activation, MCs release pro-inflammatory cytokines and chemokines, such as IL-6, IL-10, CCL1, CXCL8, TNF and GM-CSF^19,20. IL-33 also synergizes with IgE receptor activation to enhance cytokine and chemokine production in primary human MCs without IL-33 priming^19,21. We showed that IL-33 can prime human skin MCs to promote them to respond to antigenic stimulation synergistically²². On the other hand, MCs can promote IL-33 production by epithelial cells. The MC and lung epithelial cells form a feedforward loop^23,24.

High expression levels of G-couple receptors on MCs allow them to interact with neurons. MRGPRX/B2^25,26 is a newer subfamily of the large family of Mas-related G-couple receptors (MRGPR) expressed primarily on neurons^27,28. MRGPRB2 is the mouse ortholog of human MRGPRX2¹⁶. MRGPRX/B2 is predominantly expressed in neurons and MCs^26-28. MPGPRX2 and MPGPRB2 are structurally similar²⁷. Because of the similarity between the human and mouse receptors, both MPGPRX2 and MPGPRB2 can be activated by similar types of ligands, including antimicrobial host defense peptides, cationic polymer compounds, neuropeptides, and clinically important drugs such as morphine, codeine, tubocurarine, and ciprofloxacin^25-27. Activation of the MRGPRX/B2 receptor on MCs and nerve cells leads to MC degranulation, pruritus, pain, and pseudo-allergic drug reactions^25-27.

In summary, MCs are a critical cellular component in forming a complex net of cell-cell and gene-gene interactions that drive chronic inflammation and can render certain individuals resistant to medical treatments.

Mouse and human mast cell progenitors

Immature MCs develop from hematopoietic stem cells in the bone marrow before migrating to tissues. Upon reaching their destination tissues, they undergo further maturation and differentiation into connective tissue MCs (CTMCs) or mucosal MCs (MMCs)²⁹. CTMCs are frequently found close to venules and nerve endings, which effectively allows CTMCs to participate in immune responses³⁰. MMCs reside inside the epithelia layers of mucosal surfaces, such as the respiratory and gastrointestinal tracts, where MMCs play a critical role in defending against pathogens and allergens³¹. However, whether various MC subsets originate from distinct progenitor cells or a standard progenitor cell is still unclear.

MCs can be derived from both common basophils and MC progenitors and unique potential MC progenitors. Chen et al. identified MC lineage-restricted progenitors (MCPs), characterized as Lin⁻c-Kit⁺Sca-1⁻-Ly6c⁻FcεRIα⁻CD27⁻β7⁺T1/ST2⁺, in the bone marrow and proposed that MCPs are derived from multiple potential progenitors (MPPs)³². In their subsequent study, they found that MCPs can also derived from common myeloid progenitors (CMPs) but not granulocyte-monocyte progenitors (GMPs)³³. Alternatively, Arinobu et al. found that both MCs and basophils are derived from CMPs and GMPs³⁴. Furthermore, they described a subset of bi-potent progenitors in the spleen named basophil/mast cell progenitors (BMCPs). These precursor cells can potentially differentiate into either basophils or mast cells³⁴. In fact, Mifsud and colleagues indicated that the CD34⁻Flt3R⁻kit⁺Sca1⁺ blast colony-forming cells in bone marrow gave rise to colonies that contained both MCs and basophils³⁵. However, recent studies have challenged the authentic bi-potential basophil/MC progenitors of BMCPs^36,37, suggesting that BMCPs mainly gave rise to MCs rather than basophils³⁸. Moreover, indicate that most new basophils are generated in the bone marrow, not the spleen³⁹. A recent study confirmed the presence of BMCP in the mouse bone marrow⁴⁰, with a single-cell colony-forming assay demonstrating that about 10% of these bone marrow-derived BMCPs can give rise to both basophils and MCs⁴⁰. It is not clear what the difference is between pre-BMPs and these bone marrow-derived BMCPs. Recent research has discovered a population of granulocyte progenitors (GPs) that can differentiate into all granulocytes, such as MCs, basophils, neutrophils and eosinophils^33,36,41. However, further study is still needed to determine whether these GPs originate from GMPs and whether they differentiate into MCPs and BaPs.

We identified a novel population of common basophil-MC progenitors in the bone marrow with a panel of cell surface markers (Lin⁻cKit⁺Sca1⁻CD34⁺FcγRII/III⁺ FcεRIα⁺)³⁷. Phenotypically, these progenitors resemble GMPs more closely than any other progenitors. We determined that FcεR1α⁺ GMPs were more mature than GMPs and possessed a greater potential to differentiate into basophils and MCs but had not yet fully committed into bi-potential basophil-MC progenitors. Therefore, we named FcεR1α⁺ GMPs pre-basophil-MC progenitors ‘pre-BMPs’. In our previous review, we have described pre-BMPs in detail⁴². More recently, A population of less committed basophil and MC progenitors named pro-BMPs have been reported⁴³. These pro-BMPs are defined phenotypically as Lin⁻ FcεRIα⁻IL7Rα⁻Sca-1⁻c-Kit⁺CD34⁺FcγRII/III^hi E-cad^hi bone marrow cells⁴³. While Gata2 mRNA are upregulated in pro-BMPs, the IL-3 receptor has not been upregulated in these progenitors⁴³. In fact, we showed that the IL-3 complex can effectively expand the number of pre-BMPs but not pro-BMPs⁴⁴. IL-3 also expands the number of basophil progenitors in the spleen and the bone marrow⁴⁵. Upregulation of the FceR1a gene expression in early progenitors is also helpful in identifying blood-circulating human MC progenitors. Human MC progenitors can be identified as Lin⁻CD34^hi CD117 (c-Kit)^int/hi FcεRI⁺⁴⁶. Increased MC progenitors are detected in asthmatic patients with impaired lung function^46,47. Recent single-cell RNA-seq (scRNA-seq) analysis provides further evidence to support the existence of common basophil and MC progenitors in both mice and humans^38,40,48-51 and revealed novel basophil and MC progenitors, which is discussed in detail in other reviews⁴⁹. Table 1 summarizes the phenotypic characterization of various MC progenitors in mice and humans.

Table 1.

MC progenitors in mouse and human

Progenitors	Surface phenotype	Location	Ref.
Pro-BMP	Lin− E-cadherinhi FceRIa− IL7Ra− Sca-1− c-Kit+ CD34+ FcgRII/IIIhi	BM	43
Pre-BMP	Lin− c-Kit+ Sca-1− FcγRII/IIIhi CD34+ FcεR1α+	BM	37
BMCP_spleen	Lin− FcεR1α−/lo CD34+ c-kithi integrin β7hi	Spleen	34
BMCP_bone marrow	Lin− Sca-1− c-Kit+ integrin β7hi CD16/32hi	BM	40
MC	Lin− c-kit+ Sca-1− Ly6c− FcεR1α− CD27− β7+ T1/ST2+	BM	32
MCPi	Lin− CD45+ CD34+ β7hi FcεR1αlo	Intestine	34
Human MC progenitor	Lin− CD34hi c-Kitint/hi FceRI+	Blood	46
Human multi-potential progenitors	CD34+ 97A6+	PB/BM	51
Human MC progenitor	Lin− CD34lo c-Kit+ FceRI+	Blood	50

MC, mast cell; Pro-BMP, pro-basophil-MC progenitor; BM, bone marrow; PB, peripheral blood; Pre-BMP, pre-basophil-MC progenitor; BMCP, basophil/MC progenitor; MCP, MC lineage-restricted progenitor.

Mast cell subsets and genes

MC genes, encoding MC mediators, cytokine and chemokine proteins, among others, play a critical role in mediating many MC functions mentioned above. Progress in genome-wide analysis of genes using bulk and single-cell RNA-seq has enabled investigators to profile MC genes in a genome-wide fashion. We define MC identity genes by combining our MC expression profiling with those of the Immunological Genome Project⁵². Our gene expression profiling has identified MC genes expressing high levels in MCs but low levels or not expressed in basophils^37,53. Combined with those from the Immunological Genome Project, we compiled a list of 230 MC identity genes. Our MC identity gene list also includes genes that are essential for MC development and function, such as Gata2 and Lyn, which are not necessarily expressed highly in MCs relative to basophils. MC identity genes are predominantly found in gene sets encoding transcription factors, signaling molecules, proteases, enzymes, cell surface proteins, cytokines, and chemokines.

It is worth noting that many MC identity genes are consistently expressed and are not subjected to up or down regulation by external stimulations. These genes include Kit, FceR1a, and most protease genes. In contrast, some MC genes, such as cytokine and chemokine genes, are expressed at low levels and rapidly upregulated upon activation triggered by various extracellular stimulations. IgE receptor activation further induces de novo gene transcription^12,54. In our study in mouse MCs, 1089 genes were induced, and 280 genes were repressed⁵³. Through DAVID gene ontology enrichment analysis of these genes, the activation-induced genes are predominantly found in gene sets that encode cytokines/chemokines (Il5, Ccl1, Csf2, Ccl4, Ccl3, Il13, etc.), proteases and other enzymes (Ptgs2, Ggta1, Dot1l, Hs3st1, etc.), cell surface molecules (Hbegf, C5ar1, Cd44, Cd53, etc.), transcription factors (Atf3, Egr2, Nr4a1, Egr3, Nfil3, etc.)and signaling molecules (Dusp2, Dusp5, Plk3, Hipk2, etc.). The repressed genes are predominantly found in gene sets that encode receptors (Kit, Cx3cr1, Il4ra, Ms4a2, etc.), signal transduction (Syk, Mast3, Gab2, Stk4, etc.), apoptosis (Top2a, P2rx7, Atg7, Tia1, etc.), and cell proliferation (Rb1, Rbm38, Mcm7, Prkcd, etc.)⁵³. Human mast cells showed similar gene expression changes in response to antigenic stimulation. Deep cap analysis of gene expression (DeepCAGE) revealed that 260 genes were induced, and 84 genes were repressed in human skin MCs upon antigenic stimulation for two hours⁵⁵.

A clustering analysis of scRNA-seq data provides a more precise definition of MC subsets. MCs are classically divided into two subsets based on their location and function: Connective tissue CTMCs and mucosal mast MMCs. In mice, the CTMCs express carboxypeptidase A⁵⁶, mouse MC protease 4 (mMCP-4), mMCP-5, mMCP-6 and mMCP-7, whereas MMCs express two b-chymases, mMCP-1and mMCP-2. CTMCs are characterized by their constitutive presence in connective tissues, and they can maintain their presence or activity independently of T cells. In contrast, MMCs are induced in response to specific stimuli, and their development and function are regulated by T cells⁵⁷. Notably, MMCs can be induced by cytokines, such as interleukin-4, interleukin-9⁵⁸ and transforming growth factor-beta 1⁵⁹. scRNA analysis reveals two mutually exclusive MrgprB2⁺ CTMCs and MrgprB2⁻ MMCs⁶⁰. Under diseased conditions, MCs undergo dramatic changes in induced gene expression. They become activated and proliferative. These cells acquire a proinflammatory signature^61-64, an allergic signature⁶² and a molecular signature that renders them resistant to medical treatment⁶⁵. Table 2 summarizes the key MC genes.

Table 2.

Mouse mast cell genes

Pathways	Genes	Ref
Transcription factors	Creb3l1 Gata2 Mitf Meis1 Ahr Ahrr Hes1 Nfia Tcf12 Zfhx3	52,53
Cytokines and chemokines	Ccl1 Csf2 Ccl4 Ccl3 Il13 Tnf Ccl7 Tnfsf14 Il6 Il1a Ccl2 Ccl9 Csf1 Lif
Cell surface molecules	Kit Cd93 Cd96 Il1rl1 Il4ra Cxcr1 Cd3001b Cd59a Cd90 Cd103
Proteases and enzymes	Hs3st1 Pcyt1a Soat1 Tiparp Ctsg Mcpt2 Mcpt4 Mcpt9 Hs3st3a1 Hs2st1 Ndst2 Chst1 Ids Ext1 Papss2 A4galt Hs6st2 Eno2
Signaling molecules	Prkcb Plk3 Jak2 Map2k3 Ripk2 Txk Itk Fyn Nek6 Rapgef2 Bmpr2 Ednra

Transcription factors that regulate MC gene transcription

Transcription factors can be critical in differentiating MC progenitors into mature MCs. Transcription factors, including STAT5^66-68, GATA1^69,70, GATA2^71,72, IRF8⁴¹, GLI1/2⁷³, IKAROS⁷⁴, MYB⁷⁵, P1-Runx1³⁶, PLZF⁷⁶, NFIL3⁷⁷, FOG-1^78,79 and MITF^80,81 are each critical for basophil and MC differentiation (Fig. 1). Recent reviews have discussed these transcription factors in detail^42,49,82,83. Molecular mechanisms by which these transcription factors regulate MC gene transcription are still incompletely understood. During MC differentiation, at least two major molecular programs must be initiated. First, MC lineage-determining transcription factors, such as GATA2 and MITF, are upregulated in MC progenitors. Upregulation of the MC lineage-determining transcription factors promotes the transcription of MC genes that confer specialized functions to MCs. Simultaneously, MC lineage-determining transcription factors must suppress the transcription of the genes of other cell fates, especially basophil cell fates. The second major molecular event during MC differentiation is the modification of histones surrounding the enhancers of the MC genes. Here, we discuss our work investigating the mechanisms by which GATA2 and MITF regulate MC gene transcription and remodel the enhancers of the MC genes.

Figure 1.

Transcription factors that regulate basophil and MC differentiation. Transcription factors that promote and suppress the differentiation of various progenitors are depicted. The arrows indicate promoting, and the bars indicate suppressing. Pro-BMP, pro-basophil-MC progenitor; Pre-BMP, pre-basophil-MC progenitor; MCP, MC lineage-restricted progenitor; BaP, basophil progenitor.

GATA2 regulates mast cell gene transcription

GATA binding protein 2 (GATA2) is a GATA family of transcription factors member. GATA2 is critical for the survival and proliferation of hematopoietic stem cells^84,85, granulocyte-monocyte progenitor differentiation⁸⁶, and basophil and MC differentiation^71,72,82,87. GATA2 activates promoter activity of the MC carboxypeptidase A gene⁸⁸ and the baboon chymase gene⁸⁹. GATA factors bind at 1.0 and 72.8 kb upstream of the Tpsb2 gene⁹⁰. GATA2 is required for the expression of c-Kit⁹¹. We found that GATA2 is upregulated in pre-BMPs and plays an essential role in basophil and MC development⁹². Consequently, the absence of the Gata2 gene significantly reduces the number of mature MCs^71,87,93. Conventional Gata2 knockout (KO) mice cannot be used to distinguish the role of GATA2 in in maintaining the MC gene transcription in already differentiated MCs. We thus generated an inducible, conditional Gata2 KO mice to overcome this problem⁵³. We inducibly deleted the Gata2 gene in fully differentiated MCs and determined whether GATA2 is responsible for maintaining MC gene expression⁵³. We found that the upregulation of the Gata2 expression is STAT5-dependent⁹². We demonstrate that GATA2 has a profound effect on MC gene regulation. Deletion of the Gata2 gene resulted in a reduction of a broad range of MC genes⁵³. We found that haploinsufficiency of the Gata2 gene affects the generation of MCs but not basophils, implying that MCs are more dependent on GATA2 than basophils⁹³. GATA2 is also found to upregulate E-cadherin in pro-BMPs⁴³. We demonstrated that GATA2 regulates the Hdc gene encoding enzyme histidine decarboxylase^53,93. Ohneda and colleagues also reported similar findings that GATA2 is essential in maintaining MC identity at around the same time⁹⁴.

GATA2 has been reported to cooperate with other transcription factors to transcribe MC genes. GATA2 works with ETS family transcription factor PU.1 to transcribe the Il16 gene in MCs^95-97. GATA2 acts with transcription factor SMAD4 to promote the transcription of the Mcpt1 and Mcpt2 genes⁹⁸. GATA-2 interacts with AP-1 to enhance the binding of AP-1 to the IL-13 promoter region, increasing IL-13 production in MCs⁹⁹. Taken together, the published findings support that GATA2 is an essential MC lineage-determining transcription factor.

MITF regulates mast cell gene transcription

Microphthalmia-associated transcription factor (MITF) is a pleiotropic transcription factor¹⁰⁰. The role of MITF in MC differentiation was discovered by studying mice with various spontaneous mutations in the Mitf gene^80,101. MITF regulates the mouse MC protease 6 (MMCP-6) and MC tryptase genes¹⁰². Kitamura et al. demonstrated that MITF mutations significantly decrease MC numbers⁸⁰. Few MCs survive in Mitf-null mutant mice. Because of this, the role of MITF in MC lineage commitment remains poorly understood. Recent reviews have summarized the role of MITF in regulating MC differentiation^82,103. Therefore, we review our work to understand how MITF promotes MC genes while suppressing basophil genes.

We found that MITF acts downstream from GATA2⁹³. Overexpression of the Mift gene rescued Hdc gene expression in Gata2-deficient MCs⁹³. Our work further demonstrated that MITF is a dominant determinant in specifying MC vs. basophil fate. Under normal physiological conditions, the bi-potential MC basophil progenitors must decide to differentiate into either MCs or basophils, but not cells express both MC and basophil characteristics. Specifically, MCs must promote MC genes while suppressing basophil genes that confer basophil-specific functions. We hypothesized that transcription factors that can promote MC-specific genes while suppressing basophil-specific genes are likely to be expressed at high levels in MCs but at low levels in basophils. Among these potential MC transcription factor genes, we identified 15 transcription factor genes. These include Mitf, Ahrr, Ahr, Hic1, Mecom, Bhlhe40, Batf1, Eid2, Id2, Mdfic, Meis1, Mzf1, Pgr, Tal1 and Zfhx3^37,53. We tested several these transcription factor genes, including Mitf, Ahr, Ahrr and Bhlhe40⁹³. We showed that the Mitf gene, but not the Ahr, Ahrr and Bhlhe40 genes, plays a critical role in promoting MC genes or suppressing basophil genes³⁷. Mice with null Mitf gene mutation do not have functional MCs^104-106, indicating that the Mitf gene is necessary for MC differentiation. Overexpression of the Mitf gene was sufficient to rescue MC differentiation from Mitf^−/− progenitors¹⁰⁶. Recently, we demonstrated that MITF was sufficient to drive pre-BMPs to differentiate into MCs³⁷. Using Mitf^wh/wh mice, which harbor two copies of the Mitf gene with a single amino acid mutation at the basic domain (DNA binding domain) that results in deficient but demonstrable DNA binding on MC genes¹⁰¹, we observed that few surviving Mitf^wh/wh MCs lost MC gene expression, such c-Kit, IL-4Rα, and MC protease genes. Interestingly, that Mitf^wh/wh MC switched to a “basophil-like” cell, gaining basophil-expressing genes CD49b expression and Il4 mRNA expression after 28 days of culture³⁷. However, this study had the limitation that it did not distinguish whether Mitf^wh/wh BMMCs simply failed to commit to the MC lineage fully or switched from committed MCs into basophils. Our future work will attempt to resolve this issue. A similar phenomenon was observed in transcription factor IKAROS-deficient mice. Without the Ikaros gene, the basophil lineage-determine transcription factor Cebpa gene was highly upregulated in basophil progenitors and mature basophils. As a result, MC differentiation was impaired⁷⁴, supporting the idea that CEBPα suppresses Mitf gene transcription. Using a CRISPR/Cas9-based framework approach, Mo et al. demonstrated that MITF is essential for human MC differentiation¹⁰⁷. Thus, evidence supports that MITF is a master MC transcription factor promoting MC gene transcription while suppressing basophil cell fate.

In addition to studying the role of MITF in MC vs. basophil cell fate determination, we have investigated in detail how MITF promotes one of the MC genes essential in histamine synthesis. Our study suggests that MITF is induced by GATA2 and that these two transcription factors direct Hdc gene transcription in MCs in a feed-forward manner⁵³. MITF binds to the −8.8 Hdc enhancer and drives its enhancer activity. Indeed, MITF overexpression essentially restores Hdc gene expression in Gata2-deficient MCs⁵³.

Transcription factors that regulate enhancer landscape of the mast cell genes

Our knowledge of enhancers that regulate MC genes is still limited. Using the DNase I hypersensitivity assay, several enhancers in the Il4 Il13 loci were discovered to regulate the Il4 and Il13 gene transcription in basophils and MCs. Rad50 hypersensitive site (RHS)7-deficient MCs transcribe approximately 50% less of the Il4 and Il13 genes but not less of the Il5 gene¹⁰⁸. MCs deficient in consensus sequences 2 (CNS2) within the HSV region exhibit a dramatic reduction in Il4 gene transcription^109,110. A 683-bp Bgl II fragment within the HS II in the second intron of the Il4 gene was shown to exhibit MC-specific enhancer activity¹¹¹. With recent advances in the next generation of sequencing, more enhancers have been discovered for MC genes. A CNS-25 has been reported to regulate the Il9 gene in basophils and MCs¹¹². Kario et al. demonstrated a Myb enhancer regulates the differentiation of MCs and basophils⁷⁵. A recent study demonstrates that the Il6-39 kb enhancer is essential for Il6 expression in MCs⁹⁵. However, the transcriptional codes that enable transcription factor interactions within an enhancer or distal enhancers to collaboratively regulate transcription of MC genes during allergic reactions have not been deciphered.

Transcription factors regulate target genes by binding and activating enhancers. Enhancers are regulatory modules a few hundred base pairs in length located within genes or in intergenic regions. They typically comprise clusters of transcription factor-binding sites that bind sequence-specific DNA-binding transcription factors and associated factors¹¹³. Enhancers promote gene transcription by mediating the assembly of higher-order interactions of transcription factors and co-factors with promoters¹¹⁴ (Fig. 2, transcription hub). Potential enhancers can be identified by histone modifications and chromatin accessibility^115-120 (Fig. 2). For example, active enhancers can be marked by acetylation of lysine residue 27 on histone 3 (H3K27ac) modification^115-118. We have performed H3K27ac CUT&Tag-seq and ATAC-seq to identify potential enhancers^53,121.

Figure 2.

Signal-dependent transcription factors deliver signals triggered by extracellular stimulations and transduced through the PI3K, MAPK, and MyD88/IRAK/TRAF6 pathways to enhancers to form a 3D transcription hub. Antigenic stimulation (Stim) or IL-33 stimulation activates signal-dependent transcription factors (SDTFs), which then bind to stimulation-responsive enhancers to form a 3D transcription hub consisting of regular transcription factors (TFs), cofactors (COF), mediator, and essential Pol II transcriptional complexes. E=enhancer; P=promoter.

Our work implies that GATA2 can act as a “pioneer” transcription factor that shapes the enhancer landscape of MC genes⁵³. Pioneer transcription factors are defined as a special class of transcription factor proteins that can access their DNA target sites in chromatin when other factors cannot, and that continue to access the DNA prior to the time of transcriptional activation¹²². Pioneer transcription factors are known to recruit histone-modifying enzymes, such as p300, that acetylate H3K27 to the promoter and enhancer regions¹²². Our results demonstrate that GATA2 has a profound impact on the enhancer landscape of the MC genes that are associated with either typical or super-enhancers with a more significant effect on super-enhancers, which are defined as non-coding DNA regions of chromatin that were at least 12.5 kb long and associated with substantial H3K27ac modification and accessible chromatin¹²³. We demonstrated that super-enhancers have a striking 8-fold increase associating the MC genes as pivotal in primary MC functions than MC genes performing minor functions⁵³. For instance, transcription factors Gata2, Mitf genes, which have been shown to be critical for MC development and function, cytokine and chemokine genes, such as Ccl1, Ccl3, Il13, Il6 genes, cell surface molecules including the Kit, Cd96, Il1rl1, Il4ra genes, protease and enzyme genes (Mcpt2, Mcpt9, Ndst2, Hs2st1), and genes encoding signaling molecules (Prkcb, Jak2, Ripk2, Map2k3)⁵³ (Table 2) are associated with super-enhancers. Our findings are consistent with other reports that demonstrate that super-enhancers are associated with lineage-determining genes in other cell types^123-125.

Investigate further whether the enhancers we identified using the genomic approach discussed above can function in vivo in the chromatin context. We selected a rate-limiting gene, Hdc, for detailed functional enhancer analysis in mice^93,126. The Hdc gene encodes the enzyme histidine decarboxylase (HDC) that catalyzes the conversion of the amino acid histidine into histamine. We identified an enhancer, the −8.8 kb Hdc enhancer, located 8.8 kb upstream of the transcription start site (TSS) of the Hdc gene. We demonstrated that the transcription factor MITF can bind to the −8.8 kb Hdc enhancer and drive its activity. Our research also highlights that the Hdc GC box is critical for Hdc transcription and histamine synthesis in mouse and human MC lines¹²¹.

Signal-dependent transcription factors activate enhancers to respond to extracellular stimulations.

Enhancers also provide binding hubs for signal-dependent transcription factors (SDTFs) to detect signals triggered by the activation of receptors through extracellular stimulations¹²⁰. Many SDTFs are known to be activated through phosphorylation modification after various extracellular stimulations¹²⁷. SDTFs STAT5^{37,45,92,128-130}, NFAT^131-133, AP-1¹³⁴, NFκB^19,135 and EGR2^136,137 are activated in response to extracellular stimulations, such as IgE and antigen complex that crosslink the IgE receptor and IL-33 (see reviews⁸³). Other SDTFs, such as ZEB2 and EHF, have been reported to regulate MC activation^138,139. However, our knowledge of how SDTFs activate enhancers in response to extracellular stimulations is still limited.

Individual or interacting enhancers from a distance can synergistically integrate signal inputs triggered by different signaling pathways. SDTF binding sites, whether located in the same individual enhancers or distal enhancers that interact with anchoring enhancers, can bind to various SDTFs. These SDTFs contain interacting protein domains. The proximity of transcription factor binding sites within one enhancer or, as a result, enhancer-enhancer interactions can drive robust gene transcription by cooperative binding of transcription factors^140,141 (Fig. 2). It is widely recognized that SDTFs became activated through phosphorylation¹²⁰. Activated SDTFs then bind to enhancers to activate them. These activated SDTFs can also recruit other transcription factors, cofactors, and mediators, along with enhancers and promoters, to form a loop functioning as a transcription hub^113,114 (Fig. 2). A recent study provides evidence that transcription factor cooperation is more common than previously thought. This study used a high throughput analysis of cooperative transcription factor binding and found 315 transcription factor–transcription factor examples of cooperative binding, recognizing 618 novel transcription factor binding motifs¹⁴⁰. However, decoding the rules of interactions for transcription factors, cofactors, and chromatin remains challenging.

Using the Omni-ATAC-seq method, a recent research conducted by Cildir et al. found that Ca²⁺-dependent open chromatin (COC) domains within enhancers play a critical role in regulating gene expression in response to FcεRI or IL-33 stimulation¹⁴². They also identified disease-associated SNPs precisely located in COC domains and transcription factor BATF, AP-1 and ATF3-binding sites enriched in the COC domains¹⁴². However, whether these transcription factor-binding sites (TFBSs) in the COC domains indeed have a role in gene transcription in response to external stimulation awaits further research. Our group analyzed enhancers that respond to antigenic stimulation with increased H3K27ac and chromatin accessibility^22,53. Our transcription factor binding motif analysis of the antigen-stimulated enhancers revealed that GATA2, EGR2 and AP-1-binding sites are significantly enriched in these enhancers. Additional experiments are underway to investigate the role of EGR2 and AP-1 in enhancing MC gene transcription in response to antigen stimulation¹⁴³.

Our study analyzed Il13 enhancers in MCs that can respond to antigenic stimulation through IgE receptor crosslinking using histone modification marks H3K4me1 and H3K27ac ChIP-seq and chromatin accessibility assay¹⁴³. We identified two antigen stimulation-responsive Il13 enhancers: the proximal enhancer and Il13 E+6.5 enhancer (overlapping with HIS HSII/CNS1). A cluster of TFBSs is identified in the proximal Il13 enhancer through TFBSs analysis, including the GATA2, AP-1, STAT5, RUNX1 and NFATC2 binding sites. Through TFBSs mutational analysis, we found that the combinatorial GATA2, AP-1, STAT5, RUNX1, and NFATC2 binding sites at the proximal Il13 enhancer are essential for enhancer activity in response to antigenic stimulation. And the EGR2 binding site at the E+6.5 enhancer is crucial for antigenic stimulation-induced enhancer activity¹⁴³.

In another study, we focused on enhancers that respond to two extracellular stimulations. We determined whether signals generated by antigenic stimulation and signals triggered by epithelium-derived cytokine IL-33 can induce transcriptional synergy for proinflammatory cytokine genes. Viral infections are the primary trigger for exacerbating allergic inflammation^144-146. During infections, injured epithelial cells release IL-33 to act on MCs that express high levels of the IL-33 receptor (IL1RL1)^16-18. IL-33 acts synergistically with allergic stimuli, such as antigen crosslinking of the high-affinity IgE receptor, to promote the transcription of proinflammatory cytokine and chemokine genes, including IL1B, IL6, IL13, IL8, and CCL1 ^19-21,131.

We further demonstrate that IL-33 can prime human skin MCs for synergistically enhanced responses to antigenic stimulation. We propose that enhancers contain SDTF binding sites to allow SDTFs to interact with nearby SDTFs and conventional transcription factors and co-factors that bind to distal enhancers to promote transcriptional synergy of proinflammatory cytokine and chemokine genes (Fig. 2).

The role of human lung mast cells in airway inflammation

The role of human lung MCs in airway inflammation has been documented in severe asthma^64,147,148. In fact, the effectiveness of anti-IgE antibody therapy provides strong evidence that human lung MCs play a critical role in mediating chronic inflammation of the lung in difficult-to-treat asthmatic patients^149-151. However, mechanisms by which human lung MCs contribute to airway inflammation remain poorly understood.

The human lung has significantly more MCs than the mouse lung as measured by histological analysis and single-cell RNA-seq (scRNA) analysis^64,152. Human MCs are located in the airway submucosa, the outer wall of the small airway, and perivascular connective tissue^153-155. Typically, human MCs are categorized into two main subsets based on the expression of specific proteases: MC_T (Mast cell tryptase-positive) and MC_TC (Mast cell tryptase and chymase-positive)¹⁵⁶. The MC_TC subset expresses tryptase along with chymase, cathepsin G and CPA3, whereas the MC_T subset only expresses tryptas¹⁵⁶. However, recent studies using scRNA analysis revealed human MCs could be classified into seven subsets⁶⁰, although the differences between various human MC subsets resemble a continuous nature rather than an absolute distinction^63,157.

It has been well-documented that the number of MCs increases in the lungs of asthmatic patients. An increased number of MC_TC were found in the lung bronchoalveolar lavage (BAL) and the distal lung airway of the patients with severe asthma^64,147,148. MCs have been shown to infiltrate into airway smooth muscle^158,159. Recently, MC infiltration into airway epithelium has also been observed in asthmatic patients with excise-induced airway bronchoconstriction^23,24. These findings have provided a cellular basis for treating patients resistant to conventional treatment with anti-IgE to reduce MC-mediated type 2 chronic inflammation.

The exact mechanisms by which human lung MCs contribute to airway inflammation and airway constriction have not been fully understood due to the limited availability of human lung tissues. Accumulated evidence suggests that a complex network of cell-cell and gene-gene interactions is in play. Human MCs release a variety of MC mediators upon degranulation after MC activation, including preformed mediators like histamine and MC proteases such as tryptase and chymase. Additionally, human MCs synthesize mediators de novo, including leukotriene B4, leukotriene D4 and prostaglandin D2. Cytokines such as interleukin 1, interleukin 3, tumor necrosis factor, and transforming growth factor are also released^155,160. The activation of MCs can also contribute to lung inflammation by recruiting other immune cells, such as T cells, eosinophils, dendritic cells, and neutrophils. Understanding these mechanisms can shed light on the pathogenesis of airway inflammation and potentially facilitate the advancement of novel therapeutic approaches for asthma and other respiratory diseases.

One of the challenges in understanding the mechanisms of how human MCs contribute to chronic inflammation is that cytokine and chemokine gene and protein expression are not easily detected in MCs in the lung tissue or BALs of asthmatic patients. Cytokine and chemokine genes are expressed at low levels under normal conditions. Their gene expression can be upregulated rapidly upon stimulations and returned to basal levels a few hours after the stimulation, making it difficult to accurately measure cytokine and chemokine genes directly ex vivo. To overcome this challenge, we used the precision cut lung slices (PCLS) as a culture model for investigating proinflammatory cytokine gene transcription in primary lung MCs. The PCLS model, compared to the primary cell culture model, is a physiologically more relevant model for studying the whole lung in response to various stimuli¹⁶¹. This model has appropriate cell-cell interaction, resembling the human lung. We sensitized MCs in situ in PCLS culture and crosslinked IgE receptor-bound IgE with antigen. Total RNA was prepared from the treated PCLS and used for proinflammatory cytokine mRNA measurement. Fig. 3 shows that after antigenic stimulation, proinflammatory chemokine and cytokine CXCL8 and IL1B genes were upregulated by 5.5-fold and 8.2-fold, respectively. The results demonstrate that this PCLS culture system is suitable for spatial analysis of mechanisms by which human lung MCs contribute to chronic inflammation.

Figure 3.

Antigenic stimulation promotes the transcription of CXCL8 and IL1B genes in human lung slices. Human lung slices were not treated (UN) or sensitized with IgE anti-NP for 24 hours and challenged with NP-HSA for 2 h. qPCR analysis of CXCL8 (A) and IL1B (B) mRNA expression. Statistical differences were analyzed using two-tailed student’s t test. Data represents mean ± SEM of three biological samples.

Infections are the primary trigger for exacerbating allergic inflammation^144-146. Injured epithelial cells are pivotal in exacerbating the existing allergic inflammation during infections. Epithelial cell-derived cytokines IL-25, TSLP, and IL-33, released due to injury caused by infections, activate MCs to transcribe pro-inflammatory cytokine and chemokine genes^19,162. Our lab has also demonstrated that IL-33 priming and antigenic stimulation synergistically promote the transcription of proinflammatory cytokine and chemokine genes in primary human skin MC cultures²². To investigate whether IgE and IL-33 can mediate the transcription synergy of the IL13 gene in the human lung MCs, we cultured primary lung MCs from two human donors. The primary lung MCs were either untreated or primed with IL-33 for 24 hours. These cells were stimulated with IgE receptor crosslinking (IgECL) or IL-33 alone or with a combination of IL-33 and IgECL for an additional two hours. The synergistic effect between IL-33 priming and IgECL was measured by synergistic log₂ Fold Change (FC) as described in our publication²². We found that IL-33 priming for 24 hours promoted transcriptional synergy for IL13 gene transcription induced by the combined IL-33 and IgECL stimulation (log₂FC = 1.9, p < 0.05), resulting in a 5.2-fold increase in transcriptional synergy for IL13, compared to without IL-33 priming (log₂FC=0.6) (Fig. 4). Currently, we are optimizing the PCLS culture system to examine whether IL-33 priming and antigenic stimulation synergistically promote Il13 gene transcription in lung MCs in situ in the PCLS model.

Figure 4.

IL-33 priming strongly promotes IL13 transcription in response to antigenic stimulation. Human lung MCs were either untreated or primed with IL-33 for 24 hours. Then, the cells were stimulated with IgECL or IL-33 alone or with a combination of IL-33 and IgECL for an additional two hours. IL13 mRNA was analyzed using qPCR. Means +/− SEM were calculated from 3 biological samples from 3 experiments. Statistical differences between single and combined treatments were analyzed using two-way ANOVA. * P < 0.05.

Concluding Remarks

In conclusion, the pivotal role of MCs in allergic inflammation, anaphylaxis, and chronic inflammatory diseases and their interaction with various cell types highlight the complex and interconnected nature of immune responses. Further studies deciphering transcriptional codes in the critical enhancers of the MC genes would provide valuable insights into the mechanisms of how typical enhancers, super-enhancers, and promoters, along with co-factors, cooperate to maintain MC genes and induce rapid transcription of proinflammatory cytokine and chemokine genes during allergic reactions and infections. Analysis of functional SNPs within the critical enhancers will help explain individual susceptibility to allergy development and severity. This knowledge will also aid the development of potential CRISPR-based therapeutic interventions for MC-mediated diseases.

Abbreviations

MCs

Mast cells

COPD

Chronic obstructive pulmonary disease

MACS

MC activation syndrome

COVID

Coronavirus disease

Ag

Antigen

MAPK

Mitogen-activated protein kinase

MRGPR

Mas-related G-couple receptors

CTMCs

Connective tissue mast cells

MMCs

Mucosal mast cells

MCPs

Mast cell lineage-restricted progenitors

MPPs

Multiple potential progenitors

CMPs

Common myeloid progenitors

GMPs

Granulocyte-monocyte progenitors

BMCPs

Basophil/mast cell progenitors

GPs

Granulocyte progenitors

Pre-BMPs

Pre-basophil-mast cell progenitors

Pro-BMPs

Pro-basophil-mast cell progenitors

scRNA-seq

Single-cell RNA sequencing

mMCP-1

Mouse mast cell protease 1

GATA2

GATA binding protein 2

MITF

Microphthalmia-associated transcription factor

H3K4me1

Monomethylation of lysine residue 4 on histone 3

H3K27ac

Acetylation of lysine residue 27 on histone 3

HDC

Histidine decarboxylase

TSS

Transcription start site

SDTFs

Signal-dependent transcription factors

COC

Ca²⁺-dependent open chromatin

IL1RL1/ST2

Interleukin 1 receptor-like 1

BAL

Bronchoalveolar lavage

SNPs

Single-nucleotide polymorphisms