The emerging role of the hedgehog signaling pathway in immunity response and autoimmune diseases

Abstract

The Hedgehog (Hh) family is a prototypical morphogen involved in embryonic patterning, multi-lineage differentiation, self-renewal, morphogenesis, and regeneration. There are studies that have demonstrated that the Hh signaling pathway differentiates developing T cells into MHC-restricted self-antigen tolerant T cells in a concentration-dependent manner in the thymus. Whereas Hh signaling pathway is not required in the differentiation of B cells but is indispensable in maintaining the regeneration of hematopoietic stem cells (HSCs) and the viability of germinal centers (GCs) B cells. The Hh signaling pathway exerts both positive and negative effects on immune responses, which involves activating human peripheral CD4⁺ T cells, regulating the accumulation of natural killer T (NKT) cells, recruiting and activating macrophages, increasing CD4⁺Foxp3⁺ regulatory T cells in the inflammation sites to sustain homeostasis. Hedgehog signaling is involved in the patterning of the embryo, as well as homeostasis of adult tissues. Therefore, this review aims to highlight evidence for Hh signaling in the differentiation, function of immune cells and autoimmune disease. Targeting Hh signaling promises to be a novel, alternative or adjunct approach to treating tumors and autoimmune diseases.

Keywords:

• Hedgehog signaling pathway
• T cell
• B cell
• immune response
• diabetes mellitus

1. Introduction

The organs, cells, and molecules that work in a highly proficient immune surveillance machinery that recognizes and eliminates foreign substances as well as genetically altered cells, are termed the immune system. The immune system initiates early responses of innate immunity and later responses of adaptive immunity to protect human health [1,2]. Emerging evidence emphasizes the pivotal role of signaling pathways in immune responses. Several classical immune-related pathways have been discovered and their mechanisms have been studied in depth. For example, the NF-κB signaling pathway is responsible for early lymphocyte development and induces the transcription of pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs), cyclooxygenase 2 (COX2), and inducible nitric oxide synthase (iNOS) to mediate inflammatory diseases [3]. The JAK- STAT signaling pathway plays a pivotal role in immune regulation, host defense, and immune pathology by mediating cytokines [4,5]. The Wnt signaling plays an important role in lymph myelopoiesis and immune responses. They can exert their roles in T cell differentiation and effector function in various inflammatory diseases including cancer, as well as in autoimmunity and viral infections [6]. However, the immune system is intricate, and even more, aspects need to be studied and excavated because of the value of therapeutic implications.

Although, there is considerably less information available than for the Notch and WNT signaling pathways presently, the Shh signaling pathway has also been shown to contribute to lymphoid cell development and differentiation. Therefore, many researchers have focused on the Hh signaling pathway because of its newly uncovered functions in immune cell fate decisions and immune responses. Hh was first discovered by a mutagenesis screen in Drosophila, which affected the segment number and polarity in Drosophila [7]. There are three kinds of Hh proteins in mammals, namely Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh), which exhibit distinct functions. Shh has a particularly striking role in the specification of cell types in the nervous system and is also involved in the growth of the limbs and lungs. Ihh is essential to osteoblast differentiation and skeletal development. Dhh is responsible for the morphogenesis and maturation of gonads [8,9]. The Hh signaling pathway is highly conserved and possesses two transmembrane proteins—Patched (Ptch) and Smoothened (Smo) on the receiving cell [10,11]. In the absence of the Hh ligand, the transmembrane receptor Ptch inhibits Smo activity. Once the Hh protein binds to Ptch, Smo is capable of activating the transcription factor protein glioma-associated oncogene homolog (Gli) [12]. Then, Gli travels into the nucleus and switches on the transcription of Hh target genes (Figure 1). Depending on the different functions of the Hh target genes, the Hh signaling pathway is widely implicated in specifying cell fates, such as muscle cells and neuronal cells in embryos. Here, we focus on the biological function and mechanisms of Hh signaling in innate immune cells, T-cell and B-cell development and activation, and pathogenesis in autoimmune diseases. Hh proteins were initially identified in association with embryonic development. Upon Shh binding to Ptch in the Hedgehog signaling pathway, the inhibitory effect on Smo is abolished, which leads to the activation of Smo. Activated Smo signaling induces nuclear translocation of transcription factor glioma-associated oncogene (Gli1, Gli2, and Gli3) proteins, resulting in the induction of Hh-target genes, such as Ptch and Gli, to control cell proliferation, migration, differentiation, and survival [13].

Figure 1. Hedgehog signaling pathways (A) without Hedgehog ligand, (B) or with hedgehog ligand.

2. The role of Hh signaling in innate immunity

Immune responses include innate immunity and adaptive immunity. The innate immune system is the first defense that recognizes common constituents of many microorganisms and is activated immediately upon contact, irrespective of prior exposure [14]. The innate immune system consists of phagocytic cells, NK cells, the complement system, and other aspects such as barrier function and antimicrobial peptides. The cells have important roles in immune responses in the context of cancer, infection, and autoimmunity, but the differentiation and function of these cell types remain to be clearly defined. There is much evidence that WNT and NOTCH signaling pathways are emerging as regulators of hematopoietic cell development and differentiation [15]. The Hh signaling pathway is one of the cellular signaling pathways involved in embryogenesis and plays an important role in embryo development. Recent research has revealed that the Hh signaling pathway continues to function in adult tissue, such as in the differentiation and function of immune cells, cell proliferation, inflammation, and other basic processes.

2.1. Macrophages

Macrophages are phagocytes of the innate immune system. Macrophages are important innate immune cells in the body. They serve as sentinels for innate immunity and adaptive immunity, which are considered to stem from the early stage. However, the current view is that macrophages may originate in the yolk sac, and undergo self-renewal [16]. Macrophage polarization is a process of producing different functional phenotypes according to the specific surrounding environment and is generally divided into two subgroups: classically activated M1 pro-inflammatory cells and alternatively activated M2 anti-inflammatory cells, which represent the polarization of the two extremes [17–19]. Many pathological processes are associated with the phenotype of M1/M2 macrophages, and many studies have suggested that the JAK/STAT, IRF, Notch, and PI3K/Akt signaling pathways can mediate the function of macrophages. We need to explore the connection between macrophages and Hh signaling in view of complexity.

Some studies have shown that macrophages can exert an anti-tumor effect. Further evidence from studies showed that Hh is one of the major mediators involved in self-renewal and maintenance of cancer stem cells (CSCs) [20]. The result showed that the lipopolysaccharide (LPS)--activated macrophage conditioned medium (CM) itself contains high amounts of Shh, Patched-1(Ptch1) that the receptor of Shh inhibits Smo a downstream protein in the Hh pathway. When upon Shh binding the inhibition of Smo by Ptch1 is relieved. So further studies showed that the CSC phenotype was mediated by Shh-Gli ignaling pathway. The treatment of colorectal cancer (CRC) cells with CM induces significant increases in Gli activity and induces CSC phenotype in CRC cells. In addition, LPS-activated macrophage CM enhanced chemoresistance. On the contrary, these effects were blocked by the depletion of Shh in macrophage CM [21]. Tumor-derived Shh drives PD-L1 expression in tumor-associated macrophages (TAMs) to suppress tumor-infiltrating CD⁸⁺ T-cell function, leading to tumor progression finally. Shh-dependent upregulation of PD-L1 in TAMs is mediated by signal transduction and transcription activating factor 3 (STAT3), and reveals an essential role for Shh-dependent PD-L1 upregulation in TAMs in suppressing anti-tumor immunity within the TME [22].

The Shh signaling pathway plays an important role in actively orchestrating many aspects of cerebellar development and maturation. Shh molecular subgroup is one of Medulloblastoma. Shh subgroup is characterized by constitutive activation of the Shh signaling pathway leading to abnormal proliferation of the cerebellar cells. The study showed that the quantity of TAMs was abundant in Shh medulloblastoma. TAMs were significantly higher in Shh medulloblastoma than in other subgroups. This is a significant increase of monocyte chemotactic protein-1 (MCP-1/CCL2), which could recruit TAMs and promote M2 macrophage polarization in human Shh medulloblastoma. Also tumor-derived Shh directly can act on TAMs to promote M2 polarization, mediated by the transcription factor Krüppel-like factor 4 (Klf4). Therefore, there is a closely association of TAM recruitment and medulloblastoma patients’ outcomes [23,24]^. A number of studies have shown an association between Hh pathway activation and the initiation of tumor formation. The study observed an enhanced tolerance to Hh pathway inhibitors in MDA-MB-231 cells after co-culturing with M2 macrophages. Hh pathway inhibitors have demonstrated considerable cytotoxic activity in breast cancer cells. This study demonstrates the role of macrophages in Hh pathway inhibition resistance and the role of macrophage-derived IL6 in this resistance of breast cancer cells to Hh inhibition. These data indicate that antagonizing IL6 together with Hh pathway inhibitors may be a novel therapeutic strategy for breast cancer. The study suggests an important role of M2 macrophages in protecting breast cancer cells from the antitumor effects of Hh pathway inhibitors. IL6 is secreted by M2 macrophages upon Hh pathway. The IL6 therapy could therefore be a promising potential combination partner for breast cancer treated with Hh inhibitors [25].

We also noted that the expression of genes involved in Hh signaling was decreased in aged macrophages across multiple tissues. The Hh signaling in young animals increased the expression of proinflammatory cytokines, while in vitro activation of Hh signaling in old macrophages. That suppressed the expression of these inflammatory cytokines in turn. So Hh signaling could be a potential intervention axis for mitigating age-associated inflammation and related diseases. Smo, which encodes the key G protein-coupled receptor (GPCR) and is involved in the activation of Hh signaling [26,27]. Efcab7 encodes a protein involved in the localization of the EVC:EVC2 complex and positive regulation of Hh signaling. The decreased expression of Hh signaling components Smo and Efcab7 resulted in a reduction of Hh transcriptional activity. The Hh target genes Gli1 and Ptch1 have been shown to positively correlate with Hh signaling. Therefore, we inhibited Hh signaling using vismodegib, a potent Smo inhibitor, in young adult mice and the expression is increased of several genes encoding inflammatory cytokines [28]. Hh receptor Ptch1 is mainly expressed in parotid Macrophages Hh activation greatly enhances paracrine interactions between salivary gland resident macrophages, epithelial progenitors and endothelial cells through Csf1, Hgf, and C1q signaling pathways. Consistently, expression of these paracrine factors and their receptors in salivary glands decreased following irradiation but was restored by transient Hh-Gli activation [29,30].

In the absence of the Shh signaling pathway its ligand, the pathway receptor Ptch1 inhibits the signal activator Smo. Shh is an essential signaling molecule for the development and patterning of the central nervous system. The peripheral nerves have regenerative function. During regeneration, Shh signaling was activated within the entire distal region of the inferior alveolar nerve (IAN) and proximal stumps. Inhibition of Shh signaling by cyclopamine application at the transection site led to abnormal axon growth in random directions, and reduce number of macrophages meanwhile [31].

2.2. Natural killer and NKT cells

The liver lymphocyte population is enriched with natural killer (NK) cells, which are innate lymphoid cells (ILCs) that are equipped with cytotoxic granules. NKT cells for innate-like lymphocytes, they are critical immune cells for the clearance of tumors. Innate lymphoid cells (ILCs) and innate-like lymphocytes have important roles in immune responses in the context of infection, cancer, and autoimmunity [14]. ILCs regulate lymphoid tissue development and immune responses, even primarily at epithelial surfaces. Hh pathways are intricately linked. Pharmacological inhibitors of Hh signaling are in clinical trials as novel cancer therapeutics. Meanwhile, their effect on immune cell functions critical in the antitumor response should be carefully considered, NK and NKT cells in particular [14]. Forkhead box Q1(FOXQ1) expression was different in NKTCL patients than in healthy controls, with the former higher than the latter. SNK-6 cells were transfected with FOXQ1-shRNA or Shh pathway inhibitor Cyclopamine/recombinant Shh protein. Shh pathway proteins are decreased and induced apoptosis via the Shh pathway in natural killer/T-cell lymphoma (NKTCL) [32]. By identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. The results showed that hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during non-alcoholic steatohepatitis (NASH) [33]. In cultured invariant NKT (iNKT) cells, Shh enhances proliferation, inhibits apoptosis, induces activation, and stimulates expression of the profibrogenic cytokine, IL-13. Livers of transgenic mice with an overly active Hh pathway harbor increased numbers of iNKT cells. iNKT cells also express Shh. These results demonstrate that Hh ligands regulate the viability and phenotype of NKT cells [34]. Hedgehog signaling plays an important role in the functions of NK and NKT cells.

Hedgehog (Hh) signaling is activated in various types of cancer. Many Hh inhibitors show promise as anti-cancer drugs. According to evaluate the effects of Hh inhibition and oxygen concentrations on the function of activated T and NK (TNK) lymphocytes derived from patients with cancer were evaluated. Proliferation, migration, surface NKG2D expression, and cytotoxicity were all significantly inhibited, and IFN-C secretion was significantly increased upon Hh inhibitor treatment of activated TNK lymphocytes under hypoxic conditions in vitro. These results suggest that Hh signaling plays a pivotal role in activated TNK lymphocyte cell function [35]. Thirty-nine new 2-phenylindole derivatives were designed as potential anticancer agents, which of indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hh signaling activation in NIH3T3 Shh-Light II cells. These compounds of this class not only potently inhibited tubulin polymerization and cancer cell growth, but also stimulated natural killer cell cytotoxic activity and repression of Hh-dependent cancer [36].

Based on this finding, Hh signaling participates in the innate immune system and provides critical mechanisms for anti-tumor or infection immunity.

3. The role of Hh signaling adaptive immunity

The adaptive immune response takes up to a week to develop, which facilitates pathogen-specific immunologic effects or pathways, generation of immunologic memory, and regulation of host immune homeostasis [37].

3.1. The Hh signaling pathway regulates the differentiation of T cells

In the thymic stroma, thymic epithelial cells (TECs) are essential components producing the hedgehog, protein Shh to form a concentration gradient, not only promoting the differentiation of TECs in an autocrine manner [38], but also the transition of thymocytes [39]. During development, thymocytes move through distinct areas of the thymus and are exposed to different strengths of Hh signals specifying thymocytes fates.

At the stage of development from the earliest DN1 progenitors to DN2 cells, Shh, Gli2, and Gli3 are needed. In the E13.5 Gli2^-/- and Gli3^-/- embryos, differentiation decreased to DN2 cells was found [40,41], as in E13.5 shh ^-/-thymus [42]. Nevertheless, Gli1 ^-/- had the same phenotype as WT, demonstrating that Gli1 was not essential for DN1 to DN2 cells [43]. In addition, these findings could be explained by the expression pattern of the Gli transcription factors in DN1 and DN2, where Gli2 and Gli3 were highly expressed, but not Gli1^[41].

The function of the Hh signaling pathway in DN to DP has also been extensively studied. Shh is required in the successful rearrangement of the TCRβ locus to ensure that DN3 cells differentiate into DP cells [44,45]. In E16.5 shh-deficient thymus, DP cells first appeared at that time in WT, DN cells failed to rearrange the TCRβ locus, and more apoptosis was found in DN4 stages, resulting in the poor transition from DN to DP cells. However, shh negatively regulates pre-TCR-induced differentiation from DN to DP thymocytes cell in both mice and humans. This was confirmed in vitro studies using fetal thymus organ cultures (FTOCs). Thymocytes from recombination-activating gene 1 (Rag1)^-/- FTOCs were able to induce differentiation from DN3 to DP cells under the condition of anti-CD3 monoclonal antibody (mAb), which mimicked the pre-TCR signal [46]. Adding anti-Shh mAb to Rag1^-/-FTOC cells treated with anti-CD3mAb promoted thymocyte differentiation to the DP stage, while rShh arrested it [45].

In DP cells, Ihh expression is six-fold higher than that in DN cells, but the level of Gli1 transcription is lower, which reminds us that DP cells do not respond to the Ihh in autocrine manner. However, DP cells significantly increased in E16.5 Ihh± thymus, as well as CD25⁺ DN populations, which highly expressed Gli1 compared to WT [47]. These data support that Ihh produced by DP cells may negatively regulate the proliferation and differentiation of CD25⁺ DN cells, and then regulate the number of thymocytes to maintain homeostasis.

DP cells receive the regulation of the thymus microenvironment and migrate across the cortex towards the medulla to progress to the SP stage. The analysis of Gli2^ΔN2 transgenic mice, in which Hh-dependent transcription of Gli1 was highly expressed, showed a statistically significant reduction in the proportion of CD4 SP cells, coupled with an increase in the proportion of CD8 SP cells. Moreover, the persistent activation of the Hh signaling pathway allowed self-reactive T cells to escape clonal deletion; inhibited TCR mediated positive selection of CD8 T cells, altering differentiation to SP and the CD4/CD8 SP ratio; and reduced the expression of CD5 on DP and SP populations in the thymus. In contrast, Shh^-/- thymus had opposite phenotypes: DP preferred to mature to CD4SP; both positive and negative selection of a transgenic TCR were increased [48]. According to the consequences for thymocyte development of removal of the Shh signal (in the Shh^-/-thymus), or activation of Hh signaling in vivo (by Gli2ΔN2 expression) and in vitro (by Shh treatment), that strongly support the hypothesis that Hh signaling in thymocytes modulates TCR signal strength [49,50]. Collectively, this evidence indicates that the activated Hh signaling pathway gives rise to a reduction in TCR signal strength [50,51], and profoundly affects the later stages of thymocyte development.

Based on these data above, we conclude that for αβ T-cells, Hh signaling modulates TCR signal strength; plays an important role in T cell development, regulating the progression from a DN thymocyte to a DP thymocyte; participates in positive selection to ensure appropriate MHC restriction and negative selection to delete self-reactive clones. For γδ T-cells, Hh signaling promotes maturation and influences the subset distribution in the thymus [52]. In addition, Hh signaling mediates the TH1/TH2 differentiation, activation and proliferation of peripheral CD4⁺ T cells in both humans and mice subjects, and modulates the function of these effector cells [53–55].

3.2. B Cell

Hepatic stellate cell (HSCs) committed to the B-cells lineage occur mostly in bone marrow and in the fetal liver prenatally. HSCs give rise to hematopoietic multipotential progenitors (MPPs) and then lose erythroid-cell and megakaryocyte potential, retaining myeloid-cell and lymphoid-cell potential to mature into lymphoid-primed multipotent progenitors (LMPPs), from which progenitors of B cells—common lymphoid progenitors (CLPs) are produced. CLPs undergo progressive differentiation through prepro-B cells, pro-B cells, pre-B cells to immature B cells [56,57]. Immature B cells center the spleen and mature into different subsets of B cells. Feta-liver derived HSCs refer to as B-1 cells progenitors; B-2 cells including follicular B cells and marginal zone B cells are generated from bone marrow derived HSCs [58,59]. According to cell surface markers, B-1 (CD19⁺ B220lo⁻) and B-2 (CD19⁻B220⁺) cells can be distinguished [59,60]. The Hh pathway is inextricably linked with HSCs. Hh signaling modulates HSCs cell cycle regulators to balance hematopoietic homeostasis and regeneration in vivo [61]. After blocking Hh signaling in mouse bone marrow cells with the anti-Hh neutralizing Ab 5e1 or the Smo antagonist cyclopamine in ex vivo, pro-B cells with high Smo expression on the cell surface presented impaired proliferation. In additioin, depletion of the Smo protein from stromal cells impaired the generation of B-lymphoid cells from hematopoietic progenitor cells [62]. However, mutation of Gli3 in the fetal liver which showed high Shh, Ptch1 and Gli1 expression in the stroma, influenced B-cell development from the CLP populations through more mature B lineage–committed population, resulting in an overall reduction in B lineage–committed cells, the proportion of pre–B cells, and the population of CLPs in mouse. Similarly, the Shh-deficient fetal liver showed increased B lineage commitment and B-cells differentiation [63]. In peripheral lymphoid organs, Shh is produced by follicular dendritic cells (FDCs), mainly in germinal centers (GCs), and functions to protect GC B cells from Fas-induced apoptosis [64].

3.3. T Cell

Hedgehog (Hh) signaling regulates multiple aspects of animal development, tissue homeostasis and regeneration [65]. Therefore, many studies on immune responses have focused on this pathway. The glioma-associated oncogene (Gli) family is the Hh signaling pathways transcription factors, including Gli1, Gli2 and Gli3, Gli1 acts as an activator of transcription, Gli2 is both a repressor and activator, and Gli3 represses transcriptional activity [14]. After research and discovery that Gene transcription driven by the Gli2 transcriptional activator isoform (Gli2A) attenuated T-cell activation and proliferation following T-cell receptor (TCR) stimulation, and in T-cells altered gene expression profiles, impaired the TCR-induced Ca(2+) flux and nuclear expression of nuclear factor of activated Tcell2 (NFAT2), suppressed upregulation of molecules essential for activation, and attenuated signaling pathways upstream of the AP-1 and NF-κB complexes, leading to reduced activation of these important transcription factors. Inhibition of physiological Hh-dependent transcription increased NF-κB activity upon TCR ligation [66]. Hh-dependent transcription in T cells promotes Th2 transcriptional programs and differentiation, exacerbating allergic disease [67]. The Gli-dependent transcription is activated in T cells in vivo during murine antiangiogenic drug (AAD), a model for the immune pathology of asthma, and that genetic repression of Gli signaling in T cells decreases the differentiation and recruitment of Th2 cells to the lung. Hh signaling to T cells, via downstream Gli transcription factors, enhances T cell conversion to a Th2 phenotype [68]. Therefore, in the periphery, Hh signaling could function to change the threshold of T-cell activation and influence TCR-dependent T-cell fate decisions, such as TH1/TH2 differentiation [69]. The study of central nervous system (CNS)-endogenous hedgehog pathway is a signal triggered as part of the host response during the inflammatory phase of multiple sclerosis and experimental autoimmune encephalomyelitis. According to using a murine genetic model, in which the hedgehog signaling is compromised in CD4 T cells, leading to an increase in inflammatory, there is evidence that hedgehog signaling regulates the pathogenic profile of CD4 T cells by limiting their production of the inflammatory cytokines granulocyte-macrophage colony-stimulating factor and interferon-γ and by antagonizing their inflammatory program at the transcriptome level [70]. Hedgehog signaling is an important regulator of T-cell differentiation, and peripheral T-cell activation requires further investigation [65].

4. Autoimmune diseases

4.1. Insulin dependent diabetes mellitus

Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion. The range of autoimmune destruction of the B-cells of the pancreas with consequent insulin deficiency to abnormalities that result in resistance to insulin action is involved in the development of diabetes [71]. The disease in which the role of Hh signaling is being more closely examined.

Sonic hedgehog (Shh) is an intercellular signaling molecule that regulates pancreas development in mammals [72]. Hh signaling functions early during pancreas morphogenesis to regulate epithelial cells, according to beta-cell expansion and to modulate glucose metabolism by regulating insulin production in adult mice [73]. Of course, Hh signaling is not restricted to patterning in early pancreas development but also continues to signal in differentiated beta-cells of the endocrine pancreas in regulating insulin production. Thus, defective Hh signaling in the pancreas should be considered as a potential factor in the pathogenesis of diabetes [74]. Hh signaling activates the insulin gene promoter indirectly via the direct activation of islet duodenal homeobox-1(IDX-1)expression. Because IDX-1 gene expression is essential for insulin gene expression, pancreatic beta-cell development, and normal glucose homeostasis, our findings indicate that Hh signaling regulates IDX-1 expression in the endocrine pancreas. It may have provided new ideas for clinical diagnosis and treatment [75]. Constitutive activation of Shh signaling that precludes endocrine differentiation; in contrast, Manner Pancreas development requires restrained Hh signaling activation. We need focus on the effects of deregulated Hh signaling in the pancreatic mesenchyme on islet function. WhenPtch1 was deleted in these cells, Hh signaling in the pancreatic mesenchyme of mouse embryos increased. Research showed that deregulated Hh signaling in mesenchymal cells is sufficient to impair pancreatic development, affecting both endocrine and exocrine cells. Transgenic embryos displayed disrupted islet cellular composition and morphology, with a reduced β-cell portion. Thus, regulated mesenchymal Hh signaling is required for pancreas organogenesis and establishment of its proper cellular composition [76,77]. Manipulation of Shh signaling pathway can be used as reliable approach to improve the generation of functional insulin-producing cells (IPCs) from mesenchymal stromal cells (MSCs). A novel differentiation protocol was used to produce IPCs from adipose tissue–derived MSCs (ATDMSCs) based on sequential inhibition and reactivation of the Shh pathway. Then, sequential inhibition and reactivation of the Shh pathway is promoted. Insulin granule formation, glucose-stimulated insulin secretion and gene expression patterns related to the pancreatic endocrine development and function were analyzed in manipulated and unmanipulated IPCs. The early inhibition and late reactivation of the Shh signaling pathway during the differentiation of ATDMSCs improved the functional properties of IPCs, and could be considered a novel method for cell-based therapy for type 1 diabetes [78]. In contrast, deregulation of the Hh pathway impairs β-cell function by interfering with the mature β-cell differentiation state [79].

In summary, Hh signaling is implicated in protecting beta-cells from cytokine induced cytotoxicity [80]. That regulated mesenchymal Hh signaling is required for pancreas organogenesis and establishment of its proper cellular composition [81]. The Hh signaling pathway, according to several pathogenic processes, is involved in the development of diabetes.

4.2. Rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disorder that primarily affects the joints. Genome-wide association studies using single nucleotide polymorphisms have characterized more than a hundred loci associated with rheumatoid arthritis risk, most of which implicate immune mechanisms, some of which are shared with other chronic inflammatory diseases [82]. The Hh signaling pathway is a key regulator in chondrocyte growth and differentiation [83].

Rheumatoid arthritis (RA) is characterized by chronic synovitis and pannus formation associated with the activation of fibroblast-like synoviocytes (FLSs). Many research have demonstrated that both play a key role in RA pathogenesis, contribute to the cartilage injury and hyperplasia of the synovium, and contribute to the release of inflammatory cytokines. Complete Freund’s adjuvant was used to induce adjuvant induced arthritis (AIA). The protein expression of Hh signal related genes (Shh, Ptch1, Smo and Gli1) in cartilage were assayed by immune histochemistry. The mRNA levels of Hh signaling and ECM components (COII and aggrecan) were measured. The mRNA levels in cartilage tissues of AIA rats were significantly increased compared with those of sham rats, and were associated with the severity of cartilage damage. Instead, in vitro, cyclopamine effectively decreased the mRNA levels of Shh, Ptch1, Smo and Gli1, and increased the mRNA levels of COII and aggrecan in AIA chondrocytes, suggesting that the Hh signaling pathway is involved in the pathogenesis of cartilage damage in RA [76,77]. FLSs were isolated from RA synovium. Shh signaling was studied using a Smo antagonist (GDC-0449) and small interfering RNA (siRNA) targeting the Smo gene in FLSs, which showed significantly decreased proliferation compared to controls. Cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression, and decrease in cyclin-dependent kinase p21 mRNA expression in Smo-siRNA transfected cells [78]. Fibroblast-like synoviocytes acquire aggressive phenotypes characterized by enhanced migration abilities and inherent invasive qualities in rheumatoid arthritis (RA). Smo is a key component of Shh signaling and contributes to tumor cell invasion and metastasis. Investigating the role of Smo in the modulation of cell migration and exploring the underlying molecular mechanism(s), these results suggest that Smo plays an important role in RA-FLSs migration through activation of Rho GTPase signaling and may contribute to the progression of RA, thus, targeting Shh signaling may have therapeutic potential in patients with RA [84].

Annexina2 (Axna2) is an important mediating agent that induces angiogenesis in vascular diseases. Compared with healthy people, the expression of Axna2 and Axna2 receptor (Axna2R) was up-regulated in patients with RA. Axna2 promoted HUVEC proliferation by binding Axna2R according to cell experiments, and could activate Hedgehog (Hh) signaling and up-regulate the expression of Ihh and Gli. The overexpression of Axna2 could promote the development of CIA. Meanwhile, Axna2 depends on combining to Axna2R to activate and increase Hh signaling and the expression of its downstream targets VEGF, Ang-2 and MMP-2 to promote HUVEC proliferation, eventually causing angiogenesis [85]. Therefore, inhibition of Axna2 may be a potential target for the treatment of RA.

Based on these studies, the Hh signaling pathway underlies the pathogenesis of cartilage destruction in RA. The Hh signaling pathway was over-activated in the articular cartilage of CIA rats, and the up-regulated Hh signal was apparently associated with the severity of cartilage damage. Further research is needed to investigate the potential rapeutical effect of cyclopa mine and its molecular mechanisms on AIA rats. In the current investigations, we elucidated the impact of the Shh signaling pathway on FLSs proliferation and advanced the understanding of the molecular mechanisms by which the Shh pathway is linked to cell cycle regulation. and identified a new effect of Smo on RA-FLSs migration and elucidated the underlying molecular mechanisms by which the Shh pathway is linked to cell migration. Meanwhile, during the progression of RA, angiogenesis is critical in maintaining tumor-like synovial growth and an inflammatory state. It may therefore play an important role in the proliferation of RA FLSs and inhibition of angiogenesis (Figure 2 and Table 1). This research suggests a new therapeutic approach for RA. There is already research showed that saponin Platycodin D (PD) improves joint synovium inflammation and apoptosis in CIA rats. PD plays an important role in the inhibition activity of Mh7a cell. The phenomenon accompanied with the mitochondrial membrane potential decreased, the expression level of the shh signaling pathway-related protein suFu increased, the expression levels of shh and Gli decreased, and cell serum levels of TNF-a and IL-6 decreased significantly. As a result, PD prevented proliferation and migration in RA [86].

Figure 2. (A) the role of Hedgehog signaling in rheumatic diseases. Hedgehog signaling pathway is involved in the inflammatory proliferation of FLSs, the proliferation of chondrocytes, Fibroblasts and pannus. (B) Deregulation of the Hh pathway impairs β-cell function by interfering with the mature β-cell differentiation state, and may result in deregulated insulin production and secretion.

Table 1. Treatment of rheumatic diseases and diabetes by targeting the Hh signalling pathway.

Disease	Inhibitor	Target	Result	Ref
IDDM		IDX-1	Insulin gene expression,pancreatic beta-cell development,and normal glucose homeostasis	[75]
		Ptch1	Impairs pancreatic development,affects both endocrine and exocrine cells,disrupts islet cellular composition and morphology,the portion of β-cell was reduced	[76,77]
		Shh	Improve the generation of functional insulin-producing cells (IPCs) from mesenchymal stromal cells (MSCs)	[78]
		Hh	Impairs β-cell function by interfering with the mature β-cell differentiation state	[79]
RA	cyclopamine		Decreased the mRNA levels of Shh, Ptch1, Smo and Gli1, and increased the mRNA levels of COII and aggrecan in AIA chondrocytes	[76,77]
	Smo-siRNA		Decrease in cyclin-dependent kinase p21 mRNA expression	[78]
	GDC-0449	Smo	Decreased proliferation of FLSs compared to controls, cell cycle arrest was validated by the significant increase in cyclin D1 and E1 mRNA expression	[78]
		Smo	Plays an important role in RA-FLSs migration through activation of Rho GTPase signaling and may contribute to the progression of RA	[84]
	Axna2	Ihh, Gli	Axna2 depends on combining to Axna2R to activate and increase Hh signaling and the expression of its downstream targets VEGF, Ang-2 and MMP-2 to promote HUVEC proliferation, eventually causing angiogenesis	[85]

Conclusion and outlook

As a morphogen, Hh specifies embryonic αβ T-cell fates at three key checkpoints: differentiation from DN1 to DN2 cells, pre-TCR-induced transition to DP cells, and transition from DP to mature SP cells [87], and it also influences the development of γδ T-cells (Figure 3). For B cells, Hh signaling is dispensable for maintaining the regeneration of HSCs and supporting the viability of GC B cells. However, the effect of Hh on B-cell development in the fetal liver and bone marrow is controversial (Figure 3), and the mechanism remains unclear. The expression of Hh signaling components in prepro-B cells, pro-B cells, pre-B cells and immature B cells populations, and the gradient of Hh should be taken into account when exploring.

Figure 3. Hh signaling regulates the development of T cells, B cells and other immune cells, and contributes to immune responses.

Apart from its effects on lymphoid cell development, Hh signaling has been implicated in regulating immune response. On the one hand, Hh acts as a strong macrophage chemoattractant to initiate the immune response; on the other hand, Hh amplifies the macrophage proinflammatory response by stimulating the expression of OPN in NKT cells in chronic inflammatory liver.

Currently, new studies have uncovered the novel functions of Hh signaling in sustaining immune homeostasis. A large body of evidence has highlighted the critical role of Hh signaling in immune homeostasis, and has detailed how Hh signaling pathways modulate immune responses. There still exists some misunderstandings in the process of immune responses facing different antigens. Further investigations are needed to obtain a more comprehensive understanding of the role of Hh signaling in the immune network, which may shed light on therapeutic options for inflammatory diseases. Surprisingly, a pharmacological inhibitor of Hh signaling, vismodegib has been identified not only to repress the proliferation of tumor cells, but also to extensively study Hh signaling in tumor cells, whereas the impact of Hh signaling on the immune TME is a newly explored territory. Shh promotes macrophage M2 polarization and reduces effector CD⁸⁺ T-cell recruitment to the tumor [88].

Thus, it is reasonable to believe that steady progress will be achieved, which provides practical guidance in intervention of the process of inflammation, providing novel therapies and strategies for treating these diseases related to autoimmunity. A further important message is that targeting Hh signaling promises to be a novel, alternative or adjunct approach to treating tumors and autoimmune diseases. Although these drugs bear great potential as novel therapeutics for cancer therapy, their effect on immune cell functions critical in the anti-tumor response should be carefully considered. In particular, NK and NKT cells are critical immune cells for the clearance of tumors. Furthermore, the potential downstream effects of Hh signaling pathways need to be further defined.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the National Nature Science Foundation of China (No. 31960156, No.31660338); Science and Technology Support Program of Guizhou Province (QKH-ZK[2021]111, QKH[2023]506); Science and Technology Fund of Guizhou Provincial Health Commission (gzwkj2022-019); Science and Technology Bureau Project of Zunyi City (Zun shi Kehe [2021]252).