Interleukin-9 (IL-9) is a pleiotropic cytokine produced by several immune and epithelial cells. Recently, many studies have eluded the physiological and pathological roles of IL-9 and its lineage-specific helper T cell subset (Th9). In this chapter, we will focus on the immunological role of Interleukin 9 (IL-9) in allergy and autoimmunity. We will introduce the basics of IL-9 and describe the cells involved in the secretion, signaling, and regulation of IL-9. After establishing the background, we will discuss the pathogenesis and regulation of IL-9 in allergic and autoimmune diseases. We will conclude the chapter by providing an updated therapeutics that target IL-9 and their potential uses in autoimmune and allergic diseases.
- multiple sclerosis
- mast cells
- atopic dermatitis
- food allergy
Interleukin-9 (IL-9) is a pleotropic cytokine that regulates diverse immunological functions (Figure 1). This cytokine was first identified in the late 1980s as a T cell growth factor . Because of the molecular weight of IL-9, it was initially known as P40 . Later studies revealed that the observed molecular weight was due to N-link glycosylation, and actual molecular weight for this discovered molecule is 14 kDa . A similar factor was also identified from Th2 cells and mast cells where it was initially named as T-cell growth Factor III (TCGF III) and mast cell growth-enhancing activity (MEA), respectively [2, 4]. Further studies revealed that both TCGF III and MEA actually represent the P40 factor . In later years, considering its pleotropic roles and the redundant nomenclature the P40 factor was renamed as IL-9 .
The locus encoding IL9 in mouse is about 11 kb in size, and located on chromosome 13 . The Il9 locus is comprised of 5 exons and 4 introns . The Il9 locus encode for a precursor peptide of 144 amino acids, first 18 amino acids of which is signal sequence peptide. The mature IL-9 peptide, a single-chain glycoprotein of 126 amino acids, and similar to other cytokines of IL-2 family folds into a four-alpha-helix bundles . Human IL-9 locus is present on chromosome 5 in the region q31–35 . Homology between mouse and human IL-9 is about 55%, and both of them contain a conserved 10 cysteine residue to form a disulfide bond that is critical for a mature IL-9 peptide. Interestingly, three conserved non-coding sequences, CNS0, CNS1, and CNS2 are present on both mouse and human
2. IL-9, a lineage specific Th9 cytokine
T cells were originally thought to be the main source of IL-9 [12, 13, 14]. IL-9 was defined as a Th2 cytokine. The reason for this Th2 designation by many research findings included IL-9 genome. The
3. Sources of IL-9
In addition to Th9 and Th2, other immune cells have been identified as potential sources of IL-9 (Figure 2). Prominent among these immune cells is Th17 cells. Th17 cells are involved in mounting immune responses against extracellular bacteria and fungi and are implicated in autoimmunity . Activation of a Th17-associated transcription factor, retinoic acid receptor-related orphan receptor-γt (RORγt) with phorbol 12-myristate 13-acetate and ionomycin (PMA) leads to IL-9 secretion . Tregs have also be shown to secrete IL-9 both
4. IL-9 receptor signaling
IL-9 exerts its biological effect on its target cells through IL-9R receptor. The IL-9R is a heterocomplex of the alpha chain (IL-9Rα) and the common gamma chain . IL-9Rα is specific only to IL-9, whereas the gamma chain is present in the receptor complexes of several other cytokines such as IL-2, IL-4, IL-7, IL-13, IL-15, and IL-21 [37, 38, 39]. About 25% of the IL-9Rα exist in complex with the gamma chain outside IL-9 heterocomplex. IL-9Rα is of 522 amino acids in human, and 468 amino acids in mouse, and contains 11 exons . This 64 kDa glycoprotein is a member of type I hematopoietin receptor super family due to the presence of the Box1 and Box2 motifs in the intracellular domain, and WSXWS motif in the extracellular domain . Formation of a heterocomplex with the γ-chain is enhanced as IL-9 binds to IL-9Rα (Figure 2) . The binding of IL-9 to IL-9Rα results in a conformational change in IL-9R. This conformational change recruit JAK molecules to Box1 motif which results in the phosphorylation of tyrosine residues of IL-9Rα-associated JAK1 and γ-chain associated JAK3 . BOX1 motif is very critical in IL-9 mediated signaling as disruption of Box1 results in loss of phosphorylation of JAK1 and JAK3 . Activated JAK molecules then phosphorylate a tyrosine residue (Tyr407) in the IL-9Rα, which results in the phosphorylation of intermediate molecules, STAT molecules (STAT1, STAT3, and STAT5), MAPK, and IRS-PI3 pathways (Figure 3) [44, 45, 46]. Activation of these pathways contribute to the upregulation of IL-9, as well as important in the growth, differentiation, and development of the IL-9 targeted cells [47, 48].
5. IL-9 and allergic diseases
Allergic diseases including respiratory, food, and skin allergies are mainly mediated by Th2 cells through the expression of various cytokines such as IL-4, IL-5, and IL-13 (reviewed in ). The cytokine IL-9, which was initially studied in the context of Th2-mediated immune response and later associated with T-helper 9 (Th9) cells, has been shown to play an important role in allergic inflammation [50, 51]. IL-9 and its receptor IL-9Rα regulate antibody synthesis, specifically IgE, in both murine and human B cells [52, 53]. To contribute to allergic disease pathogenesis, IL-9 also promotes activation and recruitment of inflammatory cells [54, 55, 56, 57].
6. Asthma including airway allergies
Various studies have shown that IL-9 and its receptor contribute to airway allergic diseases and asthma. Sputum, serum, and lungs of patients with asthma were shown to have increased concentrations of the cytokine [58, 59, 60]. IL-9 levels were also increased in the airways of murine asthma models . IL-9Rα is expressed on human tonsillar germinal center and memory B cells, and smooth muscles in the airways. IL-9/ IL-9Rα signaling in B cells induces STAT3 and STAT5 pathways to potentiate IgE production [52, 53, 55, 62, 63]. Overexpression of IL-9 in transgenic mice or treatment with recombinant cytokine induces expansion of B-1 cells, and accumulation of mast cells in the tissues [64, 65]. IL-9 induces the release of proteases and pro-inflammatory cytokines by the mast cells to promote survival of eosinophils and increase airway permeability [66, 67]. IL-9/IL-9Rα signaling also stimulates human airway smooth muscle to secrete eotaxin1/CCL1 and induces production of IL-13 in airway epithelial cells. Eotaxin1/CCL11 and IL-13 significantly increase eosinophil recruitment and cause lung epithelial cell hypertrophy. These effects result in asthma-like symptoms, including lung inflammation, bronchial hyper-responsiveness, and mucus accumulation. Moreover, IL-9 worsens lung injury in a murine model of chronic obstructive pulmonary disease (COPD) [63, 68, 69]. The cytokine also appears to be a critical player in allergic rhinitis. Serum IL-9 in patients strongly correlates with irritative nasal symptoms including rhinorrhea . In mice, Th9 cells are significantly upregulated during allergic rhinitis and neutralization of IL-9 alleviates symptoms. Blocking IL-9 decreases the level of inflammatory cytokines (IFN-γ, IL-4, and IL-17) and eosinophils infiltration in the nasal mucosa. This causes a decrease in the frequency of sneezing and nasal rubs in experimental models of allergic rhinitis .
7. Food allergies
Studies in patients with food allergy and experimental oral hypersensitivity have shown that allergic reactions in the gastrointestinal tract are mediated by various players, including Th2-secreted cytokines, such as IL-4 and IL-9 [72, 73, 74]. Various studies have shown that IL-9 drives intestinal inflammation and plays a critical role in food allergies [75, 76]. In patients with food allergies, the severity of clinical symptoms strongly correlates with increased intestinal permeability .
8. Skin allergies
IL-9 has been identified as a potential mediator of cutaneous allergies, including atopic dermatitis (AD) and allergic contact dermatitis (ACD). Patients with atopic dermatitis have a significantly higher level of IL-9 in the serum and skin lesions . The concentration of the cytokines also positively correlates with the severity of the disease and serum IgE levels . These observations were made in both adult and pediatric patients [91, 92]. A study in a Korean population also linked IL-9 and IL-9R gene polymorphisms to AD . IL-9 induces IL-5 and IL-13 by ILC2. ILC2 and the cytokines are associated with AD pathogenesis. IL-5 and IL-13 contribute to the defective skin barrier in AD patients by downregulating tight junctions genes [94, 95]. IL-9 also promotes the secretion of the vascular endothelial growth factor (VEGF) by keratinocytes and mast cells [92, 96]. An increased level of VEGF contributes to the dilatation of capillaries, erythema, and inflammatory edema characteristics of AD [97, 98]. Moreover, IL-9 has been shown to regulate Th1-mediated allergic contact dermatitis. Patients with positive patch tests to nickel have a higher level of allergen-specific IL-9 expression in skin, peripheral blood mononuclear cells (PBMCs). Also, IL-9 potentially mediates infiltration of eosinophils in the skins as its levels strongly correlate with the cell infiltration in the tissues. This demonstrates a potential pathogenic role of the cytokine IL-9 in ACD [99, 100].
9. IL-9 and autoimmunity
The etiology or trigger of autoimmune diseases is not well understood [101, 102]. However, there is a consensus that many factors, including genetic, environmental, and cytokine dysregulation are implicated in causing aberrant immune responses that drive tissue damage [102, 103, 104]. Many studies on divergent immune responses in autoimmunity have shown dysfunction of helper T cell subsets, which include Th1, Th17, and/or Treg cells [104, 105]. Studies in the last decade have identified IL-9-secreting Th9 cells as another T helper cell subset involved in immune responses [23, 106]. The IL-9 cytokine has become the focus of many autoimmune studies [107, 108]. Initial studies showed IL-9 to be a growth factor and a Th2 cytokine [13, 108]. More recently, IL-9 has been characterized as a lineage-specific cytokine for Th9 cells . Thereafter, many immune cells involved in autoimmunity, such as Th17 and Treg cells, have demonstrated secretion of IL-9 [16, 110]. In EAE, a rodent model of MS, researchers identified Th9 and its signature cytokine, IL-9, in driving the disease process . Its close association with Th17 and TGF-β has renewed interest in the role of IL-9 in the pathogenesis of autoimmune diseases . In this section, we will examine the role of IL-9 in some autoimmune diseases such as multiple sclerosis (MS), systemic lupus erythematosus (SLE), inflammatory bowel diseases (IBD), rheumatoid arthritis (RA), and uveitis.
10. IL-9 and IL-17 dynamics in autoimmunity
The role of IL-9 in autoimmunity was illuminated when many studies reported that IL-9 and IL-17 are intricately related in driving the pathogenesis of diseases . Human and animal studies revealed that Th17 cells secrete some amount of IL-9, in addition to other proinflammatory cytokines . During the differentiation of naive T cells, TGF-β, a key driver of Th17 polarization, plays an important role in the differentiation of Th9 cells . This was well elaborated in a study by Nowak
On the other hand, IL-9 potentiates Th17 functions in an autocrine manner on Th17 cells [22, 110]. Th17 is a predominant helper T-cell subset that expresses IL-9 receptors (IL-9R)  . Through this receptor, IL-9 acts as an activator of Th17 cells . IL-9 also synergizes with TGF-β to differentiate naive T cells into Th17 cells . The presence of IL-9 in T cell cultures leads to the expansion of Th17 cells . The importance of IL-9 in Th17 cell function is emphasized in IL-9R-deficient experimental autoimmune encephalomyelitis (EAE) model. Mice that lack IL-9 signaling showed decreased Th17 cells and defective migration of Th17 cells into the CNS [22, 114]. Neutralization of IL-9 led to attenuation of disease in EAE . This unique relationship between IL-9 and Th17 provides the premise to examine the role of IL-9 in Th17-mediated autoimmune diseases.
11. Multiple sclerosis (MS)
Most autoimmune diseases like MS occur due to alteration of immune responses, which leads to tissue damage. The importance of IL-9 in MS has been enhanced through our understanding of the roles of IL-9-secreting T cells in EAE, an animal model of MS orchestrated by helper T cells . Most studies revealed IL-9 plays a pathogenic role in EAE . Th9 cells and Th17 cells were observed in the central nervous system (CNS) during EAE . Blockade of IL-9 signaling in EAE resulted in contradictory conclusions. One study reported increased severity of disease in IL9Ra KO mice on a C57BL/6 background through a loss of Treg function and increased secretion of GM-CSF . Other studies showed attenuation of disease and decreased Th17 cell infiltration into the CNS of SJL mice treated with IL-9 blocking antibody [22, 117]. This opposing view in disease outcome may be due to differences in the helper T cell composition and dysfunction driving the pathogenesis in the mouse strains. Also, IL-9 has been shown to increase chemokine CCL20, which enhances migration of Th17 into the CNS . Accumulation and activation of mast cells during the Th17-IL9 immune response could explain the feedback loop . Adoptive transfer of IL-9+ Th9 into recipient mice resulted in EAE . Th9-EAE model manifested a unique disease profile independent of Th1 and Th17 EAE models .
The role of IL-9 in MS patients is complex. A study by Roucco
Unlike other autoimmune diseases, uveitis is a heterogeneous disorder that results in inflammation of the eye . In animal models of uveitis, adoptive transfer of
Another study examined the role of IL-9 in patients with Vogt-Koyanagi-Harada (VKH) disease. VKH is a systemic autoimmunity that manifests with bilateral panuveitis . Patients with active disease had significantly higher levels of IL-9 in culture supernatants and higher IL-9 mRNA in PBMCs than did healthy controls and inactive patients . The synergy of IL-9 and IL-17 was demonstrated in the study. The secretion of IL-17 by IL-9-treated PBMCs of active patients was significantly higher compared to the controls or inactive patients . In a study that evaluated the serum of patients with Behcet’s disease, another complex autoimmune disease with uveitis, serum IL-9 was neither elevated in disease state nor correlated with disease index . More studies are needed to understand whether IL-9 signaling plays any immunological role in the eye.
13. Rheumatoid arthritis (RA)
The study of IL-9 in RA highlights its functional relationship with Tregs. In an antigen-induced animal model of arthritis, mice that lacked IL-9 had a chronic disease . Treatment with rIL-9 resolved the joint inflammation, swelling, and tissue damage. The absence of IL-9 led to impaired suppressive functions of Treg cells . Type 2 innate lymphoid cells (IL-C2) are documented to express IL-9 and have an anti-inflammatory function [128, 129]. These studies highlight the role of IL-9 in the resolution of inflammation in arthritis . In human studies, IL-9-producing IL-C2 cells were also identified in the PBMCs of RA patients [130, 131]. In a study of treatment-induced remission of RA, synovial fluid of patients showed high levels of IL-9 .
14. Systemic lupus erythematosus (SLE)
Proinflammatory cytokines are generally believed to be involved in the pathogenesis of SLE. High levels of IL-9 mRNA and Th17 cells were seen in SLE patients compared with healthy controls (HC) [132, 133]. Dantas
15. Inflammatory bowel disease (IBD)
Aberrant adaptive immune response to the gut epithelial cells involving both CD4 and CD8 is implicated in the IBD . These T cells are shown to express α4/β7 integrin, which binds to MAdcam1 on the gut epithelium [138, 139]. Gut T cells including cells that secrete IL-9 have been shown to express high levels of this integrin, and they propagate inflammation in the gut . Gene expression studies have highlighted IR4 and GATA3 expression on immune cells that reside in the epithelial lining of the gut . IRF4 is a transcription factor that drives the induction of Th9 immune responses in the gut . Animal models of colitis confirms this finding of an abundance of the IL-9-producing T cells in the gut. These T-cells-producing IL-9 are involved in breaking the intestinal barrier . In a DSS colitis model, anti-IL-9 blocking antibodies suppressed mucosal inflammation, and attenuation of disease was observed . Adoptive transfer of IL-9-producing T cells into Rag2 knockout (Rag2
Immunological assessment of patients with inflammatory bowel disease (IBD) revealed high expression of IL-9 in the lamina propria . In addition to other gut-residing T cells in IBD, CD4 cells had increased production of proinflammatory cytokines, including IL-9, which drive gut inflammation [145, 146]. Elevated levels of IL-1β and IL-9 were observed in the serum of IBD patients, and these correlated with disease prognosis . Epithelial cells of UC also showed high expression of IL-9 receptor (IL-9R) [147, 148]. This receptor expression is most pronounced in patients with active disease .
Together, these findings highlight the role of IL-9 in IBD and colitis models. IL-9 could serve as a therapeutic target for IBD. Mice treated with GATA 3 DNAzyme showed it directly reduced IL-9 production and some Th2 cytokines to attenuate disease .
16. Type I diabetes
Studies by Vasanthakumar
IL-9 appears to play both anti- and pro-inflammatory functions in autoimmunity. The functional heterogeneity of IL-9 may result from the unique cells or the microenvironment producing it. In RA, IL-9 exhibits anti-inflammatory function . Studies have elaborated the anti-inflammatory function of IL-9 as it potentiates Treg-dependent immune tolerance to allografts . In the gut, it is regarded as proinflammatory . Some studies have shown that the expression of the activation marker CD96 on Th9 cells may explain the immunological status of the secreted IL-9 . Researchers have reported that Th9 with high expression of CD96 showed a reduced ability to cause colitis compared with Th9 with low expression of CD96, which is associated with severe intestinal inflammation . More studies must be done to identify the immunological heterogeneity of IL-9.
17. IL-9 as a therapeutic target
One principle of treatment of autoimmune diseases involves inhibition of mediators of inflammation. Drugs that target proinflammatory cytokines are extensively used in the treatment of autoimmune diseases . Here we explore the use of IL-9 blockade as a therapeutic target in different disease conditions.
Medimmune LLC developed a humanized anti-IL-9 monoclonal antibody, MEDI-528 . This humanized anti-IL-9 monoclonal antibody was indicated for use in allergen-induced asthma in adults . Results from the clinical trial of Medimmune MEDI-528 showed no increased efficacy in improving respiratory functions and control of asthma compared to placebo . Preclinical studies in mice showed the efficacy of blocking IL-9 in maintaining the airway . Questions remain regarding why therapy directed at IL-9 failed to produce the desired response in humans. Heterogeneity of IL-9 sources and functions could explain the differences in airway response observed in this clinic trial.
18. Other potential IL-9 treatments
IL-9R inhibitor (rhIL-9-ETA) is a chimeric toxin targeting IL9 receptor . These IL-9R inhibitors have efficacy in targeting malignant cells in non-hodgkin’s lymphoma (NHL) and acute myeloid leukemia (AML) expressing IL9 and IL-9R . However, the efficacy of this drug has not been tested in autoimmunity. Pfizer Inc. developed a JAK/STAT pathway inhibitor, CP-690550 . It specifically targets and inhibits the activation of JAK 3 . This treatment effectively prevents transplant rejection . This drug could be beneficial in inhibiting IL-9 signaling, which depends on the JAK/STAT pathway. JAK inhibitors have been used in the treatment of RA and psoriasis . UC patients that were treated with JAK inhibitors showed decreased Th9 cells .
BNZ 132-1-40 peptide, an antagonist of IL-2, IL-9, and IL-15 from Bioniz Therapeutics is undergoing safety and tolerability testing in patients with moderate to severe alopecia areata, an autoimmune disease of the skin that leads to hair loss . However, no results from the clinical trial were available at the time of this review. Recently, FDA approved the use of BNZ-1 for the treatment of cutaneous T cell lymphoma (CTCL) . These studies suggest BNZ-1 could be used to target IL-9 in diseases .
Other potential drug options include RDP58, which targets IRF4, a transcription factor involved in Th9 induction . Interferon gamma (IFN-γ) has the ability to inhibit Th9 polarization through IL-27-dependent mechanisms . Actimmune, an IFN-γ-based therapy by Horizon Therapeutics, is FDA-approved for the treatment of chronic granulomatous disease (CGD) . The efficacy of inhibiting IL-9 by this drug could be tested in IL-9-related disorders.
The immune modulatory roles of IL-9 in health and diseases are important and provides a basis for exploring IL-9 as a therapeutic target. However, the divergent roles of IL-9 in promoting and inhibiting inflammation complicate definitive drug development. Some studies have highlighted the function of IL-9 in promoting immune tolerance. Future studies to understand cell-specific IL-9 regulation and function may resolve the conundrum of therapy development targeting IL-9. More studies in disease will broaden our knowledge about IL-9 function.
Significant progress has been made in our understanding of the functions of IL9 in health and diseases. For a long time, IL-9 was considered as a T cell growth factor, however, the identification of Th9 helper T cells has expanded our understanding on the roles IL-9 play in diseases. The pathogenic functions of IL-9 in autoimmunity and allergy suggest that IL-9 signaling can be targeted for therapy development. In this chapter, we focused on the function of IL-9 in different autoimmune diseases that include MS, SLE, RA, uveitis, and allergic conditions. We also highlighted IL-9-Th17 paradigm and its complexity in autoimmune diseases. Animal models of autoimmune diseases revealed contrasting roles of IL-9 and human studies are limited. Therefore, extensive animal and human research are necessary to elucidate the divergent immunological roles of IL-9. Such studies will be required for effective drug development that targets IL-9 signaling.
We will like to thank Kathy Kyler of the Office of the Vice President for Research and Mary Carter Ph.D. of the Writing center, University of Oklahoma Health Sciences center. Also, we will like to extend our gratitude to Dr. Jimmy Ballard and the staff of Microbiology and Immunology, OUHSC.
Conflict of interest
The authors declare no conflict of interests.