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Essential role of STAT6 in IL-4 signalling
Abstract
Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system. IL-4 activates two distinct signalling pathways through tyrosine phosphorylation of Stat6, a signal transducer and activator of transcription, and of a 170K protein called 4PS. To investigate the functional role of Stat6 in IL-4 signalling, we generated mice deficient in Stat6 by gene targeting. We report here that in the mutant mice, expression of CD23 and major histocompatibility complex (MHC) class II in resting B cells was not enhanced in response to IL-4. IL-4 induced B-cell proliferation costimulated by anti-IgM antibody was abolished. The T-cell proliferative response was also notably reduced. Furthermore, production of Th2 cytokines from T cells as well as IgE and IgG1 responses after nematode infection were profoundly reduced. These findings agreed with those obtained in IL-4 deficient mice or using antibodies to IL-4 and the IL-4 receptor. We conclude that Stat6 plays a central role in exerting IL-4 mediated biological responses.
... In mice and humans, IL4 decreases the production of IFNγ, a signature Th1 protein. Additionally, the master regulator of IL4, GATA3, indirectly cross-regulates the transcription of T-bet, the master regulator of IFNγ [21][22][23][24], promoting the development of IFNγ-negative Th2 cells which produce high levels of IL4. In contrast to data from mice and humans [25][26][27][28], antigen-specific bovine CD4+ T cell clones significantly (60-90%) co-express IFNγ and IL4 transcripts [8,9], suggesting their predominant differentiation into double-positive (IFNγ+ IL4+) Th0 cells capable of co-producing both Th1-and Th2associated hallmark proteins. ...
... In mice and humans, the presence of IL4 into the Th2 culture can enhance the expression of Th2 transcription factors, STAT6 and GATA3 [21,22], which are indispensable for the establishment of an IL4-secreting positive feedback loop. Nevertheless, our analysis based on 175 differentially expressed proteins did not predict STAT6 and GATA3 as upstream factors in rbIL4-treated bovine Th2 cells, which were observations in line with the lack of IL4 upregulation in the qPCR and flow cytometry data ( Figure 2D,E). ...
... The IPA analysis suggested that rbIL4 plays a partially different role in the bovine Th2 differentiation process compared to its counterparts in mice and humans. In mice and humans, the presence of IL4 into the Th2 culture can enhance the expression of Th2 transcription factors, STAT6 and GATA3 [21,22], which are indispensable for the establishment of an IL4-secreting positive feedback loop. Nevertheless, our analysis based on 175 differentially expressed proteins did not predict STAT6 and GATA3 as upstream factors in rbIL4-treated bovine Th2 cells, which were observations in line with the lack of IL4 upregulation in the qPCR and flow cytometry data ( Figure 2D,E). ...
Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.
... Instead of Akt, STAT6 was required for IL-4 to exhibit inhibitory activity on COX-2 induction. IL-4 belongs to the hematopoietin cytokine family, where signal transduction is achieved via the STAT pathway [27,28]. The dependence of IL-4 on STAT6 is in line with our previous findings [12]. ...
... Horseradish peroxidase-conjugated anti-mouse IgG (#1031-05) and anti-rabbit IgG (#6721) antibodies were purchased from Southern Biotech and Abcam (Cambridge, UK), respectively. IL-1β (#201-LB) was a product of R&D Systems (Minneapolis, MN, USA), and recombinant IL-4 and IL-10 were prepared in our laboratory [27]. PGE2 (#14010) and EIA kits to measure PGI2 (#515211) were Figure 6. ...
... Horseradish peroxidase-conjugated anti-mouse IgG (#1031-05) and anti-rabbit IgG (#6721) antibodies were purchased from Southern Biotech and Abcam (Cambridge, UK), respectively. IL-1β (#201-LB) was a product of R&D Systems (Minneapolis, MN, USA), and recombinant IL-4 and IL-10 were prepared in our laboratory [27]. PGE 2 (#14010) and EIA kits to measure PGI 2 (#515211) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). ...
This study aimed to explore the role of Akt protein in the induction and inhibition of prostaglandin (PG) in human follicular dendritic cell (FDC)-like cells. FDC-like cells and B cells were isolated from human tonsils. PG production was assessed using enzyme immunoassay, while the upstream cyclooxygenase-2 (COX-2) protein levels were measured using immunoblotting with FDC-like cells transfected with Akt siRNA to analyze the impact of Akt knockdown. The COX-2 expression and PG production induced with IL-1β were significantly increased by Akt knockdown. However, IL-1β did not significantly alter either total or phosphorylated Akt protein levels. Akt knockdown resulted in the augmentation of COX-2 expression induced by B cells, although the addition of B cells did not significantly modulate both total and phosphorylated Akt proteins. In contrast, IL-4 specifically exhibited a potent inhibitory effect on COX-2 protein induction and PG production via STAT6. The inhibitory activity of IL-4 was not hampered by Akt knockdown. Interestingly, COX-2 expression levels induced with IL-1β were markedly modulated with STAT1 and STAT3 knockdown. STAT1 silencing resulted in further augmentation of COX-2, whereas STAT3 silencing prohibited IL-1β from stimulating COX-2 expression. The current results suggest that Akt, IL-4, and STAT1 play inhibitory roles in PG production in FDC-like cells and expand our knowledge of the immune inflammatory milieu.
... Recent studies using mice deficient in STAT family members have revealed the essential role of STAT proteins in cytokine-mediated biologic functions (5)(6)(7)(8)(9)(10)(11)(12)(13). STAT proteins were recognized initially as transcription factors that were involved in expressions of specific genes, but not required for cell proliferation (3,14). ...
... Splenic T cells were purified as described previously (8). Briefly, splenic T cells were enriched by nylon wool column passage. ...
... The involvement of STAT proteins in lymphocyte proliferation has been discussed in several knockout mice of STAT family (7-11, 18, 20). First, proliferation of lymphocytes in responses to IL-4 and IL-12 has been shown to be impaired in Stat6-and Stat4deficient mice, respectively (7)(8)(9)(10)(11). But, it remained controversial how STAT proteins are involved in proliferation. ...
Stat3, a member of STAT, is activated by a variety of cytokines such as IL-6 family of cytokines, granulocyte CSF, epidermal growth factor, and leptin. A recent study with mice genetically deficient in the Stat3 gene has revealed its important role in the early embryogenesis. To assess the function of Stat3 in adult tissues, we disrupted the Stat3 gene specifically in T cells by conditional gene targeting using Cre-loxP system. In Stat3-deficient T cells, IL-6-induced proliferation was severely impaired. IL-6 did not enhance cell cycle progression, but prevented apoptosis of normal T cells. In contrast, IL-6 did not prevent apoptosis of Stat3-deficient T cells. Antiapoptotic protein, Bcl-2, was normally up-regulated in response to IL-6 even in Stat3-deficient T cells. These results demonstrate that Stat3 activation is involved in IL-6-dependent T cell proliferation through prevention of apoptosis independently of Bcl-2.
... STAT6 is a signaling molecule that has been shown to differentially regulate IgG isotype class-switching in mice and humans. 14,[33][34][35][36][37][38][39][40][41][42][43][44][45]47,48 We therefore set out to test whether STAT6 signaling similarly regulated class-switching in response to both transfusion and vaccination. ...
... To measure the impact of STAT6's function on class-switching in response to transfusion, we first generated a novel STAT6 KO mouse model using CRISPR/cas9 on a pure C57BL/6 background. Previously generated STAT6 KO mice 34,49 have used targeting constructs with neomycin resistance cassettes to disrupt STAT6 coding exons. 34,49 These mice were generated by electroporation into D3 ES cells derived from 129S2/SvPas mouse strain, 34,49 followed by injection into BALB/c blastocytes and multiple rounds of breeding with C57BL/6 mice to generate STAT6 KO mice on a C57BL/6 background. ...
... Previously generated STAT6 KO mice 34,49 have used targeting constructs with neomycin resistance cassettes to disrupt STAT6 coding exons. 34,49 These mice were generated by electroporation into D3 ES cells derived from 129S2/SvPas mouse strain, 34,49 followed by injection into BALB/c blastocytes and multiple rounds of breeding with C57BL/6 mice to generate STAT6 KO mice on a C57BL/6 background. 49 This results in a STAT6 deficient mouse that contains not only a neomycin cassette, but also a large swath of tightly linked 129S2 genome surrounding the STAT6 targeted locus. ...
Background:
Studies of human patients have shown that most anti-RBC alloantibodies are IgG1 or IgG3 subclasses, although it is unclear why transfused RBCs preferentially drive these subclasses over others. Though mouse models allow for the mechanistic exploration of class-switching, previous studies of RBC alloimmunization in mice have focused more on the total IgG response than the relative distribution, abundance, or mechanism of IgG subclass generation. Given this major gap, we compared the IgG subclass distribution generated in response to transfused RBCs relative to protein in alum vaccination, and determined the role of STAT6 in their generation.
Study design and methods:
WT mice were either immunized with Alum/HEL-OVA or transfused with HOD RBCs and levels of anti-HEL IgG subtypes were measured using end-point dilution ELISAs. To study the role of STAT6 in IgG class-switching, we first generated and validated novel STAT6 KO mice using CRISPR/cas9 gene editing. STAT6 KO mice were then transfused with HOD RBCs or immunized with Alum/HEL-OVA, and IgG subclasses were quantified by ELISA.
Results:
When compared with antibody responses to Alum/HEL-OVA, transfusion of HOD RBCs induced lower levels of IgG1, IgG2b, and IgG2c but similar levels of IgG3. Class switching to most IgG subtypes remained largely unaffected in STAT6 deficient mice in response to HOD RBC transfusion, with the one exception being IgG2b. In contrast, STAT6 deficient mice showed altered levels of all IgG subtypes following Alum vaccination.
Discussion:
Our results show that anti-RBC class-switching occurs via alternate mechanisms when compared with the well-studied immunogen alum vaccination.
... Theoretically, activated STAT6 then translocates to target gene promoters in the cell nucleus and thus regulate target gene expression [61,62]. Our result is consistent with earlier research reporting that STAT6 is required for mediating responses to IL-4 and IL-13 [50,[63][64][65][66]. Of interest, it was apparent that BE dose-dependently negated IL-4/13 action, thus reverting pSTAT6 to control levels. ...
... STAT6-dependent signaling has been proposed to play a crucial role in the differentiation of Th2 cells and immunoglobulin isotype conversion; promotion of proliferation and maturation of B cells; mediation of the expression of MHC-II and IgE as well as activation of mast cells in allergic inflammation [61,63,64,67]. However, there is also evidence that nonhematopoietic STAT6 is associated with epithelial tight junction dysfunction by upregulation of myosin light chain kinase (MLCK) [50]. ...
Allergic inflammation, which is the pathogenesis of allergic rhinitis and asthma, is associated with disruption of the airway epithelial barrier due to the effects of type 2 inflammatory cytokines, i.e. interleukin-4 and interleukin-13 (IL-4/13). The anti-allergic inflammatory effect of β-eudesmol (BE) on the tight junction (TJ) of the airway epithelium has not previously been reported. Herein, the barrier protective effect of BE was determined by measurement of transepithelial electrical resistance and by paracellular permeability assay in an IL-4/13-treated 16HBE14o- monolayer. Pre-treatment of BE concentration- and time- dependently inhibited IL-4/13-induced TJ barrier disruption, with the most significant effect observed at 20 μM. Cytotoxicity analyses showed that BE, either alone or in combination with IL-4/13, had no effect on cell viability. Western blot and immunofluorescence analyses showed that BE inhibited IL-4/13-induced mislocalization of TJ components, including occludin and zonula occludens-1 (ZO-1), without affecting the expression of these two proteins. In addition, the mechanism of the TJ-protective effect of BE was mediated by inhibition of IL-4/13-induced STAT6 phosphorylation, in which BE might serve as an antagonist of cytokine receptors. In silico molecular docking analysis demonstrated that BE potentially interacted with the site I pocket of the type 2 IL-4 receptor, likely at Asn-126 and Tyr-127 amino acid residues. It can therefore be concluded that BE is able to prevent IL-4/13-induced TJ disassembly by interfering with cytokine-receptor interaction, leading to suppression of STAT6-induced mislocalization of occludin and ZO-1. BE is a promising candidate for a therapeutic intervention for inflammatory airway epithelial disorders driven by IL-4/13.
... This initiates a pro-inflammatory response and exhibits potent antitumor activity [14,15]. Conversely, type 2 cytokines like IL-4 and IL-13 induce an alternative activation phenotype and elicit an anti-inflammatory response via STAT6 activation pathways [16][17][18]. Additionally, IL-10 exerts a strong anti-inflammatory effect by activating the STAT3 through IL-10R [19,20].TAMs can promote the establishment of an immunosuppressive microenvironment, stimulate angiogenesis, and facilitate tumor invasion and metastasis by promoting tumor-related angiogenesis. ...
... The IL-4/IL-13-activated STAT6 signaling pathway induces the polarization of M2-type macrophages. M2 TAMs secrete immunosuppressive cytokines such as IL-10 and TGF-β, which promote lung cancer progression [17,18,96]. TGF-β is considered a potent suppressor of antitumor immunity. ...
As one of the most common malignant tumors in the world, lung cancer has limited benefits for patients despite its diverse treatment methods due to factors such as personalized medicine targeting histological type, immune checkpoint expression, and driver gene mutations. The high mortality rate of lung cancer is partly due to the immune-suppressive which limits the effectiveness of anti-cancer drugs and induces tumor cell resistance. The currently widely recognized TAM phenotypes include the anti-tumor M1 and pro-tumor M2 phenotypes. M2 macrophages promote the formation of an immune-suppressive microenvironment and hinder immune cell infiltration, thereby inhibiting activation of the anti-tumor immune system and aiding tumor cells in resisting treatment. Analyzing the relationship between different treatment methods and macrophages in the TME can help us better understand the impact of TAMs on lung cancer and confirm the feasibility of targeted TAM therapy. Targeting TAMs to reduce the M2/M1 ratio and reverse the immune-suppressive microenvironment can improve the clinical efficacy of conventional treatment methods and potentially open up more efficient combination treatment strategies, maximizing the benefit for lung cancer patients.
... Distinct transcription factor usage in combination with the selective expression of type-2 cytokines can be used to functionally delineate these two subsets 3,6,11,12 . Th2 cell differentiation and commitment rely on the classical Th2-associated transcription factors GATA Binding Protein (GATA) 3, Signal Transducer And Activator Of Transcription (STAT) 6, STAT5, and c-Maf [13][14][15][16][17][18][19] . Although these Th2 factors work synergistically to promote Th2 lineage specification and function, some selectivity in how they promote type-2 cytokine expression has been observed. ...
... As such, we wanted to investigate if other pathways might also work independently of GATA-3 to promote IL-4 production in Tfh2 cells. The signal transducer and activator of transcription (STAT) factors STAT5 and STAT6 play important roles in the differentiation of established Th2 cells [13][14][15]18,19 . However, the role that these factors serve in Tfh2 cytokine production is less clear as STAT6 signaling is not required to gain IL-4 competency by T cells in the lymph nodes during helminth infection, and STAT5 negatively regulates Tfh cell lineage specification via IL-2 receptor signaling 12,58,[75][76][77][78] . ...
Differences in transcriptomes, transcription factor usage, and function have identified T follicular helper 2 (Tfh2) cells and T helper 2 (Th2) cells as distinct CD4+ T cell subsets in settings of type-2 inflammation. While the transcriptional programs driving Th2 cell differentiation and cytokine production are well defined, dependence on these classical Th2 programs by Tfh2 cells is less clear. Using cytokine reporter mice in combination with transcription factor inference analysis, the b-Zip transcription factor c-Maf and its targets were identified as an important regulon in both Th2 and Tfh2 cells. Conditional deletion of c-Maf in T cells confirmed its importance in type-2 cytokine expression by Th2 and Tfh2 cells. However, while c-Maf was not required for Th2-driven helminth clearance or lung eosinophilia, it was required for Tfh2-driven IgE production and germinal center formation. This differential regulation of cell mediated and humoral immunity by c-Maf was a result of redundant pathways in Th2 cells that were absent in Tfh2 cells, and c-Maf-specific mechanisms in Tfh2 cells that were absent in Th2 cells. Thus, despite shared expression by Tfh2 and Th2 cells, c-Maf serves as a unique regulator of Tfh2-driven humoral hallmarks during type-2 immunity.
... Therefore, these observations suggest that during the macrophage response to IL-4, JNK-1 serves to modulate transcriptional events and enhance the expression of selective targets. Studies in STAT-6-deficient mice [44,45] showed that STAT-6 is involved in a highly confined manner in the signaling carried out by IL-4, playing a critical role in generating many of the responses induced by IL-4. However, whereas IL-4-induced differentiation appears to be largely dependent on STAT-6, IL-4-induced proliferation and survival have been shown to be at least partially independent of STAT-6 [44,45]. ...
... Studies in STAT-6-deficient mice [44,45] showed that STAT-6 is involved in a highly confined manner in the signaling carried out by IL-4, playing a critical role in generating many of the responses induced by IL-4. However, whereas IL-4-induced differentiation appears to be largely dependent on STAT-6, IL-4-induced proliferation and survival have been shown to be at least partially independent of STAT-6 [44,45]. This finding suggests that IL-4 uses additional pathways other than STAT-6 to regulate gene expression. ...
IL(Interleukin)-4 is the main macrophage M2-type activator and induces an anti-inflammatory phenotype called alternative activation. The IL-4 signaling pathway involves the activation of STAT (Signal Transducer and Activator of Transcription)-6 and members of the MAPK (Mitogen-activated protein kinase) family. In primary-bone-marrow-derived macrophages, we observed a strong activation of JNK (Jun N-terminal kinase)-1 at early time points of IL-4 stimulation. Using selective inhibitors and a knockout model, we explored the contribution of JNK-1 activation to macrophages’ response to IL-4. Our findings indicate that JNK-1 regulates the IL-4-mediated expression of genes typically involved in alternative activation, such as Arginase 1 or Mannose receptor, but not others, such as SOCS (suppressor of cytokine signaling) 1 or p21Waf−1 (cyclin dependent kinase inhibitor 1A). Interestingly, we have observed that after macrophages are stimulated with IL-4, JNK-1 has the capacity to phosphorylate STAT-6 on serine but not on tyrosine. Chromatin immunoprecipitation assays revealed that functional JNK-1 is required for the recruitment of co-activators such as CBP (CREB-binding protein)/p300 on the promoter of Arginase 1 but not on p21Waf−1. Taken together, these data demonstrate the critical role of STAT-6 serine phosphorylation by JNK-1 in distinct macrophage responses to IL-4.
... In this study, we describe a novel human PAD caused by germline heterozygous gain-of-function (GOF) variants in the gene STAT6 found in 16 individuals from 10 unrelated families spanning three continents. Signal transducer and activator of transcription 6 (STAT6) is the main transcription factor that mediates the biological effects of IL-4, a key cytokine necessary for type 2 differentiation of T cells, B cell survival, proliferation, and class switching to IgE (Goenka and Kaplan, 2011;Takeda et al., 1996;Villarino et al., 2020;Villarino et al., 2017), as well as that of IL-13, a cytokine linked to anaphylaxis (Gowthaman et al., 2019). Affected individuals experienced early-onset severe, sometimes fatal, multisystem allergic disease which was refractory to conventional treatments. ...
... STAT6 is intimately linked to the biology of allergic inflammation. The central and most studied role of STAT6 is in mediating the biological effects of IL-4, a cytokine necessary for T H 2 differentiation, B cell survival, proliferation, and class switching to IgE (Elo et al., 2010;Takeda et al., 1996), as well as in driving M2 macrophage polarization (Ginhoux et al., 2016). In T cells, STAT6 activation induces the expression of GATA3, the master regulator of T H 2 differentiation, which in turn enhances expression of IL-4, IL-5, and IL-13, cytokines necessary for promoting allergic responses by activating mast cells and eosinophils (Sloka et al., 2011). ...
STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti–IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.
... For example, Th1 lymphocyte subpopulations respond to the binding of proinflammatory cytokines to their receptors, such as IFN-γ, IL-6, IL-12, and TNF, which will activate STATs 1, 3, 4, and NF-kB, respectively [39,164]. However, Th2 cells develop a regulatory response, mainly mediated by IL-4 binding, promoting STAT6 pathway activation [165]. Although not directly associated with Th1 and Th2 activation, STAT2 is critical for macrophage activation and type 1 interferon responses [166] and STAT5 is associated with cell proliferation and apoptosis [167]. ...
... flammatory cytokines to their receptors, such as IFN-γ, IL-6, IL-12, and TNF, which will activate STATs 1, 3, 4, and NF-kB, respectively [39,164]. However, Th2 cells develop a regulatory response, mainly mediated by IL-4 binding, promoting STAT6 pathway activation [165]. Although not directly associated with Th1 and Th2 activation, STAT2 is critical for macrophage activation and type 1 interferon responses [166] and STAT5 is associated with cell proliferation and apoptosis [167]. ...
Chagas disease, a neglected disease caused by the protozoan Trypanosoma cruzi, is endemic in 21 Latin American countries, affecting 6-8 million people. Increasing numbers of Chagas disease cases have also been reported in non-endemic countries due to migration, contamination via blood transfusions or organ transplantation, characterizing Chagas as an emerging disease in such regions. While most individuals in the chronic phase of Chagas disease remain in an asymptomatic clinical form named indeterminate, approximately 30% of the patients develop a cardiomyopathy that is amongst the deadliest cardiopathies known. The clinical distinctions between the indeterminate and the cardiac clinical forms are associated with different immune responses mediated by innate and adaptive cells. In this review, we present a collection of studies focusing on the human disease, discussing several aspects that demonstrate the association between chemokines, cytokines, and cytotoxic molecules with the distinct clinical outcomes of human infection with Trypanosoma cruzi. In addition, we discuss the role of gene polymorphisms in the transcriptional control of these immunoregulatory molecules. Finally, we discuss the potential application of cytokine expression and gene polymorphisms as markers of susceptibility to developing the severe form of Chagas disease, and as targets for disease control.
... Dupilumab is a recombinant human IgG4 monoclonal antibody that inhibits IL-4 signaling via the type I receptor (IL-4Rα/γc) and both IL-4 and IL-13 signaling via the type II receptor (IL-4Rα /IL-13Rα). As a consequence, recruitment and activation of eosinophils are inhibited [17,18]. Mepolizumab is a monoclonal antibody of the IgG1 kappa class, directed against IL-5, thereby inhibiting IL-5 signaling and reducing the production and survival of eosinophils. ...
... Lastly, STAT6 is activated in response to IL-4 and IL-13 [23,24] via phosphorylation at Tyr641 [25] and, in Daudi cells (also called Burkitt lymphoma) after stimulation with IFN-α, STAT6 forms an unusual heterodimer with STAT5 protein [26]. ...
Renal tumors comprise ~7% of all malignant pediatric tumors. Approximately 90% of pediatric kidney tumors comprise Wilms tumors, and the remaining 10% include clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, renal cell carcinoma and other rare renal tumors. Over the last 30 years, the role of cytokines and their receptors has been considerably investigated in both cancer progression and anti-cancer therapy. However, more effective immunotherapies require the cytokine profiling of each tumor type and comprehensive understanding of tumor biology. In this study, we aimed to investigate the activation of signaling pathways in response to cytokines in three pediatric kidney tumor cell lines, in WT-CLS1 and WT-3ab cells (both are Wilms tumors), and in G-401 cells (a rhabdoid kidney tumor, formerly classified as Wilms tumor). We observed that interferon-alpha (IFN-α) and interferon-gamma (IFN-γ) very strongly induced the activation of the STAT1 protein, whereas IL-6 and IFN-α activated STAT3 and IL-4 activated STAT6 in all examined tumor cell lines. STAT protein activation was examined by flow cytometry and Western blot using phospho-specific anti-STAT antibodies which recognize only activated (phosphorylated) STAT proteins. Nuclear translocation of phospho-STAT proteins upon activation with specific cytokines was furthermore confirmed by immunofluorescence. Our results also showed that both IFN-α and IFN-γ caused upregulation of major histocompatibility complex (MHC) class I proteins, however, these cytokines did not have any effect on the expression of MHC class II proteins. We also observed that pediatric kidney tumor cell lines exhibit the functional expression of an additional cytokine signaling pathway, the tumor necrosis factor (TNF)-α-mediated activation of nuclear factor kappa B (NF-κB). In summary, our data show that human pediatric renal tumor cell lines are responsive to stimulation with various human cytokines and could be used as in vitro models for profiling cytokine signaling pathways.
... plays a central role in allergic inflammation. STAT6 mediates IL-4 signaling, a key cytokine responsible for Th2 cell differentiation, B-cell survival, proliferation and IgE class switching.56,57 In T cells, STAT6 activation induces the expression of the transcription factor GATA3, which subsequently results in the enhanced expression of IL-4, IL-5, and IL-13 promoting allergic responses by activating mast cells and eosinophils.58 ...
Inborn errors of immunity (IEIs) encompass a diverse spectrum of genetic disorders that disrupt the intricate mechanisms of the immune system, leading to a variety of clinical manifestations. Traditionally associated with an increased susceptibility to recurrent infections, IEIs have unveiled a broader clinical landscape, encompassing immune dysregulation disorders characterized by autoimmunity, severe allergy, lymphoproliferation, and even malignancy. This review delves into the intricate interplay between IEIs and the JAK–STAT signaling pathway, a critical regulator of immune homeostasis. Mutations within this pathway can lead to a wide array of clinical presentations, even within the same gene. This heterogeneity poses a significant challenge, necessitating individually tailored therapeutic approaches to effectively manage the diverse manifestations of these disorders. Additionally, JAK–STAT pathway defects can lead to simultaneous susceptibility to both infection and immune dysregulation. JAK inhibitors, with their ability to suppress JAK–STAT signaling, have emerged as powerful tools in controlling immune dysregulation. However, questions remain regarding the optimal selection and dosing regimens for each specific condition. Hematopoietic stem cell transplantation (HSCT) holds promise as a curative therapy for many JAK–STAT pathway disorders, but this procedure carries significant risks. The use of JAK inhibitors as a bridge to HSCT has been proposed as a potential strategy to mitigate these risks.
... IL-21R signaling within GC B cells is rewired towards a robust yet transient p-STAT3 induction (60), yielding a considerable degree of similarity between IL-21R-and STAT3-deficient GC B-cell responses (60,80). Conversely, IL-4 signal activates the STAT6 pathway (81). ...
Interleukin 21 (IL-21) is a pleiotropic cytokine that is overproduced in multiple autoimmune settings. Provision of IL-21 from follicular helper T cells is an important component of T-cell help within germinal centers (GC), and the last few years have seen a resurgence of interest in IL-21 biology in the context of the GC environment. While it has been more than a decade since T cell-derived IL-21 was found to upregulate B-cell expression of the GC master transcription factor B-cell lymphoma 6 (Bcl-6) and to promote GC expansion, several recent studies have collectively delivered significant new insights into how this cytokine shapes GC B-cell selection, proliferation, and fate choice. It is now clear that IL-21 plays an important role in GC zonal polarization by contributing to light zone GC B-cell positive selection for dark zone entry as well as by promoting cyclin D3-dependent dark zone inertial cycling. While it has been established that IL-21 can contribute to the modulation of GC output by aiding the generation of antibody-secreting cells (ASC), recent studies have now revealed how IL-21 signal strength shapes the fate choice between GC cycle re-entry and ASC differentiation in vivo. Both provision of IL-21 and sensitivity to this cytokine are finely tuned within the GC environment, and dysregulation of this pathway in autoimmune settings could alter the threshold for germinal center B-cell selection and differentiation, potentially promoting autoreactive B-cell responses.
... The native E2 protein influenced the CD4 + T cells to a Th2 profile, considering results from Stat3 and Stat6. Interestingly, IL-2, acting via Stat5A and Stat5B, is an important negative regulator of Th17 cell differentiation (17), and Stat6 is a key for IL-4 signaling (18). We examined Stat activation in CD4 + T cells with stimulation in the presence or absence of sE2 or sE2 F442NYT (Fig. 3A). ...
The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4⁺T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4⁺T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4⁺T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4⁺T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.
IMPORTANCE
The nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.
... STAT family members are key players in the differentiation of certain helper T cell subsets (35). For instance, STAT1 and STAT4 are crucial for the differentiation of Th1 cells (36), STAT6 is necessary for Th2 cell differentiation (37), and STAT3 and STAT5 are required for Th17 and iTreg differentiation, respectively (38)(39)(40). Regarding Th2 cell differentiation, STAT3 is also required for Th2 cell differentiation (17). ...
Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4 ⁺ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1 ⁺ and granzyme ⁺ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.
... Importantly, ERK1/2 activation also regulates the production of anti-inflammatory mediators during efferocytosis, promoting the polarization of macrophages toward a proresolving phenotype, which contributes to sustained efferocytosis and the resolution of inflammation [32,33]. One crucial transcription factor influenced by ERK1/2 activation to regulate macrophage polarization is Stat6 [34,35]. The phosphorylation of Stat6 promotes the transcription of genes involved in macrophage proresolving and efferocytosis response, such as Gas6 and MERTK [36,37]. ...
Background
Efferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation, prevents the release of inflammatory molecules, and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remain ill-defined.
Methods
We used GFP bone marrow chimeric knockout (KO) mice to demonstrate that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing MERTK in the brain to restrict efferocytosis of resident microglia and peripheral-derived monocyte/macrophages.
Results
Single-cell RNAseq identified MERTK expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis and overall protein expression of p-MERTK, p-ERK, and p-Stat6. The percentage of GFP ⁺ monocyte/macrophages and resident microglia engulfing NeuN ⁺ or TUNEL ⁺ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to the wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with MERTK-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Selective inhibitors of ERK and Stat6 attenuated this effect, confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway.
Conclusions
Our findings implicate the ERK/Stat6/MERTK axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.
... STAT6 is an intracellular transcription factor downstream of IL4 and IL4R/JAK-kinase signalling cascade and a central node of immune polarization and a key modulator for the risk of allergic disease in humans and mice [3,46]. Translocation of STAT6 to the nucleus, activates a pattern of gene expression mediating Th2 cell differentiation, M2 macrophage polarization, promotion of B cell survival and IgE class switching [47][48][49][50]. ...
Purpose of review
Allergy and atopic features are now well recognized manifestations of many inborn errors of immunity (IEI), and indeed may be the hallmark in some, such as DOCK8 deficiency. In this review, we describe the current IEI associated with atopy, using a comprehensive literature search and updates from the IUIS highlighting clinical clues for underlying IEI such as very early onset of atopic disease or treatment resistance to enable early and accurate genetic diagnosis.
Recent findings
We focus on recently described genes, their categories of pathogenic mechanisms and the expanding range of potential therapies.
Summary
We highlight in this review that patients with very early onset or treatment resistant atopic disorders should be investigated for an IEI, as targeted and effective therapies exist. Early and accurate genetic diagnosis is crucial in this cohort to reduce the burden of disease and mortality.
... STAT6 is activated by the combining of inflammatory cytokines IL-4 and its receptor. STAT6 phosphorylation induces dimerization and nuclear translocation to activate gene transcription of inflammatory cytokines, such as TSLP, IL-33, and chemokines, such as C-C motif ligand 26 (CCL26) [31][32][33][34]. ...
Various allergic diseases such as atopic dermatitis (AD), allergic rhinitis, and asthma are considered incurable conditions that have yet to be fully conquered. Paedoksan (PDS), an herbal preparation consisting of 14 medicines, displays effective anti-inflammatory and anti-allergic properties, yet its underlying molecular mechanism is unknown. This study aims to uncover PDS’s mechanism for treating allergic diseases and suggest its therapeutic potential. Through a network pharmacological prediction, its impact on signal transducer and activator of transcription 6 (STAT6) regulation, a sub-mechanism of interleukin 4 (IL-4), a major inflammatory cytokine involved in degranulation and allergy, was investigated in RBL-2H3 cells and an atopic mouse model. PDS inhibits immunoglobulin E (IgE)-induced degranulation and STAT6 phosphorylation evoked by IL-4 in granulocytes. The downregulation of phospho-STAT6 and thymic stromal lymphopoietin (TSLP) by PDS was confirmed in 2,4-dinitrochlorobenzene (DNCB)-induced mouse skin. The results demonstrate that PDS exhibited remarkable effects on degranulation and STAT6 phosphorylation in RBL-2H3 cells, as well as in an atopic mouse model. Furthermore, the main active components from PDS based on chromatographic analysis showed good accordance with PDS’s effects on RBL-2H3 cells. In summary, these findings collectively suggest that PDS holds the potential to effectively suppress inflammatory and allergic reactions by obstructing the target IL-4 protein and its downstream effects, as elucidated through a network pharmacological analysis.
... The primary STAT activated in response to IL-4 and IL-13 signaling is STAT6, which is essential for the expression of proteins encoded by IL-4-responsive genes, including class II major histocompatibility (MHC) molecules, 75 CD23, 76 germline immunoglobulin ε and γ1, 77 IL-4, 75 and IL-4Rα chain. 75,76 Following the assembly of the IL-4 cytokine/receptor complex and the consequent activation of JAKs, tyrosine residues within the IL-4Rα intracellular domain are phosphorylated. 65,[78][79][80] Three highly conserved tyrosine residues in the IL-4Rα chain intracellular domain (Y575, Y603, and Y631 in humans and in mice) serve as docking sites for STAT6, 39,40,81 which binds through its conserved SH2 domain ( Figure 2B). ...
The structurally and functionally related interleukin‐4 (IL‐4) and IL‐13 cytokines play pivotal roles in shaping immune activity. The IL‐4/IL‐13 axis is best known for its critical role in T helper 2 (Th2) cell‐mediated Type 2 inflammation, which protects the host from large multicellular pathogens, such as parasitic helminth worms, and regulates immune responses to allergens. In addition, IL‐4 and IL‐13 stimulate a wide range of innate and adaptive immune cells, as well as non‐hematopoietic cells, to coordinate various functions, including immune regulation, antibody production, and fibrosis. Due to its importance for a broad spectrum of physiological activities, the IL‐4/IL‐13 network has been targeted through a variety of molecular engineering and synthetic biology approaches to modulate immune behavior and develop novel therapeutics. Here, we review ongoing efforts to manipulate the IL‐4/IL‐13 axis, including cytokine engineering strategies, formulation of fusion proteins, antagonist development, cell engineering approaches, and biosensor design. We discuss how these strategies have been employed to dissect IL‐4 and IL‐13 pathways, as well as to discover new immunotherapies targeting allergy, autoimmune diseases, and cancer. Looking ahead, emerging bioengineering tools promise to continue advancing fundamental understanding of IL‐4/IL‐13 biology and enabling researchers to exploit these insights to develop effective interventions.
... The large diversity of downstream effects is driven by a family of four different receptor-associated kinases (JAK1, JAK2, JAK3, and TYK2) and five STAT molecules (STAT1, STAT2, STAT3, STAT5A/B, and STAT6). For instance, JAK1, JAK3, and their interactions with STAT3, STAT5, and STAT6 are necessary for the pathogenic IL-4 signaling in AD [68][69][70], and STAT6 drives IgE class switching [71,72]. The JAK-STAT signaling pathway is thus a desirable target for its numerous pathogenic roles in AD, leading to the development of multiple small-molecule Janus kinase inhibitors (JAK-i). ...
Purpose of Review
Atopic dermatitis (AD) remains a dermatological disease that imposes a significant burden on society. Air pollution has previously been linked to both the onset and severity of atopic dermatitis. As air pollution remains a critical environmental factor impacting human health, this review seeks to provide an overview of the relationship between different air pollutants and AD.
Recent Findings
AD can develop from multiple causes that can be broadly grouped into epidermal barrier dysfunction and immune dysregulation. Air pollution imposes significant health risks and includes a wide variety of pollutant types. AD has been linked to outdoor air pollutants such as particulate matter (PM), volatile organic compounds (VOC), gaseous compounds, and heavy metals. Exposure to indoor pollutants such as tobacco smoke and fungal molds has also been associated with an increased incidence of AD. While different pollutants impact distinct molecular pathways in the cell, they mostly converge on ROS product, DNA damage, and dysregulated T-cell activity and cytokine production.
Summary
The presented review suggests a strengthening tie between air pollution and AD. It points to opportunities for further studies to clarify, as well as potential therapeutic opportunities that leverage the mechanistic relationships between air pollution and AD.
... The primary function of STAT6 is in the M2 differentiation of macrophages [106]. In addition, STAT6 plays a role in the T helper 2 response as well as IgE (Immunoglobulin E) production [107]. Like STAT2 and STAT4, the role of STAT6 remains largely unknown, though there is some evidence that STAT6 is associated with supporting the cancer stem cell niche in OvCa. ...
Ovarian cancer (OvCa) is a deadly gynecologic malignancy that presents many clinical challenges due to late-stage diagnoses and the development of acquired resistance to standard-of-care treatment protocols. There is an increasing body of evidence suggesting that STATs may play a critical role in OvCa progression, resistance, and disease recurrence, and thus we sought to compile a comprehensive review to summarize the current state of knowledge on the topic. We have examined peer reviewed literature to delineate the role of STATs in both cancer cells and cells within the tumor microenvironment. In addition to summarizing the current knowledge of STAT biology in OvCa, we have also examined the capacity of small molecule inhibitor development to target specific STATs and progress toward clinical applications. From our research, the best studied and targeted factors are STAT3 and STAT5, which has resulted in the development of several inhibitors that are under current evaluation in clinical trials. There remain gaps in understanding the role of STAT1, STAT2, STAT4, and STAT6, due to limited reports in the current literature; as such, further studies to establish their implications in OvCa are necessitated. Moreover, due to the deficiency in our understanding of these STATs, selective inhibitors also remain elusive, and therefore present opportunities for discovery.
... We identified among the downregulated pathways VEGF, Insulin, MAPK, Hpyoxia, PPAR, TGF-beta, and H 2 O 2 signaling. We focused, however, on those genes that are linked to JAK-STAT signaling among the genes repressed in GR LysMCre ATMs (Supplementary Fig. S5A), because IL-4 signaling through STAT6 is a major regulator of anti-inflammatory macrophage polarization 41,42 . We therefore assessed public ChIP-seq datasets for GR and STAT6 binding in macrophage cultures and found a large overlap between GR and STAT6 binding sites from independent datasets ( Supplementary Fig. S5B), i.e., GR binding in response to 45 min of dexamethasone stimulation 43 and STAT6 binding in response to 30 or 60 min IL-4 exposure 44 . ...
Insulin resistance (IR) during obesity is linked to adipose tissue macrophage (ATM)-driven inflammation of adipose tissue. Whether anti-inflammatory glucocorticoids (GCs) at physiological levels modulate IR is unclear. Here, we report that deletion of the GC receptor (GR) in myeloid cells, including macrophages in mice, aggravates obesity-related IR by enhancing adipose tissue inflammation due to decreased anti-inflammatory ATM leading to exaggerated adipose tissue lipolysis and severe hepatic steatosis. In contrast, GR deletion in Kupffer cells alone does not alter IR. Co-culture experiments show that the absence of GR in macrophages directly causes reduced phospho-AKT and glucose uptake in adipocytes, suggesting an important function of GR in ATM. GR-deficient macrophages are refractory to alternative ATM-inducing IL-4 signaling, due to reduced STAT6 chromatin loading and diminished anti-inflammatory enhancer activation. We demonstrate that GR has an important function in macrophages during obesity by limiting adipose tissue inflammation and lipolysis to promote insulin sensitivity.
... For example, many DEGs were seen to harbor binding sites for particular transcription factors. These included STAT6, which is involved in IL4 responses, [29] and IRF1, which has a key role in the antiviral response [30]. Similarly, many of these genes are targets for gga-mir-155, a microRNA variety already known to be implicated in various immune responses [31,32]. ...
The purpose of the current study was to examine transcriptomic-based profiling of differentially expressed innate immune genes between indigenous and commercial chickens. In order to compare the transcriptome profiles of the different chicken breeds, we extracted RNA from blood samples of the Isfahan indigenous chicken (as indigenous) and Ross broiler chicken (as commercial) breeds. RNA-Seq yielded totals of 36,763,939 and 31,545,002 reads for the indigenous and commercial breeds, respectively, with clean reads then aligned to the chicken reference genome (Galgal5). Overall, 1327 genes were significantly differentially expressed, of which 1013 genes were upregulated in the commercial versus the indigenous breed, while 314 were more highly expressed in the indigenous birds. Furthermore, our results demonstrated that the SPARC, ATP6V0D2, IL4I1, SMPDL3A, ADAM7, TMCC3, ULK2, MYO6, THG1L and IRG1 genes were the most significantly expressed genes in the commercial birds and the PAPPA, DUSP1, PSMD12, LHX8, IL8, TRPM2, GDAP1L1, FAM161A, ABCC2 and ASAH2 genes were the most significant in the indigenous chickens. Of notable finding in this study was that the high-level gene expressions of heat-shock proteins (HSPs) in the indigenous breeds could serve as a guideline for future genetic improvement. This study identified genes with breed-specific expression, and comparative transcriptome analysis helped understanding of the differences in underlying genetic mechanisms between commercial and local breeds. Therefore, the current results can be used to identify candidate genes for further breed improvement.
... Following parasitic helminth infection, the type 2 cytokines IL-4 and IL-13 signal through the shared IL-4Rα chain leading to the activation of the transcription factor signal transducer and activator of transcription (STAT)-6 [15]. Activated STAT-6 binds to the promotor regions of multiple genes and promotes their expression, resulting in polarization and proliferation of alternatively activated macrophages (AAMs). ...
Helminths are parasitic worms that coevolve with their host, usually resulting in long-term persistence through modulating host immunity. The multifarious mechanisms altering the immune system induced by helminths have significant implications on the control of coinfecting pathogens such as viruses. Here, we explore the recent literature to highlight the main immune alterations and mechanisms that affect the control of viral coinfection. Insights from these mechanisms are valuable in the understanding of clinical observations in helminth-prevalent areas and in the design of new therapeutic and vaccination strategies to control viral diseases.
... [13][14][15][16] STAT6 is the central transcription factor that mediates the biological effects of IL-4 and IL-13. [17][18][19] The binding of IL-4 and IL-13 to IL-4R receptor (IL-4R) complexes triggers the phosphorylation of conserved tyrosine residues in the cytoplasmic domain of the common IL-4 receptor alpha (IL-4Ra) subunit by the receptor-associated JAKs. 20,21 STAT6 is subsequently recruited through the binding of its tandem Src homology 2 (SH2) domains to the IL-4Ra phosphotyrosine docking sites. ...
Background:
Inborn errors of immunity (IEI) have been implicated in causing immune dysregulation, including allergic diseases. The signal transducer and activator of transcription 6 (STAT6) is a key regulator of allergic responses.
Objective:
We sought to characterize a novel gain-of-function (GOF) STAT6 mutation identified in a child with severe allergic manifestations.
Methods:
We performed whole-exome and targeted gene sequencing, lymphocyte characterization, and molecular and functional analyses of mutated STAT6.
Results:
We report a child with a missense mutation in the DNA binding domain of STAT6 (c.1114G>A, p.E372K) who presented with severe atopic dermatitis, eosinophilia and elevated IgE. Naive lymphocytes from the affected patient displayed increased TH2 and suppressed TH1 and TH17 cell responses. The mutation augmented both basal and cytokine-induced STAT6 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reversed STAT6 hyperresponsiveness to IL-4, normalized TH1, and TH17, suppressed the eosinophilia and improved the patient's atopic dermatitis.
Conclusions:
We identified a novel IEI due to STAT6 GOF mutation that gave rise to severe allergic dysregulation. Janus kinase inhibitor therapy could represent an effective targeted treatment for this disorder.
... This results in allergic inflammation, for example asthma, atopic dermatitis, rhinitis, eczema and food allergies (230). Th2 polarization is induced by IL-4 via STAT6 (232,233) and IL-2 via STAT5 (234) and the master transcription factor is GATA-3, which also acts as an epigenetic modifier (235,236). Th2 cells express chemokine receptors including CCR3, CCR4 and CCR8 that react to chemokines secreted from epithelial cells which mediates Th2 cell recruitment to the site of allergen exposure (230). ...
... Les sources d'IL-4 sont multiples puisqu'elles peuvent provenir des cellules NK T, des mastocytes ou des basophiles [201,202]. Ces différents signaux sont responsables, à travers la phosphorylation activatrice de STAT 6, de l'expression du facteur de transcription GATA-3 luimême responsable de la synthèse des cytokines IL-4, IL-5 et IL-13 [203][204][205]. GATA-3 régule également sa propre expression ce qui permet le maintien du phénotype Th2. ...
Au cours de ma thèse, je me suis intéressé à l'influence de la dynamique membranaire sur la fonctionnalité de P2X7 dans les lymphocytes T en lien avec les différences de développement du LED chez les souris lupiques MRL/lpr et B6/lpr. Ainsi, j'ai émis l'hypothèse que le taux de cholestérol circulant pourrait influencer l'organisation des microdomaines membranaires de signalisation des lymphocytes T normaux et pathologiques et être responsable des différences de sensibilité du récepteur P2X7 à l'ATP4-. J'ai donc recherché, in vitro et in vivo, l'existence de liens entre la régulation du métabolisme du cholestérol et la fonctionnalité du récepteur P2X7 selon l'état d'activation et de différenciation des lymphocytes T. Pour ce faire, j'ai mis au point les techniques de modulation et de quantification des taux de cholestérol membranaire ainsi que les protocoles permettant de déterminer la localisation du récepteur P2X7 au sein des domaines membranaires. J'ai ainsi pu démontrer, in vitro, sur des lignées lymphocytaires T tumorales, l'influence du taux de cholestérol membranaire, basal ou modulé, sur les fonctions cellulaires induites par le récepteur P2X7. J'ai également pu mettre en évidence, ex vivo, une variation des taux de cholestérol membranaire selon l'état d'activation et de différenciation des lymphocytes T normaux ou pathologiques et que ces différences impactent la fonctionnalité de P2X7. De plus, j'ai montré chez des souris normales B6 soumises à un régime enrichi en cholestérol que le taux de cholestérol membranaire des lymphocytes T et surtout leur sensibilité à l'ATP4- est dépendante du taux de cholestérol circulant. Enfin, des traitements hypo et hyper cholestérolémiants sont actuellement réalisés pour évaluer l'impact du taux de cholestérol circulant sur la sévérité des pathologies auto-immunes des souris MRL/lpr et B6/lpr.
... Activation of STAT6 is a key signaling in macrophage function, required for the alternative activation of macrophages (M2φ) [27], and indispensable for IL4-mediated activation of target gene transcription as TNFα and interleukin-8 (IL8) [28,29]. Thus, we questioned whether IL4 presentation via EMF-assisted SPION-IL4 could lead to the phosphorylation of STAT6 (pSTAT6) and to M2φ priming via the IL4/STAT6 pathway. ...
The persistence of inflammatory mediators in tissue niches significantly impacts regenerative outcomes and contributes to chronic diseases. Interleukin-4 (IL4) boosts pro-healing phenotypes in macrophages (Mφ) and triggers the activation of signal transducer and activator of transcription 6 (STAT6). Since the IL4/STAT6 pathway reduces Mφ responsiveness to inflammation in a targeted and precise manner, IL4 delivery offers personalized possibilities to overcome inflammatory events. Despite its therapeutic potential, the limited success of IL4-targeted delivery is hampered by inefficient vehicles. Magnetically assisted technologies offer precise and tunable nanodevices for the delivery of cytokines by combining contactless modulation, high tissue penetration, imaging features, and low interference with the biological environment. Although superparamagnetic iron oxide nanoparticles (SPION) have shown clinical applicability in imaging, SPION-based approaches have rarely been explored for targeted delivery and cell programming. Herein, we hypothesized that SPION-based carriers assist in efficient IL4 delivery to Mφ, favoring a pro-regenerative phenotype (M2φ). Our results confirmed the efficiency of SPION-IL4 and Mφ responsiveness to SPION-IL4 with evidence of STAT6-mediated polarization. SPION-IL4-treated Mφ showed increased expression of M2φ associated-mediators (IL10, ARG1, CCL2, IL1Ra) when compared to the well-established soluble IL4. The ability of SPION-IL4 to direct Mφ polarization using sophisticated magnetic nanotools is valuable for resolving inflammation and assisting innovative strategies for chronic inflammatory conditions.
Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value.
T cells are fundamental components in tumour immunity and cancer immunotherapies, which have made immense strides and revolutionized cancer treatment paradigm. However, recent studies delineate the predicament of T cell dysregulation in tumour microenvironment and the compromised efficacy of cancer immunotherapies. CRISPR screens enable unbiased interrogation of gene function in T cells and have revealed functional determinators, genetic regulatory networks, and intercellular interactions in T cell life cycle, thereby providing opportunities to revamp cancer immunotherapies. In this review, we briefly described the central roles of T cells in successful cancer immunotherapies, comprehensively summarised the studies of CRISPR screens in T cells, elaborated resultant master genes that control T cell activation, proliferation, fate determination, effector function, and exhaustion, and highlighted genes (BATF, PRDM1, and TOX) and signalling cascades (JAK-STAT and NF-κB pathways) that extensively engage in multiple branches of T cell responses. In conclusion, this review bridged the gap between discovering element genes to a specific process of T cell activities and apprehending these genes in the global T cell life cycle, deepened the understanding of T cell biology in tumour immunity, and outlined CRISPR screens resources that might facilitate the development and implementation of cancer immunotherapies in the clinic.
Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3⁺ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms.
Spinocerebellar ataxia 3 (SCA3) is an incurable, neurodegenerative genetic disorder that leads to progressive cerebellar ataxia and other parkinsonian‐like pathologies because of loss of cerebellar neurons. The role of an expanded polyglutamine aggregate on neural progenitor cells is unknown. Here, we show that SCA3 patient‐specific induced neural progenitor cells (iNPCs) exhibit proliferative defects. Moreover, SCA3 iNPCs have reduced autophagic expression compared to control. Furthermore, although SCA3 iNPCs continue to proliferate, they do not survive subsequent passages compared to control iNPCs, indicating the likelihood that SCA3 iNPCs undergo rapid senescence. Exposure to interleukin‐4 (IL‐4), a type 2 cytokine produced by immune cells, resulted in an observed increase in expression of autophagic programs and a reduction in the proliferation defect observed in SCA3 iNPCs. Our results indicate a previously unobserved role of SCA3 disease ontology on the neural stem cell pool and a potential therapeutic strategy using IL‐4 to ameliorate or delay disease pathology in the SCA3 neural progenitor cell population.
Various disciplines cooperate to find novel approaches to cure impaired body functions by repairing, replacing, or regenerating cells, tissues, or organs. The possibility that a stable differentiated cell can reprogram itself opens the door to new therapeutic strategies against a multitude of diseases caused by the loss or dysfunction of essential, irreparable, and specific cells. One approach to cell therapy is to induce reprogramming of adult cells into other functionally active cells. Understanding the factors that cause or contribute to T cell plasticity is not only of clinical importance but also expands the knowledge of the factors that induce cells to differentiate and improves the understanding of normal developmental biology. The present review focuses on the advances in the conversion of peripheral CD4+ T cells, the conditions of their reprogramming, and the methods proposed to control such cell differentiation.
Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.
Food allergy affects more than 500 million people in the world, and its prevalence is increasing at an alarming rate causing serious public health concerns; however, prevention and treatment methods are still under investigation and are relatively scarce so far. Insights on pathophysiology reveal a complex interplay of the immune cells (e.g., DCs, T cells, and B cells) resulting in allergy or tolerance. Studies have shown that melatonin metabolisms are altered in patients with allergic diseases, suggesting that melatonin might impact allergic diseases. Notably, melatonin can orchestrate the differentiation and function of immune cells. Additionally, the disease severities of many allergic diseases and the function of the immune system exhibit circadian rhythmicity. Therefore, melatonin, a rhythm regulator, may also act indirectly on the immune system through the circadian clock to regulate food allergies. Herein, we reviewed the impacts of melatonin on food allergy and its underlying regulatory mechanisms, providing a theoretical reference for melatonin as effective means of prevention and treatment for food allergy in the future.
T follicular helper cells comprise a specialized, heterogeneous subset of immune-competent T helper cells capable of influencing B cell responses in lymphoid tissues. In physiology, for example in response to microbial challenges or vaccination, this interaction chiefly results in the production of protecting antibodies and humoral memory. In the context of kidney transplantation, however, immune surveillance provided by T follicular helper cells can take a life of its own despite matching of human leukocyte antigens and employing the latest immunosuppressive regiments. This puts kidney transplant recipients at risk of subclinical and clinical rejection episodes with a potential risk for allograft loss. In this review, the current understanding of immune surveillance provided by T follicular helper cells is briefly described in physiological responses to contrast those pathological responses observed after kidney transplantation. Sensitization of T follicular helper cells with the subsequent emergence of detectable donor-specific human leukocyte antigen antibodies, non-human leukocyte antigen antibodies their implication for kidney transplantation and lessons learnt from other transplantation “settings” with special attention to antibody-mediated rejection will be addressed.
IL-4 has long been established as a key regulator of Th cells and for promoting effective B cell survival and isotype class switching. Yet, despite having been extensively studied, the specific role of IL-4 in generating humoral memory in vivo is unclear. In this review, we explore the recent studies that unravel the cellular sources and spatiotemporal production of IL-4, the relationship between IL-4 and IL-21 during germinal center responses and the formation of Ab-secreting cells, and the current understanding of whether IL-4 promotes or suppresses memory B cell generation in vitro and in vivo.
Sebaceous glands are holocrine glands that produce sebum, which primarily contains lipids that help maintain the barrier function of the skin. Dysregulated lipid production contributes to the progression of some diseases characterized by dry skin, including atopic dermatitis. Although the lipid production of sebaceous glands has been well studied, few studies have assessed their role in skin immune responses. We found that sebaceous glands and sebocytes expressed interleukin (IL)-4 receptor and produced high levels of Th2-associated inflammatory mediators following IL-4 treatment, suggesting immunomodulatory effects. Galectin-12 is a lipogenic factor expressed in sebocytes that affects their differentiation and proliferation. Using galectin-12-knockdown sebocytes, we demonstrated that galectin-12 regulated immune response in cells exposed to IL-4 and promoted CCL26 expression by upregulating PPARγ. Moreover, galectin-12 suppresses the expression of endoplasmic reticulum (ER) stress-response molecules and CCL26 upregulation by IL-4 was reversed following sebocyte treatment with inducers of ER stress, suggesting that galectin-12 controls IL-4 signaling by suppressing ER stress. Using galectin-12-knockout mice, we demonstrated that galectin-12 positively regulated the IL-4-induced enlargement of sebaceous glands and development of an atopic dermatitis-like phenotype. Thus, galectin-12 regulates the skin immune response through promoting PPARγ expression and suppressing ER stress in sebaceous glands.
Fetal and child's immune systems differ from those of adults. Developing immune systems exhibit increased or decreased sensitivity to drugs, infection, or toxicants compared to adult immune systems. Understanding fetal and neonatal immune systems will help predict toxicity or the pathogenesis or prognosis of diseases. In this study, we evaluated whether the innate and adaptive immune system of fetal and young minipigs could respond to external stimuli compared to a medium-treated group and analyzed several immunological parameters for developmental immunotoxicity according to developmental stages. We performed a hematological analysis of fetal cord bloods and the bloods of neonatal and 4-week-old piglets. Splenocytes were isolated at each developmental stage and treated with lipopolysaccharide (LPS), R848, and concanavalin A (ConA). Various cytokines were measured in the cell supernatants. Total antibody production was also evaluated in serum. The percentage of lymphocytes was dominant in gestational weeks (GW) 10 and 12 and started to decrease from postnatal day (PND) 0. From PND0, the percentage of neutrophils increased. Interleukin (IL)-1β, IL-6, and interferon (IFN)-α were induced from GW10 in response to LPS and R848 stimulation. Th1 cytokine induction was detected from PND0 upon ConA stimulation, whereas Th2 cytokine release was observed from GW10. IgM and IgG production was sustained at low levels at fetal stages and was significantly increased after birth. This study reconfirmed that the fetal immune system could respond to external stimuli and that hematological analysis, cytokine evaluation, and antibody subclass measurement can be useful parameters for developmental immunotoxicity using minipigs.
Today, treatment options for cancer patients typically include surgery, radiation therapy, immunotherapy, and chemotherapy. While these therapies have saved lives and reduced pain and suffering, cancer still takes millions of lives every year around the world. Researchers are now developing advanced therapeutic strategies such as immunotherapy, targeted therapy, and combination nanotechnology for drug delivery. In addition, the identification of new biomarkers will potentiate early-stage diagnosis. Molecular Targets and Cancer presents information about cancer diagnosis and therapy in a simple way. It covers several aspects of the topic with updated information on par with medical board levels. The book features contributions from experts and includes an overview of cancer from basic biology and pathology, classifications, surveillance, prevention, diagnosis, types of cancer, treatment and prognosis. The first part of this book introduces the reader to cancer epidemiology, genetic alterations in cancer, exogenous and endogenous factors in carcinogenesis, roles for growth factors in cancer progression, cell signaling in cancer, transcription factors in cancer, and cancer genetics and epigenetics. This comprehensive guide is a valuable resource for oncologists, researchers, and all medical professionals who work in cancer care and research.
High-affinity allergen-specific IgE is essential for the severe allergic anaphylaxis response. High-affinity Abs are formed by successive rounds of selection of Ag-specific B cells in the germinal center (GC); however, several studies have shown that IgE+ GC B cells are impaired in their ability to undergo selection in the GC. A pathway, known as the "indirect switching pathway" for IgE, has been described whereby Ag-specific B cells initially switch to the IgG1 isotype and undergo affinity selection in the GC, with a secondary switch to the IgE isotype after affinity selection. In previous work, using a food allergy model in mice, we investigated how high-affinity IgE develops in the GC, but we did not test the indirect switching model. In this study, we analyzed the importance of the indirect switching pathway by constructing IgG1-cre Bcl6-fl/fl mice. In these mice, once B cells switch to IgG1, they delete Bcl6 and thus cannot enter or persist in the GC. When we tested IgG1-cre Bcl6-fl/fl mice with our food allergy model, we found that, as expected, IgG1 Abs had decreased affinity, but unexpectedly, the affinity of IgE for allergen was unchanged. IgG1-cre Bcl6-fl/fl mice underwent anaphylaxis in response to allergen, consistent with the formation of high-affinity IgE. Thus, in a food allergy response, high-affinity IgE can be efficiently formed in the absence of indirect switching to IgG1, either by direct selection of IgE+ GC B cells or indirect selection of IgM+ GC B cells that later switch to IgE.
Retinoic acid possesses potent immunomodulatory properties in various cell types, including macrophages. In this study, we first investigated the effects at the transcriptional and functional levels of exogenous retinoic acid in murine bone marrow-derived macrophages (BMDMs) in the presence or absence of interleukin 4 (IL4), a cytokine with potent anti-inflammatory properties. We examined the effect of IL4 on vitamin A homeostasis in macrophages by quantifying retinoid synthesis and secretion. Our RNAseq data show that exogenous retinoic acid synergizes with IL4 to regulate anti-inflammatory pathways such as oxidative phosphorylation and phagocytosis. Efferocytosis and lysosomal degradation assays validated gene expression changes at the functional level. IL4 treatment altered the expression of several genes involved in vitamin A transport and conversion to retinoic acid. Radiolabeling experiments and mass spectrometry assays revealed that IL4 stimulates retinoic acid production and secretion in a signal transducer and activator of transcription 6 (STAT6)-dependent manner. In summary, our studies highlight the key role of exogenous and endogenous retinoic acid in shaping the anti-inflammatory response of macrophages.
L'immunothérapie spécifique (ou désensibilisation) aux venins d'hyménoptères est un traitement qui permet de prévenir la récidive d’une anaphylaxie chez les patients allergiques au venin de guêpe ou d’abeille. Une augmentation très rapide des doses est souvent utilisée lors de la phase initiale de ce traitement dont la bonne tolérance n’est pas bien expliquée. Le but de ce travail était de décrire les changements précoces du système immunitaire pendant l’initiation de l’immunothérapie aux venins d’hyménoptères pouvant expliquer cette bonne tolérance. Nous avons inclus 29 patients traités pour une allergie au venin d’hyménoptères avec une initiation de traitement par « ultra-rush » en 3h30. Des prélèvements sanguins ont été pratiqués avant le début du traitement, à 1h30 et juste avant la dernière injection de venin. L’évolution de la tryptase sanguine a été analysée. L'activation des polynucléaires basophiles ainsi que l'expression FcεRI à leur surface ont été analysées par intensité moyenne de fluorescence par cytométrie en flux. Pour évaluer l'évolution de la réactivité des polynucléaires basophiles, un test d'activation des basophiles (TAB) a été réalisé à chaque temps. L’évolution des populations lymphocytaires T et myéloïdes a été également analysée par cytométrie en flux. Nous avons montré une diminution significative de la tryptase sérique pendant l’ultra-rush, de même qu’une diminution significative de l’activation des polynucléaires basophiles et une diminution de l’expression de FcεRI à leur surface. Etonnamment, le TAB a montré une réponse in vitro des basophiles significativement plus élevée à l'extrait de venin à la fin de « l'ultra-rush » par rapport à avant le début du traitement. Nous avons également montré une augmentation significative des cellules dendritiques et une diminution significative des lymphocytes « Natural Killer » (NK) dans le sang. Concernant les populations lymphocytaires T, nous avons montré une ugmentation significative des populations lymphocytaires T dans le sang, sauf pour les Lymphocytes T CD4+et CD8+ naïfs. En conclusion, l’augmentation des doses de venin par « ultra-rush » est bien tolérée grâce à une inhibition des polynucléaires basophiles impliquant une diminution de l’expression de FcεRI à leur surface. L'ultra-rush entraîne également des modifications précoces dans la réponse immunitaire innée et adaptative.
The JAK–STAT pathway is the central intracellular signal transduction cascade initiated by ligand binding to numerous growth factor and cytokine receptors, and since its discovery two decades ago it has been the subject of extensive investigation. The pathway is negatively regulated by the SOCS family proteins, which restrict the extent of the cytokine response, thus preventing excessive and deleterious inflammatory and immune signaling. There are now many examples of human mutations and disease conditions where the JAK–STAT–SOCS axis is perturbed, and we have witnessed the advent of JAK inhibitors in the clinic. This review explores our current understanding of how this unique pathway functions.
The activation of a latent DNA binding factor by interleukin-4 (IL-4), the IL-4 nuclear activated factor (IL-4 NAF), occurs within minutes of IL-4 binding to its receptor. Molecular characterization of IL-4 NAF by ultraviolet light cross-linking experiments revealed a single protein of 120-130 kDa in contact with the DNA target site. Glycerol gradient sedimentation analysis indicated a molecular mass of IL-4 NAF consistent with a monomer that is capable of binding DNA. The IL-4 NAF target site is a palindromic sequence that is also recognized by the interferon-induced transcription factor, p91/STAT1α. However, IL-4 NAF and p91/STAT1α display distinguishable DNA binding specificities that may generate one level of specificity in the expression of target genes. Previous studies suggested the involvement of the insulin receptor substrate-1 (IRS-1) in the IL-4 signal transduction pathway. Although IRS-1 is involved in the stimulation of mitogenesis, our results demonstrate that activation of IL-4 NAF is independent of IRS-signaling proteins. The results of this study indicate that IL-4 stimulates bifurcating signal pathways that can direct mitogenesis via the IRS-signaling proteins and specific gene expression via the IL-4 NAF.
The lymphokine B-cell stimulatory factor 1 (BSF-1) has been shown to greatly enhance the differentiation of lipopolysaccharide-activated B cells into IgG1- and IgE-secreting cells in vitro. To determine whether in vivo IgG1 and IgE antibody responses are BSF-1 dependent, the ability of a monoclonal rat IgG1 anti-BSF-1 antibody, 11B11, to affect polyclonal IgG1 and IgE production in mice infected with the nematode parasite Nippostrongylus brasiliensis or injected with a purified goat antibody to mouse IgD was studied. 11B11-containing ascites fluid or purified 11B11 strongly inhibited IgE production in both systems but did not affect IgG1 production, while control ascites or normal rat IgG1 had no IgE-inhibitory activity. These results indicate an important physiologic role for BSF-1 in the generation of IgE antibody responses and suggest means for limiting the production of antibodies responsible for allergic reactions without inhibiting protective antibody responses.
Interleukin-4 (IL-4) was shown to induce a potent mitogenic response in the IL-3-dependent myeloid progenitor cell line, FDCP-2. Although IL-4 could not sustain long-term growth of FDCP-2 cells, it enhanced their growth in serum-free medium containing IL-3. IL-4 triggered prominent tyrosine phosphorylation of a substrate(s) migrating at 170 kDa and less striking phosphorylation of several other proteins, including the IL-4 receptor. By contrast, IL-3 induced distinct tyrosine phosphorylation of proteins migrating at 145, 97, 70, 55 and 52 kDa in the same cell line. IL-4 treatment of FDCP-2 cells caused a dramatically strong association of phosphatidylinositol 3-kinase (PI 3-kinase) both with the 170 kDa tyrosine phosphorylated substrate and with the IL-4 receptor itself. By contrast, IL-3 triggered only weak association of PI 3-kinase activity with the 97 kDa substrate. While IL-4 did not affect cellular raf, IL-3 stimulation did induce a shift in its mobility presumably due to serine/threonine phosphorylation. Taken together, our results indicate that IL-4 and IL-3 activate distinct phosphorylation cascades in the same cell background; this may reflect a difference in the biological function of these two cytokines.
Resting T cells proliferate in response to B cell stimulatory factor 1 (BSF-1; interleukin 4) plus phorbol myristate acetate (PMA). This response is obtained with highly purified T cells and is density independent, suggesting that accessory cells are not required. Both L3T4+ and Lyt-2+ T cells respond to BSF-1 plus PMA. Although BSF-1 alone does not cause T cell proliferation, it maintains the viability of small, dense T cells, indicating that it acts on resting T cells. Furthermore, BSF-1 is required early in the proliferative response of resting T cells to BSF-1 plus PMA, further supporting the concept that it acts on G0 or early G1 cells. However, BSF-1 is also needed late in the first round of division of T cells stimulated with BSF-1 plus PMA. Removing BSF-1 at 24 h of stimulation prevents entry into S phase. These results indicate that BSF-1 is involved in both the induction of competence and in the progression phases of T cell division.
BSF-1 prepares resting BALB/c, DBA/2, and BDF1 B cells to enter S phase more promptly in response to subsequent culture with anti-IgM-based stimulants. It prepares DBA/2 and BDF1 B cells to respond to LPS, but its preparative effect for LPS responses of BALB/c B cells is both inconstant and meager. Preparation mediated by BSF-1 requires extended contact of B cells with the stimulant for full effect. Half-maximal preparation requires approximately 12 h of contact, as judged by delayed addition of BSF-1 or by inhibition of BSF-1 action with anti-BSF-1 mAbs. BSF-1 preparative action on resting DBA/2 B cells is mimicked by anti-Lyb-2.1 antibody. B cell blasts prepared by culture with BSF-1 and anti-IgM show modest responses to high concentrations of BSF-1; large B cells directly isolated from the spleen are not stimulated to enter S phase by BSF-1. These results lead us to conclude that BSF-1 functions principally as an activation factor for resting B cells.
The present studies demonstrate that both T-cell-derived supernatants containing B-cell growth factor (BCGF or BSF) and a partially purified preparation of the B-cell growth factor (BSF-p1) induce an increase in the expression of IA and IE-encoded antigens on small resting B cells. This increase is detectable by 6-8 hr after initiation of culture and is relatively selective, since levels of surface immunoglobulin and H-2 antigens do not increase to the same extent. Although interferon-gamma induces increased expression of Ia antigens on macrophages and dividing neoplastic B cells, it does not induce an increase in the expression of Ia antigens on resting B cells. These results demonstrate that BSF-p1 may play two roles: (i) it acts on resting B cells to increase the levels of Ia antigen expression; and (ii) it sustains the growth of B cells that have been previously activated with mitogens, antigens, or anti-Ig.
The interleukin 4 (IL-4) signaling pathway involves activation, by tyrosine phosphorylation, of two distinct substrates, a signal-transducing factor (STF-IL4) and the IL-4-induced phosphotyrosine substrate (4PS). It is not known whether the IL-4-mediated activation of these substrates occurs via related or distinct signaling pathways. We report that 32D cells, an IL-3-dependent myeloid progenitor cell line in which no phosphorylated 4PS is found, activate high levels of STF-IL4 in response to IL-4. Consistent with the known requirement for 4PS or insulin receptor substrate 1 (IRS-1) in IL-4-mediated mitogenesis, activation of STF-IL4 in 32D cells is not sufficient for IL-4-inducible c-myc expression. In addition, we have examined the ability of 32D cells transfected with different truncation mutants of the human IL-4 receptor to activate Jak-3 kinase and STF-IL4 in response to human IL-4. As in the case of 4PS/IRS-1, we have found that activation of both Jak-3 and STF-IL4 requires the presence of the IL-4 receptor region comprising aa 437-557. The finding that the same region of the IL-4 receptor is required for the induction of both 4PS/IRS-1 and STF-IL4 suggests that the IL-4-stimulated activation of these two substrates might involve common factors.
By searching a database of expressed sequences, we identified a member of the signal transducers and activators of transcription
(Stat) family of proteins. Human and murine full-length cDNA clones were obtained and sequenced. The sequence of the human
cDNA was identical to the recently published sequence for interleukin-4 (IL-4)-Stat (J. Hou, U. Schindler, W.J. Henzel, T.C.
Ho, M. Brasseur, and S. L. McKnight, Science 265:1701-1706, 1994), while the murine Stat6 amino acid and nucleotide sequences
were 83 and 84% identical to the human sequences, respectively. Using Stat6-specific antiserum, we demonstrated that Stat6
is rapidly tyrosine phosphorylated following stimulation of appropriate cell lines with IL-4 or IL-3 but is not detectably
phosphorylated following stimulation with IL-2, IL-12, or erythropoietin. In contrast, IL-2, IL-3, and erythropoietin induce
the tyrosine phosphorylation of Stat5 while IL-12 uniquely induces the tyrosine phosphorylation of Stat4. Inducible tyrosine
phosphorylation of Stat6 requires the membrane-distal region of the IL-4 receptor alpha chain. This region of the receptor
is not required for cell growth, demonstrating that Stat6 tyrosine phosphorylation does not contribute to mitogenesis.
The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.
The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen-presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology.
Several specific conclusions can be drawn from these studies: 1. IL-4 is required for the generation of both primary polyclonal and secondary antigen-specific IgE responses in vivo. 2. IL-4 is required to maintain established, ongoing, antigen-specific and polyclonal IgE responses. 3. Most, but not all, polyclonal IgE production during a secondary immune response is IL-4-dependent. Memory B cells that have already switched to IgE at the DNA level may no longer require stimulation with IL-4 to be induced to secrete IgE. 4. The generation of a secondary IgE response is not dependent upon the presence of IL-4 during primary immunization. However, if IL-4 is not present during primary immunization, it is required during secondary immunization for the generation of an IgE response. 5. IL-4 does not appear to be required for the generation of in vivo IgG1 responses, and in at least some instances, does not contribute significantly to the generation of IgG1 responses in vivo. 6. A late-acting form of T-cell help other than IL-4 appears to be required for the generation of an IgE, but not an IgG1 response. 7. An antibody that inhibits IL-4 binding to IL-4 receptors affects Ig isotype selection in the same way as an antibody that neutralizes IL-4. 8. IFN-gamma can act in both spontaneous and induced immune responses to suppress IgE production. 9. IFN-gamma can also suppress IgG1 production and stimulate IgG2a production. However, IFN-gamma appears to suppress polyclonal IgG1 responses more than antigen-specific IgG1 responses, and it enhances, but is not required for, the generation of IgG2a responses. 10. IFN-alpha appears to resemble IFN-gamma in its ability to inhibit IgE and enhance IgG2a responses in GaM delta-injected mice, but it requires the presence of IFN-gamma to suppress IgG1 production in these mice. 11. Both IFN-alpha and IFN-gamma appear to be able to decrease IgE production in some human patients. 12. There is no direct evidence that IL-5 contributes to the generation of in vivo antibody responses. Two general conclusions may also be drawn.(ABSTRACT TRUNCATED AT 400 WORDS)
Interleukin-4 (IL-4) promotes the growth and differentiation of many hematopoietic cells in vitro; in particular, it directs
the immunoglobulin (Ig) class switch to IgG1 and IgE. Mice homozygous for a mutation that inactivates the IL-4 gene were generated
to test the requirement for IL-4 in vivo. In the mutant mice T and B cell development was normal, but the serum levels of
IgG1 and IgE were strongly reduced. The IgG1 dominance in a T cell-dependent immune response was lost, and IgE was not detectable
upon nematode infection. Thus, some but not all of the in vitro properties of IL-4 are critical for the physiology of the
immune system in vivo.
A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.
We have isolated and sequenced a cDNA clone encoding the human lymphocyte receptor for IgE (Fc epsilon R). The deduced protein sequence reveals that Fc epsilon R consists of 321 amino acids, without any signal sequence, and is oriented with its N-terminus on the cytoplasmic side and its C-terminus on the outside of the cell. This molecule shows striking sequence homology with chicken asialoglycoprotein receptor (hepatic lectin), suggesting a possible role for Fc epsilon R in endocytosis. Fc epsilon R mRNA is expressed in B cells, B cell lines, and macrophage cell lines. It is not expressed in T cells or T cell lines, with the exception of an HTLV-transformed T cell line. mRNAs expressed in a macrophage line and in the latter T cell line differ in size from mRNA expressed in B cells. Human BSF-1 (or IL-4) induces the expression of Fc epsilon R mRNA in B cells, but not in T cells.
Recent work in both the human and murine systems has demonstrated that IL-4 is capable of specifically inducing the synthesis of the low affinity receptor for IgE (Fc epsilon RII). In addition, in conjunction with LPS, IL-4 will induce IgG1 and IgE synthesis. To analyze the correlation between Fc epsilon RII induction and IgE secretion, Fc epsilon RII and IgE levels were measured by RIA on murine splenic B cells stimulated with LPS and IL-4 over 7 days of culture. Treatment with LPS and IL-4 gave a 20- to 50-fold (day 3) "superinduction" of Fc epsilon RII levels compared with a 3- to 5-fold induction with IL-4 alone; removal of IL-4 resulted in a rapid decline in Fc epsilon RII levels. The cells expressing high Fc epsilon RII levels were determined to be blasts. Superinduction of Fc epsilon RII occurs at 10 U/ml IL-4 and remains relatively constant in the range of 10 to 1000 U/ml. In contrast, with increasing IL-4, IgE levels increase, reaching microgram levels at day 7 with 300 U/ml IL-4. Triggering the cells with anti-Ig, as expected, gave no Ig secretion, and in addition, Fc epsilon RII superinduction by IL-4 and anti-Ig was not seen. PMA is known to block Ig secretion induced by LPS. Concentrations of PMA that totally abrogated IgE secretion had no effect on Fc epsilon RII superinduction, indicating that the latter phenomena can be separated from IL-4-induced Ig secretion. Superinduction also results in higher levels of Fc epsilon RII fragment release into the media. Thus, attempts were made to influence IgE secretion by adding additional purified Fc epsilon RII fragment to the culture. The purified fragment did not have a significant influence on IgE levels in this system.
The Fc epsilon receptor II (Fc epsilon RII, CD23) functions in B cell growth and differentiation and in IgE-mediated immunity. The Fc epsilon RII structure expressed on various cell types has been analyzed identifying two species, Fc epsilon RIIa and Fc epsilon RIIb. Sequence analysis of the cloned cDNAs revealed that they differ only at the N-terminal cytoplasmic region, but share the same C-terminal extracellular region. These Fc epsilon RII species appear to be generated utilizing different transcriptional initiation sites and alternative RNA splicing. Fc epsilon RIIa is constitutively expressed only in normal B cells and B cell lines, whereas Fc epsilon RIIb expression is detectable in various cell types, such as monocytes and eosinophils. Normally, Fc epsilon RIIb is undetectable in B cells and monocytes, and can be induced by interleukin-4. Moreover, Fc epsilon RIIb is expressed on peripheral blood lymphocytes in atopic individuals. These findings may explain the difference in Fc epsilon RIIa and Fc epsilon RIIb function in B cells and the effector phase of IgE-mediated immunity.
B-cell stimulatory factor 1 (BSF-1) is a T-cell-derived lymphokine that acts together with low concentrations of anti-IgM antibodies to stimulate resting B cells to enter the G1 phase of the cell cycle and to synthesize DNA. We show here that supernatants from EL-4 cells, rich in BSF-1 activity, and BSF-1 purified by high-pressure liquid chromatography (HPLC-BSF-1) act on resting B cells, in the absence of anti-IgM antibodies, to prepare them to respond to anti-IgM and BSF-1. A 24-hour preculture with BSF-1 speeds the entry into S phase of B cells subsequently cultured with anti-IgM and BSF-1 by approximately equal to 12 hours and causes substantial increase in cell volume of all resting B cells. Both of these effects, stimulated either by EL-4 supernatants or by HPLC-BSF-1, are inhibited by a monoclonal anti-BSF-1 antibody. These results lead us to propose that BSF-1 should be regarded as a B-cell activation factor.
To investigate the role of NF-IL6 in vivo, we have generated NF-IL6 (-/-) mice by gene targeting. NF-IL6 (-/-) mice were highly susceptible to infection by Listeria monocytogenes. Electron microscopic observation revealed the escape of a larger number of pathogens from the phagosome to the cytoplasm in activated macrophages from NF-IL6 (-/-) mice. Furthermore, the tumor cytotoxicity of macrophages from NF-IL6 (-/-) mice was severely impaired. However, cytokines involved in macrophage activation, such as TNF and IFN gamma, were induced normally in NF-IL6 (-/-) mice. Nitric oxide (NO) formation was induced to a similar extent in macrophages from both wild-type and NF-IL6 (-/-) mice. These results demonstrate the crucial role of NF-IL6 in macrophage bactericidal and tumoricidal activities as well as the existence of a NO-independent mechanism of these activities. We also demonstrate that NF-IL6 is essential for the induction of G-CSF in macrophages and fibroblasts.
The protein IRS-1 acts as an interface between signalling proteins with Src-homology-2 domains (SH2 proteins) and the receptors for insulin, IGF-1, growth hormone, several interleukins (IL-4, IL-9, IL-13) and other cytokines. It regulates gene expression and stimulates mitogenesis, and appears to mediate insulin/IGF-1-stimulated glucose transport. Thus, survival of the IRS-1-/- mouse with only mild resistance to insulin was surprising. This dilemma is provisionally resolved with our discovery of a second IRS-signalling protein. We purified and cloned a likely candidate called 4PS from myeloid progenitor cells and, because of its resemblance to IRS-1, we designate it IRS-2. Alignment of the sequences of IRS-2 and IRS-1 revealed a highly conserved amino terminus containing a pleckstrin-homology domain and a phosphotyrosine-binding domain, and a poorly conserved carboxy terminus containing several tyrosine phosphorylation motifs. IRS-2 is expressed in many cells, including tissues from IRS-1-/- mice, and may be essential for signalling by several receptor systems.
To understand the molecular bases for cytokine redundancy and pleiotropy, we have compared the Stat proteins activated in peripheral blood lymphocytes (PBLs) by cytokines with shared and distinct actions. Interleukin-2 (IL-2) rapidly activated Stat5 in fresh PBL, and Stat3 and Stat5 in preactivated PBL. IL-7 and IL-15 induced the same complexes as IL-2, a feature explained by the existence of similar tyrosine-phosphorylated motifs in the cytoplasmic domains of IL-2R beta and IL-7R that can serve as docking sites for Stat proteins. IL-13 Induced the same complexes as IL-4, a finding explained by our studies implicating IL-4R as a shared component of the receptors. These studies demonstrate that a single cytokine can activate different combinations of Stat proteins under different physiological conditions, and also indicate two mechanisms by which distinct cytokines can activate the same Stat protein.
Achsah Keegan and colleagues consider the signaling mechanisms utilized by the interleukin 4 (IL-4) receptor and review evidence suggesting that these mechanisms can account for the known responses of hematopoietic and non-hematopoietic cells to IL-4. Most of these data have been obtained from analyses of the ability of IL-4 to regulate the growth of IL-3-dependent myeloid cell lines. These results have implicated a pathway of activation homologous to that utilized by insulin and insulin-like growth factor 1 (IGF-1). However, it is possible that the regulation of growth responses through the IL-4 receptor (and other receptors), and the differentiative events elicited in lymphocytes, may not be mediated by the same post-receptor events.
Interleukin-4 (IL-4) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. IL-4 may mediate its biological effects, at least in part, by activating a tyrosine-phosphorylated DNA binding protein. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the IL-4 receptor provided evidence for direct coupling of receptor and transcription factor during the IL-4 Stat activation cycle. Such observations indicate that IL-4 Stat has the same functional domain for both receptor coupling and dimerization.
Interleukin-4 (IL-4) treatment of 32D cells overexpressing insulin receptor substrate 1 (IRS-1) causes prompt tyrosine phosphorylation of IRS-1. Transfection of truncation mutants of the human IL-4 (huIL-4) receptor into 32D-IRS-1 cells demonstrated that the region from amino acid 437-557 is important for IL-4 signaling. This region of the IL-4 receptor (IL-4R) contains the motif 488PL-X4-NPXYXSXSD502 (insulin/IL-4R [I4R]) found in the insulin and insulin-like growth factor 1 receptors. Mutation of Y497 to F yielded receptors that caused little or no IRS-1 phosphorylation in response to huIL-4 when expressed in 32D-IRS-1 cells. Most cell lines expressing Y497F also failed to proliferate in response to huIL-4. Furthermore, a glutathione-S-transferase fusion protein containing the I4R motif-bound IRS-1, tyrosine kinase(s), and other unidentified phosphoproteins with molecular sizes of 140, 80, and 55 kd. Thus, the central tyrosine of the I4R motif has a major role in IL-4-mediated signal transduction in 32D cells.
Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.
Murine T-helper clones are classified into two distinct subsets (Th1 and Th2) on the basis of their patterns of lymphokine secretion. Th1 clones secrete interleukin-2 (IL-2), tumour necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), whereas Th2 clones secrete IL-4, IL-5 and IL-10 (ref. 1). These subsets are reciprocally regulated by IL-4, IL-10 and IFN-gamma and differentially promote antibody or delayed-type hypersensitivity responses. To evaluate whether IL-4 is required for mounting Th2 responses, we generated IL-4-mutant mice (IL-4-/-) and assessed the cytokine secretion pattern of T cells both from naive and Nippostrongylus brasiliensis infected mice. CD4+ T cells from naive IL-4-/- mice failed to produce Th2-derived cytokines after in vitro stimulation. The levels of Th2 cytokines IL-5, IL-9 and IL-10 from CD4+ T cells obtained after nematode infection were significantly reduced. The reduced IL-5 production in IL-4-/- mice correlated with reduced helminth-induced eosinophilia, which has been shown to be dependent on IL-5 in vivo. We conclude that IL-4 is required for the generation of the Th2-derived cytokines and that immune responses dependent on these cytokines are impaired.
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