Indispensable role of Stat5a in Stat6-independent Th2 cell differentiation and allergic airway inflammation

It is well-recognized that Stat6 plays a critical role in Th2 cell differentiation and the induction of allergic inflammation. We have previously shown that Stat5a is also required for Th2 cell differentiation and allergic airway inflammation. However, it is the relative importance and redundancy of Stat6 and Stat5a in Th2 cell differentiation and allergic airway inflammation are unknown. In this study we addressed these issues by comparing Stat5a-deficient (Stat5a(-/-)) mice, Stat6(-/-) mice, and Stat5a- and Stat6 double-deficient (Stat5a(-/-) Stat6(-/-)) mice on the same genetic background. Th2 cell differentiation was severely decreased in Stat6(-/-)CD4+ T cells, but Stat6-independent Th2 cell differentiation was still significantly observed in Stat6(-/-)CD4+ T cells. However, even in the Th2-polarizing condition (IL-4 plus anti-IFN-gamma mAb), no Th2 cells developed in Stat5a(-/-)Stat6(-/-) CD4+ T cells. Moreover, Ag-induced eosinophil and lymphocyte recruitment in the airways was severely decreased in Stat5a(-/-)Stat6(-/-) mice compared with that in Stat6(-/-) mice. These results indicate that Stat5a plays an indispensable role in Stat6-independent Th2 cell differentiation and subsequent Th2 cell-mediated allergic airway inflammation.     

STAT6 expression in multiple cell types mediates the cooperative development of allergic airway disease

Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6(-/-) mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6(-/-) mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6(-/-) mice even when STAT6(+/+) BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2(-/-) mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4(+)CD25(+)Foxp3(+) T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6(-/-) mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6(-/-) mice were equally efficient in suppressing CD4(+) T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.     

Enhanced Th2 cell-mediated allergic inflammation in Tyk2-deficient mice

Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-gamma and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-alphabeta, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2(-/-)) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2(-/-) mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2(-/-) mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4(+) T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2(-/-) mice. Adoptive transfer experiments revealed that CD4(+) T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2(-/-) mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2(-/-) mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2(-/-) mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways.     

IL-33 induces antigen-specific IL-5+ T cells and promotes allergic-induced airway inflammation independent of IL-4

Type 2 cytokines (IL-4, IL-5, and IL-13) play a pivotal role in helminthic infection and allergic disorders. CD4(+) T cells which produce type 2 cytokines can be generated via IL-4-dependent and -independent pathways. Although the IL-4-dependent pathway is well documented, factors that drive IL-4-independent Th2 cell differentiation remain obscure. We report here that the new cytokine IL-33, in the presence of Ag, polarizes murine and human naive CD4(+) T cells into a population of T cells which produce mainly IL-5 but not IL-4. This polarization requires IL-1R-related molecule and MyD88 but not IL-4 or STAT6. The IL-33-induced T cell differentiation is also dependent on the phosphorylation of MAPKs and NF-kappaB but not the induction of GATA3 or T-bet. In vivo, ST2(-/-) mice developed attenuated airway inflammation and IL-5 production in a murine model of asthma. Conversely, IL-33 administration induced the IL-5-producing T cells and exacerbated allergen-induced airway inflammation in wild-type as well as IL-4(-/-) mice. Finally, adoptive transfer of IL-33-polarized IL-5(+)IL-4(-)T cells triggered airway inflammation in naive IL-4(-/-) mice. Thus, we demonstrate here that, in the presence of Ag, IL-33 induces IL-5-producing T cells and promotes airway inflammation independent of IL-4.     

STAT1-activating cytokines limit Th17 responses through both T-bet-dependent and -independent mechanisms

Given the association with autoimmune disease, there is great interest in defining cellular factors that limit overactive or misdirected Th17-type inflammation. Using in vivo and in vitro models, we investigated the molecular mechanisms for cytokine-mediated inhibition of Th17 responses, focusing on the role of STAT1 and T-bet in this process. These studies demonstrate that, during systemic inflammation, STAT1- and T-bet-deficient T cells each exhibit a hyper-Th17 phenotype relative to wild-type controls. However, IL-17 production was greater in the absence of T-bet, and when both STAT1 and T-bet were deleted, there was no further increase, with the double-deficient cells instead behaving more like STAT1-deficient counterparts. Similar trends were observed during in vitro priming, with production of Th17-type cytokines greater in T-bet(-/-) T cells than in either STAT1(-/-) or STAT1(-/-) T-bet(-/-) counterparts. The ability of IFN-γ and IL-27 to suppress Th17 responses was reduced in T-bet-deficient cells, and most importantly, ectopic T-bet could suppress signature Th17 gene products, including IL-17A, IL-17F, IL-22, and retinoic acid-related orphan receptor γT, even in STAT1-deficient T cells. Taken together, these studies formally establish that, downstream of IFN-γ, IL-27, and likely all STAT1-activating cytokines, there are both STAT1 and T-bet-dependent pathways capable of suppressing Th17 responses.     

Impaired GATA3-dependent chromatin remodeling and Th2 cell differentiation leading to attenuated allergic airway inflammation in aging mice

Age-related changes in lymphocytes are most prominent in the T cell compartment. There have been substantial numbers of reports on T cell function in aged mice and humans, such as on the production of Th1 and Th2 cytokines, but the results show considerable variation and contradictions. In the present study, we used 8- to 12-mo-old aging mice and a well-established in vitro Th1/Th2 cell differentiation culture system to identify molecular defects in Th1/Th2 cell differentiation that can be detected in the relatively early stages of aging. The capability to differentiate into Th2 cells is reduced in aging mouse CD4(+) T cells. Decreased activation of the ERK MAPK cascade upon TCR stimulation, but normal intracellular-free calcium ion concentration mobilization and normal IL-4-induced STAT6 activation were observed in aging mouse CD4(+) T cells. In addition, reduced expression of GATA3 was detected in developing Th2 cells. Chromatin remodeling of the Th2 cytokine gene locus was found to be impaired. Th2-dependent allergic airway inflammation was milder in aging mice compared with in young adult mice. These results suggest that the levels of Th2 cell differentiation and resulting Th2-dependent immune responses, including allergic airway inflammation, decline during aging through defects in the activation of the ERK MAPK cascade, expression of GATA3 protein and GATA3-dependent chromatin remodeling of the Th2 cytokine gene locus. In the present study, we provide the first evidence indicating that a chromatin-remodeling event in T cells is impaired by aging.     

High expression of IL-22 suppresses antigen-induced immune responses and eosinophilic airway inflammation via an IL-10-associated mechanism

Allergic inflammation in the airway is generally considered a Th2-type immune response. However, Th17-type immune responses also play important roles in this process, especially in the pathogenesis of severe asthma. IL-22 is a Th17-type cytokine and thus might play roles in the development of allergic airway inflammation. There is increasing evidence that IL-22 can act as a proinflammatory or anti-inflammatory cytokine depending on the inflammatory context. However, its role in Ag-induced immune responses is not well understood. This study examined whether IL-22 could suppress allergic airway inflammation and its mechanism of action. BALB/c mice were sensitized and challenged with OVA-Ag to induce airway inflammation. An IL-22-producing plasmid vector was delivered before the systemic sensitization or immediately before the airway challenge. Delivery of the IL-22 gene before sensitization, but not immediately before challenge, suppressed eosinophilic airway inflammation. IL-22 gene delivery suppressed Ag-induced proliferation and overall cytokine production in CD4(+) T cells, indicating that it could suppress Ag-induced T cell priming. Antagonism of IL-22 by IL-22-binding protein abolished IL-22-induced immune suppression, suggesting that IL-22 protein itself played an essential role. IL-22 gene delivery neither increased regulatory T cells nor suppressed dendritic cell functions. The suppression by IL-22 was abolished by deletion of the IL-10 gene or neutralization of the IL-10 protein. Finally, IL-22 gene delivery increased IL-10 production in draining lymph nodes. These findings suggested that IL-22 could have an immunosuppressive effect during the early stage of an immune response. Furthermore, IL-10 plays an important role in the immune suppression by IL-22.     

A mouse model of airway disease: oncostatin M-induced pulmonary eosinophilia, goblet cell hyperplasia, and airway hyperresponsiveness are STAT6 dependent, and interstitial pulmonary fibrosis is STAT6 independent

Oncostatin M (OSM), a pleiotropic cytokine of the gp130 cytokine family, has been implicated in chronic allergic inflammatory and fibrotic disease states associated with tissue eosinophilia. Mouse (m)OSM induces airway eosinophilic inflammation and interstitial pulmonary fibrosis in vivo and regulates STAT6 activation in vitro. To determine the requirement of STAT6 in OSM-induced effects in vivo, we examined wild-type (WT) and STAT6-knockout (STAT6(-/-)) C57BL/6 mouse lung responses to transient ectopic overexpression of mOSM using an adenoviral vector (AdmOSM). Intratracheal AdmOSM elicited persistent eosinophilic lung inflammation that was abolished in STAT6(-/-) mice. AdmOSM also induced pronounced pulmonary remodeling characterized by goblet cell hyperplasia and parenchymal interstitial fibrosis. Goblet cell hyperplasia was STAT6 dependent; however, parenchymal interstitial fibrosis was not. OSM also induced airway hyperresponsiveness in WT mice that was abolished in STAT6(-/-) mice. OSM stimulated an inflammatory signature in the lungs of WT mice that demonstrated STAT6-dependent regulation of Th2 cytokines (IL-4, IL-13), chemokines (eotaxin-1/2, MCP-1, keratinocyte chemoattractant), and extracellular matrix modulators (tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase-13), but STAT6-independent regulation of IL-4Rα, total lung collagen, collagen-1A1, -1A2 mRNA, and parenchymal collagen and α smooth muscle actin accumulation. Thus, overexpression of mOSM induces STAT6-dependent pulmonary eosinophilia, mucous/goblet cell hyperplasia, and airway hyperresponsiveness but STAT6-independent mechanisms of lung tissue extracellular matrix accumulation. These results also suggest that eosinophil or neutrophil accumulation in mouse lungs is not required for OSM-induced lung parenchymal collagen deposition and that OSM may have unique roles in the pathogenesis of allergic and fibrotic lung disease.     

STAT1 expression in dendritic cells, but not T cells, is required for immunity to Leishmania major

The generation of Th1 responses is important for resistance to intracellular pathogens, including the parasite, Leishmania major. Although IFN-gammaR/STAT1 signaling promotes a Th1 response via the up-regulation of T-bet, the requirement for STAT1 in Th1 cell differentiation remains controversial. Although in some cases Th1 cells develop independently of STAT1, STAT1(-/-) mice fail to develop a Th1 response during L. major infection. However, the interpretation of this result is complicated by the role STAT1 plays in Ag presentation and, more importantly, in elimination of parasites by macrophages, because both defective Ag presentation and increased parasite burden can influence Th cell development. To resolve this issue, we assessed the ability of STAT1(-/-) T cells to become Th1 cells and protect mice against L. major following adoptive transfer into STAT1-sufficient mice. We found that whereas T-bet is critical for the differentiation of protective Th1 cells during L. major infection, IFN-gammaR and STAT1 are dispensable. Given that a STAT1-independent Th1 cell response was generated by STAT1-sufficient APCs, but not by STAT1(-/-) cells, we next addressed whether dendritic cells (DCs) require STAT1 signaling to effectively present Ag. We found that STAT1(-/-) DCs had impaired up-regulation of MHC and costimulatory molecules, and, as a consequence, the absence of STAT1 resulted in reduced Th1 cell priming. Taken together, these results demonstrate that T cell expression of STAT1 is not required for the development of Th1 cells protective against L. major and instead stress the importance of STAT1 signaling in DCs for the optimal induction of Th1 responses.     

Ras activation in T cells determines the development of antigen-induced airway hyperresponsiveness and eosinophilic inflammation

The central role for Th2 cells in the development of Ag-induced airway hyperresponsiveness and eosinophilic inflammation is well documented. We have reported a crucial role for TCR-induced activation of the Ras/extracellular signal-regulated kinase mitogen-activated protein kinase cascade in Th2 cell differentiation. Here, we show that the development of both OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation in a mouse asthma model are attenuated in transgenic mice by the overexpression of enzymatically inactive Ras molecules in T cells. In addition, reduced levels of IL-5 production and eosinophilic inflammation induced by nematode infection (Nippostrongylus brasiliensis or Heligmosomoides polygyrus) were detected. Thus, the level of Ras activation in T cells appears to determine Th2-dependent eosinophilic inflammation and Ag-induced airway hyperresponsiveness.     

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

Background

Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.

Methods

Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.

Results

OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.

Conclusions

Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.     

Preventative Effect of an Herbal Preparation (HemoHIM) on Development of Airway Inflammation in Mice via Modulation of Th1/2 Cells Differentiation

HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4⁺ T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4⁺ T cells displayed increased Th1 (IFN-γ⁺ cell) as well as decreased Th2 (IL-4⁺ cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance.