T cells are necessary for ILC2 activation in house dust mite‐induced allergic airway inflammation in mice

Allergic asthma is a chronic inflammation of the airways mediated by an adaptive type 2 immune response. Upon allergen exposure, group 2 innate lymphoid cells (ILC2s) can be rapidly activated and represent an early innate source of IL‐5 and IL‐13. Here, we used a house dust mite (HDM)‐driven asthma mouse model to study the induction of ILC2s in allergic airway inflammation. In BALF, lungs, and lymph nodes, ILC2 activation is critically dependent on prior sensitization with HDM. Importantly, T cells are required for ILC2 induction, whereby T‐cell activation precedes ILC2 induction. During HDM‐driven allergic airway inflammation the accumulation of ILC2s in BALF is IL‐33 independent, although infiltrating ILC2s produce less cytokines in Il33^?/? mice. Transfer of in vitro polarized OVA‐specific OT‐II Th2 cells alone or in combination with Th17 cells followed by OVA and HDM challenge is not sufficient to induce ILC2, despite significant eosinophilic inflammation and T‐cell activation. In this asthma model, ILC2s are therefore not an early source of Th2 cytokines, but rather contribute to type 2 inflammation in which Th2 cells play a key role. Taken together, ILC2 induction in HDM‐mediated allergic airway inflammation in mice critically depends on activation of T cells.     

T cells and ILC2s are major effector cells in influenza‐induced exacerbation of allergic airway inflammation in mice

Influenza virus infection is an important cause of severe asthma exacerbations, but it remains unclear how a Th1‐mediated antiviral response triggers a prototypical Th2 disease. We investigated CD4⁺ T cells and group 2 innate lymphoid cells (ILC2s) in influenza virus‐infected mice. We found that ILC2s accumulated in the lung rapidly after influenza virus infection, but the induction of IL‐5 and IL‐13 secretion was delayed and concomitant with T cell activation. In an influenza‐induced exacerbation of allergic airway inflammation model we noticed an initial reduction of ILC2 numbers and cytokine production in broncho‐alveolar lavage compared to chronic house dust mite (HDM)‐mediated airway inflammation alone. ILC2s phenotype was characterized by low T1/ST2, ICOS, KLRG1, and CD25 expression, resembling naïve ILC2s. The contribution of ILC2s to type 2 cytokine production in the early stage of the influenza‐induced exacerbation was limited. In contrast, T cells showed increased IL‐4 and IL‐5 production when exposed to both HDM and influenza virus. Upon virus clearance, ILC2s regained an activated T1/ST2^highICOS^highKLRG1^highCD25^high phenotype paired with cytokine production and were major contributors to the type 2 cytokine milieu. Collectively, our data indicate that both T cells and ILC2s contribute to influenza‐induced exacerbation of allergic airway inflammation, but with different kinetics.     

An ST2‐dependent role of bone marrow‐derived group 2 innate lymphoid cells in pulmonary fibrosis

Recent evidence supports that bone marrow (BM)‐derived hematopoietic progenitor cells play an important role in lung injury and fibrosis. While these cells give rise to multiple cell types, the ST2 (Il1rl1)‐expressing group 2 innate lymphoid cells (ILC2s) derived from BM progenitors have been implicated in tissue repair and remodeling, including in lung fibrosis. To further investigate the precise role of BM‐derived ILC2s in the pathogenesis of fibrotic lung disease, their importance in the bleomycin‐induced lung fibrosis model was evaluated by analyzing the effects of selective ST2 deficiency in the BM compartment. The results showed that while ST2‐sufficient control mice exhibited activation of lung IL‐33/ST2 signaling, ILC2 recruitment, IL‐13 induction, and fibrosis, these responses were significantly diminished in ST2‐deficient‐BM chimera mice, with selective loss of ST2 expression only in the BM. This diminished response to bleomycin was similar to that seen in ST2 global knockout mice, suggesting the predominant importance of ST2 from the BM compartment. In wild‐type mice, ILC2 recruitment to the lung was accompanied by a concomitant decrease in ST2⁺ BM cells. ST2‐deficient BM cells were unresponsive to IL‐33‐induced ILC2 maturation. Finally, lineage‐negative wild‐type, but not ST2‐deficient BM cells from bleomycin‐treated mice stimulated lung fibroblast type I collagen expression, which was associated with elevated TGFβ expression in the BM cells. Taken together, these findings suggested that the BM‐derived ILC2s were recruited to fibrotic lung through the IL‐33/ST2 pathway, and contributed to fibroblast activation to promote lung fibrosis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Antigen presenting cell‐derived IL‐6 restricts Th2‐cell differentiation

The identification of DC‐derived signals orchestrating activation of Th1 and Th17 immune responses has advanced our understanding on how these inflammatory responses develop. However, whether specific signals delivered by DCs also participate in the regulation of Th2 immune responses remains largely unknown. In this study, we show that administration of antigen‐loaded, IL‐6‐deficient DCs to naïve mice induced an exacerbated Th2 response, characterized by the differentiation of GATA‐3‐expressing T lymphocytes secreting high levels of IL‐4, IL‐5, and IL‐13. Coinjection of wild type and IL‐6‐deficient bone marrow‐derived dendritic cells (BMDCs) confirmed that IL‐6 exerted a dominant, negative influence on Th2‐cell development. This finding was confirmed in vitro, where exogenously added IL‐6 was found to limit IL‐4‐induced Th2‐cell differentiation. iNKT cells were required for optimal Th2‐cell differentiation in vivo although their activation occurred independently of IL‐6 secretion by the BMDCs. Collectively, these observations identify IL‐6 secretion as a major, unsuspected, mechanism whereby DCs control the magnitude of Th2 immunity.     

Increased ILC3s associated with higher levels of IL‐1β aggravates inflammatory arthritis in mice lacking phagocytic NADPH oxidase

The role of redox regulation in immune‐mediated arthritis has been previously described. However, the relationship between innate immune cells, including innate lymphoid cells (ILCs) and phagocyte‐derived ROS, in this process remains unclear. Here, we characterize ILCs and measure the IL‐1 family cytokines along with other cytokines relevant to ILC functions and development in serum‐induced arthritic joints in wild type and phagocytic NADPH oxidase (NOX2)‐deficient Ncf1^?/? mice. We found more severe serum‐induced joint inflammation and increased NCR⁺ ILC3s in inflamed joints of Ncf1^?/? mice. Furthermore, in vitro stimulation with IL‐1β on Tbet⁺ ILC1s from joints facilitated their differentiation into ROR‐γt⁺ ILC3s. Moreover, treatment with IL‐1 antagonists effectively lowered the proportions of NCR⁺ ILC3s and IL‐17A producing ILC3s in Ncf1^?/? arthritic mice and ameliorated the joint inflammation. These results suggest that NOX2 is an essential regulator of ILC transdifferentiation and may mediate this process in a redox‐dependent manner through IL‐1β production in the inflammatory joint. Our findings shed important light on the role of ILCs in the initiation and progression in tissue inflammation and delineate a novel innate immune cell‐mediated pathogenic mechanism through which redox regulation may determine the direction of immune responses in joints.     

Unexpected involvement of IL‐13 signalling via a STAT6 independent mechanism during murine IgG2a development following viral vaccination

In this study, recombinant pox viral vaccination was shown to induce highly elevated IgG2a and low IgG1 antibody expression in mice lacking IL‐4 or STAT6, whilst IL‐13^?/? mice exhibited elevated IgG1, but very low IgG2a. These findings revealed that IL‐13 and IL‐4 differentially regulated antibody development. To understand this further, when STAT6^?/? mice were given a vaccine co‐expressing IL‐13Rα2 that temporarily sequestered IL‐13, significantly reduced IgG2a expression, was detected. These findings for the first time demonstrated that IL‐13 regulated IgG2a differentiation utilising an alternative IL‐13R signalling pathway independent of STAT6 (IL‐13Rα2 pathway). This was further corroborated by the (i) elevated IL‐13Rα2 expression detected on STAT6^?/? lung MHCII⁺ CD11c⁺ cells 24 h post IL‐13 inhibitor vaccination and ii) significant up‐regulation of IL‐13Rα2 expression on spleen and lung derived MHCII⁺CD11c⁺ following inhibition of STAT6 signalling in vitro, or vaccination with IL‐4R/STAT6 antagonist in vivo. When T follicular helper (Tfh) cells which regulate antibody differentiation were assessed post vaccination, although no difference in IL‐4 expression was observed, greatly reduced IFN‐γ expression was detected in IL‐13^?/? and STAT6^?/? mice compared to wild‐type. These findings support the notion that the balance of IL‐13 level at the vaccination site can differentially regulate T and B‐cell immune outcomes.     

Interferon‐γ constrains cytokine production of group 2 innate lymphoid cells

Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin‐5 (IL‐5), which supports eosinophil responses in various tissues; they also produce IL‐13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL‐33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon‐γ (IFN‐γ). Interferon‐γ severely inhibited IL‐5 and IL‐13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α‐galactosylceramide (α‐GalCer) to induce NKT cells to produce IL‐33 and IFN‐γ. Intraperitoneal injection of α‐GalCer in mice induced NKT cell activation resulting in IL‐5 and IL‐13 production by ILC2s. Administration of anti‐IFN‐γ together with α‐GalCer significantly enhanced the production of IL‐5 and IL‐13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL‐33 in Il33^?/? mice pre‐treated with α‐GalCer. Hence, IFN‐γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

ILC2 frequency and activity are inhibited by glucocorticoid treatment via STAT pathway in patients with asthma

Background

Group 2 innate lymphoid cells (ILC2s) were closely associated with asthma. However, there were no perspective studies about the effects of glucocorticoid on ILC2s in asthma patients. Our objective was to perform a perspective study and evaluate the ILC2 activity after glucocorticoid therapy in asthma patients.

Methods

The asthma and asthma with allergic rhinitis patients were treated with glucocorticoid for 3 months. The circulating ILC2 levels were evaluated. The effects of glucocorticoid on ILC2s and possible signalling pathways were investigated in vitro.

Results

The patients were well‐controlled, and the high ILC2 levels were significantly decreased at 1 and 3 months after treatment. Peripheral blood monocytes from allergic patients produced dramatic IL‐5, IL‐13 and IL‐9 in response to IL‐25, IL‐33 plus IL‐2, and glucocorticoid significantly decreased their levels. Moreover, ILC2s were identified to be the predominant source of IL‐5, IL‐13 and IL‐9, and glucocorticoid treatment was able to reverse their high levels. STAT3, STAT5, STAT6, JAK3 and MEK signalling pathways were proved to be involved in regulating ILC2 activity under the glucocorticoid treatment.

Conclusion

The data suggested that glucocorticoid administration could be effective in treating asthma by regulating ILC2s via MEK/JAK‐STAT signalling pathways. This provides a new understanding of glucocorticoid application in regard to allergic diseases.     

IL‐6 controls susceptibility to helminth infection by impeding Th2 responsiveness and altering the Treg phenotype in vivo

IL‐6 plays a pivotal role in favoring T‐cell commitment toward a Th17 cell rather than Treg‐cell phenotype, as established through in vitro model systems. We predicted that in the absence of IL‐6, mice infected with the gastrointestinal helminth Heligmosomoides polygyrus would show reduced Th17‐cell responses, but also enhanced Treg‐cell activity and consequently greater susceptibility. Surprisingly, worm expulsion was markedly potentiated in IL‐6‐deficient mice, with significantly stronger adaptive Th2 responses in both IL‐6^?/? mice and BALB/c recipients of neutralizing anti‐IL‐6 monoclonal Ab. Although IL‐6‐deficient mice showed lower steady‐state Th17‐cell levels, IL‐6‐independent Th17‐cell responses occurred during in vivo infection. We excluded the Th17 response as a factor in protection, as Ab neutralization did not modify immunity to H. polygyrus infection in BALB/c mice. Resistance did correlate with significant changes to the associated Treg‐cell phenotype however, as IL‐6‐deficient mice displayed reduced expression of Foxp3, Helios, and GATA‐3, and enhanced production of cytokines within the Treg‐cell population. Administration of an anti‐IL‐2:IL‐2 complex boosted Treg‐cell proportions in vivo, reduced adaptive Th2 responses to WT levels, and fully restored susceptibility to H. polygyrus in IL‐6‐deficient mice. Thus, in vivo, IL‐6 limits the Th2 response, modifies the Treg‐cell phenotype, and promotes host susceptibility following helminth infection.     

Card9‐dependent IL‐1β regulates IL‐22 production from group 3 innate lymphoid cells and promotes colitis‐associated cancer

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell‐specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL‐1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis‐associated cancer (CAC) model. Card9^?/? mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL‐1β generation and defective IL‐1β controlled IL‐22 production from group 3 innate lymphoid cells. Consistent with the key role of immune‐derived IL‐22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9^?/? mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9‐controlled, ILC3‐mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9‐mediated innate immunity in inflammation‐associated carcinogenesis.     

Characterization of ST2 transgenic mice with resistance to IL‐33

IL‐33, a member of the IL‐1 family, activates MAPK and NF‐κB through its receptor ST2L and IL‐1RAcP. ST2, a member of the IL‐1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL‐33 and to inhibit the IL‐33/ST2L/IL‐1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL‐33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal MΦ, splenomegaly, accumulation of periodic acid‐Schiff‐positive material in the lung, and high concentrations of serum IL‐5 and IL‐13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal MΦ from IL‐33‐stimulated mice, mRNA expression of M2 MΦ marker genes were increased compared with thioglycollate‐elicited peritoneal MΦ. The IL‐33‐stimulation also increased the secretion of IL‐6 from MΦ. However, when the IL‐33 was preincubated with ST2 prior to its addition to the MΦ cultures, the secretion of IL‐6 was attenuated. These data suggest that, though IL‐33 induced the Th2‐type immune responses and infiltration of M2 type MΦ into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.     

Immunisation route‐dependent expression of IL‐4/IL‐13 can modulate HIV‐specific CD8+ CTL avidity

All HIV‐1 ‘systemic vaccine trials’ in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8⁺ T cells generated higher levels of IL‐4/IL‐13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL‐4^?/? and IL‐13^?/? KO mice have shown that the capacity to express IFN‐γ, IL‐4 and/or IL‐13 by K^dGag_197–205‐specific CTL differed between these groups and was inversely correlated with CTL avidity (IL‐13^?/?>IL‐4^?/?>BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL‐13^?/? and wild type mice. When IL‐13 was reconstituted in IL‐13^?/? splenocytes in vitro, their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL‐13 can greatly enhance CTL avidity. In contrast, extracellular IL‐4 appears to be important in maintaining long‐term Th1/Th2 balance in CTL, even though expression of IL‐4 by CTL markedly reduced avidity. STAT6^?/? mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K^dGag_197–205‐specific CTL was also regulated by IL‐4/IL‐13.     

T‐cell‐derived,but not B‐cell‐derived,IL‐10 suppresses antigen‐specific T‐cell responses in Litomosoides sigmodontis‐infected mice

IL‐10, a cytokine with pleiotropic functions is produced by many different cells. Although IL‐10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth‐induced immune evasion. Here, we show that B cells and T cells produce IL‐10 during murine Litomosoides sigmodontis infection. IL‐10‐deficient mice produced increased amounts of L. sigmodontis‐specific IFN‐γ and IL‐13 suggesting a suppressive role for IL‐10 in the initiation of the T‐cell response to infection. Using cell type‐specific IL‐10‐deficient mice, we dissected different functions of T‐cell‐ and B‐cell‐derived IL‐10. Litomosoides sigmodontis‐specific IFN‐γ, IL‐5, and IL‐13 production increased in the absence of T‐cell‐derived IL‐10 at early and late time points of infection. In contrast, B‐cell‐specific IL‐10 deficiency did not lead to significant changes in L. sigmodontis‐specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag‐specific cellular responses during L. sigmodontis infection is suppressed by T‐cell‐derived IL‐10 and not by B‐cell‐derived IL‐10.