Group 2 innate lymphoid cells utilize the IRF4-IL-9 module to coordinate epithelial cell maintenance of lung homeostasis.

Group 2 innate lymphoid cells (ILC2s) have an important role in acute allergic lung inflammation. Given their distribution and function, lung ILC2s are hypothesized to coordinate epithelial responses to the external environment; however, how barrier surveillance is linked to ILC2 activation remains unclear. Here, we demonstrate that alveolar type II cells are the main source of interleukin (IL)-33 and thymic stromal lymphopoietin (TSLP) generated in response to chitin or migratory helminths. IL-33 and TSLP synergistically induce an interferon regulatory factor 4 (IRF4)-IL-9 program in ILC2s, and autocrine IL-9 promotes rapid IL-5 and IL-13 production required for optimal epithelial responses in the conducting airways. Thus, ILC2s link alveolar function to regulation of airway flow, revealing a key interaction between resident lymphoid and structural cells that might underlie similar organizational hierarchies in other organs.

A protective role for periostin and TGF-ß in IgE-mediated allergy and airway hyperresponsiveness.

BACKGROUND: The pathophysiology of asthma involves allergic inflammation and remodelling in the airway and airway hyperresponsiveness (AHR) to cholinergic stimuli, but many details of the specific underlying cellular and molecular mechanisms remain unknown. Periostin is a matricellular protein with roles in tissue repair following injury in both the skin and heart. It has recently been shown to be up-regulated in the airway epithelium of asthmatics and to increase active TGF-ß. Though one might expect periostin to play a deleterious role in asthma pathogenesis, to date its biological role in the airway is unknown. OBJECTIVE: To determine the effect of periostin deficiency on airway responses to inhaled allergen. METHODS: In vivo measures of airway responsiveness, inflammation, and remodelling were made in periostin deficient mice and wild-type controls following repeated intranasal challenge with Aspergillus fumigatus antigen. In vitro studies of the effects of epithelial cell-derived periostin on murine T cells were also performed. RESULTS: Surprisingly, compared with wild-type controls, periostin deficient mice developed increased AHR and serum IgE levels following allergen challenge without differences in two outcomes of airway remodelling (mucus metaplasia and peribronchial fibrosis). These changes were associated with decreased expression of TGF-ß1 and Foxp3 in the lungs of periostin deficient mice. Airway epithelial cell-derived periostin-induced conversion of CD4(+) CD25(-) cells into CD25(+) , Foxp3(+) T cells in vitro in a TGF-ß dependent manner. CONCLUSIONS AND CLINICAL RELEVANCE: Allergen-induced increases in serum IgE and bronchial hyperresponsiveness are exaggerated in periostin deficient mice challenged with inhaled aeroallergen. The mechanism of periostin's effect as a brake on allergen-induced responses may involve augmentation of TGF-ß-induced T regulatory cell differentiation.

Control of intestinal Nod2-mediated peptidoglycan recognition by epithelium-associated lymphocytes.

Innate immune recognition of the bacterial cell wall constituent peptidoglycan by the cytosolic nucleotide-binding oligomerization domain 2 (Nod2) receptor has a pivotal role in the maintenance of intestinal mucosal homeostasis. Whereas peptidoglycan cleavage by gut-derived lysozyme preserves the recognition motif, the N-acetylmuramoyl-L-alanine amidase activity of the peptidoglycan recognition protein 2 (PGLYRP-2) destroys the Nod2-detected muramyl dipeptide structure. PGLYRP-2 green fluorescent protein (GFP) reporter and wild-type mice were studied by flow cytometry and quantitative RT-PCR to identify Pglyrp-2 expression in cells of the intestinal mucosa and reveal a potential regulatory function on epithelial peptidoglycan recognition. CD3(+)/CD11c(+) T lymphocytes revealed significant Pglyrp-2 expression, whereas epithelial cells and intestinal myeloid cells were negative. The mucosal Pglyrp-2-expressing lymphocyte population demonstrated a mixed T-cell receptor (TCR) αß or γδ phenotype with predominant CD8α and less so CD8ß expression, as well as significant staining for the activation markers B220 and CD69, presenting a typical intraepithelial lymphocyte phenotype. Importantly, exposure of peptidoglycan to PGLYRP-2 significantly reduced Nod2/Rip2-mediated epithelial activation. Also, moderate but significant alterations of the intestinal microbiota composition were noted in Pglyrp-2-deficient animals. PGLYRP-2 might thus have a significant role in regulation of the enteric host-microbe homeostasis.

Accumulation of eosinophils in intestine-draining mesenteric lymph nodes occurs after Trichuris muris infection.

Eosinophils have recently been demonstrated capable of localizing to lymph nodes that drain mucosal surfaces, in particular during T helper 2 (Th2) responses. Resistance of mice to infection with the gastrointestinal nematode Trichuris muris depends critically on mounting of a Th2 response and represents a useful model system to investigate Th2 responses. Following infection of resistant BALB/c mice with T. muris, we observed accumulation of eosinophils in intestine-draining mesenteric lymph nodes (MLNs). The accumulation of MLN eosinophils was initiated during the second week of infection and peaked during worm expulsion. In contrast, we detected a comparably late and modest increase in eosinophil numbers in the MLNs of infected susceptible AKR mice. MLN eosinophils localized preferentially to the medullary region of the lymph node, displayed an activated phenotype and contributed to the interleukin-4 (IL-4) response in the MLN. Despite this, mice genetically deficient in eosinophils efficiently generated IL-4-expressing CD4(+) T cells, produced Th2 cytokines and mediated worm expulsion during primary T. muris infection. Thus, IL-4-expressing eosinophils accumulate in MLNs of T. muris-infected BALB/c mice but are dispensable for worm expulsion and generation of Th2 responses, suggesting a distinct or subtle role of MLN eosinophils in the immune response to T. muris infection.

Modulation of blood fluke development in the liver by hepatic CD4+ lymphocytes.

We have identified an alternate developmental pathway in the life cycle of the trematode pathogen Schistosoma mansoni. This pathway is used in immunodeficient hosts in which the parasite fails to receive appropriate signals from the host immune system. Helminth development is altered at an early stage during infection, resulting in the appearance of attenuated forms that prolong survival of host and parasite. Hepatic CD4+ T lymphocyte populations are an integral component of the immune signal recognized by the parasite.

Deletion of a coordinate regulator of type 2 cytokine expression in mice.

Mechanisms that underlie the patterning of cytokine expression in T helper (T(H)) cell subsets remain incompletely defined. An evolutionarily conserved approximately 400-bp noncoding sequence in the intergenic region between the genes Il4 and Il13, designated conserved noncoding sequence 1 (CNS-1), was deleted in mice. The capacity to develop T(H)2 cells was compromised in vitro and in vivo in the absence of CNS-1. Despite the profound effect in T cells, mast cells from CNS-1(-/-) mice maintained their capacity to produce interleukin 4. A T cell-specific element critical for the optimal expression of type 2 cytokines may represent the evolution of a regulatory sequence exploited by adaptive immunity.

Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter.

Effector T cells mediate adaptive immunity and immunopathology, but methods for tracking such cells in vivo are limited. We engineered knockin mice expressing IL-4 linked via a viral IRES element with enhanced green fluorescent protein (EGFP). Reporter T cells primed under Th2 conditions showed sensitive and faithful EGFP expression and maintained endogenous IL-4. After Nippostrongylus infection, reporter expression demonstrated the evolution of type 2 immunity from tissue lymphocytes and thence to lymph node CD4(+) T cells, which subsequently migrated into tissue. The appearance of EGFP(+) CD4(+) T cells in tissue, but not in lymph nodes, was Stat6-dependent. Transferred EGFP(+) CD4(+) T cells from infected animals conferred protection against Nippostrongylus to immunodeficient mice. These mice will provide a valuable reagent for assessing immunity in vivo.

Early transcription and silencing of cytokine genes underlie polarization of T helper cell subsets.

Naive CD4+ T cells activated through TCR/CD28 under Th1 or Th2 conditions expressed canonical cytokine patterns irrespective of cell division. Only cells that had divided fewer than four times were capable of reexpressing alternative cytokines when restimulated under opposing conditions. Although T cells transcribed both IFN-gamma and IL-4 within hours in a Stat4-/Stat6-independent manner, neither T-bet nor GATA-3 was induced optimally without Stat signals, and polarized cytokine expression was not sustained. Cytokine genes were positioned apart from heterochromatin in resting T cell nuclei, consistent with rapid expression. After polarization, the majority of silenced cytokine alleles were repositioned to heterochromatin. Naive T cells transit through sequential stages of cytokine activation, commitment, silencing, and physical stabilization during polarization into differentiated effector subsets.

Functional plasticity of the LACK-reactive Vbeta4-Valpha8 CD4(+) T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB / c mice.

Early production of IL-4 by LACK-reactive Vbeta4-Valpha8 CD4(+) T cells instructs aberrant Th2 cell development and susceptibility to Leishmania major in BALB / c mice. This was demonstrated using Vbeta4(+)-deficient BALB / c mice as a result of chronic infection with MMTV (SIM), a mouse mammary tumor virus expressing a Vbeta4-specific superantigen. The early IL-4 response was absent in these mice which develop a Th1 response to L. major. Here, we studied the functional plasticity of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells using BALB/ c mice inoculated with L. major shortly after infection with MMTV (SIM), i. e. before deletion of Vbeta4(+) cells. These mice fail to produce the early IL-4 response to L. major and instead exhibit an IFN-gamma response that occurs within LACK-reactive Vbeta4-Valpha8 CD4(+) T cells. Neutralization of IFN-gamma restores the production of IL-4 by these cells. These data suggest that the functional properties of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells are not irreversibly fixed.

Coordinate regulation of the IL-4, IL-13, and IL-5 cytokine cluster in Th2 clones revealed by allelic expression patterns.

The cytokines IL-4, IL-13, and IL-5 are markers for the Th2 subset of effector T cells and are often expressed together. These cytokine genes are organized within 140 kb of orthologous DNA in both mouse and human. Using IL-4-expressing CD4+ T cell clones derived from F1 mice, we identified allelic polymorphisms for each of these cytokines and assessed the parental identity of the cytokine mRNAs. Both monoallelic and biallelic expression occurred for each gene and for an additional gene, IL-3, that lies with GM-CSF over 450 kb telomeric on the same chromosome. When coexpressed in T cell clones, IL-4 was expressed from the same allele as IL-13 or IL-5 in 81% of instances. In contrast, there was only 52% concordance of these three cytokines at the allelic level among clones that expressed IL-3. Independent expression of the cytokine alleles occurs commonly in T cells, but the clustered locus encompassing IL-4, IL-13, and IL-5 is subject to coordinate regulation.

Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8+ T cells.

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-gamma-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-gamma-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.

Faithful expression of the human 5q31 cytokine cluster in transgenic mice.

Interleukins -4, -5, and -13, cardinal cytokines produced by Th2 cells, are coordinately expressed and clustered in 150-kb syntenic regions on mouse chromosome 11 and human chromosome 5q31. We analyzed two sets of human yeast artificial chromosome transgenic mice that contained the 5q31 cytokines to assess whether conserved sequences required for their coordinate and cell-specific regulation are contained within the cytokine cluster itself. Human IL-4, IL-13, and IL-5 were expressed under Th2, but not Th1, conditions in vitro. Each of these cytokines was produced during infection with Nippostrongylus brasiliensis, a Th2-inducing stimulus, and human IL-4 was generated after activation of NK T cells in vivo. Consistently fewer cells produced the endogenous mouse cytokines in transgenic than in control mice, suggesting competition for stable expression between the mouse and human genes. These data imply the existence of both conserved trans-activating factors and cis-regulatory elements that underlie the coordinate expression and lineage specificity of the type 2 cytokine genes in lymphocytes.

In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease.

In contrast to intact BALB/c mice, BALB/c mice rendered deficient in Vbeta4+ CD4+ T cells develop a Th1 response to infection with Leishmania major and are resistant. Vbeta4-deficient BALB/c mice are unable to generate the early IL-4 transcription occurring in Vbeta4 Valpha8 CD4+ T cells of BALB/c mice within 1 day of infection. Here we demonstrate that treatment of Vbeta4-deficient BALB/c mice with IL-4 during the first 64 h after infection instructs Th2 cell development and susceptibility to infection. The demonstrated inability of IL-4 to reverse the resistant phenotype of BALB/c mice treated with anti-CD4 mAb the day before infection suggest that these effects of IL-4 require its interaction with CD4+ T cells. In contrast to draining lymph node cells from BALB/c mice, cells from Vbeta4-deficient BALB/c mice remain responsive to IL-12 following infection. Strikingly, administration of IL-4 to Vbeta4-deficient BALB/c mice renders their lymph node cells unresponsive to IL-12 by down-regulating IL-12R beta2-chain expression. This study directly demonstrates that in BALB/c mice IL-4 is necessary and sufficient to initiate the molecular events steering Th2 cell maturation and susceptibility to L. major.

Baseline airway hyperreactivity in A/J mice is not mediated by cells of the adaptive immune system.

Human asthma is characterized by increased airway hyperreactivity to a variety of bronchoconstricting agents. Aberrant type 2 immune responses in the lung have been associated with airway hyperreactivity in both human asthma and in murine models of allergic airways disease. Despite their intrinsically elevated basal airway reactivity to smooth muscle constricting agents, A/J mice demonstrated no inherent inflammatory cell infiltration nor elevation of type 2 cytokines in the lung. Crossed bone marrow reconstitution experiments between A/J and MHC congenic B10.A mice revealed enhanced airway reactivity only in A/J recipients, irrespective of whether they had been reconstituted with A/J or B10. A hemopoietic cells. Further, A/J-derived bone marrow cells did not affect the reactivity of B10.A recipients. Although mice on RAG-deficient and IL-4-deficient backgrounds demonstrate substantial abrogation of allergen-induced airway hyperreactivity, these gene deletions had no impact on the elevated baseline reactivity when backcrossed onto A/J mice. Thus, in these mice, basal airway hyperreactivity is maintained independently of type 2 immunity induced by allergens.

Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species sequence comparisons.

Long-range regulatory elements are difficult to discover experimentally; however, they tend to be conserved among mammals, suggesting that cross-species sequence comparisons should identify them. To search for regulatory sequences, we examined about 1 megabase of orthologous human and mouse sequences for conserved noncoding elements with greater than or equal to 70% identity over at least 100 base pairs. Ninety noncoding sequences meeting these criteria were discovered, and the analysis of 15 of these elements found that about 70% were conserved across mammals. Characterization of the largest element in yeast artificial chromosome transgenic mice revealed it to be a coordinate regulator of three genes, interleukin-4, interleukin-13, and interleukin-5, spread over 120 kilobases.

Robust B cell immunity but impaired T cell proliferation in the absence of CD134 (OX40).

CD134 (OX40) is a member of the TNF receptor family that is expressed on activated T lymphocytes. T cells from mice that lack expression of CD134 made strong responses to a range of challenges, but they showed impaired proliferation in response to direct stimulation through the TCR with monoclonal anti-CD3epsilon Ab. CD134-deficient mice controlled infection with Leishmania major, Nippostrongylus brasiliensis, and Theiler's murine encephalomyelitis virus, and they made overtly normal Ab responses to a variety of antigens. Thus, CD134 is not essential for many T cell responses in vivo, nor is it required for the provision of help to B cells. Nonetheless, a subtle role in the regulation of T cell reactivity is suggested by the effect of CD134 deficiency on in vitro T cell responses.

Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4+ T cells.

Naive Itk-deficient CD4+ T cells were unable to establish stable IL-4 production, even when primed in Th2-inducing conditions. In contrast, IFNgamma production was little affected. Failure to express IL-4 occurred even among cells that had gone through multiple cell divisions and was associated with a delay in the kinetics and magnitude of NFATc nuclear localization. IL-4 production was restored genetically by retroviral reconstitution of Itk or biochemically by augmenting the calcium flux with ionomycin. In vivo, Itk-deficient mice were unable to establish functional Th2 cells. Development of protective Th1 cells was unimpeded. These data define a nonredundant role for Itk in modulating signals from the TCR/CD28 pathways that are specific for the establishment of stable IL-4 but not IFNgamma expression.