Loricrin and Cytokeratin Disorganisation in Severe Forms of Periodontitis.

OBJECTIVE: The aim of this research was to investigate the role of the cornified epithelium, the outermost layer of the oral mucosa, engineered to prevent water loss and microorganism invasion, in severe forms of periodontitis (stage III or IV, grade C). METHODS: Porphyromonas gingivalis, a major periodontal disease pathogen, can affect cornified epithelial protein expression through chronic activation of signal transducer and activator of transcription 6 (Stat6). We used a mouse model, Stat6VT, that mimics this to determine the effects of barrier defect on P gingivalis-induced inflammation, bone loss, and cornified epithelial protein expression, and compared histologic and immunohistologic findings with tissues obtained from human controls and patients with stage III and IV, grade C disease. Alveolar bone loss in mice was assessed using micro-computerised tomography, and soft tissue morphology was qualitatively and semi-quantitatively assessed by histologic examination for several proteins, including loricrin, filaggrin, cytokeratin 1, cytokeratin 14, a proliferation marker, a pan-leukocyte marker, as well as morphologic signs of inflammation. Relative cytokine levels were measured in mouse plasma by cytokine array. RESULTS: In the tissues from patients with periodontal disease, there were greater signs of inflammation (rete pegs, clear cells, inflammatory infiltrates) and a decrease and broadening of expression of loricrin and cytokeratin 1. Cytokeratin 14 expression was also broader and decreased in stage IV. P gingivalis-infected Stat6VT mice showed greater alveolar bone loss in 9 out of 16 examined sites, and similar patterns of disruption to human patients in expression of loricrin and cytokeratins 1 and 14. There were also increased numbers of leukocytes, decreased proliferation, and greater signs of inflammation compared with P gingivalis-infected control mice. CONCLUSIONS: Our study provides evidence that changes in epithelial organisation can exacerbate the effects of P gingivalis infection, with similarities to the most severe forms of human periodontitis.

Interleukin-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration in allergic airway inflammation.

Allergic asthma is a chronic lung disease characterized by airway hyperresponsiveness and cellular infiltration that is exacerbated by immunoglobulin E-dependent mast cell (MC) activation. Interleukin-9 (IL-9) promotes MC expansion during allergic inflammation but precisely how IL-9 expands tissue MCs and promotes MC function is unclear. In this report, using multiple models of allergic airway inflammation, we show that both mature MCs (mMCs) and MC progenitors (MCp) express IL-9R and respond to IL-9 during allergic inflammation. IL-9 acts on MCp in the bone marrow and lungs to enhance proliferative capacity. Furthermore, IL-9 in the lung stimulates the mobilization of CCR2+ mMC from the bone marrow and recruitment to the allergic lung. Mixed bone marrow chimeras demonstrate that these are intrinsic effects in the MCp and mMC populations. IL-9-producing T cells are both necessary and sufficient to increase MC numbers in the lung in the context of allergic inflammation. Importantly, T cell IL-9-mediated MC expansion is required for the development of antigen-induced and MC-dependent airway hyperreactivity. Collectively, these data demonstrate that T cell IL-9 induces lung MC expansion and migration by direct effects on the proliferation of MCp and the migration of mMC to mediate airway hyperreactivity.

ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Allergic airway recall responses require IL-9 from resident memory CD4+ T cells.

Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. Yet, the identity of long-term memory cells that mediate allergic recall responses is not well defined. In this report, using a mouse model of chronic allergen exposure followed by an allergen-free rest period, we characterized a subpopulation of CD4+ T cells that secreted IL-9 as an obligate effector cytokine. IL-9-secreting cells had a resident memory T cell phenotype, and blocking IL-9 during a recall challenge or deleting IL-9 from T cells significantly diminished airway inflammation and airway hyperreactivity. T cells secreted IL-9 in an allergen recall-specific manner, and secretion was amplified by IL-33. Using scRNA-seq and scATAC-seq, we defined the cellular identity of a distinct population of T cells with a proallergic cytokine pattern. Thus, in a recall model of allergic airway inflammation, IL-9 secretion from a multicytokine-producing CD4+ T cell population was required for an allergen recall response.

An IL-9-pulmonary macrophage axis defines the allergic lung inflammatory environment.

Despite IL-9 functioning as a pleiotropic cytokine in mucosal environments, the IL-9-responsive cell repertoire is still not well defined. Here, we found that IL-9 mediates proallergic activities in the lungs by targeting lung macrophages. IL-9 inhibits alveolar macrophage expansion and promotes recruitment of monocytes that develop into CD11c+ and CD11c- interstitial macrophage populations. Interstitial macrophages were required for IL-9-dependent allergic responses. Mechanistically, IL-9 affected the function of lung macrophages by inducing Arg1 activity. Compared with Arg1-deficient lung macrophages, Arg1-expressing macrophages expressed greater amounts of CCL5. Adoptive transfer of Arg1+ lung macrophages but not Arg1- lung macrophages promoted allergic inflammation that Il9r-/- mice were protected against. In parallel, the elevated expression of IL-9, IL-9R, Arg1, and CCL5 was correlated with disease in patients with asthma. Thus, our study uncovers an IL-9/macrophage/Arg1 axis as a potential therapeutic target for allergic airway inflammation.

Comparative Analysis of Alternative Splicing Profiles in Th Cell Subsets Reveals Extensive Cell Type-Specific Effects Modulated by a Network of Transcription Factors and RNA-Binding Proteins.

Alternative splicing (AS) plays an important role in the development of many cell types; however, its contribution to Th subsets has been clearly defined. In this study, we compare mice naive CD4+ Th cells with Th1, Th2, Th17, and T regulatory cells and observed that the majority of AS events were retained intron, followed by skipped-exon events, with at least 1200 genes across cell types affected by AS events. A significant fraction of the AS events, especially retained intron events from the 72-h time point, were no longer observed 2 wk postdifferentiation, suggesting a role for AS in early activation and differentiation via preferential expression of specific isoforms required during T cell activation, but not for differentiation or effector function. Examining the protein consequence of the exon-skipping events revealed an abundance of structural proteins encoding for intrinsically unstructured peptide regions, followed by transmembrane helices, ß strands, and polypeptide turn motifs. Analyses of expression profiles of RNA-binding proteins (RBPs) and their cognate binding sites flanking the discovered AS events revealed an enrichment for specific RBP recognition sites in each of the Th subsets. Integration with publicly available chromatin immunoprecipitation sequencing datasets for transcription factors support a model wherein lineage-determining transcription factors impact the RBP profile within the differentiating cells, and this differential expression contributes to AS of the transcriptome via a cascade of cell type-specific posttranscriptional rewiring events.

Selectin Dependence of Allergic Skin Inflammation Is Diminished by Maternal Atopy.

Allergic skin inflammation requires the influx of inflammatory cells into the skin. Extravasation of leukocytes into the skin requires interactions between endothelial selectins and their glycan ligands on the surface of leukocytes. Selectin-ligand formation requires the activity of several glycosyltransferases, including Fut7 In this report, we tested the importance of Fut7 for the development of allergic skin inflammation in the Stat6VT transgenic mouse model. We observed that Fut7 deficiency was protective but did not eliminate disease. Segregation of the data by gender of the parent that transmitted the Stat6VT transgene, but not by gender of the pups, which were analyzed for disease, revealed that the protective effects of Fut7 deficiency were significantly greater when dams were Stat6VT negative. In contrast, in mice from litters of Stat6VT+ dams, Fut7 deficiency resulted in only modest protection. These findings indicate that pups from atopic dams exhibit a greater propensity for allergic disease, similar to observations in humans, and that the effect of maternal atopy is due to enhanced selectin-independent mechanisms of leukocyte recruitment in their offspring. Together, these results demonstrate that Fut7 deficiency can be protective in a model of atopic dermatitis but that maternal atopy diminishes these protective effects, suggesting alternative pathways for leukocyte recruitment in the absence of Fut7 enzyme activity. These observations have implications for understanding how the environment in utero predisposes for the development of allergic disease.

Pancreas transplantation for cystic fibrosis: A frequently missed opportunity.

Cystic fibrosis (CF) is an inherited autosomal recessive disorder. Despite optimized therapy, the majority of affected individuals ultimately die of respiratory failure. As patients with CF are living longer, extra-pulmonary manifestations may develop including pancreatic failure, which manifests as exocrine insufficiency, and CF-related diabetes (CFRD). Both of these can be managed through pancreas transplantation. Pancreas transplantation is usually performed in combination with another organ, most often with a kidney transplant for end-stage diabetic nephropathy. In the CF patient population, the two settings where inclusion of a pancreas transplant should be considered would be in combination with a lung transplant for CF pulmonary disease, or in combination with a liver for CF-related liver disease with cirrhosis. This report will discuss this topic in detail, including a review of the literature regarding combinations of lung/pancreas and liver/pancreas transplant.

Predictive biomarker modeling of pediatric atopic dermatitis severity based on longitudinal serum collection.

The pathogenesis of atopic dermatitis (AD) results from complex interactions between environmental factors, barrier defects, and immune dysregulation resulting in systemic inflammation. Therefore, we sought to characterize circulating inflammatory profiles in pediatric AD patients and identify potential signaling nodes which drive disease heterogeneity and progression. We analyzed a sample set of 87 infants that were at high risk for atopic disease based on atopic dermatitis diagnoses. Clinical parameters, serum, and peripheral blood mononuclear cells (PBMCs) were collected upon entry, and at one and four years later. Within patient serum, 126 unique analytes were measured using a combination of multiplex platforms and ultrasensitive immunoassays. We assessed the correlation of inflammatory analytes with AD severity (SCORAD). Key biomarkers, such as IL-13 (rmcorr=0.47) and TARC/CCL17 (rmcorr=0.37), among other inflammatory signals, significantly correlated with SCORAD across all timepoints in the study. Flow cytometry and pathway analysis of these analytes implies that CD4 T cell involvement in type 2 immune responses were enhanced at the earliest time point (year 1) relative to the end of study collection (year 5). Importantly, forward selection modeling identified 18 analytes in infant serum at study entry which could be used to predict change in SCORAD four years later. We have identified a pediatric AD biomarker signature linked to disease severity which will have predictive value in determining AD persistence in youth and provide utility in defining core systemic inflammatory signals linked to pathogenesis of atopic disease.

STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus.

T helper cell differentiation requires lineage-defining transcription factors and factors that have shared expression among multiple subsets. BATF is required for development of multiple Th subsets but functions in a lineage-specific manner. BATF is required for IL-9 production in Th9 cells but in contrast to its function as a pioneer factor in Th17 cells, BATF is neither sufficient nor required for accessibility at the Il9 locus. Here we show that STAT5 is the earliest factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cultures. The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows acquisition of the IL-9-secreting phenotype. STAT5 and BATF convert Th17 cells into cells that mediate IL-9-dependent effects in allergic airway inflammation and anti-tumor immunity. Thus, BATF requires the STAT5 signal to mediate plasticity at the Il9 locus.

Expression Efficiency of Multiple Il9 Reporter Alleles Is Determined by Cell Lineage.

Generation of allelic gene reporter mice has provided a powerful tool to study gene function in vivo. In conjunction with imaging technologies, reporter mouse models facilitate studies of cell lineage tracing, live cell imaging, and gene expression in the context of diseases. Although there are several advantages to using reporter mice, caution is important to ensure the fidelity of the reporter protein representing the gene of interest. In this study, we compared the efficiency of two Il9 reporter strains Il9citrine and Il9GFP in representing IL-9-producing CD4+ TH9 cells. Although both alleles show high specificity in IL-9-expressing populations, we observed that the Il9GFP allele visualized a much larger proportion of the IL-9-producing cells in culture than the Il9citrine reporter allele. In defining the mechanistic basis for these differences, chromatin immunoprecipitation and chromatin accessibility assay showed that the Il9citrine allele was transcriptionally less active in TH9 cells compared with the wild-type allele. The Il9citrine allele also only captured a fraction of IL-9-expressing bone marrow-derived mast cells. In contrast, the Il9 citrine reporter detected Il9 expression in type 2 innate lymphoid cells at a greater percentage than could be identified by IL-9 intracellular cytokine staining. Taken together, our findings demonstrate that the accuracy of IL-9 reporter mouse models may vary with the cell type being examined. These studies demonstrate the importance of choosing appropriate reporter mouse models that are optimal for detecting the cell type of interest as well as the accuracy of conclusions.

Bcl6 and Blimp1 reciprocally regulate ST2+ Treg-cell development in the context of allergic airway inflammation.

BACKGROUND: Bcl6 is required for the development of T follicular helper cells and T follicular regulatory (Tfr) cells that regulate germinal center responses. Bcl6 also affects the function of regulatory T (Treg) cells. OBJECTIVE: The goal of this study was to define the functions of Bcl6 in Treg cells, including Tfr cells, in the context of allergic airway inflammation. METHODS: We used a model of house dust mite sensitization to challenge wild-type, Bcl6fl/fl Foxp3-Cre, and Prdm1 (Blimp1)fl/fl Foxp3-Cre mice to study the reciprocal roles of Bcl6 and Blimp1 in allergic airway inflammation. RESULTS: In the house dust mite model, Tfr cells repress the production of IgE and Bcl6+ Treg cells suppress the generation of type 2 cytokine-producing cells in the lungs. In mice with Bcl6-deficient Treg cells, twice as many ST2+ (IL-33R+) Treg cells develop as are observed in wild-type mice. ST2+ Treg cells in the context of allergic airway inflammation are Blimp1 dependent, express type 2 cytokines, and share features of visceral adipose tissue Treg cells. Bcl6-deficient Treg cells are more susceptible, and Blimp1-deficient Treg cells are resistant, to acquiring the ST2+ Treg-cell phenotype in vitro and in vivo in response to IL-33. Bcl6-deficient ST2+ Treg cells, but not Bcl6-deficient ST2+ conventional T cells, strongly promote allergic airway inflammation when transferred into recipient mice. Lastly, ST2 is required for the exacerbated allergic airway inflammation in Bcl6fl/fl Foxp3-Cre mice. CONCLUSIONS: During allergic airway inflammation, Bcl6 and Blimp1 play dual roles in regulating Tfr-cell activity in the germinal center and in the development of ST2+ Treg cells that promote type 2 cytokine responses.

BATF-Interacting Proteins Dictate Specificity in Th Subset Activity.

The basic leucine zipper (bZIP) transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells, but not IL-9 or IL-17 in the reciprocal subset. The mechanism for this restricted activity is unclear. In this report, we define BATF binding partners that contribute to Th subset-specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. Using primary mouse T cell culture, we observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest, but significant, increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, cotransduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cells. Taken together, our results reveal that JunB, c-Jun, and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.

A conserved enhancer regulates Il9 expression in multiple lineages.

Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.

Paracrine IL-2 Is Required for Optimal Type 2 Effector Cytokine Production.

IL-2 is a pleiotropic cytokine that promotes the differentiation of Th cell subsets, including Th1, Th2, and Th9 cells, but it impairs the development of Th17 and T follicular helper cells. Although IL-2 is produced by all polarized Th subsets to some level, how it impacts cytokine production when effector T cells are restimulated is unknown. We show in this article that Golgi transport inhibitors (GTIs) blocked IL-9 production. Mechanistically, GTIs blocked secretion of IL-2 that normally feeds back in a paracrine manner to promote STAT5 activation and IL-9 production. IL-2 feedback had no effect on Th1- or Th17-signature cytokine production, but it promoted Th2- and Th9-associated cytokine expression. These data suggest that the use of GTIs results in an underestimation of the presence of type 2 cytokine-secreting cells and highlight IL-2 as a critical component in optimal cytokine production by Th2 and Th9 cells in vitro and in vivo.

STAT3 Activation Impairs the Stability of Th9 Cells.

Th9 cells regulate multiple immune responses, including immunity to pathogens and tumors, allergic inflammation, and autoimmunity. Despite ongoing research into Th9 development and function, little is known about the stability of the Th9 phenotype. In this study, we demonstrate that IL-9 production is progressively lost in Th9 cultures during several rounds of differentiation. The loss of IL-9 is not due to an outgrowth of cells that do not secrete IL-9, as purified IL-9 secretors demonstrate the same loss of IL-9 in subsequent rounds of differentiation. The loss of IL-9 production correlates with increases in phospho-STAT3 levels within the cell, as well as the production of IL-10. STAT3-deficient Th9 cells have increased IL-9 production that is maintained for longer in culture than IL-9 in control cultures. IL-10 is responsible for STAT3 activation during the first round of differentiation, and it contributes to instability in subsequent rounds of culture. Taken together, our results indicate that environmental cues dictate the instability of the Th9 phenotype, and they suggest approaches to enhance Th9 activity in beneficial immune responses.