Catherine Ptaschinski

Epigenetic control of Foxp3 by SMYD3 H3K4 histone methyltransferase controls iTreg development and regulates pathogenic T-cell responses during pulmonary viral infection

The generation of regulatory T (Treg) cells is driven by Foxp3 and is responsible for dampening inflammation and reducing autoimmunity. In this study, the epigenetic regulation of inducible Treg (iTreg) cells was examined and an H3K4 histone methyltransferase, SMYD3 (SET and MYND Domain 3), which regulates the expression of Foxp3 by a TGFβ1/Smad3 (transforming growth factor-β1/Smad3)-dependent mechanism, was identified. Using chromatin immunoprecipitation assays, SMYD3 depletion led to a reduction in H3K4me3 in the promoter region and CNS1 (conserved noncoding DNA sequence) of the foxp3 locus. SMYD3 abrogation affected iTreg cell formation while allowing dysregulated interleukin-17 production. In a mouse model of respiratory syncytial virus (RSV) infection, a model in which iTreg cells have a critical role in regulating lung pathogenesis, SMYD3−/− mice demonstrated exacerbation of RSV-induced disease related to enhanced proinflammatory responses and worsened pathogenesis within the lung. Our data highlight a novel activation role for the TGFβ-inducible SMYD3 in regulating iTreg cell formation leading to increased severity of virus-related disease.

IPS-1 signaling has a nonredundant role in mediating antiviral responses and the clearance of respiratory syncytial virus.

The cytosolic RNA helicases melanoma differentiation–associated gene 5 and retinoic acid–inducible gene-I and their adaptor IFN-β promoter stimulator (IPS-1) have been implicated in the recognition of viral RNA and the production of type I IFN. Complementing the endosomal TLR, melanoma differentiation–associated gene 5, and retinoic acid–inducible gene-I provides alternative mechanisms for viral detection in cells with reduced phagocytosis or autophagy. The infection route of respiratory syncytial virus (RSV)—via fusion of virus particles with the cell membrane—points to IPS-1 signaling as the pathway of choice for downstream antiviral responses. In the current study, viral clearance and inflammation resolution were indeed strongly affected by the absence of an initial IPS-1–mediated IFN-β response. Despite the blunted inflammatory response in IPS-1–deficient alveolar epithelial cells, pulmonary macrophages, and CD11b+ dendritic cells (DC), the lungs of RSV-infected IPS-1–knockout mice showed augmented recruitment of inflammatory neutrophils, monocytes, and DC. Interestingly, pulmonary CD103+ DC could functionally compensate for IPS-1 deficiency with the upregulation of certain inflammatory cytokines and chemokines, possibly via TLR3 and TLR7 signaling. The increased inflammation and reduced viral clearance in IPS-1–knockout mice was accompanied by increased T cell activation and IFN-γ production. Experiments with bone marrow chimeras indicated that RSV-induced lung pathology was most severe when IPS-1 expression was lacking in both immune and nonimmune cell populations. Similarly, viral clearance was rescued upon restored IPS-1 signaling in either the nonimmune or the immune compartment. These data support a nonredundant function for IPS-1 in controlling RSV-induced inflammation and viral replication.

RSV-Induced H3K4 Demethylase KDM5B Leads to Regulation of Dendritic Cell-Derived Innate Cytokines and Exacerbates Pathogenesis In Vivo.

Respiratory syncytial virus (RSV) infection can result in severe disease partially due to its ability to interfere with the initiation of Th1 responses targeting the production of type I interferons (IFN) and promoting a Th2 immune environment. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. RSV-infected bone marrow-derived DCs (BMDCs) upregulated expression of Kdm5b/Jarid1b H3K4 demethylase. Kdm5b-specific siRNA inhibition in BMDC led to a 10-fold increase in IFN-β as well as increases in IL-6 and TNF-α compared to control-transfected cells. The generation of Kdm5b fl/fl-CD11c-Cre+ mice recapitulated the latter results during in vitro DC activation showing innate cytokine modulation. In vivo, infection of Kdm5b fl/fl-CD11c-Cre+ mice with RSV resulted in higher production of IFN-γ and reduced IL-4 and IL-5 compared to littermate controls, with significantly decreased inflammation, IL-13, and mucus production in the lungs. Sensitization with RSV-infected DCs into the airways of naïve mice led to an exacerbated response when mice were challenged with live RSV infection. When Kdm5b was blocked in DCs with siRNA or DCs from Kdm5b fl/fl-CD11c-CRE mice were used, the exacerbated response was abrogated. Importantly, human monocyte-derived DCs treated with a chemical inhibitor for KDM5B resulted in increased innate cytokine levels as well as elicited decreased Th2 cytokines when co-cultured with RSV reactivated CD4+ T cells. These results suggest that KDM5B acts to repress type I IFN and other innate cytokines to promote an altered immune response following RSV infection that contributes to development of chronic disease.

MicroRNA function in mast cell biology: protocols to characterize and modulate microRNA expression.

MicroRNAs (miRNAs) are small noncoding RNA molecules that can modulate mRNA levels through RNA-induced silencing complex (RISC)-mediated degradation. Recognition of target mRNAs occurs through imperfect base pairing between an miRNA and its target, meaning that each miRNA can target a number of different mRNAs to modulate gene expression. miRNAs have been proposed as novel therapeutic targets and many studies are aimed at characterizing miRNA expression patterns and functions within a range of cell types. To date, limited research has focused on the function of miRNAs specifically in mast cells; however, this is an emerging field. In this chapter, we will briefly overview miRNA synthesis and function and the current understanding of miRNAs in hematopoietic development and immune function, emphasizing studies related to mast cell biology. The chapter will conclude with fundamental techniques used in miRNA studies, including RNA isolation, real-time PCR and microarray approaches for quantification of miRNA expression levels, and antagomir design to interfere with miRNA function.

Epigenetic regulation of IL-12-dependent T cell proliferation

It is well established that the cytokine IL‐12 and the transcription factor STAT4, an essential part of the IL‐12 signaling pathway, are critical components of the Th1 differentiation process in T cells. In response to pathogenic stimuli, this process causes T cells to proliferate rapidly and secrete high amounts of the cytokine IFN‐γ, leading to the Th1 proinflammatory phenotype. However, there are still unknown components of this differentiation pathway. We here demonstrated that the expression of the histone methyltransferase Mll1 is driven by IL‐12 signaling through STAT4 in humans and mice and is critical for the proper differentiation of a naϊve T cell to a Th1 cell. Once MLL1 is up‐regulated by IL‐12, it regulates the proliferation of Th1 cells. As evidence of this, we show that Th1 cells from Mll1+/− mice are unable to proliferate rapidly in a Th1 environment in vitro and in vivo. Additionally, upon restimulation with cognate antigen Mll1+/−, T cells do not convert to a Th1 phenotype, as characterized by IFN‐γ output. Furthermore, we observed a reduction in IFN‐γ production and proliferation in human peripheral blood stimulated with tetanus toxoid by use of a specific inhibitor of the MLL1/menin complex. Together, our results demonstrate that the MLL1 gene plays a previously unrecognized but essential role in Th1 cell biology and furthermore, describes a novel pathway through which Mll1 expression is regulated.

Factors Affecting the Immunity to Respiratory Syncytial Virus: From Epigenetics to Microbiome

Respiratory syncytial virus (RSV) is a common pathogen that infects virtually all children by 2 years of age and is the leading cause of hospitalization of infants worldwide. While most children experience mild symptoms, some children progress to severe lower respiratory tract infection. Those children with severe disease have a much higher risk of developing childhood wheezing later in life. Many risk factors are known to result in exacerbated disease, including premature birth and early age of RSV infection, when the immune system is relatively immature. The development of the immune system before and after birth may be altered by several extrinsic and intrinsic factors that could lead to severe disease predisposition in children who do not exhibit any currently known risk factors. Recently, the role of the microbiome and the resulting metabolite profile has been an area of intense study in the development of lung disease, including viral infection and asthma. This review explores both known risk factors that can lead to severe RSV-induced disease as well as emerging topics in the development of immunity to RSV and the long-term consequences of severe infection.

Hox5 Paralogous Genes Modulate Th2 Cell Function during Chronic Allergic Inflammation via Regulation of Gata3

Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. Th2 cells contribute to the development of disease through release of the cytokines IL-4, IL-5, and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. The molecular mechanisms behind the disease pathology remain largely unknown. In this study we investigated a potential regulatory role for the Hox5 gene family, Hoxa5, Hoxb5, and Hoxc5, genes known to be important in lung development within mesenchymal cell populations. We found that Hox5-mutant mice show exacerbated pathology compared with wild-type controls in a chronic allergen model, with an increased Th2 response and exacerbated lung tissue pathology. Bone marrow chimera experiments indicated that the observed enhanced pathology was mediated by immune cell function independent of mesenchymal cell Hox5 family function. Examination of T cells grown in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression, and elevated production of IL-4, IL-5, and IL-13 in Hox5-deficient T cells compared with wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus and decreased Gata3 and IL-4 expression, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of Th2 immune cell function that demonstrates a redeployment of mesenchyme-associated developmental genes.

Hox5 genes direct elastin network formation during alveologenesis by regulating myofibroblast adhesion

Significance Hox5 genes play critical roles in embryonic lung development, but mutants die at birth, preventing investigation of potential postnatal functions for these genes. Surprisingly, we show that the highest expression levels of Hox5 genes occur 1–2 weeks after birth. We created a conditional allele for Hoxa5 that allowed us to generate and study mutants for all three Hox5 genes during postnatal development. Hox5 mutants have poorly developed alveoli and expanded distal airspaces resulting from an abrogated elastin network. These defects arise from the inability of Hox5 mutant fibroblasts to adhere to the fibronectin matrix due to loss of integrins Itga5/b1. Thus, our data highlight redundant roles for all three Hox5 genes in regulating fibroblast adhesion and elastogenesis during alveologenesis. Hox5 genes (Hoxa5, Hoxb5, Hoxc5) are exclusively expressed in the lung mesenchyme during embryogenesis, and the most severe phenotypes result from constitutive loss of function of all three genes. Because Hox5 triple null mutants exhibit perinatal lethality, the contribution of this paralogous group to postembryonic lung development is unknown. Intriguingly, expression of all three Hox5 genes peaks during the first 2 weeks after birth, reaching levels far exceeding those measured at embryonic stages, and surviving Hoxa5 single and Hox5 AabbCc compound mutants exhibit defects in the localization of alveolar myofibroblasts. To define the contribution of the entire Hox5 paralogous group to this process, we generated an Hoxa5 conditional allele to use with our existing null alleles for Hoxb5 and Hoxc5. Postnatally, mesenchymal deletion of Hoxa5 in an Hoxb5/Hoxc5 double-mutant background results in severe alveolar simplification. The elastin network required for alveolar formation is dramatically disrupted in Hox5 triple mutants, while the basal lamina, interstitial matrix, and fibronectin are normal. Alveolar myofibroblasts remain Pdgfrα+/SMA+ double positive and present in normal numbers, indicating that the irregular elastin network is not due to fibroblast differentiation defects. Rather, we observe that SMA+ myofibroblasts of Hox5 triple mutants are morphologically abnormal both in vivo and in vitro with highly reduced adherence to fibronectin. This loss of adhesion is a result of loss of the integrin heterodimer Itga5b1 in mutant fibroblasts. Collectively, these data show an important role for Hox5 genes in lung fibroblast adhesion necessary for proper elastin network formation during alveologenesis.

Chapter 2 – Acute and Chronic Inflammation Induces Disease Pathogenesis

The recognition of pathogenic insults can be accomplished by a number of mechanisms that function to initiate inflammatory responses and mediate clearance of invading pathogens. This initial response, when functioning optimally, will lead to minimal leukocyte accumulation and activation for the clearance of the inciting agent and have little effect on homeostatic function. However, often the inciting agent elicits a very strong inflammatory response, either due to host recognition systems or due to the agents ability to damage host tissue. Thus, the host innate immune system mediates the damage and tissue destruction in an attempt to clear the inciting agent from the system. These initial acute responses can have long-term and even irreversible effects on tissue function. If the initial responses are not sufficient to facilitate the clearance of the foreign pathogen or material, the response shifts toward a more complex and efficient process mediated by lymphocyte populations that respond to specific residues displayed by the foreign material. Normally, these responses are coordinated and only minimally alter physiologic function of the tissue. However, in unregulated responses the initial reaction can become acutely catastrophic, leading to local or even systemic damage to the tissue or organs, resulting in degradation of normal physiologic function. Alternatively, the failure to regulate the response or clear the inciting agent could lead to chronic and progressively more pathogenic responses. Each of these potentially devastating responses has specific and often overlapping mechanisms that have been identified and lead to the damage within tissue spaces. A series of events take place during both acute and chronic inflammation that lead to the accumulation of leukocytes and damage to the local environment.