Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell-Mediated Cytotoxicity.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell-mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation in vivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

Chromodomain helicase DNA-binding 4 (CHD4) regulates early B cell identity and V(D)J recombination.

B lymphocytes develop from uncommitted precursors into immunoglobulin (antibody)-producing B cells, a major arm of adaptive immunity. Progression of early progenitors to antibody-expressing cells in the bone marrow is orchestrated by the temporal regulation of different gene programs at discrete developmental stages. A major question concerns how B cells control the accessibility of these genes to transcription factors. Research has implicated nucleosome remodeling ATPases as mediators of chromatin accessibility. Here, we describe studies of chromodomain helicase DNA-binding 4 (CHD4; also known as Mi-2ß) in early B cell development. CHD4 comprises multiple domains that function in nucleosome mobilization and histone binding. CHD4 is a key component of Nucleosome Remodeling and Deacetylase, or NuRD (Mi-2) complexes, which assemble with other proteins that mediate transcriptional repression. We review data demonstrating that CHD4 is necessary for B lineage identity: early B lineage progression, proliferation in response to interleukin-7, responses to DNA damage, and cell survival in vivo. CHD4-NuRD is also required for the Ig heavy-chain repertoire by promoting utilization of distal variable (VH ) gene segments in V(D)J recombination. In conclusion, the regulation of chromatin accessibility by CHD4 is essential for production of antibodies by B cells, which in turn mediate humoral immune responses to pathogens and disease.

A longitudinal study of within-person trajectories in quality of life in patients receiving early palliative care.

PURPOSE: To reveal the within-person trajectories in quality of life (QOL) in patients receiving early palliative care. Previous studies have mostly focused aggregated trajectories, based on all research participants combined, whereas this study focused on within-person trajectories in QOL and on whether the variability in QOL trajectories across patients is substantial enough to raise doubts about aggregated trajectories. METHODS: Twenty-five older persons in early palliative care completed the McGill Quality of Life Questionnaire multiple times. Reliable change analyses provided estimates of the occurrence of statistically significant within-person change. RESULTS: There was reliable, within-person variation in QOL scores across time, more so for physical than for psychological aspects of well-being. Changes in QOL scores occurred for most patients but the trajectories were not linear and there was no common trend in the nonlinear patterns. CONCLUSIONS: Reliable change across time can be identified in persons receiving early palliative care. However, the trajectories are notably variable and patient-specific. The high degrees of within-person variability across time suggests the importance of repeated QOL assessments and of dynamic tailoring of clinical treatments.

This study describes the quality of life (QOL) trajectories of 25 persons in early palliative care for life-limiting chronic conditions. Previous studies have mostly focused on aggregated trajectories for all research participants combined. In contrast, this study focused on within-person trajectories in QOL and on whether the variability in QOL trajectories across patients is substantial enough to question the meaningfulness of aggregated trajectories. There was both between-patient and within-person variation in QOL scores across time and no common trend. For some patients, the change was for improving scores across time. For other patients the change was for worsening scores across time. And for still other patients, the changes were reliable but not linear. The trajectories are thus quite variable and patient-specific. The variability in QOL trajectories is substantial enough to raise concerns about aggregated trajectories. The high degrees of within-person variability across time suggests the importance of repeated QOL assessments and of dynamic tailoring of clinical treatments.

Single cell analysis of host response to helminth infection reveals the clonal breadth, heterogeneity, and tissue-specific programming of the responding CD4+ T cell repertoire.

The CD4+ T cell response is critical to host protection against helminth infection. How this response varies across different hosts and tissues remains an important gap in our understanding. Using IL-4-reporter mice to identify responding CD4+ T cells to Nippostrongylus brasiliensis infection, T cell receptor sequencing paired with novel clustering algorithms revealed a broadly reactive and clonally diverse CD4+ T cell response. While the most prevalent clones and clonotypes exhibited some tissue selectivity, most were observed to reside in both the lung and lung-draining lymph nodes. Antigen-reactivity of the broader repertoires was predicted to be shared across both tissues and individual mice. Transcriptome, trajectory, and chromatin accessibility analysis of lung and lymph-node repertoires revealed three unique but related populations of responding IL-4+ CD4+ T cells consistent with T follicular helper, T helper 2, and a transitional population sharing similarity with both populations. The shared antigen reactivity of lymph node and lung repertoires combined with the adoption of tissue-specific gene programs allows for the pairing of cellular and humoral responses critical to the orchestration of anti-helminth immunity.

Prognostic accuracy of a peripheral blood transcriptome signature in chronic hypersensitivity pneumonitis.

The prognostic value of peripheral blood mononuclear cell (PBMC) expression profiles, when used in patients with chronic hypersensitivity pneumonitis (CHP), as an adjunct to traditional clinical assessment is unknown. RNA-seq analysis on PBMC from 37 patients with CHP at initial presentation determined that (1) 74 differentially expressed transcripts at a 10% false discovery rate distinguished those with (n=10) and without (n=27) disease progression, defined as absolute FVC and/or diffusing capacity of the lungs for carbon monoxide (DLCO) decline of ≥10% and increased fibrosis on chest CT images within 24 months, and (2) classification models based on gene expression and clinical factors strongly outperform models based solely on clinical factors (baseline FVC%, DLCO% and chest CT fibrosis).

CHD4 is essential for transcriptional repression and lineage progression in B lymphopoiesis.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

Dimensionality, Item Response Theory, Effect Size Attenuation, and Test Bias Analyses of the Self-Importance of Moral Identity Scale (SIMIS).

The extent to which morality and being a moral person are important to one's identity is most commonly assessed using Aquino and Reed's (2002) Self-Importance of Moral Identity Scale (SIMIS). This study provided detailed psychometric examinations of the structure and discrimination levels of the SIMIS in a large (N = 2108) and heterogeneous sample. Results indicated that the SIMIS is clearly 2-dimensional, as expected. The Internalization and Symbolization subscales provided sufficient, and sometimes high levels of test information across the latent trait continuums. There were no redundant items and no bias based on gender. The most notable, albeit minor, shortcomings were that there are too many response options and that test information (discrimination power) was diminished at high levels of the Internalization latent trait continuum, apparently due to skewness. The fluctuating levels of measurement precision resulted in slightly greater attenuations in effect sizes for Internalization than for Symbolization across data for 31 other measures. The present findings from a large dataset and a variety of modern, revealing statistical methods provided relatively consistent, favorable findings for the measure.

Forkhead box protein 3 demethylation is associated with tolerance induction in peanut-induced intestinal allergy.

BACKGROUND: Regulatory T (Treg) cells play an essential role in the maintenance of immune homeostasis in allergic diseases. OBJECTIVES: We sought to define the mechanisms underlying induction of tolerance to peanut protein and prevention of the development of peanut allergy. METHODS: High or low doses of peanut extract were administered to pups every day for 2 weeks before peanut sensitization and challenge. After challenge, symptoms, Treg cell numbers, and forkhead box protein 3 (Foxp3), TH2 and TH17 cytokine, and Tgfß expression in mesenteric lymph node (MLN) CD4+ T cells and jejunum were monitored. Treg cell suppressive activity and Foxp3 methylation in MLN CD4+ T cells were assayed. RESULTS: Feeding high but not low doses of peanut before sensitization induced tolerance, as demonstrated by prevention of diarrhea and peanut-specific IgE responses, increases in the percentage of CD4+CD25+FoxP3+ cells in MLNs, and Foxp3 mRNA and protein expression in CD4+ cells from MLNs or jejunum. Feeding high doses of peanut before sensitization decreased percentages of CD3+CD4+IL-13+ and CD3+CD4+IL-17+ cells in MLNs and decreased Il13 and Il17a and increased Tgfß mRNA expression in the jejunum; numbers of CD103+ dendritic cells in MLNs were significantly increased. Treg cell suppression was shown to be antigen specific. Foxp3 methylation was increased in peanut extract-sensitized and challenged mice, whereas in tolerized mice levels were significantly reduced. CONCLUSIONS: Feeding high doses of peanut to pups induced tolerance to peanut protein. Foxp3 demethylation was associated with tolerance induction, indicating that Treg cells play an important role in the regulation of peanut sensitivity and maintenance of immune homeostasis.

Overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung.

Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year round, but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore, in the current study, we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next, we determined the effect of GDF15 on viral replication, antiviral responses, and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally, we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein overexpression in mice led to exaggerated inflammatory responses to HRV, increased infectious particle release, and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover, GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly, Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation, which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e., HRV) infection in cigarette smoke-exposed airways with GDF15 overproduction.

Regulation of toll-like receptor signaling by the SF3a mRNA splicing complex.

The innate immune response plays a key role in fighting infection by activating inflammation and stimulating the adaptive immune response. However, chronic activation of innate immunity can contribute to the pathogenesis of many diseases with an inflammatory component. Thus, various negatively acting factors turn off innate immunity subsequent to its activation to ensure that inflammation is self-limiting and to prevent inflammatory disease. These negatively acting pathways include the production of inhibitory acting alternate proteins encoded by alternative mRNA splice forms of genes in Toll-like receptor (TLR) signaling pathways. We previously found that the SF3a mRNA splicing complex was required for a robust innate immune response; SF3a acts to promote inflammation in part by inhibiting the production of a negatively acting splice form of the TLR signaling adaptor MyD88. Here we inhibit SF3a1 using RNAi and subsequently perform an RNAseq study to identify the full complement of genes and splicing events regulated by SF3a in murine macrophages. Surprisingly, in macrophages, SF3a has significant preference for mRNA splicing events within innate immune signaling pathways compared with other biological pathways, thereby affecting the splicing of specific genes in the TLR signaling pathway to modulate the innate immune response.

Inducible and naturally occurring regulatory T cells enhance lung allergic responses through divergent transcriptional pathways.

BACKGROUND: Regulatory T cells attenuate development of asthma in wild-type (WT) mice, with both naturally occurring regulatory T (nTreg) cells and inducible regulatory T (iTreg) cells exhibiting suppressive activity. When transferred into CD8-deficient (CD8-/-) recipients, both cell types enhanced development of allergen-induced airway hyperresponsiveness. OBJECTIVE: We sought to determine whether the pathways leading to enhancement of lung allergic responses by transferred nTreg and iTreg cells differed. METHODS: nTreg cells (CD4+CD25+) were isolated from WT mice and iTreg cells were generated from WT CD4+CD25- T cells after activation in the presence of TGF-ß and transferred into sensitized CD8-/- recipients before challenge. Development of airway hyperresponsiveness, cytokine levels, and airway inflammation were monitored. RESULTS: Transfer of nTreg cells enhanced lung allergic responses, as did transfer of iTreg cells. Although anti-IL-13 reduced nTreg cell-mediated enhancement, it was ineffective in iTreg cell-mediated enhancement; conversely, anti-IL-17, but not anti-IL-13, attenuated the enhancement by iTreg cells. Recovered iTreg cells from the lungs of CD8-/- recipients were capable of IL-17 production and expressed high levels of signature genes of the TH17 pathway, RORγt and Il17, whereas reduced expression of the Treg cell key transcription factor forkhead box p3 (Foxp3) was observed. In vitro exogenous IL-6-induced IL-17 production in iTreg cells, and in vivo conversion of transferred iTreg cells was dependent on recipient IL-6. CONCLUSIONS: iTreg cells, similar to nTreg cells, exhibit functional plasticity and can be converted from suppressor cells to pathogenic effector cells, enhancing lung allergic responses, but these effects were mediated through different pathways.

Cell Origin Dictates Programming of Resident versus Recruited Macrophages during Acute Lung Injury.

Two populations of alveolar macrophages (AMs) coexist in the inflamed lung: resident AMs that arise during embryogenesis, and recruited AMs that originate postnatally from circulating monocytes. The objective of this study was to determine whether origin or environment dictates the transcriptional, metabolic, and functional programming of these two ontologically distinct populations over the time course of acute inflammation. RNA sequencing demonstrated marked transcriptional differences between resident and recruited AMs affecting three main areas: proliferation, inflammatory signaling, and metabolism. Functional assays and metabolomic studies confirmed these differences and demonstrated that resident AMs proliferate locally and are governed by increased tricarboxylic acid cycle and amino acid metabolism. Conversely, recruited AMs produce inflammatory cytokines in association with increased glycolytic and arginine metabolism. Collectively, the data show that even though they coexist in the same environment, inflammatory macrophage subsets have distinct immunometabolic programs and perform specialized functions during inflammation that are associated with their cellular origin.

Nurse-led navigation to provide early palliative care in rural areas: a pilot study.

BACKGROUND: Few services are available to support rural older adults living at home with advancing chronic illness. The objective of this project was to pilot a nurse-led navigation service to provide early palliative support for rural older adults and their families living at home with advancing chronic illness. METHODS: Twenty-five older adults and 11 family members living with advancing chronic illness received bi-weekly home visits by a nurse navigator over a 2-year period. Navigation services included symptom management, education, advance care planning, advocacy, mobilization of resources, and psychosocial support. The nurse navigator collected longitudinal data on older adult and family needs, and older adult quality of life and healthcare utilization. RESULTS: Satisfaction with the service was high. There was no attrition over the 2-year period except through death, and few cancelled visits, indicating a high degree of acceptability of the intervention. The navigator addressed complex, multi-faceted needs through connecting health, social, and informal community resources. Participants who indicated a preferred place of death were able to die in that preferred place (n = 7). Emergency room use by participants was minimal and largely unpreventable by the nurse navigator. Longitudinal health-related quality of life scores for many participants were poor, lending further support to the need for more focused attention to this upstream palliative population. CONCLUSIONS: Using a nurse navigator to facilitate early palliative care for rural older adults living with advanced chronic illness is a promising innovation for meeting the needs of this population. Further research is required to evaluate outcomes on a larger scale.

Generalisability theory analyses of concept mapping assessment scores in a problem-based medical curriculum.

CONTEXT: In problem-based learning (PBL), students construct concept maps that integrate different concepts related to the PBL case and are guided by the learning needs generated in small-group tutorials. Although an instrument to measure students' concept maps in PBL programmes has been developed, the psychometric properties of this instrument have not yet been assessed. OBJECTIVES: This study evaluated the generalisability of and sources of variance in medical students' concept map assessment scores in a PBL context. METHODS: Medical students (Year 4, n = 116) were asked to construct three integrated concept maps in which the content domain of each map was to be focused on a PBL clinical case. Concept maps were independently evaluated by four raters based on five criteria: valid selection of concepts; hierarchical arrangement of concepts; degree of integration; relationship to the context of the problem, and degree of student creativity. Generalisability theory was used to compute the reliability of the concept map scores. RESULTS: The dependability coefficient, which indicates the reliability of scores across the measured facets for making absolute decisions, was 0.814. Students' concept map scores (universe scores) accounted for the largest proportion of total variance (47%) across all score comparisons. Rater differences accounted for 10% of total variance, and the student × rater interaction accounted for 25% of total variance. The variance attributable to differences in the content domain of the maps was negligible (2%). The remaining 16% of the variance reflected unexplained sources of error. Results from the D study suggested that a dependability level of 0.80 can be achieved by using three raters who each score two concept map domains, or by using five raters who each score only one concept map domain. CONCLUSIONS: This study demonstrated that concept mapping assessment scores of medical students in PBL have high reliability. Results suggested that greater improvements in dependability might be made by increasing the number of raters rather than by increasing the number of concept map domains.

Steroidogenic enzyme Cyp11a1 regulates Type 2 CD8+ T cell skewing in allergic lung disease.

Allergic asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic airway inflammation and airway hyperresponsiveness. Numbers of CD8(+)IL-13(+) T cells are increased in asthmatics and during the development of experimental asthma in mice. In an atopic environment rich in IL-4, these CD8(+) T cells mediate asthmatic responses, but the mechanisms regulating the conversion of CD8(+) effector T cells from IFN-γ- to pathogenic IL-13-producing effector cells that contribute to an asthma phenotype have not been defined. Here, we show that cholesterol side-chain cleavage P450 enzyme, Cyp11a1, is a key regulator of CD8(+) T-cell conversion. Expression of the gene, protein, and enzymatic activity of Cyp11a1 were markedly increased in CD8(+) T cells differentiated in the presence of IL-2 plus IL-4 compared with cells differentiated in IL-2 alone. Inhibition of Cyp11a1 enzymatic activity with aminoglutethimide or reduction in the expression of Cyp11a1 using short hairpin RNA prevented the IL-4-induced conversion of IFN-γ- to IL-13-producing cells without affecting expression of the lineage-specific transcription factors T-box expressed in T cells (T-bet) or GATA binding protein 3 (GATA3). Adoptive transfer of aminoglutethimide-treated CD8(+) T cells into sensitized and challenged CD8-deficient recipients failed to restore airway hyperresponsiveness and inflammation. We demonstrate that Cyp11a1 controls the phenotypic conversion of CD8(+) T cells from IFN-γ to IL-13 production, linking steroidogenesis in CD8(+) T cells, a nonclassical steroidogenic tissue, to a proallergic differentiation pathway.

Type I IFNs downregulate myeloid cell IFN-γ receptor by inducing recruitment of an early growth response 3/NGFI-A binding protein 1 complex that silences ifngr1 transcription.

The ability of type I IFNs to increase susceptibility to certain bacterial infections correlates with downregulation of myeloid cell surface IFNGR, the receptor for the type II IFN (IFN-γ), and reduced myeloid cell responsiveness to IFN-γ. In this study, we show that the rapid reductions in mouse and human myeloid cell surface IFNGR1 expression that occur in response to type I IFN treatment reflect a rapid silencing of new ifngr1 transcription by repressive transcriptional regulators. Treatment of macrophages with IFN-ß reduced cellular abundance of ifngr1 transcripts as rapidly and effectively as actinomycin D treatment. IFN-ß treatment also significantly reduced the amounts of activated RNA polymerase II (pol II) and acetylated histones H3 and H4 at the ifngr1 promoter and the activity of an IFNGR1-luc reporter construct in macrophages. The suppression of IFNGR1-luc activity required an intact early growth response factor (Egr) binding site in the proximal ifngr1 promoter. Three Egr proteins and two Egr/NGFI-A binding (Nab) proteins were found to be expressed in bone macrophages, but only Egr3 and Nab1 were recruited to the ifngr1 promoter upon IFN-ß stimulation. Knockdown of Nab1 in a macrophage cell line prevented downregulation of IFNGR1 and prevented the loss of acetylated histones from the ifngr1 promoter. These data suggest that type I IFN stimulation induces a rapid recruitment of a repressive Egr3/Nab1 complex that silences transcription from the ifngr1 promoter. This mechanism of gene silencing may contribute to the anti-inflammatory effects of type I IFNs.

Stepwise epigenetic and phenotypic alterations poise CD8+ T cells to mediate airway hyperresponsiveness and inflammation.

The functional plasticity of CD8(+) T cells in an atopic environment, encompassing a spectrum from IFN-γ- to IL-13-producing cells, is pivotal in the development of allergic airway hyperresponsiveness and inflammation, and yet remains mechanistically undefined. We demonstrate that CD8(+) T cell IL-13 induction proceeded through a series of distinct IL-4/GATA3-regulated stages characterized by gene expression and epigenetic changes. In vivo, CD8(+) T cells exposed to an environment rich in IL-4 displayed epigenetic changes at the GATA3 and IL-13 promoter indicative of transcriptional activation and IL-13 production. In vitro, IL-4 triggered the stepwise molecular conversion of CD8(+) T cells from IFN-γ to IL-13 production. During the initial stage, IL-4 suppressed T-bet and induced GATA3 expression, characterized by enhanced activating histone modifications and RNA polymerase II (Pol II) recruitment to the GATA3 locus. Notably, recruitment of GATA3 and RNA Pol II to the IL-13 promoter was also detected at this initial stage. However, enhanced IL-13 transcription only occurred at a later stage after TCR stimulation, indicating that IL-4-induced GATA3 recruitment poises the IL-13 locus for TCR-mediated transcription. Thus, both in vivo and in vitro, an atopic (IL-4) environment poises CD8(+) T cells via stepwise epigenetic and phenotypic mechanisms for pathogenic conversion to IL-13 production, which is ultimately triggered via an allergen-mediated TCR stimulus.

Role of macrophage receptor with collagenous structure in innate immune tolerance.

Macrophages play a key role in host defense against microbes, in part, through phagocytosis. Macrophage receptor with collagenous structure (MARCO) is a scavenger receptor on the cell surface of macrophages that mediates opsonin-independent phagocytosis. The goal of our study is to investigate the role of MARCO in LPS or lipotechoic acid-induced macrophage tolerance. Although it has been established that expression of MARCO and phagocytosis is increased in tolerant macrophages, the transcriptional regulation and biological role of MARCO in tolerant macrophages have not been investigated. In this study, we confirm that tolerized mouse bone marrow-derived macrophages (BMDM) selectively increase expression of MARCO (both transcript and cell surface receptor) and increase phagocytosis. We found that H3K4me3 dynamic modification of a promoter site of MARCO was increased in tolerized BMDM. Blocking methylation by treatment with 5-aza-2'-deoxycytidine resulted in reduced H3K4me3 binding in the promoter of MARCO, decreased expression of MARCO, and impaired phagocytosis in tolerized BMDM. However, 5-aza-2'-deoxycytidine had no effect on the inflammatory component of innate immune tolerance. In aggregate, we found that histone methylation was critical to MARCO expression and phagocytosis in tolerized macrophages, but did not affect the inflammatory component of innate immune tolerance.

Dissecting childhood asthma with nasal transcriptomics distinguishes subphenotypes of disease.

BACKGROUND: Bronchial airway expression profiling has identified inflammatory subphenotypes of asthma, but the invasiveness of this technique has limited its application to childhood asthma. OBJECTIVES: We sought to determine whether the nasal transcriptome can proxy expression changes in the lung airway transcriptome in asthmatic patients. We also sought to determine whether the nasal transcriptome can distinguish subphenotypes of asthma. METHODS: Whole-transcriptome RNA sequencing was performed on nasal airway brushings from 10 control subjects and 10 asthmatic subjects, which were compared with established bronchial and small-airway transcriptomes. Targeted RNA sequencing nasal expression analysis was used to profile 105 genes in 50 asthmatic subjects and 50 control subjects for differential expression and clustering analyses. RESULTS: We found 90.2% overlap in expressed genes and strong correlation in gene expression (ρ = .87) between the nasal and bronchial transcriptomes. Previously observed asthmatic bronchial differential expression was strongly correlated with asthmatic nasal differential expression (ρ = 0.77, P = 5.6 × 10(-9)). Clustering analysis identified TH2-high and TH2-low subjects differentiated by expression of 70 genes, including IL13, IL5, periostin (POSTN), calcium-activated chloride channel regulator 1 (CLCA1), and serpin peptidase inhibitor, clade B (SERPINB2). TH2-high subjects were more likely to have atopy (odds ratio, 10.3; P = 3.5 × 10(-6)), atopic asthma (odds ratio, 32.6; P = 6.9 × 10(-7)), high blood eosinophil counts (odds ratio, 9.1; P = 2.6 × 10(-6)), and rhinitis (odds ratio, 8.3; P = 4.1 × 10(-6)) compared with TH2-low subjects. Nasal IL13 expression levels were 3.9-fold higher in asthmatic participants who experienced an asthma exacerbation in the past year (P = .01). Several differentially expressed nasal genes were specific to asthma and independent of atopic status. CONCLUSION: Nasal airway gene expression profiles largely recapitulate expression profiles in the lung airways. Nasal expression profiling can be used to identify subjects with IL13-driven asthma and a TH2-skewed systemic immune response.

Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.

Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.