Kelly Maurer

Immune dysregulation in severe influenza

Among previously healthy children with severe influenza, the mechanisms leading to increased pathology are not understood. We hypothesized that children with severe influenza would have high levels of circulating cytokines. To examine this, we recruited patients with severe influenza and examined plasma cytokine levels as well as the ability of peripheral blood cells to respond to stimuli. Ten patients with severe influenza were enrolled during the 2005–2007 influenza seasons. We evaluated plasma cytokine levels, circulating NK cells, and responses to TLR ligands during the illness. We compared these patients with five patients with moderate influenza, six patients with respiratory syncytial virus (RSV), and 24 noninfected controls. Patients with influenza showed depressed responses to TLR ligands when compared with RSV patients and healthy controls (P<0.05). These normalized when retested during a convalescent phase. Plasma levels of IL‐6, IL‐12, and IFN‐γ were elevated in influenza patients compared with controls (P<0.05). A compromised ability to produce TNF‐α was reproduced by in vitro infection, and the magnitude of the effect correlated with the multiplicity of infection and induction of IFN regulatory factor 4 expression. Aberrant, systemic, innate responses to TLR ligands during influenza infection may be a consequence of specific viral attributes such as a high inoculum or rapid replication and may underlie the known susceptibility of influenza‐infected patients to secondary bacterial infections.

The SLE Transcriptome Exhibits Evidence of Chronic Endotoxin Exposure and Has Widespread Dysregulation of Non-Coding and Coding RNAs

Background Gene expression studies of peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE) have demonstrated a type I interferon signature and increased expression of inflammatory cytokine genes. Studies of patients with Aicardi Goutières syndrome, commonly cited as a single gene model for SLE, have suggested that accumulation of non-coding RNAs may drive some of the pathologic gene expression, however, no RNA sequencing studies of SLE patients have been performed. This study was designed to define altered expression of coding and non-coding RNAs and to detect globally altered RNA processing in SLE. Methods Purified monocytes from eight healthy age/gender matched controls and nine SLE patients (with low-moderate disease activity and lack of biologic drug use or immune suppressive treatment) were studied using RNA-seq. Quantitative RT-PCR was used to validate findings. Serum levels of endotoxin were measured by ELISA. Results We found that SLE patients had diminished expression of most endogenous retroviruses and small nucleolar RNAs, but exhibited increased expression of pri-miRNAs. Splicing patterns and polyadenylation were significantly altered. In addition, SLE monocytes expressed novel transcripts, an effect that was replicated by LPS treatment of control monocytes. We further identified increased circulating endotoxin in SLE patients. Conclusions Monocytes from SLE patients exhibit globally dysregulated gene expression. The transcriptome is not simply altered by the transcriptional activation of a set of genes, but is qualitatively different in SLE. The identification of novel loci, inducible by LPS, suggests that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism.

Cytokine-Induced Monocyte Characteristics in SLE

Monocytes in SLE have been described as having aberrant behavior in a number of assays. We examined gene expression and used a genome-wide approach to study the posttranslational histone mark, H4 acetylation, to examine epigenetic changes in SLE monocytes. We compared SLE monocyte gene expression and H4 acetylation with three types of cytokine-treated monocytes to understand which cytokine effects predominated in SLE monocytes. We found that γ-interferon and α-interferon both replicated a broad range of the gene expression changes seen in SLE monocytes. H4 acetylation in SLE monocytes was overall higher than in controls and there was less correlation of H4ac with cytokine-treated cells than when gene expression was compared. A set of chemokine genes had downregulated expression and H4ac. Therefore, there are significant clusters of aberrantly expressed genes in SLE which are strongly associated with altered H4ac, suggesting that these cells have experienced durable changes to their epigenome.

The Role of MicroRNAs and Human Epidermal Growth Factor Receptor 2 in Proliferative Lupus Nephritis.

To understand the roles of microRNAs (miRNAs) in proliferative lupus nephritis (LN).

Interferon regulatory factor 1 and histone H4 acetylation in systemic lupus erythematosus

Histone acetylation modulates gene expression and has been described as increased in systemic lupus erythematosus (SLE). We investigated interferon regulatory factor 1 (IRF1) interactions that influence H4 acetylation (H4ac) in SLE. Intracellular flow cytometry for H4 acetylated lysine (K) 5, K8, K12, and K16 was performed. Histone acetylation was defined in monocytes and T cells from controls and SLE patients. RNA-Seq studies were performed on monocytes to look for an imbalance in histone acetyltransferases and histone deacetylase enzyme expression. Expression levels were validated using real-time quantitative RT-PCR. IRF1 induction of H4ac was evaluated using D54MG cells overexpressing IRF1. IRF1 protein interactions were studied using co-immunoprecipitation assays. IRF1-dependent recruitment of histone acetyltransferases to target genes was examined by ChIP assays using p300 antibody. Flow cytometry data showed significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation in SLE monocytes. HDAC3 and HDAC11 gene expression were decreased in SLE monocytes. PCAF showed significantly higher gene expression in SLE than controls. IRF1-overexpressing D54MG cells were associated with significantly increased H4K5, H4K8, and H4K12 acetylation compared to vector-control D54MG cells both globally and at specific target genes. Co-immunoprecipitation studies using D54MG cells revealed IRF1 protein-protein interactions with PCAF, P300, CBP, GCN5, ATF2, and HDAC3. ChIP experiments demonstrated increased p300 recruitment to known IRF1 targets in D54MG cells overexpressing IRF1. In contrast, p300 binding to IRF1 targets decreased in D54MG cells with IRF1 knockdown. SLE appears to be associated with an imbalance in histone acetyltransferases and histone deacetylase enzymes favoring pathologic H4 acetylation. Furthermore, IRF1 directly interacts with chromatin modifying enzymes, supporting a model where recruitment to specific target genes is mediated in part by IRF1.

Monocyte polarization: the relationship of genome-wide changes in H4 acetylation with polarization

The character of monocytes is both molded by and contributes to ongoing immune responses. We hypothesized that monocyte polarization could have durable qualities and these would be mediated partly by changes in the chromatin. We defined genome-wide expression and histone H4 acetylation (H4ac) changes after γ-interferon (IFN), α-IFN and interleukin-4 treatment. To identify genes with altered potential for expression, we stimulated polarized monocytes and identified genes up- or downregulated after polarization and stimulation but not either treatment alone. We also defined durability after an 18-h or 3-day washout. Genes uniquely regulated after the combination of polarization and stimulus were durably altered, with 51% of the effects being durable. This gene set was highly enriched for cytokine-induced alterations in H4ac, with P-values ranging from 10−24 to 10−37. Certain regulons defined by patterns of expression were also associated with altered H4ac, with P-values ranging from 10−4 to 10−29. Networking software revealed a high density of mitogen-activated protein (MAP) kinase nodes in these clusters. Therefore, some changes in monocyte gene expression were sustained over a 3-day period. These durably altered gene sets were enriched for changes in H4ac and were associated with potential MAP kinase effects.

B cell development in chromosome 22q11.2 deletion syndrome.

Chromosome 22q11.2 deletion syndrome is a common immune deficiency associated with thymic hypoplasia. Most patients did not survive until the mid-1980s and now there is a growing adult population. B cell and immunoglobulin defects have been described and appear to be increased in the adult population. We used flow cytometry, B cell stimulation and repertoire analysis to understand B cell function. B cell production at early stages appeared to be normal in patients but adult patients exhibited a deficit of switched memory B cells. Follicular helper T cells were present at higher percentages in patients and they exhibited a more activated phenotype in patients compared to controls. In spite of that, somatic hypermutation was decreased in patients compared to controls at all ages. Fewer mutations per clone were seen, strongly implicating aberrant T cell help. Therefore, patients with chromosome 22q11.2 deletion syndrome have a progressive decrease in switched memory B cells and evidence of compromised T cell help. In children, evidence of compromised T cell help is limited to decreased somatic hypermutation. With age, greater manifestations become apparent even though a minority of patients have hypogammaglobulinemia. As this population ages, this has important implications for management.

Endotoxin tolerance in monocytes can be mitigated by α2-interferon

Endotoxin tolerance is characterized by diminished expression of inflammatory cytokines after sequential exposure to Toll‐like receptor stimuli. Many mechanisms contribute to tolerance; however, chromatin remodeling appears to be the most significant regulator. The type II interferon, IFN‐γ, has been recognized as being able to reverse or abrogate the establishment of tolerance. Type I interferons have not been investigated previously, and they bind a distinct receptor. We found that α2‐interferon was able to abrogate or diminish tolerance by endotoxin, as defined by measuring mRNA levels at recognized tolerance targets. We also found that α2‐interferon treatment during tolerization was associated with increased H3K4me3 and H3K4me2 levels at promoters of tolerance targets in THP1 cells. These marks were normalized after exposure of the cells to α2‐interferon. Interferon regulatory factor 1 is a transcription factor activated and induced by types I and II interferons. We found recruitment of this transcription factor paralleled tolerance and inhibition of tolerance at target genes. Therefore, there are at least 2 distinct pathways by which endotoxin tolerance may be mitigated. A type I interferon, in spite of binding to a different receptor, was just as able to inhibit tolerance as the type II interferon and also appeared to act by modifying chromatin at tolerance target genes.

Overall downregulation of mRNAs and enrichment of H3K4me3 change near genome-wide association study signals in systemic lupus erythematosus: Cell-specific effects

This study was designed to define gene expression and H3K4me3 histone modifications in T cells, B cells, and monocytes in systemic lupus erythematosus (SLE). Array studies of total peripheral blood mononuclear cells have demonstrated gene expression signatures related to neutrophils, interferon, and other inflammatory pathways. It is not clear how consistent these effects are across different cell types. In this study, RNA-seq and chromatin immunoprecipitation-seq were utilized to identify gene expression patterns and H3K4me3 histone modifications related to promoter activation in SLE. Across the three cell types, there was 55% concordance for gene expression changes related to SLE. Key conserved pathways were ribosome biogenesis among upregulated genes and heat shock response among downregulated genes. ETS family transcription factors (TFs) and STAT1 were revealed as common regulators by position weight matrices. When epigenetic changes were leveraged with gene expression, the pivotal TFs ATF3 and FOS were defined with ATF3 also cross-referencing with gene expression-identified TFs. Genome-wide association study (GWAS) single nucleotide polymorphisms associated with SLE were cross-referenced with both mRNA and H3K4me3 changes in SLE. Baseline mRNA expression and H3K4me3 peak height was higher at sites that cross-referenced with GWAS signals, however, all three cell types exhibited an overall decrease in expression of GWAS-associated RNAs differentially expressed in SLE. H3K4me3 changes in SLE were also enriched in GWAS-associated sites. In summary, the SLE disease process is associated with both shared and cell-specific changes in gene expression and epigenetics. Surprisingly, GWAS-associated RNAs were overall markedly decreased across all three cell types. TF analysis identified ATF3, FOS, STAT1, and ETS family members as critical, all pathways with a recognized relationship to the SLE disease process. GWAS signals clearly mark both cell-type specific changes in SLE as well as concordant changes across all three cell types. Interpretation of single nucleotide polymorphism effects in SLE will require tissue-specific mechanistic studies and therapeutics will require mechanistic studies in multiple cell types.

SERPINB2 is regulated by dynamic interactions with pause-release proteins and enhancer RNAs

HIGHLIGHTSSERPINB2 is regulated by enhancer RNAs.Enhancer RNAs interact with pause‐release proteins CDK9 and NELF.Manipulation of enhancer RNAs regulates expression of SERPINB2. ABSTRACT The SERPINB2 gene is strongly upregulated in inflammatory states. In monocytes, it can constitute up to 1% of total cellular protein. It functions in protection from proteotoxic stress and plays a role in angioedema. The purpose of this study was to define the roles of enhancer RNAs embedded in the SERPIN gene complex. We found that the upstream enhancer RNAs upregulated SERPINB2 and the enhancer RNAs were expressed prior to those of SERPINB2 mRNA. Studies of the SERPINB2 promoter demonstrated the presence of an RNA polymerase II pause‐inducing protein, NELF. Stimulation with LPS led to recruitment of the pause‐releasing kinase P‐TEFb and departure of the pause‐inducing protein NELF. RNA immunoprecipitation revealed that NELF and the CDK9 component of P‐TEFb bound to the enhancer RNAs after stimulation with distinct kinetics. Knock‐down of the enhancer RNAs compromised stimulus induction of promoter and enhancer chromatin changes. Conversely, over‐expression was associated with enhanced recruitment of c‐JUN and increased expression of SERPINB2 mRNA expression. This study is the first to associate enhancer RNAs with SERPINB2 and is the first demonstration of acquisition of NELF binding by enhancer RNAs on chromatin.