IRF1 is a transcription factor that participates in interferon signaling. Previous studies of IRF1 binding have utilized in vitro assays. We used ChIP-seq in human monocytes to better define the recognition motif for IRF1. The newly identified 18bp motif (RAAASNGAAAGTGAAASY) is a refinement of the 13bp IRF1 motif commonly used. We utilized the 18bp consensus motif and identified 345 potential target genes. To compare the 18bp motif with the 13bp motif, we compared putative gene targets. Only 56 potential gene targets were defined by both consensus motifs. To compare biological effects of interferon on the 13bp and the 18bp consensus targets, we mined expression data from cells exposed to interferons or transfected with IRF1. In all cases, the 18bp consensus motif was more strongly associated with transcriptional responses than the 13bp motif. Therefore, the new 18bp consensus motif appears to have a greater association with biological activities of IRF1.

Systemic lupus erythematosus is a polygenic disorder affecting approximately 1:1000 adults. Recent data have implicated interferons in the pathogenesis and the expression of many genes downstream of interferons are regulated at the level of histone modifications. We examined H4 acetylation and gene expression in monocytes from patients with systemic lupus erythematosus to define alterations to the epigenome. Monocytes from 14 controls and 24 SLE patients were used for analysis by chromatin immunoprecipitation for H4 acetylation and gene expression arrays. Primary monocytes treated with μ-interferon were used as a comparator. Data were analyzed for concordance of H4 acetylation and gene expression. Network analyses and transcription factor analyses were performed to identify potential pathways. H4 acetylation was significantly altered in monocytes from patients with systemic lupus erythematosus. Sixty three percent of genes with increased H4 acetylation had the potential for regulation by IRF1. IRF1 binding sites were also upstream of nearly all genes with both increased H4 acetylation and gene expression. μ-interferon was a significant contributor to both expression and H4 acetylation patterns but the greatest concordance was seen in the enrichment of certain transcription factor binding sites upstream of genes with increased H4 acetylation in SLE and genes with increased H4 acetylation after μ-interferon treatment.

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.

In systemic lupus erythematosus, TNFα is elevated in the serum and correlates with disease activity and triglyceride levels. The stimuli that drive TNFα in this setting are incompletely understood. This study was designed to evaluate monocyte chromatin at the TNFα locus to identify semi-permanent changes that might play a role in altered expression of TNFα. SLE patients with relatively quiescent disease (mean Physician Global Assessment=0.6) and healthy controls were recruited for this study. TNFα expression was measured by intracellular cytokine staining of different monocyte subsets in patients (n=24) and controls (n=12). Histone acetylation at the TNFα locus was measured by chromatin immunoprecipitation using a normalized quantitative PCR in patients (n=46) and controls (n=24). There were no differences in the overall fractions of cells expressing CD14 in SLE patients compared to controls, however, the fraction of DR+/CD16+ cells expressing CD14 was slightly higher as was true in the monocyte subset defined by DR+/CD11b+. Within the monocyte population defined by physical characteristics and DR+/CD14+, TNFα expressing cells were more frequent in SLE patients compared to controls. Both the fraction of positive cells and the mean fluorescence intensity were higher in patients than controls. Consistent with this was the finding that monocytes from patients had increased TNFα transcripts and more highly acetylated histones at the TNFα locus compared to controls. Furthermore, patients with the highest levels of TNFα histone acetylation were more likely to have had consistently elevated erythrocyte sedimentation rates, and to have required cytotoxic use. Histone acetylation, associated with increased transcriptional competence of TNFα, may play a role in certain inflammatory aspects of the disease.

The cell autonomous response to viral infection is carefully regulated to induce type I interferons (IFNs), which in turn induce the establishment of an antiviral state. Leucine-rich repeat (in Flightless I) interacting protein-1 (LRRFIP1) and LRRFIP2 are 2 related proteins that have been identified as interacting with MyD88 and Flightless I homolog, a leucine-rich repeat protein. LRRFIP2 positively regulates NFκB and macrophage cytokine production after lipopolysaccharide, but less is known about LRRFIP1. We hypothesized that LRRFIP1 could be more important in antiviral responses, as overexpression led to type I IFN production in a pilot study. The induction of type I IFNs occurred even in the absence of virus, but was enhanced by the presence of virus. Conversely, knockdown of LRRFIP1 compromised IFN expression. We found that LRRFIP1 was rapidly recruited to influenza-containing early endosomes in a p38-dependent fashion. This was specific for virus-containing endosomes as there was almost no colocalization of LRRFIP1 with early endosomes in the absence of virus. Further, LRRFIP1 was recruited to RNA-containing vesicles. Taken together, these data suggest that LRRFIP1 participates in cell responses to virus at early time points and is important for type I IFN induction.

Summary

We hypothesized that sirolimus, an mTOR inhibitor, may be effective in patients with autoimmune lymphoproliferative syndrome (ALPS) and treated patients who were intolerant to or failed other therapies. Four patients were treated for autoimmune cytopenias; all had a rapid complete or near complete response. Two patients were treated for autoimmune arthritis and colitis, demonstrating marked improvement. Three patients had complete resolution of lymphadenopathy and splenomegaly and all patients had a reduction in double negative T cells, a population hallmark of the disease. Based on these significant responses, we recommend that sirolimus be considered as second-line therapy for patients with steroid-refractory disease.

Tumor necrosis factor alpha (TNF-α) is an important mediator of inflammation, apoptosis, and the development of secondary lymphoid structures. Multiple polymorphic microsatellites have been identified in and around the gene, and there are also multiple single-base pair biallelic polymorphisms in the introns and promoter. The TNF-α −308 promoter polymorphism is a G-to-A transition which has been statistically associated with various autoimmune disorders. Some studies have found that it may directly mediate the increased transcription of TNF-α in some circumstances. This study characterizes proteins interacting at the polymorphic promoter site. Affinity purification of binding proteins and confirmatory chromatin immunoprecipitation assays were used to identify the proteins. Electrophoretic mobility shift analyses and surface plasmon resonance were used to define binding characteristics. Proteins interacting at this site include GCF2/LRRFIP1 and Ets-1. GCF2/LRRFIP1 appears to act as a repressor and occupies the −308 site in cells that do not make TNF-α. Cells competent to produce TNF-α have Ets-1 bound to the −308 promoter site. Active transcription is accompanied by NF-κB and c-Jun binding to the proximal promoter. Thus, dynamic changes on the TNF-α promoter, particularly at the −308 site, accompany the transition from repressed to active transcription. GCF2/LRRFIP1 is the first TNF-α repressor identified.

Patients with defects in phagocytic function are predisposed to intracellular microorganisms and typically have early dissemination of the infection. Recognition of the underlying disorder and aggressive antimicrobial therapy has been beneficial for the patients. Improved understanding of the pathophysiology has also affected patient management by allowing specific, targeted immunomodulatory intervention. The disorders described in this review are not common but have had a significant impact on our understanding of the role of phagocytic cells in host defense. Conversely, understanding the role of the neutrophil and macrophage in infection has benefited not just the patients described in this review but also other patients with similar disease processes.

The roles of cytokines in the progression of human immunodeficiency virus (HIV)-associated disease are controversial. The patterns of innate cytokine production have been postulated to shift from TH1- to TH2-type cytokines with the progression of HIV-associated disease. Although there have been studies of cytokines in children and adults, no data are available on cytokine production in healthy or HIV-infected adolescents. We analyzed and characterized cytokine mRNA and protein levels for gamma interferon, interleukin 2 (IL-2), IL-4, and tumor necrosis factor alpha and protein levels of IL-6 in both stimulated and unstimulated peripheral blood mononuclear cells obtained from a large longitudinal, observational cohort study of HIV-seropositive and -seronegative adolescents. We correlated cytokine results with viral load and CD4+-T-cell counts as critical markers of disease progression in HIV-infected adolescents. These data were used to examine hypotheses related to the TH1-to-TH2 cytokine shift in a sample of HIV-infected adolescents. Five hundred twenty subjects participating in the REACH (Reaching for Excellence in Adolescent Care and Health) Project of the Adolescent Medicine HIV/AIDS Research Network contributed blood samples. Samples selected for the cross-sectional data set analyzed had to meet selection criteria developed to minimize the potential confounding effects of acute intercurrent illnesses or infections, recent vaccination for hepatitis, and altered hormone status and to optimize congruence of cytokine measurements with assays of viral load and CD4+-T-cell counts. Group differences in the proportions of subjects with detectable levels of each cytokine marker were compared. In the subset of subjects with detectable cytokine values, differences in detected values were compared across subgroups defined by HIV serostatus and among HIV-seropositive subjects by three viral load classifications. The study sample was 65% HIV seropositive, 71% African-American, and 75% female with a mean age of 17.4 years. HIV-seropositive subjects were relatively healthy with mean and median CD4+-T-cell counts of 534 and 499 cells/mm3, respectively. Only 8.1% of subjects had CD4+-T-cell counts below 200 cells/mm3, and 25% had viral loads that were below the threshold of detection (<400 copies/ml). Detailed analyses of these data indicate that there were no differences in cytokines detected in HIV-seropositive and HIV-seronegative adolescents, and there was no apparent relationship between the cytokine measurements and the viral load or CD4+-T-cell categorization, the parameters selected as markers of HIV-associated disease status. These adolescents, including the HIV-seropositive subjects, were relatively healthy, and the HIV-infected subjects were at an early stage in the course of their HIV-associated disease. On the basis of our data, we conclude that, early in the course of HIV-associated disease in adolescents, there are no detectable shifts from TH1 to TH2 cytokine production.

Regulatory T cells are found primarily in the CD4+ CD25+ fraction of T cells and play an important role in the prevention of autoimmunity. We examined CD4+ CD25+ T cells in 33 healthy children and adults and compared them to a population with an inherited form of thymic hypoplasia and a predisposition to autoimmune disease. Absolute numbers of CD4+ CD25+ T cells were markedly higher in healthy infants than in infants with chromosome 22q11.2 deletion syndrome.

Lipopolysaccharide (LPS) is a very potent inducer of tumor necrosis factor alpha (TNF-α) expression from monocytes and macrophages. Another inflammatory cytokine, gamma interferon (IFN-γ), can potentiate the effects of LPS, but the mechanism is not thoroughly understood. Previous reports emphasized the ability of IFN-γ to upregulate CD14 expression (the receptor for LPS), and nearly all studies have utilized sequential stimulation with IFN-γ followed by LPS to exploit this phenomenon. This study demonstrates that IFN-γ can upregulate the effect of LPS at the level of transcription. Human monoblastic Mono-Mac-6 cells produced up to threefold-greater levels of TNF-α when simultaneously stimulated with LPS and IFN-γ compared to treatment with LPS alone. RNase protection studies showed a similar increase in RNA beginning as early as within 30 min. The synthesis of TNF-α mRNA in IFN-γ- and LPS-treated Mono-Mac-6 cells was also temporally prolonged even though the message turnover rate was identical to that seen in LPS stimulated cells. The modulatory effect of IFN-γ may be mediated by Jak2.

Quantitation of cytokine production is a valuable adjunct to standard immunologic assays in defining several pathologic processes. Nevertheless, there is little agreement about which tissues should be assayed, which type of assay should be performed, and which stimulation protocol should be used. As these types of assays enter the clinical arena, there is need for standardization. There is also a need to maximize the amount of information which may be derived from a single sample. We compared secreted interleukin 4 (IL-4), IL-2, IL-6, tumor necrosis factor alpha (TNF-α), and gamma interferon proteins as measured by enzyme-linked immunosorbent assay with intracellular cytokine production (IL-2 and gamma interferon) as detected by flow cytometry and quantitative competitive PCR for IL-2, IL-4, TNF-α, and gamma interferon mRNA and cDNA. Results from unstimulated cells and cells stimulated with phorbol myristate acetate, phytohemagglutinin, and phorbol myristate acetate plus phytohemagglutin were compared. All three methodologies detected significant stimulation of cytokine production. The combination of phytohemagglutinin and phorbol myristate acetate was overall the most-potent stimulus.

Chromosome 22q11.2 deletion syndrome is a common syndrome typically consisting of variable cardiac defects, hypoparathyroidism, developmental delay, and immunodeficiency. The hemizygous deletion has variable effects on the immune system even within the same kindred, and the extent of the immunodeficiency is difficult to predict. Some patients have shown improvement over time; however, this is the first prospective longitudinal study of the dynamic nature of the immunodeficiency. Nineteen patients were studied prospectively between 1994 and 1997. The results of the newborn immunologic studies in the chromosome 22q11.2 deletion group were significantly different from those of a group of newborns with cardiac disease due to other causes. Peripheral blood T-cell numbers were decreased in the chromosome 22q11.2 deletion group, although T-cell function was largely preserved. The group as a whole demonstrated few changes in the first year of life, but a subset of patients with markedly diminished T-cell numbers did demonstrate improvement. Therefore, improvement in peripheral blood T-cell counts is variable in chromosome 22q11.2 deletion syndrome. The patients with the lowest T-cell counts improved the most in the first year of life.

We wished to determine the prevalence of immunoglobulin A (IgA) deficiency in patients with the chromosome 22q11.2 deletion syndrome. A total of 32 patients with the chromosome 22q11.2 deletion were examined for IgA deficiency. We report a 13% (n = 4) prevalence of IgA deficiency in patients with this syndrome. The odds ratio of IgA deficiency in this population is 14.20 (P < 0.0001). This confirms the occurrence of significant humoral deficits in this predominantly cellular immunodeficiency.

The clinical presentations of adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency are widely variable and include clinical and immunologic findings compatible with common variable immunodeficiency. The screening of 44 patients with common variable immunodeficiency failed to identify any individuals with deficiencies of these enzymes.

Allogeneic hematopoietic cell transplantation (HCT) has been employed for 40 years to ameliorate or cure primary immune deficiency (PID) diseases, including severe combined immune deficiency (SCID) and non-SCID PID. There is a critical need for evaluation of the North American experience of different HCT approaches for these diseases, in order to identify best practices and plan future investigative clinical trials. A conference of experts in HCT treatment of PID has recommended: (1) a comprehensive cross-sectional and retrospective analysis of HCT survivors with SCID; (2) a prospective study of SCID patients receiving HCT, with comparable baseline and follow-up testing across participating centers; (3) a pilot study of newborn screening for SCID to identify affected infants prior to compromise by infection; and (4) for the non-SCID diseases, Wiskott-Aldrich syndrome and Chronic Granulomatous Disease, studies of the natural history of disease in patients who do or do not receive HCT. To accomplish these goals, collaboration by a consortium of institutions in North America is proposed. Participation of immunologists and HCT physicians having interest in PID and experts in laboratory methods, clinical outcomes assessment, databases and analysis will be required for the success of these studies.

Background

22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder, affecting an estimated 1 : 2000–4000 live births. Patients with 22q11.2DS have a broad spectrum of phenotypic abnormalities which generally includes congenital cardiac abnormalities, palatal anomalies, and immunodeficiency. Additional findings, such as skeletal anomalies and autoimmune disorders, can confer significant morbidity in a subset of patients. 22q11.2DS is a contiguous gene DS and over 40 genes are deleted in patients; thus deletion of several genes within this region contributes to the clinical features. Mutations outside or on the remaining 22q11.2 allele are also known to modify the phenotype.

Methods

We utilised whole exome, targeted exome and/or Sanger sequencing to examine the genome of 17 patients with 22q11.2 deletions and phenotypic features found in <10% of affected individuals.

Results and conclusions

In four unrelated patients, we identified three novel mutations in SNAP29, the gene implicated in the autosomal recessive condition cerebral dysgenesis, neuropathy, ichthyosis and keratoderma (CEDNIK). SNAP29 maps to 22q11.2 and encodes a soluble SNARE protein that is predicted to mediate vesicle fusion at the endoplasmic reticulum or Golgi membranes. This work confirms that the phenotypic variability observed in a subset of patients with 22q11.2DS is due to mutations on the non-deleted chromosome, which leads to unmasking of autosomal recessive conditions such as CEDNIK, Kousseff, and a potentially autosomal recessive form of Opitz G/BBB syndrome. Furthermore, our work implicates SNAP29 as a major modifier of variable expressivity in 22q11.2 DS patients.