A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders.

Allergen-specific type 2 helper T (TH2) cells play a central role in initiating and orchestrating the allergic and asthmatic inflammatory response pathways. One major factor limiting the use of such atopic disease-causing T cells as both therapeutic targets and clinically useful biomarkers is the lack of an accepted methodology to identify and differentiate these cells from overall nonpathogenic TH2 cell types. We have described a subset of human memory TH2 cells confined to atopic individuals that includes all allergen-specific TH2 cells. These cells are terminally differentiated CD4+ T cells (CD27- and CD45RB-) characterized by coexpression of CRTH2, CD49d, and CD161 and exhibit numerous functional attributes distinct from conventional TH2 cells. Hence, we have denoted these cells with this stable allergic disease-related phenotype as the TH2A cell subset. Transcriptome analysis further revealed a distinct pathway in the initiation of pathogenic responses to allergen, and elimination of these cells is indicative of clinical responses induced by immunotherapy. Together, these findings identify a human TH2 cell signature in allergic diseases that could be used for response-monitoring and designing appropriate immunomodulatory strategies.

Acute hyperglycemia impairs IL-6 expression in humans.

Normal glucose metabolism is critical to immune function but the effects of short-term hyperglycemia on immunity are not well described. To study this phenomenon, we induced hyperglycemia in healthy subjects for 2 h with intravenous dextrose and octreotide. An RNA-seq analysis of whole blood RNA demonstrated alterations in multiple immune pathways and transcripts during acute hyperglycemia including decreased transcription of IL-6, an important component of both innate and adaptive immune responses. Additional in vitro studies of human peripheral blood mononuclear cells (PBMCs) exposed to high glucose confirmed decreased IL-6 expression, most prominently in CD14(+)CD16(+) intermediate monocytes. Hyperglycemia also reduced IL-17A expression suggesting further impairment of immune responses during acute hyperglycemia. These findings demonstrate multiple defective immune responses in acute hyperglycemia and suggest a novel role for intermediate monocytes as metabolically sensitive innate immune cells.

Neuroinvasive West Nile Infection Elicits Elevated and Atypically Polarized T Cell Responses That Promote a Pathogenic Outcome.

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.

Comparison of biomarkers for systemic juvenile idiopathic arthritis.

BACKGROUND: Differentiation of systemic juvenile idiopathic arthritis (SJIA) fever from other childhood fevers is often delayed due to the lack of reliable, specific biomarkers. We hypothesized that PD-L1 expression is dysregulated in SJIA monocytes and compared it to other candidate SJIA biomarkers. METHODS: This pilot study enrolled children with fever without source and compared PD-L1 expression on myeloid cells to C-reactive protein, erythrocyte sedimentation rate, leukocyte counts, S100A12, S100A8, S100A9, calprotectin, and procalcitonin. Logistic regression models were fit to test SJIA diagnosis with each marker used as an independent predictor. Receiver operating characteristic curves and area under curve were calculated. Gene expression profiling on a subset of samples was performed. RESULTS: Twenty subjects (10 active SJIA, 10 febrile non-SJIA) were enrolled. S100 proteins were significantly elevated in SJIA with >80% sensitivity and >90% specificity. PD-L1 expression was significantly lower in SJIA. Other markers were not specific for SJIA. On exploratory gene analysis, 106 genes were significant for SJIA association, and several of these are associated with immune response pathways. CONCLUSION: In this small cohort, S100 proteins were specific diagnostic biomarkers for SJIA in children with fever. Decreased PD-L1 surface expression on circulating myeloid cells in SJIA suggests possible mechanism for loss of peripheral immune regulation.

Avidity-dependent programming of autoreactive T cells in T1D.

Fate determination for autoreactive T cells relies on a series of avidity-dependent interactions during T cell selection, represented by two general types of signals, one based on antigen expression and density during T cell development, and one based on genes that interpret the avidity of TCR interaction to guide developmental outcome. We used proinsulin-specific HLA class II tetramers to purify and determine transcriptional signatures for autoreactive T cells under differential selection in type 1 diabetes (T1D), in which insulin (INS) genotypes consist of protective and susceptible alleles that regulate the level of proinsulin expression in the thymus. Upregulation of steroid nuclear receptor family 4A (NR4A) and early growth response family genes in proinsulin-specific T cells was observed in individuals with susceptible INS-VNTR genotypes, suggesting a mechanism for avidity-dependent fate determination of the T cell repertoire in T1D. The NR4A genes act as translators of TCR signal strength that guide central and peripheral T cell fate decisions through transcriptional modification. We propose that maintenance of an NR4A-guided program in low avidity autoreactive T cells in T1D reflects their prior developmental experience influenced by proinsulin expression, identifying a pathway permissive for autoimmunity.

A novel approach to tracking antigen-experienced CD4 T cells into functional compartments via tandem deep and shallow TCR clonotyping.

Extensive diversity in the human repertoire of TCRs for Ag is both a cornerstone of effective adaptive immunity that enables host protection against a multiplicity of pathogens and a weakness that gives rise to potential pathological self-reactivity. The complexity arising from diversity makes detection and tracking of single Ag-specific CD4 T cells (ASTs) involved in these immune responses challenging. We report a tandem, multistep process to quantify rare TCRß-chain variable sequences of ASTs in large polyclonal populations. The approach combines deep high-throughput sequencing (HTS) within functional CD4 T cell compartments, such as naive/memory cells, with shallow, multiple identifier-based HTS of ASTs identified by activation marker upregulation after short-term Ag stimulation in vitro. We find that clonotypes recognizing HLA class II-restricted epitopes of both pathogen-derived Ags and self-Ags are oligoclonal and typically private. Clonotype tracking within an individual reveals private AST clonotypes resident in the memory population, as would be expected, representing clonal expansions (identical nucleotide sequence; "ultraprivate"). Other AST clonotypes share CDR3ß amino acid sequences through convergent recombination and are found in memory populations of multiple individuals. Tandem HTS-based clonotyping will facilitate studying AST dynamics, epitope spreading, and repertoire changes that arise postvaccination and following Ag-specific immunotherapies for cancer and autoimmune disease.

In active relapsing-remitting multiple sclerosis, effector T cell resistance to adaptive T(regs) involves IL-6-mediated signaling.

Patients with multiple sclerosis (MS) manifest demyelination and neurodegeneration mediated in part by CD4(+) T cells that have escaped regulation. Resistance of pathogenic effector T cells (T(effs)) to suppression by regulatory T cells (T(regs)) has been demonstrated in several autoimmune diseases. Although impairment in T(reg) number and function has been observed in relapsing-remitting MS (RRMS), T(eff) resistance has not been well studied in this disease. To determine whether T(eff) resistance contributes to failed tolerance in RRMS, we performed T(reg) suppression assays with T(effs) from either RRMS patients not on immunomodulatory therapy or healthy individuals. T(eff) resistance was present in the T(effs) of RRMS patients with active disease but not from patients with inactive disease. Interleukin-6 (IL-6) and phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) promote T(eff) resistance to T(regs), and we found an increase in IL-6 receptor α (IL-6Rα) expression and elevated IL-6 signaling as measured by pSTAT3 in our RRMS subjects. Further, the impaired suppression in RRMS subjects correlated with an increase in IL-6Rα surface expression on CD4(+) T cells and an increase in pSTAT3 in response to IL-6. To address whether the enhanced pSTAT3 contributed to T(eff) resistance in active RRMS patients, we blocked STAT3 phosphorylation and found that impaired suppression was reversed. Therefore, enhanced IL-6R signaling through pSTAT3, in some cases through increased IL-6Rα expression, contributed to T(eff) resistance in active RRMS. These markers may aid in determining disease activity and responsiveness to immunomodulatory therapies in RRMS.

Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers.

Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.

Molecular signatures associated with Mx1-mediated resistance to highly pathogenic influenza virus infection: mechanisms of survival.

Understanding the role of host factors during lethal influenza virus infection is critical to deciphering the events that determine the fate of the host. One such factor is encoded by the Mx1 gene, which confers resistance to influenza virus infection. Here, we compared pathology and global gene expression profiles in lung tissue from BALB/c (Mx1(-)) and BALB · A2G-Mx1 mice (Mx1(+/+)) infected with the fully reconstructed 1918 pandemic influenza virus. Mx1(+/+) mice showed less tissue damage than Mx(-) animals, and pathology and mortality were further reduced by treating the mice with interferon prior to infection. Using global transcriptional profiling, we identified distinct molecular signatures associated with partial protection, complete protection, and the contribution of interferon to the host response. In the absence of interferon treatment, partial protection was characterized by the generation of an acute response with the upregulation of genes associated with apoptosis, reactive oxygen species, and cell migration. Complete protection was characterized by the downregulation of cytokine and chemokine genes previously associated with influenza virus pathogenesis. The contribution of interferon treatment to total protection in virus-infected Mx1(+/+) mice was characterized by the altered regulation of cell cycle genes. These genes were upregulated in Mx1(+/+) mice treated with interferon but downregulated in the absence of interferon treatment. Our results suggest that Mx1(+/+) mice generate a protective antiviral response by controlling the expression of key modulator molecules associated with influenza virus lethality.

Functional genomics reveals the induction of inflammatory response and metalloproteinase gene expression during lethal Ebola virus infection.

Ebola virus is the etiologic agent of a lethal hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Previous studies with Zaire Ebola virus (ZEBOV), mouse-adapted virus (MA-ZEBOV), and mutant viruses (ZEBOV-NP(ma), ZEBOV-VP24(ma), and ZEBOV-NP/VP24(ma)) allowed us to identify the mutations in viral protein 24 (VP24) and nucleoprotein (NP) responsible for acquisition of high virulence in mice. To elucidate specific molecular signatures associated with lethality, we compared global gene expression profiles in spleen samples from mice infected with these viruses and performed an extensive functional analysis. Our analysis showed that the lethal viruses (MA-ZEBOV and ZEBOV-NP/VP24(ma)) elicited a strong expression of genes 72 h after infection. In addition, we found that although the host transcriptional response to ZEBOV-VP24(ma) was nearly the same as that to ZEBOV-NP/VP24(ma), the contribution of a mutation in the NP gene was required for a lethal phenotype. Further analysis indicated that one of the most relevant biological functions differentially regulated by the lethal viruses was the inflammatory response, as was the induction of specific metalloproteinases, which were present in our newly identify functional network that was associated with Ebola virus lethality. Our results suggest that this dysregulated proinflammatory response increased the severity of disease. Consequently, the newly discovered molecular signature could be used as the starting point for the development of new drugs and therapeutics. To our knowledge, this is the first study that clearly defines unique molecular signatures associated with Ebola virus lethality.

Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection.

Periodic outbreaks of highly pathogenic avian H5N1 influenza viruses and the current H1N1 pandemic highlight the need for a more detailed understanding of influenza virus pathogenesis. To investigate the host transcriptional response induced by pathogenic influenza viruses, we used a functional-genomics approach to compare gene expression profiles in lungs from 129S6/SvEv mice infected with either the fully reconstructed H1N1 1918 pandemic virus (1918) or the highly pathogenic avian H5N1 virus Vietnam/1203/04 (VN/1203). Although the viruses reached similar titers in the lung and caused lethal infections, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus. VN/1203-infected animals also exhibited an earlier and more potent inflammatory response. This response included induction of genes encoding components of the inflammasome. VN/1203 was also able to disseminate to multiple organs, including the brain, which correlated with changes in the expression of genes associated with hematological functions and lipoxin biogenesis and signaling. Both viruses elicited expression of type I interferon (IFN)-regulated genes in wild-type mice and to a lesser extent in mice lacking the type I IFN receptor, suggesting alternative or redundant pathways for IFN signaling. Our findings suggest that VN/1203 is more pathogenic in mice as a consequence of several factors, including the early and sustained induction of the inflammatory response, the additive or synergistic effects of upregulated components of the immune response, and inhibition of lipoxin-mediated anti-inflammatory responses, which correlated with the ability of VN/1203 to disseminate to extrapulmonary organs.

MicroRNA expression and virulence in pandemic influenza virus-infected mice.

The worst known H1N1 influenza pandemic in history resulted in more than 20 million deaths in 1918 and 1919. Although the underlying mechanism causing the extreme virulence of the 1918 influenza virus is still obscure, our previous functional genomics analyses revealed a correlation between the lethality of the reconstructed 1918 influenza virus (r1918) in mice and a unique gene expression pattern associated with severe immune responses in the lungs. Lately, microRNAs have emerged as a class of crucial regulators for gene expression. To determine whether differential expression of cellular microRNAs plays a role in the host response to r1918 infection, we compared the lung cellular "microRNAome" of mice infected by r1918 virus with that of mice infected by a nonlethal seasonal influenza virus, A/Texas/36/91. We found that a group of microRNAs, including miR-200a and miR-223, were differentially expressed in response to influenza virus infection and that r1918 and A/Texas/36/91 infection induced distinct microRNA expression profiles. Moreover, we observed significant enrichment in the number of predicted cellular target mRNAs whose expression was inversely correlated with the expression of these microRNAs. Intriguingly, gene ontology analysis revealed that many of these mRNAs play roles in immune response and cell death pathways, which are known to be associated with the extreme virulence of r1918. This is the first demonstration that cellular gene expression patterns in influenza virus-infected mice may be attributed in part to microRNA regulation and that such regulation may be a contributing factor to the extreme virulence of the r1918.

Knowledge based identification of essential signaling from genome-scale siRNA experiments.

BACKGROUND: A systems biology interpretation of genome-scale RNA interference (RNAi) experiments is complicated by scope, experimental variability and network signaling robustness. Over representation approaches (ORA), such as the Hypergeometric or z-score, are an established statistical framework used to associate RNA interference effectors to biologically annotated gene sets or pathways. These methods, however, do not directly take advantage of our growing understanding of the interactome. Furthermore, these methods can miss partial pathway activation and may be biased by protein complexes. Here we present a novel ORA, protein interaction permutation analysis (PIPA), that takes advantage of canonical pathways and established protein interactions to identify pathways enriched for protein interactions connecting RNAi hits. RESULTS: We use PIPA to analyze genome-scale siRNA cell growth screens performed in HeLa and TOV cell lines. First we show that interacting gene pair siRNA hits are more reproducible than single gene hits. Using protein interactions, PIPA identifies enriched pathways not found using the standard Hypergeometric analysis including the FAK cytoskeletal remodeling pathway. Different branches of the FAK pathway are distinctly essential in HeLa versus TOV cell lines while other portions are uneffected by siRNA perturbations. Enriched hits belong to protein interactions associated with cell cycle regulation, anti-apoptosis, and signal transduction. CONCLUSION: PIPA provides an analytical framework to interpret siRNA screen data by merging biologically annotated gene sets with the human interactome. As a result we identify pathways and signaling hypotheses that are statistically enriched to effect cell growth in human cell lines. This method provides a complementary approach to standard gene set enrichment that utilizes the additional knowledge of specific interactions within biological gene sets.