Modeling host genetic regulation of influenza pathogenesis in the collaborative cross.

Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.

The signature of a T-cell response to KSHV persists across space and time in individuals with epidemic and endemic KS from Uganda.

Inadequate T-cell control of Kaposi sarcoma-associated herpesvirus (KSHV) infection predisposes to development of Kaposi sarcoma (KS), but little is known about the T-cell response to KSHV. Postulating that KS tumors contain abundant KSHV-specific T-cells, we performed transcriptional profiling and T-cell receptor (TCR) repertoire analysis of tumor biopsies from 144 Ugandan adults with KS. We show that CD8+ T-cells and M2-polarized macrophages dominate the tumor micro-environment (TME). The TCR repertoire of KS tumor infiltrating lymphocytes (TIL) is shared across non-contiguous tumors and persists across time. Clusters of T-cells with predicted shared specificity for uncharacterized antigens, potentially encoded by KSHV, comprise ~25% of KS TIL, and are shared across tumors from different time points and individuals. Single-cell RNA-sequencing of blood identifies a non-proliferating effector memory phenotype and captured the TCRs in 14,698 putative KSHV-specific T-cells. These results suggest that a polyspecific KSHV-specific T-cell response inhibited by M2 macrophages exists within the KS TME, and provide a foundation for studies to define its specificity at a large scale.

Genomic analysis reveals pre- and postchallenge differences in a rhesus macaque AIDS vaccine trial: insights into mechanisms of vaccine efficacy.

We have employed global transcriptional profiling of whole blood to identify biologically relevant changes in cellular gene expression in response to alternative AIDS vaccine strategies with subsequent viral challenge in a rhesus macaque vaccine model. Samples were taken at day 0 (prechallenge), day 14 (peak viremia), and week 12 (set point) from animals immunized with replicating adenovirus type 5 host range (Ad5hr) recombinant viruses expressing human immunodeficiency virus HIV(env)(89.6P), simian immunodeficiency virus SIV(gag)(239), or SIV(nef)(239) alone or in combination with two intramuscular boosts with HIV(89.6P)gp140ΔCFI protein (L. J. Patterson et al., Virology 374:322-337, 2008), and each treatment resulted in significant control of viremia following simian-human immunodeficiency virus SHIV(89.6P) challenge (six animals per group plus six controls). At day 0, 8 weeks after the last treatment, the microarray profiles revealed significant prechallenge differences between treatment groups; data from the best-protected animals led to identification of a network of genes related to B cell development and lymphocyte survival. At peak viremia, expression profiles of the immunized groups were extremely similar, and comparisons to control animals reflected immunological differences other than effector T cell functions. Suggested protective mechanisms for vaccinated animals included upregulation of interleukin-27, a cytokine known to inhibit lentivirus replication, and increased expression of complement components, which may synergize with vaccine-induced antibodies. Divergent expression profiles at set point for the immunized groups implied distinct immunological responses despite phenotypic similarities in viral load and CD4(+) T cell levels. Data for the gp140-boosted group provided evidence for antibody-dependent, cell-mediated viral control, whereas animals immunized with only the replicating Ad5hr recombinants exhibited a different evolution of the B cell compartment even at 3 months postchallenge. This study demonstrates the sensitivity and discrimination of gene expression profiling of whole blood as an analytical tool in AIDS vaccine trials, providing unique insights into in vivo mechanisms and potential correlates of protection.

Host regulatory network response to infection with highly pathogenic H5N1 avian influenza virus.

During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.

Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus.

The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.

Mavorixafor, an Orally Bioavailable CXCR4 Antagonist, Increases Immune Cell Infiltration and Inflammatory Status of Tumor Microenvironment in Patients with Melanoma.

Purpose: Mavorixafor is an oral, selective inhibitor of the CXCR4 chemokine receptor that modulates immune cell trafficking. A biomarker-driven phase Ib study (NCT02823405) was conducted in 16 patients with melanoma to investigate the hypothesis that mavorixafor favorably modulates immune cell profiles in the tumor microenvironment (TME) and to evaluate the safety of mavorixafor alone and in combination with pembrolizumab. Experimental Design: Serial biopsies of melanoma lesions were assessed after 3 weeks of mavorixafor monotherapy and after 6 weeks of combination treatment for immune cell markers by NanoString analysis for gene expression and by multiplexed immunofluorescent staining for in situ protein expression. Serum samples taken at biopsy timepoints were evaluated for key chemokine and cytokine alterations using the Myriad Rules Based Medicine multiplex immunoassays. Results: Within the TME, mavorixafor alone increased CD8+ T-cell infiltration, granzyme B signal, antigen presentation machinery, and both tumor inflammatory signature (TIS) and IFNγ gene expression signature scores. Increases in the key serum cytokines CXCL9 and CXCL10 were further enhanced when mavorixafor was combined with pembrolizumab. Adverse events (AE), as assessed by the investigator according to NCI Common Terminology Criteria for Adverse Events (v4.03), related to either mavorixafor or pembrolizumab (≥15%) were diarrhea, fatigue, maculopapular rash, and dry eye. Reported AEs were all ≤ grade 3. Conclusion/Discussion: Treatment with single-agent mavorixafor resulted in enhanced immune cell infiltration and activation in the TME, leading to increases in TIS and IFNγ gene signatures. Mavorixafor as a single agent, and in combination with pembrolizumab, has an acceptable safety profile. These data support further investigation of the use of mavorixafor for patients unresponsive to checkpoint inhibitors. Significance: Despite survival improvements in patients with melanoma treated with checkpoint inhibitor therapy, a significant unmet medical need exists for therapies that enhance effectiveness. We propose that mavorixafor sensitizes the melanoma tumor microenvironment and enhances the activity of checkpoint inhibitors, and thereby may translate to a promising treatment for broader patient populations.

Lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes.

The enormous toll on human life during the 1918-1919 Spanish influenza pandemic is a constant reminder of the potential lethality of influenza viruses. With the declaration by the World Health Organization of a new H1N1 influenza virus pandemic, and with continued human cases of highly pathogenic H5N1 avian influenza virus infection, a better understanding of the host response to highly pathogenic influenza viruses is essential. To this end, we compared pathology and global gene expression profiles in bronchial tissue from macaques infected with either the reconstructed 1918 pandemic virus or the highly pathogenic avian H5N1 virus A/Vietnam/1203/04. Severe pathology was observed in respiratory tissues from 1918 virus-infected animals as early as 12 hours after infection, and pathology steadily increased at later time points. Although tissues from animals infected with A/Vietnam/1203/04 also showed clear signs of pathology early on, less pathology was observed at later time points, and there was evidence of tissue repair. Global transcriptional profiles revealed that specific groups of genes associated with inflammation and cell death were up-regulated in bronchial tissues from animals infected with the 1918 virus but down-regulated in animals infected with A/Vietnam/1203/04. Importantly, the 1918 virus up-regulated key components of the inflammasome, NLRP3 and IL-1beta, whereas these genes were down-regulated by A/Vietnam/1203/04 early after infection. TUNEL assays revealed that both viruses elicited an apoptotic response in lungs and bronchi, although the response occurred earlier during 1918 virus infection. Our findings suggest that the severity of disease in 1918 virus-infected macaques is a consequence of the early up-regulation of cell death and inflammatory related genes, in which additive or synergistic effects likely dictate the severity of tissue damage.

Computational identification of hepatitis C virus associated microRNA-mRNA regulatory modules in human livers.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of chronic liver disease by infecting over 170 million people worldwide. Recent studies have shown that microRNAs (miRNAs), a class of small non-coding regulatory RNAs, are involved in the regulation of HCV infection, but their functions have not been systematically studied. We propose an integrative strategy for identifying the miRNA-mRNA regulatory modules that are associated with HCV infection. This strategy combines paired expression profiles of miRNAs and mRNAs and computational target predictions. A miRNA-mRNA regulatory module consists of a set of miRNAs and their targets, in which the miRNAs are predicted to coordinately regulate the level of the target mRNA. RESULTS: We simultaneously profiled the expression of cellular miRNAs and mRNAs across 30 HCV positive or negative human liver biopsy samples using microarray technology. We constructed a miRNA-mRNA regulatory network, and using a graph theoretical approach, identified 38 miRNA-mRNA regulatory modules in the network that were associated with HCV infection. We evaluated the direct miRNA regulation of the mRNA levels of targets in regulatory modules using previously published miRNA transfection data. We analyzed the functional roles of individual modules at the systems level by integrating a large-scale protein interaction network. We found that various biological processes, including some HCV infection related canonical pathways, were regulated at the miRNA level during HCV infection. CONCLUSION: Our regulatory modules provide a framework for future experimental analyses. This report demonstrates the utility of our approach to obtain new insights into post-transcriptional gene regulation at the miRNA level in complex human diseases.

Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors.

Disruption of the TSC1 or TSC2 gene leads to the development of tumors in multiple organs, most commonly affecting the kidney, brain, lung, and heart. Recent genetic and biochemical studies have identified a role for the tuberous sclerosis gene products in phosphoinositide 3-kinase signaling. On growth factor stimulation, tuberin, the TSC2 protein, is phosphorylated by Akt, thereby releasing its inhibitory effects on p70S6K. Here we demonstrate that primary tumors from tuberous sclerosis complex (TSC) patients and the Eker rat model of TSC expressed elevated levels of phosphorylated mammalian target of rapamycin (mTOR) and its effectors: p70S6K, S6 ribosomal protein, 4E-BP1, and eIF4G. In the Eker rat, short-term inhibition of mTOR by rapamycin was associated with a significant tumor response, including induction of apoptosis and reduction in cell proliferation. Surprisingly, these changes were not accompanied by significant alteration in cyclin D1 and p27 levels. Our data provide in vivo evidence that the mTOR pathway is aberrantly activated in TSC renal pathology and that treatment with rapamycin appears effective in the preclinical setting.

Expression quantitative trait Loci for extreme host response to influenza a in pre-collaborative cross mice.

Outbreaks of influenza occur on a yearly basis, causing a wide range of symptoms across the human population. Although evidence exists that the host response to influenza infection is influenced by genetic differences in the host, this has not been studied in a system with genetic diversity mirroring that of the human population. Here we used mice from 44 influenza-infected pre-Collaborative Cross lines determined to have extreme phenotypes with regard to the host response to influenza A virus infection. Global transcriptome profiling identified 2671 transcripts that were significantly differentially expressed between mice that showed a severe ("high") and mild ("low") response to infection. Expression quantitative trait loci mapping was performed on those transcripts that were differentially expressed because of differences in host response phenotype to identify putative regulatory regions potentially controlling their expression. Twenty-one significant expression quantitative trait loci were identified, which allowed direct examination of genes associated with regulation of host response to infection. To perform initial validation of our findings, quantitative polymerase chain reaction was performed in the infected founder strains, and we were able to confirm or partially confirm more than 70% of those tested. In addition, we explored putative causal and reactive (downstream) relationships between the significantly regulated genes and others in the high or low response groups using structural equation modeling. By using systems approaches and a genetically diverse population, we were able to develop a novel framework for identifying the underlying biological subnetworks under host genetic control during influenza virus infection.

Aberrant beta-catenin signaling in tuberous sclerosis.

The pathology associated with tuberous sclerosis complex (TSC) shows diverse phenotypes that suggest abnormal signaling of multiple pathways. Besides the negative regulatory role of the TSC1/TSC2 proteins on mTOR, we have reported an effect on beta-catenin signaling at the level of the degradation complex in vitro. The TSC1/TSC2 complex associates with GSK3 and Axin and promotes beta-catenin degradation to inhibit Wnt-stimulated TCF/LEF-dependent transcription. Here, we show that beta-catenin and its effectors, cyclin D1 and connexin 43, were up-regulated in TSC-related angiomyolipomas and lymphangioleiomyomatosis. This was supported by the failure of three disease-causing TSC2 missense mutants to inhibit Wnt signaling. Further, the interaction between TSC1/TSC2 and components of the beta-catenin degradation complex was dependent on Wnt stimulation such that binding of tuberin to GSK3 and Axin was reduced in the presence of Wnt whereas the tuberin-Dishevelled interaction was increased. GSK3 activity played a role in regulating the assembly/stability of the degradation complex. Inhibition of GSK3 by lithium chloride reduced its association with TSC1 whereas disruption of GSK3-phosphorylation sites in TSC1 reduced interaction between TSC2 and TSC1. Collectively, our data provide further evidence that beta-catenin signaling plays a role in TSC pathogenesis in vivo and suggest a novel role of GSK3 in modulating the TSC1/TSC2 complex through TSC1 phosphorylation.