Longsi Ran

Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.

Defining the origins and evolution of the chemokine/chemokine receptor system

The chemokine system has a critical role in mammalian immunity, but the evolutionary history of chemokines and chemokine receptors are ill-defined. We used comparative whole genome analysis of fruit fly, sea urchin, sea squirt, pufferfish, zebrafish, frog, and chicken to identify chemokines and chemokine receptors in each species. We report 127 chemokine and 70 chemokine receptor genes in the 7 species, with zebrafish having the most chemokines, 63, and chemokine receptors, 24. Fruit fly, sea urchin, and sea squirt have no identifiable chemokines or chemokine receptors. This study represents the most comprehensive analysis of the chemokine system to date and the only complete characterization of chemokine systems outside of mouse and human. We establish a clear evolutionary model of the chemokine system and trace the origin of the chemokine system to ∼650 million years ago, identifying critical steps in their evolution and demonstrating a more extensive chemokine system in fish than previously thought.

Gene Expression Analysis of Host Innate Immune Responses during Lethal H5N1 Infection in Ferrets

ABSTRACT How viral and host factors contribute to the severe pathogenicity of the H5N1 subtype of avian influenza virus infection in humans is poorly understood. We identified three clusters of differentially expressed innate immune response genes in lungs from H5N1 (A/Vietnam/1203/04) influenza virus-infected ferrets by oligonucleotide microarray analysis. Interferon response genes were more strongly expressed in H5N1-infected ferret lungs than in lungs from ferrets infected with the less pathogenic H3N2 subtype. In particular, robust CXCL10 gene expression in H5N1-infected ferrets led us to test the pathogenic role of signaling via CXCL10s cognate receptor, CXCR3, during H5N1 influenza virus infection. Treatment of H5N1-infected ferrets with the drug AMG487, a CXCR3 antagonist, resulted in a reduction of symptom severity and delayed mortality compared to vehicle treatment. We contend that unregulated host interferon responses are at least partially responsible for the severity of H5N1 infection and provide evidence that attenuating the CXCR3 signaling pathway improves the clinical course of H5N1 infection in ferrets.

Interferon-Mediated Immunopathological Events Are Associated with Atypical Innate and Adaptive Immune Responses in Patients with Severe Acute Respiratory Syndrome

ABSTRACT It is not understood how immune inflammation influences the pathogenesis of severe acute respiratory syndrome (SARS). One area of strong controversy is the role of interferon (IFN) responses in the natural history of SARS. The fact that the majority of SARS patients recover after relatively moderate illness suggests that the prevailing notion of deficient type I IFN-mediated immunity, with hypercytokinemia driving a poor clinical course, is oversimplified. We used proteomic and genomic technology to systematically analyze host innate and adaptive immune responses of 40 clinically well-described patients with SARS during discrete phases of illness from the onset of symptoms to discharge or a fatal outcome. A novel signature of high IFN-α, IFN-γ, and IFN-stimulated chemokine levels, plus robust antiviral IFN-stimulated gene (ISG) expression, accompanied early SARS sequelae. As acute illness progressed, SARS patients entered a crisis phase linked to oxygen saturation profiles. The majority of SARS patients resolved IFN responses at crisis and expressed adaptive immune genes. In contrast, patients with poor outcomes showed deviated ISG and immunoglobulin gene expression levels, persistent chemokine levels, and deficient anti-SARS spike antibody production. We contend that unregulated IFN responses during acute-phase SARS may culminate in a malfunction of the switch from innate immunity to adaptive immunity. The potential for the use of the gene signatures we describe in this study to better assess the immunopathology and clinical management of severe viral infections, such as SARS and avian influenza (H5N1), is therefore worth careful examination.

Host adaptive immunity deficiency in severe pandemic influenza

IntroductionPandemic A/H1N1/2009 influenza causes severe lower respiratory complications in rare cases. The association between host immune responses and clinical outcome in severe cases is unknown.MethodsWe utilized gene expression, cytokine profiles and generation of antibody responses following hospitalization in 19 critically ill patients with primary pandemic A/H1N1/2009 influenza pneumonia for identifying host immune responses associated with clinical outcome. Ingenuity pathway analysis 8.5 (IPA) (Ingenuity Systems, Redwood City, CA) was used to select, annotate and visualize genes by function and pathway (gene ontology). IPA analysis identified those canonical pathways differentially expressed (P < 0.05) between comparison groups. Hierarchical clustering of those genes differentially expressed between groups by IPA analysis was performed using BRB-Array Tools v.3.8.1.ResultsThe majority of patients were characterized by the presence of comorbidities and the absence of immunosuppressive conditions. pH1N1 specific antibody production was observed around day 9 from disease onset and defined an early period of innate immune response and a late period of adaptive immune response to the virus. The most severe patients (n = 12) showed persistence of viral secretion. Seven of the most severe patients died. During the late phase, the most severe patient group had impaired expression of a number of genes participating in adaptive immune responses when compared to less severe patients. These genes were involved in antigen presentation, B-cell development, T-helper cell differentiation, CD28, granzyme B signaling, apoptosis and protein ubiquitination. Patients with the poorest outcomes were characterized by proinflammatory hypercytokinemia, along with elevated levels of immunosuppressory cytokines (interleukin (IL)-10 and IL-1ra) in serum.ConclusionsOur findings suggest an impaired development of adaptive immunity in the most severe cases of pandemic influenza, leading to an unremitting cycle of viral replication and innate cytokine-chemokine release. Interruption of this deleterious cycle may improve disease outcome.

Gene Expression Profiling of Host Response in Models of Acute HIV Infection

HIV infection is characterized by a host response composed of adaptive and innate immunity that partially limits viral replication; however, it ultimately fails in eradicating the virus. To model host gene expression during acute HIV infection, we infected cynomolgus macaques with the SIV/HIV-1 chimeric virus, SHIV89.6P, and profiled gene expression in peripheral blood over a 5-wk period using a high density cDNA microarray. We demonstrate that viral challenge induced a widespread suppression of genes regulating innate immunity, including the LPS receptors, CD14 and TLR4. An overexpression of 16 IFN-stimulated genes was also observed in response to infection; however, it did not correlate with control over viral titers. A statistical analysis of the dataset identified 10 genes regulating apoptosis with differential expression during the first 2 wk of infection (p < 0.004). Quantitative real-time PCR verified transcriptional increases in IFN-α-inducible genes and decreases in genes regulating innate immunity. Therefore, the persistence of high viral loads despite an extensive IFN response suggests that HIV can resist in vivo IFN treatment despite published reports of in vitro efficacy. The transcriptional suppression of genes regulating innate immunity may allow HIV to evade acute host responses and establish a chronic infection and may reduce innate host defense against opportunistic infections.

Modeling host responses in ferrets during A/California/07/2009 influenza infection.

Immune responses during infection with pandemic H1N1 2009 influenza A virus (2009-H1N1) are still poorly understood. Using an experimental infection model in ferrets, we examined the pathological features and characterized the host immune responses by using microarray analysis, during infection with 2009-H1N1 A/California/07/2009 and seasonal A/Brisbane/59/2007. Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of interferon-stimulated genes were expressed early, correlated to lung pathology, and abruptly decreased expression on day 7 following infection of A/California/07/2009. Interestingly, the drop in innate immune gene expression was replaced by a significant increase of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-influenza antibodies were first observed on day 7, commensurate with the viral clearance. We propose that lung pathology in humans occurs during the innate phase of host immunity and a delay or failure to switch to the adaptive phase may contribute to morbidity and mortality during severe 2009-H1N1 infections.

Regulation of CCR2 chemokine receptor mRNA stability.

During inflammatory and immunological responses, leukocytes respond to external stimuli by altering the stability of cytokine and cytokine receptor messages. Change in message stability is an effective mechanism for rapidly regulating steady state levels of mRNA. Cytokine messages containing A‐U‐rich elements located in the 3′ untranslated region (ARE) are the best studied examples of this process. AREs have been shown to act as targeting motifs for degradation of cytokine and transcription factor messages. We have recently observed that the interleukin‐8 (IL‐8) receptor messages, IL‐8RA and B (CXCR1 and CXCR2), also undergo changes in stability in response to the inflammatory stimulator lipopolysaccharide (LPS). To determine whether regulation of message stability is a common mechanism for modulation of chemokine receptor mRNA we explored whether the stability of the CC chemokine receptor message for CCR2 (monocyte chemotactic protein‐1 receptor) is also regulated by LPS. We found that LPS induces a rapid loss of steady state levels of CCR2 message through message degradation. Furthermore, LPS stimulated the decay of Poly(A) CCR2 mRNA faster than total CCR2 RNA, indicating that deadenylation is the first step in LPS‐induced CCR2 RNA degradation. We conclude from these experiments that LPS stimulates the rapid degradation of CCR2 messages through a two‐step process, deadenylation followed by degradation of the message body. In contrast to the results obtained for CCR2 mRNA, macrophage inflammatory protein‐1α messages, which contain an ARE motif, were stabilized by LPS stimulation, indicating that chemokine and chemokine receptor mRNA stability are regulated by different and opposing mechanisms. J. Leukoc. Biol.62: 653–660; 1997.

Identification of a novel mechanism for endotoxin-mediated down-modulation of CC chemokine receptor expression.

In the present study, we explored the molecular mechanisms by which bacterial endotoxin (LPS) mediates the down‐regulation of CCR2 receptors on human monocytes. We found that LPS induced a marked reduction in CCR2 cell surface protein levels which was blocked by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A. The effector mechanism underlying LPS‐induced CCR2 down‐modulation appears to involve the enzymatic activity of proteinases since Western blot analysis of LPS‐stimulated monocytes revealed the degradation of a 38‐kDa species corresponding to the CCR2B monomer. In RBL cells expressing the CCR2B‐green fluorescent protein (GFP) fusion chemokine receptor, LPS stimulated the internalization and degradation of CCR2. The serine proteinase inhibitor N‐α‐p‐tosyl‐L‐lysine chloromethyl ketone blocked LPS‐induced down‐modulation of CCR2 in monocytes and CCR2B‐GFP in RBL cells. This work describes a previously uncharacterized mechanism for CC chemokine receptor down‐modulation that is dependent upon tyrosine kinase activation and serine proteinase‐mediated receptor degradation and may provide further insight into the mechanisms of leukocyte regulation during immunological and inflammatory responses.

Sequencing, Annotation, and Characterization of the Influenza Ferret Infectome

ABSTRACT Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas.