Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7- and cell contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and it is suppressed by exosome release inhibitors. Further, purified concentrated HCV RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen-sensing within infected cells and as a host strategy to induce an unopposed innate response in replication-nonpermissive by-stander cells.
Innate immune responses are characterized by precise gene expression whereby gene subsets are temporally induced to limit infection, although the mechanisms involved are incompletely understood. We show that antiviral immunity in Drosophila requires the transcriptional pausing pathway, including Negative Elongation Factor (NELF) that pauses RNA Polymerase II (Pol II) and Positive Elongation Factor b (P-TEFb), which releases paused Pol II to produce full length transcripts. We identify a set of genes that is rapidly transcribed upon arbovirus infection, including components of antiviral pathways (RNA silencing, autophagy, JAK/STAT, Toll, and Imd) and various Toll receptors. Many of these genes require P-TEFb for expression and exhibit pausing-associated chromatin features. Furthermore, transcriptional pausing is critical for antiviral immunity in insects, as NELF and P-TEFb are required to restrict viral replication in adult flies and vector mosquito cells. Thus, transcriptional pausing primes virally-induced genes to facilitate rapid gene induction and robust antiviral responses.
From plants to humans, the ability to control infection at the level of an individual cell – a process termed cell-autonomous immunity – equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell’s interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic and inflammasome-related antimicrobial activities within the cytosol as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies (GWAS) and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of Guanylate Binding Proteins (GBPs) with tuberculosis susceptibility and Crohn’s colitis.
Despite numerous attempts over many years to develop an HIV vaccine based on classical strategies, none has convincingly succeeded to date. A number of approaches are being pursued in the field, including building upon possible efficacy indicated by the recent RV144 clinical trial, which combined two HIV vaccines. Here, we argue for an approach based, in part, on understanding the HIV envelope spike and its interaction with broadly neutralizing antibodies (bnAbs) at the molecular level and using this understanding to design immunogens as possible vaccines. BnAbs can protect against virus challenge in animal models and many such antibodies have been isolated recently. We further propose that studies focused on how best to provide T cell help to B cells that produce bnAbs are crucial for optimal immunization strategies. The synthesis of rational immunogen design and immunization strategies, together with iterative improvements, offers great promise for advancing toward an HIV vaccine.
Commensal bacteria are necessary for the development and maintenance of a healthy immune system. Harnessing the ability of microbiota to affect host immunity is considered an important therapeutic strategy for many mucosal and non-mucosal immune-related conditions, such as inflammatory bowel diseases (IBD), celiac disease, metabolic syndrome, diabetes and microbial infections. In addition to well-established immunostimulatory effects of the microbiota, the presence of individual mutualistic commensal bacteria with immunomodulatory effects has been described. These organisms are permanent members of the commensal microbiota and affect host immune homeostasis in specific ways. Identification of individual examples of such immunomodulatory commensals and understanding their mechanisms of interaction with the host will be invaluable in designing therapeutic strategies to reverse intestinal dysbiosis and recover immunological homeostasis.
Innate lymphoid cells (ILCs) are a recently described group of innate immune cells that can regulate immunity, inflammation, and tissue repair in multiple anatomical compartments, particularly the barrier surfaces of the skin, airways, and intestine. Broad categories of ILCs have been defined based on transcription factor expression and the ability to produce distinct patterns of effector molecules. Recent studies have revealed that ILC populations can regulate commensal bacterial communities, contribute to resistance to helminth and bacterial pathogens, promote inflammation, and orchestrate tissue repair and wound healing. This review will examine the phenotype and function of murine and human ILCs and discuss the critical roles these innate immune cells play in health and disease.
Insects rely on innate immune responses controlled by the immune deficiency (IMD), Toll and other immune signaling pathways, to combat infection by a broad spectrum of pathogens. These pathways signal to downstream NF-κB family transcription factors which control specific anti-pathogen action via direct transcriptional control of immune effectors, hematopoiesis and melanization. Here we show that in the Anopheles malaria vector, IMD and Toll pathways mediate species-specific defenses against Plasmodium and bacteria through the transcriptional regulation of splicing factors Caper and IRSF1 that, in turn, determine the production of pathogen-specific splice variant repertoires of the hypervariable pattern recognition receptor AgDscam. This mechanism represents an additional level of immune response regulation that may provide a previously unrecognized level of plasticity to the insect immune pathway-regulated anti-pathogen defenses.
Chronic infections with persistent pathogens such as helminths, mycobacteria, Plasmodium and hepatitis viruses affect more than a third of the human population and are associated with increased susceptibility to other pathogens as well as reduced vaccine efficacy. Although these observations suggest an impact of chronic infections in modulating immunity to unrelated antigens, little is known regarding the underlying mechanisms. Here, we summarize evidence of the most prevalent infections affecting immunity to unrelated pathogens and vaccines, and discuss potential mechanisms of how different bystander chronic infections might impact immune responses. We suggest that bystander chronic infections affect different stages of host responses and may impact transmission of other pathogens, recognition and innate immune responses, priming and differentiation of adaptive effector responses, as well as the development and maintenance of immunological memory. Further understanding of the immunological effects of co-infection should provide opportunities to enhance vaccine efficacy and control infectious diseases.
HIV-1-specific antibodies and CD8+ cytotoxic T cells are detected in most HIV-1-infected people. Yet, HIV-1 infection is not eradicated. Contributing to the failure to mount a sterilizing immune response may be the inability of antigen-presenting dendritic cells (DCs) to sense HIV-1 during acute infection, and thus unable to effectively prime naïve, HIV-1-specific T cells. Recent findings related to DC-expressed innate immune factors, including SAMHD1, TREX1, and TRIM5, provide a molecular basis for understanding why DCs fail to adequately sense invasion by this deadly pathogen and suggest experimental approaches to improve T cell priming to HIV-1 in prophylactic vaccination protocols.
Immunosuppression therapy following organ transplantation is a significant factor in the development and progression of Kaposi’s sarcoma-associated herpesvirus (KSHV)-induced post-transplant Kaposi’s sarcoma (KS). Switching from cyclosporine to the mTOR inhibitor rapamycin is reported to promote KS regression without allograft rejection. Examining the underlying molecular basis for this clinical observation, we find that KSHV infection selectively upregulates mTOR signaling in primary human lymphatic endothelial cells (LEC) but not blood endothelial cells (BEC), and sensitizes LEC to rapamycin-induced apoptosis. Viral transcriptome analysis revealed that while infected BEC display conventional latency, KSHV-infected LEC support a radically different program involving widespread deregulation of both latent and lytic genes. ORF45, a lytic gene selectively expressed in infected LEC, is required for mTOR activation and critical for rapamycin sensitivity. These studies reveal the existence of a unique herpesviral gene expression program corresponding to neither canonical latency nor lytic replication with important pathogenetic and therapeutic consequences.
Type 1 interferon (IFN) inhibits the release of HIV-1 virus particles via poorly defined mechanisms. Here, we show that IFNα induces retention of viral particles on the surface of fibroblasts, T cells, or primary lymphocytes infected with HIV-1 lacking the Vpu protein. Retained particles are tethered to cell surfaces, can be endocytosed, appear fully assembled, exhibit mature morphology, and can be detached by protease. Strikingly, expression of the HIV-1 Vpu protein attenuates the ability of human cells to adhere to, and thereby retain, nascent HIV-1 particles upon IFNα treatment. Vpu also counteracts the IFNα-induced retention of virus-like particles assembled from the Ebola virus matrix protein. Furthermore, levels of IFNα that suppress replication of Vpu-defective HIV-1 have little effect on wild-type HIV-1. Thus, we propose that HIV-1 expresses Vpu to counteract an IFNα-induced, general host defense that inhibits dissemination of enveloped virions from the surface of infected cells.
The life cycle of several viruses involves host or virally encoded small noncoding RNAs, which play important roles in posttranscriptional regulation. Small noncoding RNAs include microRNAs (miRNAs), which modulate the transcriptome, and small interfering RNAs (siRNAs), which are involved in pathogen defense in plants, worms, and insects. We show that insect and mammalian poxviruses induce the degradation of host miRNAs. The virally encoded poly(A) polymerase, which polyadenylates viral transcripts, also mediates 3′ polyadenylation of host miRNAs, resulting in their degradation by the host machinery. In contrast, siRNAs, which are protected by 2′O-methylation (2′OMe), were not targeted by poxviruses. These findings suggest that poxviruses may degrade host miRNAs to promote replication and that virus-mediated small RNA degradation likely contributed to 2′OMe evolution.
HIV-1 assembly and release occurs at the plasma membrane in T lymphocytes, while intracellular sites of virus assembly or accumulation are apparent in macrophages. The host protein tetherin (BST-2) inhibits HIV release from the plasma membrane by retaining viral particles at the cell surface, but the role of tetherin at intracellular HIV assembly sites is unclear. We determined that tetherin is significantly upregulated upon macrophage infection and localizes to an intracellular virus-containing compartment (VCC). Tetherin localized at the virus-VCC membrane interface, suggesting that tetherin physically tethers virions in VCCs. Tetherin knockdown diminished and redistributed VCCs within macrophages and promoted HIV release and cell-cell transmission. The HIV Vpu protein, which downregulates tetherin from the plasma membrane, did not fully overcome tetherin-mediated restriction of particle release in macrophages. Thus, tetherin is essential for VCC formation, and may account for morphologic differences in the apparent HIV assembly sites in macrophages versus T cells.
The interferon-induced host restriction factor tetherin poses a barrier for SIV transmission from primates to humans. Following cross-species transmission, the chimpanzee precursor of pandemic HIV-1 switched from the accessory protein Nef to Vpu to effectively counteract human tetherin. As we report here, the experimental reintroduction of HIV-1 into its original chimpanzee host resulted in a virus that can use both Vpu and Nef to antagonize chimpanzee tetherin. Functional analyses demonstrated that alterations in and near the highly conserved ExxxLL motif in the C-terminal loop of Nef were critical for the reacquisition of anti-tetherin activity. Strikingly, just two amino acid changes allowed HIV-1 Nef to counteract chimpanzee tetherin and promote virus release. Our data demonstrate that primate lentiviruses can reacquire lost accessory gene functions during a single in vivo passage and suggest that other functional constraints keep Nef ready to regain anti-tetherin activity.
Nitric oxide (NO) defends against intracellular pathogens but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with < 2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.
The uptake of apoptotic cells by phagocytes is defined as efferocytosis. In this issue of Cell Host & Microbe, Martin et al. and Yang et al. report that macrophage- and neutrophil-mediated efferocytosis of apoptotic cells containing mycobacteria is an innate antibacterial effector mechanism.
Type I interferons (IFNs) are considered to be the universal mechanism by which viral infections are controlled. However, many IFN-stimulated genes (ISGs) rely on antiviral pathways that are toxic to host cells, which may be detrimental in non-renewable cell types, such as neurons. We show that dorsal root ganglionic (DRG) neurons produce little type I IFNs in response to infection with a neurotropic virus, herpes simplex type 1 (HSV-1). Further, type I IFN treatment fails to completely block HSV-1 replication and to induce IFN-primed cell death in neurons. We find that DRG neurons require autophagy to limit HSV-1 replication both in vivo and in vitro. In contrast, mucosal epithelial cells and other mitotic cells respond robustly to type I IFNs and do not require autophagy to control viral replication. These findings reveal a fundamental difference in the innate antiviral strategies employed by neurons and mitotic cells to control HSV-1 infection.
Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to death mechanisms, leaving the mechanisms underlying this observation unresolved. We find that following apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.
Regulation of intestinal dietary fat absorption is critical to maintaining energy balance. While intestinal microbiota clearly impact the host’s energy balance, their role in intestinal absorption and extra-intestinal metabolism of dietary fat is less clear. Using in vivo imaging of fluorescent fatty acid (FA) analogs delivered to gnotobiotic zebrafish hosts, we reveal that microbiota stimulate FA uptake and lipid droplet (LD) formation in the intestinal epithelium and liver. Microbiota increase epithelial LD number in a diet-dependent manner. The presence of food led to the intestinal enrichment of bacteria from the phylum Firmicutes. Diet-enriched Firmicutes and their products were sufficient to increase epithelial LD number, whereas LD size was increased by other bacterial types. Thus, different members of the intestinal microbiota promote FA absorption via distinct mechanisms. Diet-induced alterations in microbiota composition might influence fat absorption, providing mechanistic insight into how microbiota-diet interactions regulate host energy balance.
Reactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer membrane / periplasmic proteins including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling.
Epstein-Barr Virus (EBV), which is associated with multiple human tumors, persists as a minichromosome in the nucleus of B-lymphocytes and induces malignancies through incompletely understood mechanisms. Here, we present a large-scale functional genomic analysis of EBV. Our experimentally generated nucleosome positioning maps and viral protein binding data were integrated with over 700 publicly available high-throughput sequencing data sets for human lymphoblastoid cell lines mapped to the EBV genome. We found that viral lytic genes are coexpressed with cellular cancer-associated pathways, suggesting that the lytic cycle may play an unexpected role in virus-mediated oncogenesis. Host regulators of viral oncogene expression and chromosome structure were identified and validated, revealing a role for the B-cell-specific protein Pax5 in viral gene regulation and the cohesin complex in regulating higher order chromatin structure. Our findings provide a deeper understanding of latent viral persistence in oncogenesis and establish a valuable viral genomics resource for future exploration.
Historically landmark experiments showed that capsule switching is critical for Streptococcus pneumonia survival. Further studies demonstrated that capsule ‘transformation’ occurs via DNA uptake. In this issue of Cell Host and Microbe, Bikard et al. (2012) show that CRISPR-Cas systems inhibit DNA uptake, selecting for the outgrowth of CRISPR-defective pneumococci.
Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease, resist infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic lifecycle of P. falciparum, a subset of erythrocyte microRNAs translocate into the parasite. Two microRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite mRNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical microRNA activity which may represent a unique host defense strategy against complex eukaryotic pathogens.
Similarities in innate immune signaling exist between mammals and the nematode Caenorhabditis elegans. Now, Ziegler et al. (2009) and Ren et al. (2009) demonstrate that a protein kinase C δ homolog in C. elegans is involved in innate immunity, providing evidence that the conservation of immune signaling networks extends further than previously thought.
Recent work has illuminated three critical aspects of the cell biology of HIV-1 particle genesis. First, we have come to understand which and how cellular membranes are selected as platforms for particle assembly, and consequently rudimentary movies of virion genesis are available. Second, an understanding of how the ESCRT pathway enables virion budding is accruing, and its various roles in the life of cells have become more fully appreciated. Third, it has become apparent that a host inhibitor can block HIV-1 particle release, and that antagonism of this inhibitor underlies the ability of HIV and SIV accessory genes to facilitate particle release. Here, I review recent progress in these three areas.