Tiffany A. Reese

Chitin induces accumulation in tissue of innate immune cells associated with allergy

Allergic and parasitic worm immunity is characterized by infiltration of tissues with interleukin (IL)-4- and IL-13-expressing cells, including T-helper-2 cells, eosinophils and basophils. Tissue macrophages assume a distinct phenotype, designated alternatively activated macrophages. Relatively little is known about the factors that trigger these host responses. Chitin, a widespread environmental biopolymer of N-acetyl-β-d-glucosamine, provides structural rigidity to fungi, crustaceans, helminths and insects. Here, we show that chitin induces the accumulation in tissue of IL-4-expressing innate immune cells, including eosinophils and basophils, when given to mice. Tissue infiltration was unaffected by the absence of Toll-like-receptor-mediated lipopolysaccharide recognition but did not occur if the injected chitin was pre-treated with the IL-4- and IL-13-inducible mammalian chitinase, AMCase, or if the chitin was injected into mice that overexpressed AMCase. Chitin mediated alternative macrophage activation in vivo and the production of leukotriene B4, which was required for optimal immune cell recruitment. Chitin is a recognition element for tissue infiltration by innate cells implicated in allergic and helminth immunity and this process can be negatively regulated by a vertebrate chitinase.

Type 2 immunity is controlled by IL-4/IL-13 expression in hematopoietic non-eosinophil cells of the innate immune system

Nippostrongylus brasiliensis infection and ovalbumin-induced allergic lung pathology are highly interleukin (IL)-4/IL-13 dependent, but the contributions of IL-4/IL-13 from adaptive (T helper [Th]2 cells) and innate (eosinophil, basophils, and mast cells) immune cells remain unknown. Although required for immunoglobulin (Ig)E induction, IL-4/IL-13 from Th2 cells was not required for worm expulsion, tissue inflammation, or airway hyperreactivity. In contrast, innate hematopoietic cell–derived IL-4/IL-13 was dispensable for Th2 cell differentiation in lymph nodes but required for effector cell recruitment and tissue responses. Eosinophils were not required for primary immune responses. Thus, components of type 2 immunity mediated by IL-4/IL-13 are partitioned between T cell–dependent IgE and an innate non-eosinophil tissue component, suggesting new strategies for interventions in allergic immunity.

Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter

Parasites make it hard to fight viruses Microbial co-infections challenge the immune system—different pathogens often require different flavors of immune responses for their elimination or containment (see the Perspective by Maizels and Gause). Two teams studied what happens when parasitic worms and viruses infect mice at the same time. Reese et al. found that parasite co-infection woke up a dormant virus. Osborne et al. found that mice already infected with parasitic worms were worse at fighting off viruses. In both cases, worms skewed the immune response so that the immune cells and the molecules they secreted created an environment favorable for the worm at the expense of antiviral immunity. Science, this issue p. 573 and p. 578; see also p. 517 Coinfection with intestinal parasites leads to altered antiviral immunity in mice. Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi’s sarcoma–associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a “two-signal” model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Identification of novel microRNA-like molecules generated from herpesvirus and host tRNA transcripts.

ABSTRACT We applied deep sequencing technology to small RNA fractions from cells lytically infected with murine gammaherpesvirus 68 (γHV68) in order to define in detail small RNAs generated from a cluster of tRNA-related polycistronic structures located at the left end of the viral genome. We detected 10 new candidate microRNAs (miRNAs), six of which were confirmed by Northern blot analysis, leaving four as provisional. In addition, we determined that previously identified and annotated viral miRNA molecules were not necessarily represented as the most abundant sequence originating from a transcript. Based on these new small RNAs and previously reported γHV68 miRNAs, we were able to further describe and annotate the distinctive γHV68 tRNA-miRNA structures. We used this deep sequencing data and computational analysis to identify similar structures in the mouse genome and validated that these host structures also give rise to small RNAs. This reveals a possible convergent usage of tRNA/polymerase III (pol III) transcripts to generate small RNAs from both mammalian and viral genomes.

Sequential Infection with Common Pathogens Promotes Human-like Immune Gene Expression and Altered Vaccine Response

Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth and compared their blood immune signatures to mock-infected mice before and after vaccination against yellow fever virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore, basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans.

Differential enzymatic activity of common haplotypic versions of the human acidic Mammalian chitinase protein.

Mouse models have shown the importance of acidic mammalian chitinase activity in settings of chitin exposure and allergic inflammation. However, little is known regarding genetic regulation of AMCase enzymatic activity in human allergic diseases. Resequencing the AMCase gene exons we identified 8 non-synonymous single nucleotide polymorphisms including three novel variants (A290G, G296A, G339T) near the gene area coding for the enzyme active site, all in linkage disequilibrium. AMCase protein isoforms, encoded by two gene-wide haplotypes, and differentiated by these three single nucleotide polymorphisms, were recombinantly expressed and purified. Biochemical analysis revealed the isoform encoded by the variant haplotype displayed a distinct pH profile exhibiting greater retention of chitinase activity at acidic and basic pH values. Determination of absolute kinetic activity found the variant isoform encoded by the variant haplotype was 4-, 2.5-, and 10-fold more active than the wild type AMCase isoform at pH 2.2, 4.6, and 7.0, respectively. Modeling of the AMCase isoforms revealed positional changes in amino acids critical for both pH specificity and substrate binding. Genetic association analyses of AMCase haplotypes for asthma revealed significant protective associations between the variant haplotype in several asthma cohorts. The structural, kinetic, and genetic data regarding the AMCase isoforms are consistent with the Th2-priming effects of environmental chitin and a role for AMCase in negatively regulating this stimulus.

Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection

Variation in the presentation of hereditary immunodeficiencies may be explained by genetic or environmental factors. Patients with mutations in HOIL1 (RBCK1) present with amylopectinosis-associated myopathy with or without hyper-inflammation and immunodeficiency. We report that barrier-raised HOIL-1-deficient mice exhibit amylopectin-like deposits in the myocardium but show minimal signs of hyper-inflammation. However, they show immunodeficiency upon acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to HOIL-1 function in hematopoietic cells and macrophages in production of protective cytokines. In contrast, HOIL-1-deficient mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ-herpesvirus 68 (MHV68), and these infections conferred a hyper-inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of HOIL-1, IL-6, Caspase-1 and Caspase-1;Caspase-11-deficient mice following Listeria infection. Thus chronic herpesvirus infection generates signs of auto-inflammation and complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype-phenotype studies. DOI: http://dx.doi.org/10.7554/eLife.04494.001

Making Mouse Models That Reflect Human Immune Responses

Humans are infected with a variety of acute and chronic pathogens over the course of their lives, and pathogen-driven selection has shaped the immune system of humans. The same is likely true for mice. However, laboratory mice we use for most biomedical studies are bred in ultra-hygienic environments, and are kept free of specific pathogens. We review recent studies that indicate that pathogen infections are important for the basal level of activation and the function of the immune system. Consideration of these environmental exposures of both humans and mice can potentially improve mouse models of human disease.

Latent gammaherpesvirus 68 infection induces distinct transcriptional changes in different organs

ABSTRACT Previous studies identified a role for latent herpesvirus infection in cross-protection against infection and exacerbation of chronic inflammatory diseases. Here, we identified more than 500 genes differentially expressed in spleens, livers, or brains of mice latently infected with gammaherpesvirus 68 and found that distinct sets of genes linked to different pathways were altered in the spleen compared to those in the liver. Several of the most differentially expressed latency-specific genes (e.g., the gamma interferon [IFN-γ], Cxcl9, and Ccl5 genes) are associated with known latency-specific phenotypes. Chronic herpesvirus infection, therefore, significantly alters the transcriptional status of host organs. We speculate that such changes may influence host physiology, the status of the immune system, and disease susceptibility.

Pervasive Transcription of a Herpesvirus Genome Generates Functionally Important RNAs

ABSTRACT Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi’s sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely “noise” generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription. IMPORTANCE The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle. The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.