Daniel L. Popkin

Gamma Interferon Blocks Gammaherpesvirus Reactivation from Latency

ABSTRACT Establishment of latent infection and reactivation from latency are critical aspects of herpesvirus infection and pathogenesis. Interfering with either of these steps in the herpesvirus life cycle may offer a novel strategy for controlling herpesvirus infection and associated disease pathogenesis. Prior studies show that mice deficient in gamma interferon (IFN-γ) or the IFN-γ receptor have elevated numbers of cells reactivating from murine gammaherpesvirus 68 (γHV68) latency, produce infectious virus after the establishment of latency, and develop large-vessel vasculitis. Here, we demonstrate that IFN-γ is a powerful inhibitor of reactivation of γHV68 from latency in tissue culture. In vivo, IFN-γ controls viral gene expression during latency. Importantly, depletion of IFN-γ in latently infected mice results in an increased frequency of cells reactivating virus. This demonstrates that IFN-γ is important for immune surveillance that limits reactivation of γHV68 from latency.

An Slfn2 mutation causes lymphoid and myeloid immunodeficiency due to loss of immune cell quiescence

Here we describe a previously unknown form of inherited immunodeficiency revealed by an N-ethyl-N-nitrosourea–induced mutation called elektra. Mice homozygous for this mutation showed enhanced susceptibility to bacterial and viral infection and diminished numbers of T cells and inflammatory monocytes that failed to proliferate after infection and died via the intrinsic apoptotic pathway in response to diverse proliferative stimuli. They also had a greater proportion of T cells poised to replicate DNA, and their T cells expressed a subset of activation markers, suggestive of a semi-activated state. We positionally ascribe the elektra phenotype to a mutation in the gene encoding Schlafen-2 (Slfn2). Our findings identify a physiological role for Slfn2 in the defense against pathogens through the regulation of quiescence in T cells and monocytes.

Role of Lymphocytic Choriomeningitis Virus (LCMV) in Understanding Viral Immunology: Past, Present and Future

Lymphocytic choriomeningitis virus (LCMV) is a common infection of rodents first identified over eighty years ago in St. Louis, MO, U.S.A. It is best known for its application in immunological studies. The history of LCMV closely correlates with the development of modern immunology. With the use of LCMV as a model pathogen several key concepts have emerged: Major Histocompatibility Complex (MHC) restriction, T cell memory, persistent infections, T cell exhaustion and the key role of immune pathology in disease. Given the phenomenal infrastructure within this field (e.g., defined immunodominant and subdominant epitopes to all T cell receptor specificities as well as the cognate tetramers for enumeration in vivo) the study of LCMV remains an active and productive platform for biological research across the globe to this day. Here we present a historical primer that highlights several breakthroughs since the discovery of LCMV. Next, we highlight current research in the field and conclude with our predictions for future directions in the remarkable field of LCMV research.

Disruption of MyD88 signaling suppresses hemophagocytic lymphohistiocytosis in mice

Hemophagocytic lymphohistiocytosis (HLH) is a rare inflammatory disorder with a poor prognosis for affected individuals. To find a means of suppressing the clinical phenotype, we investigated the cellular and molecular mechanisms leading to HLH in Unc13d(jinx/jinx) mice, in which cytolytic function of NK and CD8(+) T cells is impaired. Unc13d(jinx/jinx) mutants infected with lymphochoriomeningitis virus (LCMV) present typical clinical features of HLH, including splenomegaly, elevated serum IFNγ, and anemia. Proteins mediating cell-cell contact, cytokine signaling or Toll-like receptor (TLR) signaling were analyzed. We show that neither the integrin CD18, which is involved in adhesion between antigen-presenting cells and effector T cells, nor tumor necrosis factor (TNF) made nonredundant contributions to the disease phenotype. Disruption of IFNγ signaling reduced immune cell activation in Unc13d(jinx/jinx) mice, but also resulted in uncontrolled viral proliferation and exaggerated release of inflammatory cytokines. Abrogating the function of myeloid differentiation primary response gene 88 (MyD88) in Unc13d(jinx/jinx) mice suppressed immune cell activation and controlled cytokine production in an IL-1 receptor 1 (IL-1R1)-independent way. Our findings implicate MyD88 as the key initiator of myeloid and lymphoid proliferation in HLH, and suggest that blockade of this signaling molecule may reduce immunopathology in patients.

ENU-induced phenovariance in mice: inferences from 587 mutations

BackgroundWe present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.FindingsIn the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.ConclusionsCollectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Flt3 permits survival during infection by rendering dendritic cells competent to activate NK cells

A previously unappreciated signal necessary for dendritic cell (DC)-mediated activation of natural killer (NK) cells during viral infection was revealed by a recessive N-ethyl-N-nitrosourea-induced mutation called warmflash (wmfl). Wmfl homozygotes displayed increased susceptibility to mouse cytomegalovirus (MCMV) infection. In response to MCMV infection in vivo, delayed NK cell activation was observed, but no intrinsic defects in NK cell activation or function were identified. Rather, coculture experiments demonstrated that NK cells are suboptimally activated by wmfl DCs, which showed impaired cytokine production in response to MCMV or synthetic TLR7 and TLR9 ligands. The wmfl mutation was identified in the gene encoding the Fms-like tyrosine kinase 3 (Flt3). Flt3 ligand (Flt3L) is transiently induced in the serum upon infection or TLR activation. However, antibody blockade reveals no acute requirement for Flt3L, suggesting that the Flt3L → Flt3 axis programs the development of DCs, making them competent to support NK effector function. In the absence of Flt3 signaling, NK cell activation is delayed and survival during MCMV infection is markedly compromised.

Genetic vs Environmental Factors That Correlate With Rosacea: A Cohort-Based Survey of Twins

IMPORTANCE To our knowledge, this is the first study on rosacea to formally define genetic and environmental contributions. OBJECTIVES To study a cohort of identical and fraternal twins to determine whether genetic factors contribute to rosacea development and, if genetic factors are present, quantitatively estimate the genetic contribution, as well as to identify environmental factors that correlate with rosacea by controlling for genetic susceptibility. DESIGN, SETTING, AND PARTICIPANTS Identical and fraternal twins were surveyed regarding risk factors implicated in rosacea. Faculty dermatologists determined a rosacea score for each twin participant according to the National Rosacea Society (NRS) grading system. Data were collected at the annual Twins Days Festival in Twinsburg, Ohio, on August 4-5, 2012, and August 2-3, 2013. Analysis was conducted for several months after each meeting. A cohort of 550 twin individuals, with most from Ohio, Pennsylvania, and the northeastern United States, participated. MAIN OUTCOMES AND MEASURES The NRS score and rosacea subtype were assessed using the NRS grading system and physical examination by board-certified dermatologists. RESULTS Among the 275 twin pairs (550 individuals), there were 233 identical twin pairs with a mean rosacea score of 2.46 and 42 fraternal twin pairs with a mean rosacea score of 0.75. We observed a higher association of NRS scores between identical vs fraternal twins (r = 0.69 vs r = 0.46; P = .04), demonstrating a genetic contribution. Using the ACE model (proportion of variance in a trait heritable secondary to additive genetics [A] vs the proportions due to a common environment [C] and unique environment [E]), we calculated this genetic contribution to be 46%. A higher NRS score was also significantly associated with the following factors: age (r = 0.38; P < .001) and lifetime UV radiation exposure (r = 0.26; P < .001). These associations remained after use of propensity score matching to adjust for multicollinearity. Other correlated variables included body mass index (r = 0.21; P < .001), smoking (r = 0.10; P < .02), alcohol consumption (r = 0.11; P = .01), cardiovascular comorbidity (r = 0.17; P < .001), and skin cancer comorbidity (r = 0.19; P < .001). CONCLUSIONS AND RELEVANCE The study of twins allows us to separate genetic susceptibility and the influence of environmental factors affecting rosacea. We found that approximately half of the contribution to the NRS score could be accounted for by genetics and the other half by environment. We identified correlations between rosacea and UV radiation exposure, alcohol, smoking, skin cancer history, cardiac comorbidity, and age. These findings may help improve current management and expectations of individuals affected by rosacea.

Murine cytomegalovirus infection inhibits tumor necrosis factor alpha responses in primary macrophages.

ABSTRACT Despite robust host immune responses the betaherpesvirus murine cytomegalovirus (MCMV) is able to establish lifelong infection. This capacity is due at least in part to the virus utilizing multiple immune evasion mechanisms to blunt host responses. Macrophages are an important cell for MCMV infection, dissemination, and latency despite expression in the host of multiple cytokines, including tumor necrosis factor alpha (TNF-α), that can induce an antiviral state in macrophages or other cells. In this study, we found that MCMV infection of bone marrow-derived macrophages inhibited TNF-α-induced ICAM-1 surface expression and mRNA expression in infected cells via expression of immediate early and/or early viral genes. MCMV infection blocked TNF-α-induced nuclear translocation of NF-κB. This inhibition of TNF-α signaling was explained by a decrease in TNF receptor 1 (TNFR1) and TNFR2 that was due to decreased mRNA for the latter. These findings provide a mechanism by which MCMV can evade the effects of an important host cytokine in macrophages.

Slc15a4, a Gene Required for pDC Sensing of TLR Ligands, Is Required to Control Persistent Viral Infection

Plasmacytoid dendritic cells (pDCs) are the major producers of type I IFN in response to viral infection and have been shown to direct both innate and adaptive immune responses in vitro. However, in vivo evidence for their role in viral infection is lacking. We evaluated the contribution of pDCs to acute and chronic virus infection using the feeble mouse model of pDC functional deficiency. We have previously demonstrated that feeble mice have a defect in TLR ligand sensing. Although pDCs were found to influence early cytokine secretion, they were not required for control of viremia in the acute phase of the infection. However, T cell priming was deficient in the absence of functional pDCs and the virus-specific immune response was hampered. Ultimately, infection persisted in feeble mice. We conclude that pDCs are likely required for efficient T cell priming and subsequent viral clearance. Our data suggest that reduced pDC functionality may lead to chronic infection.

Murine cytomegalovirus paralyzes macrophages by blocking IFNγ-induced promoter assembly

Macrophages (Mϕ) are activated by IFNγ and are important cellular targets for infection by human and murine cytomegalovirus (MCMV), making it advantageous for CMVs to block IFNγ-induced Mϕ differentiation. We found that MCMV infection inhibited IFNγ regulation of many genes in Mϕ. MCMV infection blocked IFNγ responses at the level of transcription without blocking Janus kinase/signal transducer and activator of transcription pathway activation and targeted IFN response factor 1- and class II transactivator-dependent and independent promoters. MCMV did not alter basal transcription from IFNγ-responsive promoters and left the majority of cellular transcripts unchanged even after 48 h of infection. The effects of MCMV infection were specific to chromosomal rather than transiently transfected promoters. Characterization of the IFNγ-responsive chromosomal class II transactivator promoter revealed that MCMV infection blocked IFNγ-induced promoter assembly, allowing the virus to transcriptionally paralyze infected Mϕ responses while allowing basal transcription to proceed.