Evasion Of Innate Immune Response Research Articles

Mechanisms of pathogenicity for the emerging fungus Candida auris.

Candida auris recently emerged as an urgent public health threat, causing outbreaks of invasive infections in healthcare settings throughout the world. This fungal pathogen persists on the skin of patients and on abiotic surfaces despite antiseptic and decolonization attempts. The heightened capacity for skin colonization and environmental persistence promotes rapid nosocomial spread. Following skin colonization, C. auris can gain entrance to the bloodstream and deeper tissues, often through a wound or an inserted medical device, such as a catheter. C. auris possesses a variety of virulence traits, including the capacity for biofilm formation, production of adhesins and proteases, and evasion of innate immune responses. In this review, we highlight the interactions of C. auris with the host, emphasizing the intersection of laboratory studies and clinical observations.

Ebola virus VP35 perturbs type I interferon signaling to facilitate viral replication

As one of the deadliest viruses, Ebola virus (EBOV) causes lethal hemorrhagic fevers in humans and nonhuman primates. The suppression of innate immunity leads to robust systemic virus replication of EBOV, leading to enhanced transmission. However, the mechanism of EBOV-host interaction is not fully understood. Here, we identified multiple dysregulated genes in early stage of EBOV infection through transcriptomic analysis, which are highly clustered to Jak-STAT signaling. EBOV VP35 and VP30 were found to inhibit type I interferon (IFN) signaling. Moreover, exogenous expression of VP35 blocks the phosphorylation of endogenous STAT1, and suppresses nuclear translocation of STAT1. Using serial truncated mutations of VP35, N-terminal 1–220 amino acid residues of VP35 were identified to be essential for blocking on type I IFN signaling. Remarkably, VP35 of EBOV suppresses type I IFN signaling more efficiently than those of Bundibugyo virus (BDBV) and Marburg virus (MARV), resulting in stable replication to facilitate the pathogenesis. Altogether, this study enriches understanding on EBOV evasion of innate immune response, and provides insights into the interplay between filoviruses and host.

How the initial discovery of modified RNA enabled evasion of innate immune responses and facilitated the development of RNA therapeutics.

Besides being the physical link between DNA and proteins, RNAs play several other key roles, including RNA catalysis and gene regulation. Recent advances in the design of lipid nanoparticles have facilitated the development of RNA-based therapeutics. However, chemically and in vitro transcribed RNAs can activate innate immunity, leading to the production of proinflammatory cytokines and interferons, a response similar to the one induced by viral infections. Since these responses are undesirable for certain therapeutic applications, it is important to develop ways to block the sensing of exogenous RNAs by immune cells, such as monocytes, macrophages and dendritic cells. Fortunately, RNA sensing can be blocked by chemical modifications of certain nucleotides, particularly uridine, a finding that has facilitated the development of RNA-based therapeutics such as small interfering RNAs and mRNA vaccines. Here, I provide a backstory on how improved understanding of RNA sensing by innate immunity can be applied to develop more effective RNA therapeutics.

A multiomics analysis of direct interkingdom dynamics between influenza A virus and Streptococcus pneumoniae uncovers host-independent changes to bacterial virulence fitness.

For almost a century, it has been recognized that influenza A virus (IAV) infection can promote the development of secondary bacterial infections (SBI) mainly caused by Streptococcus pneumoniae (Spn). Recent observations have shown that IAV is able to directly bind to the surface of Spn. To gain a foundational understanding of how direct IAV-Spn interaction alters bacterial biological fitness we employed combinatorial multiomic and molecular approaches. Here we show IAV significantly remodels the global transcriptome, proteome and phosphoproteome profiles of Spn independently of host effectors. We identified Spn surface proteins that interact with IAV proteins (hemagglutinin, nucleoprotein, and neuraminidase). In addition, IAV was found to directly modulate expression of Spn virulence determinants such as pneumococcal surface protein A, pneumolysin, and factors associated with antimicrobial resistance among many others. Metabolic pathways were significantly altered leading to changes in Spn growth rate. IAV was also found to drive Spn capsule shedding and the release of pneumococcal surface proteins. Released proteins were found to be involved in evasion of innate immune responses and actively reduced human complement hemolytic and opsonizing activity. IAV also led to phosphorylation changes in Spn proteins associated with metabolism and bacterial virulence. Validation of proteomic data showed significant changes in Spn galactose and glucose metabolism. Furthermore, supplementation with galactose rescued bacterial growth and promoted bacterial invasion, while glucose supplementation led to enhanced pneumolysin production and lung cell apoptosis. Here we demonstrate that IAV can directly modulate Spn biology without the requirement of host effectors and support the notion that inter-kingdom interactions between human viruses and commensal pathobionts can promote bacterial pathogenesis and microbiome dysbiosis.

Overexpression Bombyx mori HEXIM1 Facilitates Immune Escape of Bombyx mori Nucleopolyhedrovirus by Suppressing BmRelish-Driven Immune Responses.

Bombyx mori nucleopolyhedrovirus (BmNPV), a typical arthropod-specific enveloped DNA virus, is one of the most serious pathogens in silkworm farming, but the potential mechanisms of the evasion of innate immune responses from BmNPV infection are still poorly understood. HEXIM1 is an RNA-binding protein, best known as an inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation by RNA polymerase II. In this study, Bombyx mori HEXIM1 (BmHEXIM1) was cloned and characterized, and its expression was found to be remarkably upregulated after BmNPV infection. Furthermore, BmHEXIM1 was detected to increase the proliferation of BmNPV, and its full length is essential for assisting BmNPV immune escape by suppressing BmRelish-driven immune responses. This study brought new insights into the mechanisms of immune escape of BmNPV and provided theoretical guidance for the breeding of BmNPV-resistant silkworm varieties.

Evidence against the Human Metapneumovirus G, SH, and M2-2 Proteins as Bona Fide Interferon Antagonists.

ABSTRACTThe production of type I interferon (IFN) is the hallmark of the innate immune response. Most, if not all, mammalian viruses have a way to circumvent this response. Fundamental knowledge on viral evasion of innate immune responses may facilitate the design of novel antiviral therapies. To investigate how human metapneumovirus (HMPV) interacts with the innate immune response, recombinant viruses lacking G, short hydrophobic (SH), or M2-2 protein expression were assessed for IFN induction in A549 cells. HMPV lacking G or SH protein expression induced similarly low levels of IFN, compared to the wild-type virus, whereas HMPV lacking M2-2 expression induced significantly more IFN than the wild-type virus. However, sequence analysis of the genomes of M2-2 mutant viruses revealed large numbers of mutations throughout the genome. Over 70% of these nucleotide substitutions were A-to-G and T-to-C mutations, consistent with the properties of the adenosine deaminase acting on RNA (ADAR) protein family. Knockdown of ADAR1 by CRISPR interference confirmed the role of ADAR1 in the editing of M2-2 deletion mutant virus genomes. More importantly, Northern blot analyses revealed the presence of defective interfering RNAs (DIs) in M2-2 mutant viruses and not in the wild-type virus or G and SH deletion mutant viruses. DIs are known to be potent inducers of the IFN response. The presence of DIs in M2-2 mutant virus stocks and hypermutated virus genomes interfere with studies on HMPV and the innate immune response and should be addressed in future studies.IMPORTANCE Understanding the interaction between viruses and the innate immune response is one of the barriers to the design of antiviral therapies. Here, we investigated the role of the G, SH, and M2-2 proteins of HMPV as type I IFN antagonists. In contrast to other studies, no IFN-antagonistic functions could be observed for the G and SH proteins. HMPV with a deletion of the M2-2 protein did induce type I IFN production upon infection of airway epithelial cells. However, during generation of virus stocks, these viruses rapidly accumulated DIs, which are strong activators of the type I IFN response. Additionally, the genomes of these viruses were hypermutated, which was prevented by generating stocks in ADAR knockdown cells, confirming a role for ADAR in hypermutation of HMPV genomes or DIs. These data indicate that a role of the HMPV M2-2 protein as a bona fide IFN antagonist remains elusive.

Human respiratory syncytial virus methyl transferase: a potential antiviral target?

Human respiratory syncytial virus (HRSV) causes bronchiolitis and pneumonia. The role of methyltransferase (MTase) activity of HRSV polymerase in viral replication is unknown. Literature reviews of similar viral MTases and homology- modeling of RSV MTase bound to GTP and S-adenosylmethionine (SAM) have shown sequence similarity and the conserved catalytic residues (K-D-K-E) and the SAM-binding (GXGXG) domain. Combined with the recent reports of the importance of 2’O methylation of viral RNAs in the host innate immune response evasion, and its proposed role in viral replication, HRSV MTase holds promise as a potential antiviral target. Further biological validation of HRSV MTase could facilitate the discovery of novel HRSV antivirals targeting MTase enzyme activity.

Staphylococcus epidermidis from prosthetic joint infections induces lower IL-1β release from human neutrophils than isolates from normal flora.

The aim of this study was to test the hypothesis that Staphylococcus epidermidis isolated from prosthetic joint infections (PJIs) differs from S.epidermidis isolated from normal flora in terms of its capacity to induce activation of caspase-1 and release of IL-1β in human neutrophils. The amount of active caspase-1 was determined over 6h by detecting Ac-YVAD-AMC fluorescence in human neutrophils incubated with S.epidermidis isolates from PJIs (ST2) or normal flora. The amount of IL-1β was detected by ELISA in neutrophil supernatants after 6h of incubation. Mean IL-1β release was lower after incubation with S.epidermidis from PJIs compared to isolates from normal flora, but no statistically significant difference was found in active caspase-1. Substantial inter-individual differences in both active caspase-1 and IL-1β were noted. These results suggest that evasion of innate immune response, measured as reduced capacity to induce release of IL-1β from human neutrophils, might be involved in the predominance of ST2 in S.epidermidis PJIs, but that other microbe-related factors are probably also important.

Hepatitis B Virus Evasion From Cyclic Guanosine Monophosphate–Adenosine Monophosphate Synthase Sensing in Human Hepatocytes

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver disease and cancer worldwide. The mechanisms of viral genome sensing and the evasion of innate immune responses by HBV infection are still poorly understood. Recently, the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) was identified as a DNA sensor. In this study, we investigated the functional role of cGAS in sensing HBV infection and elucidate the mechanisms of viral evasion. We performed functional studies including loss-of-function and gain-of-function experiments combined with cGAS effector gene expression profiling in an infectious cell culture model, primary human hepatocytes, and HBV-infected human liver chimeric mice. Here, we show that cGAS is expressed in the human liver, primary human hepatocytes, and human liver chimeric mice. While naked relaxed-circular HBV DNA is sensed in a cGAS-dependent manner in hepatoma cell lines and primary human hepatocytes, host cell recognition of viral nucleic acids is abolished during HBV infection, suggesting escape from sensing, likely during packaging of the genome into the viral capsid. While the hepatocyte cGAS pathway is functionally active, as shown by reduction of viral covalently closed circular DNA levels in gain-of-function studies, HBV infection suppressed cGAS expression and function in cell culture models and humanized mice. Conclusion: HBV exploits multiple strategies to evade sensing and antiviral activity of cGAS and its effector pathways.

Modulation of mRNA Translation and Cell Viability by Influenza A Virus Derived Nonstructural Protein 1.

Translation of in vitro transcribed messenger RNA (mRNA) is known to be compromised by cell's innate immune responses. Herein we show that when mRNA encoding nonstructural protein 1 (NS1), an immune evasion gene derived from influenza A virus, is co-delivered with mRNA encoding green fluorescent protein (GFP), higher GFP expression can be observed in four different interferon competent cell types within 6 h, indicating NS1's wide host range property and rapid counter response to the cells' innate immune response. Enhanced mRNA translation correlates with reduced interferon production in all tested cell types and substituting a small portion of luciferase mRNA with NS1 mRNA enhances luciferase production compared to the same dose composing of only luciferase mRNA although in a cell type specific manner. Toxicity caused by transfection of unmodified mRNA is mitigated with the delivery of NS1 mRNA and is observed only in NS1 without cleavage and polyadenylation specificity factor 30 kda (CPSF30) inhibition function. Conversely, delivery of mRNA encoding NS1 with CPSF30 inhibition function aggravated toxicity. Overall, we demonstrate that NS1 enhanced mRNA transfection through active evasion of innate immune responses and modulated cellular viability during mRNA transfection.

FRET studies examining the interaction of hepatitis C NS3 helicase and human DExD/H box helicases

The innate immune response to viral infection is the intracellular first line of defense against invading pathogens. One strategy that viruses, such as hepatitis C virus (HCV), use to establish a chronic infection is to short circuit this innate immune response. HCV encodes a multi‐functional non‐structural protein 3 (NS3), which is a protease covalently tethered to a C‐terminal helicase. One role of the NS3 protease in innate immune response evasion is to cleave mitochondrial anti‐viral signaling protein (MAVS). The function of the NS3 helicase is essential for replication but it is unclear why it is covalently linked to the protease.We propose that HCV NS3 helicase works to evade the innate immune response by binding to pathogen associated molecular patterns (PAMPs) in the HCV genome and interacting with the RIG‐I like receptor LGP2, to localize the NS3 protease near MAVs for cleavage.In order to study the interaction between human and viral helicases we used a process called Förster resonant energy transfer (FRET) where an excited fluorescent molecule (donor) transfers its energy to a nearby fluorescent molecule of a different type (acceptor) which in turn emits a red shifted fluorescent light. This process occurs in distances smaller than 10 nm between the donor and acceptor which indicates obvious interaction.We generated two sets of plasmids encoding donor (mCFP) and acceptor (mYFP) fused to viral and human helicases respectively. The donor plasmid set encodes recombinant proteins in which all or part of HCV NS3 and its cofactor NS4A were fused to a mCFP fluorescent protein. The first expresses only the NS3 helicase domain (mCFP‐NS3h) fused to mCFP. In the second, mCFP was fused with full‐length NS3 (mCFP‐NS3). In the third, mCFP was fused to NS3 and only the NS3 cofactor domain of NS4A (lacking the membrane anchor and C‐terminus, i.e. mCFP‐NS3‐4Acd), and in the fourth, mCFP was fused to NS3 and full length NS4A (mCFP‐NS3‐NS4A). The acceptor plasmid set encodes recombinant mYFP fused to DDX1, DDX3, DDX5, and DHX 58 (LGP2). HEK293T cells were co‐transfected with each donor plasmid and acceptor plasmid. The live cells were imaged using an optical micro‐spectroscope (OptiMis) which consists of a two photon excitation scanning optical microscope with high spectral resolution. In a separate set of experiments each of the donor plasmids were co‐transfected with a plasmid expressing RFP fused to MAVs and a nuclear localization signal.The fluorescently labeled recombinant NS3 proteins were visualized in real‐time in HEK293T cells. The RFP MAVs re‐localized to the nucleus only when it was co‐transfected with plasmids that encoded NS4A, in addition to the NS3 protease. FRET was observed between all recombinant HCV NS3 proteins and LGP2, but not between any recombinant HCV NS3 proteins and closely related DDX 1, DDX3, and DDX5. Our data indicates that the NS3 fusion proteins are biologically active and that NS3 interacts with LGP2 via its helicase domain.

Evasion of Innate Immune Responses by the Highly Virulent Cryptococcus gattii by Altering Capsule Glucuronoxylomannan Structure.

Cryptococcus neoformans causes life-threatening diseases mainly in immunosuppressed hosts such as AIDS patients; C. gattii causes disseminated infections even in healthy hosts. To identify the possible molecular mechanisms underlying this difference in virulence, we investigated the survival and histopathology of lung tissue in wild-type and CD4-depleted mice infected with C. neoformans H99 and C. gattii JP02 (the highly virulent strain isolated in Japan); we then compared dendritic cell (DC) cytokine release responses to different cell fractions from these two strains. JP02-infected mice exhibited shorter survival and fewer inflammatory cells in the lung than H99-infected control mice. Depletion of CD4-related cellular immunity reduced survival of H99-infected mice but had no effect on the survival or inflammatory cell infiltration in JP02-infected mice, suggesting that JP02 evades immune detection. To identify the molecule(s) conferring this difference, we measured cytokine production from murine DCs co-cultured with H99 and JP02 in vitro. The levels of inflammatory cytokines from DCs treated with intact JP02 cells, the extracted capsule, secreted extracellular polysaccharides, and purified glucuronoxylomannan (GXM) were markedly lower than those induced by intact H99 cells and corresponding H99 fractions. Structural analysis of GXM indicated that JP02 altered one of two O-acetyl groups detected in the H99 GXM. Deacetylated GXM lost the ability to induce inflammatory cytokine release from DCs, implicating these O-acetyl groups in immune recognition. We conclude that the highly virulent C. gattii processes a structural alteration in GXM that allows this pathogen to evade the immune response and therefore elimination.

Cell Wall-Anchored Surface Proteins of Staphylococcus aureus: Many Proteins, Multiple Functions.

Staphylococcus aureus persistently colonizes about 20% of the population and is intermittently associated with the remainder. The organism can cause superficial skin infections and life-threatening invasive diseases. The surface of the bacterial cell displays a variety of proteins that are covalently anchored to peptidoglycan. They perform many functions including adhesion to host cells and tissues, invasion of non-phagocytic cells, and evasion of innate immune responses. The proteins have been categorized into distinct classes based on structural and functional analysis. Many surface proteins are multifunctional. Cell wall-anchored proteins perform essential functions supporting survival and proliferation during the commensal state and during invasive infections. The ability of cell wall-anchored proteins to bind to desquamated epithelial cells is important during colonization, and the binding to fibrinogen is of particular significance in pathogenesis.

Induction of interferon-gamma and downstream pathways during establishment of fetal persistent infection with bovine viral diarrhea virus

Development of transplacental infection depends on the ability of the virus to cross the placenta and replicate within the fetus while counteracting maternal and fetal immune responses. Unfortunately, little is known about this complex process. Non-cytopathic (ncp) strains of bovine viral diarrhea virus (BVDV), a pestivirus in the Flaviviridae family, cause persistent infection in early gestational fetuses (<150 days; persistently infected, PI), but are cleared by immunocompetent animals and late gestational fetuses (>150 days; transiently infected, TI). Evasion of innate immune response and development of immunotolerance to ncp BVDV have been suggested as possible mechanisms for the establishment of the persistent infection. Previously we have observed a robust temporal induction of interferon (IFN) type I (innate immune response) and upregulation of IFN stimulated genes (ISGs) in BVDV TI fetuses. Modest chronic upregulation of ISGs in PI fetuses and calves reflects a stimulated innate immune response during persistent BVDV infection. We hypothesized that establishing persistent fetal BVDV infection is also accompanied by the induction of IFN-gamma (IFN-γ). The aims of the present study were to determine IFN-γ concentration in blood and amniotic fluid from control, TI and PI fetuses during BVDV infection and analyze induction of the IFN-γ downstream pathways in fetal lymphoid tissues. Two experiments with in vivo BVDV infections were completed. In Experiment 1, pregnant heifers were infected with ncp BVDV type 2 on day 75 or 175 of gestation or kept naïve to generate PI, TI and control fetuses, respectively. Fetuses were collected by Cesarean section on day 190. In Experiment 2, fetuses were collected on days 82, 89, 97, 192 and 245 following infection of pregnant heifers on day 75 of gestation. The results were consistent with the hypothesis that ncp BVDV infection induces IFN-γ secretion during acute infection in both TI and PI fetuses and that lymphoid tissues such as spleen, liver and thymus, serve both as possible sources of IFN-γ and target organs for its effects. Notably, induction of IFN-γ coincides with a decrease in BVDV RNA concentrations in PI fetal blood and tissues. This is the first report indicating the possible presence of an adaptive immune response in persistent BVDV infections, which may be contributing to the observed reduction of viremia in PI fetuses.

Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain

By using a cultured neuroblastoma cell line, the present authors recently showed that the N protein of virulent rabies virus fixed strain Nishigahara (Ni), but not that of the attenuated derivative Ni-CE, mediates evasion of induction of type I interferon (IFN). In this study, to determine whether Ni N protein indeed fulfills this function in vivo, the abilities to suppress IFN responses in the mouse brain of Ni-CE and the virulent chimeric virus CE(NiN), which has the N gene from Ni in the genetic background of Ni-CE, were compared. It was demonstrated that CE(NiN) propagates and spreads more efficiently than does Ni-CE in the brain and that IFN response in brains infected with CE(NiN) is weaker than in those infected with Ni-CE. It was also shown that amino acids at positions 273 and 394 in the N protein, which are known as pathogenic determinants, affect the ability of the viruses to suppress IFN response in the brain. These findings strongly suggest that, in the brain, rabies virus N protein plays important roles in evasion of innate immune responses and thereby in efficient propagation and spread of virus leading to lethal outcomes of infection.

Staphylococcus aureus innate immune evasion is lineage-specific: A bioinfomatics study

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections.

Conserved Charged Amino Acids within Sendai Virus C Protein Play Multiple Roles in the Evasion of Innate Immune Responses

One of the accessory proteins of Sendai virus (SeV), C, translated from an alternate reading frame of P/V mRNA has been shown to function at multiple stages of infection in cell cultures as well as in mice. C protein has been reported to counteract signal transduction by interferon (IFN), inhibit apoptosis induced by the infection, enhance the efficiency of budding of viral particles, and regulate the polarity of viral genome-length RNA synthesis to maximize production of infectious particles. In this study, we have generated a series of SeV recombinants containing substitutions of highly conserved, charged residues within the C protein, and characterized them together with previously-reported C′/C(−), 4C(−), and F170S recombinant viruses in infected cell cultures in terms of viral replication, cytopathogenicity, and antagonizing effects on host innate immunity. Unexpectedly, the amino acid substitutions had no or minimal effect on viral growth and viral RNA synthesis. However, all the substitutions of charged amino acids resulted in the loss of a counteracting effect against the establishment of an IFN-α-mediated anti-viral state. Infection by the virus (Cm2′) containing mutations at K77 and D80 induced significant IFN-β production, severe cytopathic effects, and detectable amounts of viral dsRNA production. In addition to the Cm2′ virus, the virus containing mutations at E114 and E115 did not inhibit the poly(I:C)-triggered translocation of cellular IRF-3 to the nucleus. These results suggest that the C protein play important roles in viral escape from induction of IFN-β and cell death triggered by infection by means of counteracting the pathway leading to activation of IRF-3 as well as of minimizing viral dsRNA production.

The Presence of CD14 Overcomes Evasion of Innate Immune Responses by VirulentFrancisella tularensisin Human Dendritic Cells In Vitro and Pulmonary Cells In Vivo

Francisella tularensis is a Gram-negative bacterium that causes acute, lethal disease following inhalation. We have previously shown that viable F. tularensis fails to stimulate secretion of proinflammatory cytokines following infection of human dendritic cells (hDC) in vitro and pulmonary cells in vivo. Here we demonstrate that the presence of the CD14 receptor is critical for detection of virulent F. tularensis strain SchuS4 by dendritic cells, monocytes, and pulmonary cells. Addition of soluble CD14 (sCD14) to hDC restored cytokine production following infection with strain SchuS4. In contrast, addition of anti-CD14 to monocyte cultures inhibited the ability of these cells to respond to strain SchuS4. Addition of CD14 or blocking CD14 following SchuS4 infection in dendritic cells and monocytes, respectively, was not due to alterations in phagocytosis or replication of the bacterium in these cells. Administration of sCD14 in vivo also restored cytokine production following infection with strain SchuS4, as assessed by increased concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-12p70, and IL-6 in the lungs of mice receiving sCD14 compared to mock-treated controls. In contrast to homogenous cultures of monocytes or dendritic cells infected in vitro, mice treated with sCD14 in vivo also exhibited controlled bacterial replication and dissemination compared to mock-treated controls. Interestingly, animals that lacked CD14 were not more susceptible or resistant to pulmonary infection with SchuS4. Together, these data support the hypothesis that the absence or low abundance of CD14 on hDC and in the lung contributes to evasion of innate immunity by virulent F. tularensis. However, CD14 is not required for development of inflammation during the last 24 to 48 h of SchuS4 infection. Thus, the presence of this receptor may aid in control of virulent F. tularensis infections at early, but not late, stages of infection.

Quantitative analysis of cellular proteome alterations in human influenza A virus‐infected mammalian cell lines

Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

Evasion of Innate Immune Responses: Evidence for Mannose Binding Lectin Inhibition of Tumor Necrosis Factor Alpha Production by Macrophages in Response toBlastomyces dermatitidis

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-alpha) production. Since macrophage production of TNF-alpha is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-alpha production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-alpha production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-alpha production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF-alpha production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-alpha production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-beta-Glucan stimulated TNF-alpha production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-beta-glucan antibody inhibited TNF-alpha production by PM. With anti-1,3-beta-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-beta-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-beta-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-beta-glucan by anti-1,3-beta-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-beta-glucan sites or sterically masking 1,3-beta-glucan sites, thus preventing 1,3-beta-glucan stimulation of PM for TNF-alpha production.