Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler

<p>Abstract</p> <p>Background</p> <p>The rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage.</p> <p>Results</p> <p>We have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats <it>Pteropus vampyrus </it>and <it>Myotis lucifugus</it>. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in <it>P. vampyrus</it>, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli.</p> <p>Conclusion</p> <p>The statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.</p

Nucleic Acid Scavenging Polymers Inhibit Extracellular DNA-Mediated Innate Immune Activation without Inhibiting Anti-Viral Responses

<div><p>Toll-like receptor (TLR) family members, 3, 7 and 9 are key components in initiation and progression of autoimmune disorders such as systemic lupus erythematosus (SLE). These TLRs are often referred to as nucleic acid-sensing TLRs based on their ability to recognize DNAs or RNAs produced by pathogens or damaged cells. During autoimmune disease progression these receptors recognize self nucleic acids as well as self nucleic acid-containing complexes and contribute to inflammatory cytokine production and subsequent enhancement of serum autoantibody levels. We have recently discovered that nucleic-acid scavenging polymers (NASPs) can neutralize the proinflammatory effects of nucleic acids. Here, we begin to explore what effects such NASPs have on normal immune function. We show that such NASPs can inhibit TLR activation without affecting nucleic acid-independent T cell activation. Moreover, we observe that stimulation of immune cells by encapsulated nucleic acids, such as those found in viral particles, is unaffected by NASPs. Thus NASPs only limit the activation of the immune system by accessible extra-cellular nucleic acid and do not engender non-specific immune suppression. These important findings suggest that NASPs represent a new approach toward anti-inflammatory drug development as these agents can potentially be utilized to block overt autoimmune disorders and inflammation while allowing normal immune responses to occur.</p></div

Polymers regulate B cell proliferation and antibody production post nucleic acid stimulation.

<p>(A) Polymers block B cell proliferation post CpG but not LPS stimulation. B cells were isolated and CFSE labeled. They were then cultured in the presence of LPS and CpG as well as 20 µg/ml of CDP or PAMAM polymer. Proliferation was assessed using CFSE dilution using flow cytometry. Flow plots show cell proliferation in the presence or absence of stimulation. Graph (right) complies proliferation data for stimulations in the presence or absence of polymers. Data are representative of 3 independent experiments. n = 9 mice per group. p<0.05. (B) Polymers block IgM antibody production post CpG but not LPS stimulation. B cells were cultured in the presence of CpG and LPS as well as 20 µg/ml of CDP or PAMAM polymer. Supernatants were collect 72 hours post stimulation and IgM levels were assessed via ELISA. Data are representative of 3 independent experiments. *p<0.05.</p

Polymers do not affect DC activation by encapsulated viruses.

<p>(A) Polymers do not affect TNFα production post VSV stimulation. DCs were generated form bone marrow cells of WT animals cultured in the presence of IL4 and GMCSF for 7 days. Cells were then stimulated with live VSV at a multiplicity of infection (MOI) of 1 in the presence of 20 µg/mL PAMAM-G3. Culture supernatants were analyzed for secretion of IL6 by ELISA, 24 h later. (B) Polymers do not affect TNFα production post vaccinia stimulation. DCs were generated form bone marrow cells of WT animals cultured in the presence of IL4 and GMCSF for 7 days. Cells were then stimulated with live vaccinia at a multiplicity of infection (MOI) of 1 in the presence of 20 µg/mL PAMAM-G3. Culture supernatants were analyzed for secretion of IL6 by ELISA, 24 h later. (C) Polymers do not affect IFNα production post VSV stimulation. pDCs were generated from bone marrow cells of WT animals in the presence of Flt-3L for 10 days. Cells were then stimulated with live VSV at an MOI of 1 in the presence of 20 µg/mL PAMAM-G3. Supernatants were analyzed for the presence of IFNα by ELISA 24 h later. Data are presented as mean+/− SD and are representative of at least 3 independent experiments. n = 9 mice per group. (D) Polymers do not affect IL6 production post VSV stimulation as compared to TLR inhibitors. DCs were generated form bone marrow cells of WT animals cultured in the presence of IL4 and GMCSF for 7 days. Cells were then stimulated with live VSV at an MOI of 1 in the presence of 20 µg/mL PAMAM-G3, CLI-095 and IRS954. Supernatants were analyzed for the presence of IL6 by ELISA 24 h later. Data are presented as mean+/− SD and are representative of at least 3 independent experiments. n = 9 mice per group. *p<0.05.</p

Polymers regulate cell surface receptor expression on B cells and DCs post nucleic acid stimulation.

<p>(A) Polymers block cell surface receptor expression post CpG but not LPS stimulation. BMDC were cultured as previously described. DCs were then cultured in the presence of LPS and CpG as well as 20 µg/ml of each polymer (CDP and PAMAM). CD86 and MHCII cell surface receptor expression was assessed 24 hours later using flow cytometry. Data are representative of 3 independent experiments. n = 9 mice per group. *p<0.05. (B) Polymers block cell surface receptor expression post CpG but not LPS stimulation. B cells were isolated and cultured as previously described. B cells were then cultured in the presence of LPS and CpG as well as 20 µg/ml of each polymer. CD86 and MHCII cell surface receptor expression was assessed 24 hours later using flow cytometry. Data are representative of 3 independent experiments. n = 9 mice per group. *p<0.05.</p

Pre-treatment of DCs with CDP and PAMAM does not affect T cell activation and proliferation by DCs.

<p>(A) Co-culturing of DCs with polymers does not result in impaired OVA uptake. BMDCs were cultured in the presence of 20 µg/ml CDP or PAMAM-G3 as well as 1 mg/ml OVA-FITC for 30 minutes. OVA uptake was then assessed by flow cytometry. (B) Pre-treatment of DCs with polymers does not affect the ability of DCs to uptake OVA antigen. BMDCs were cultured in the presence of 20 µg/ml CDP or PAMAM-G3. Cell were then washed and pulsed with OVA-FITC for 30 minutes. OVA uptake was then assessed by flow cytometry. Data are representative of at least three independent experiments. Data from three different experiments are compiled in MFI graph. *p<0.05.</p

Mice deficient in caspase-1 are partially protected from acute weight loss after intramuscular immunization.

<p>Groups of adult C57BL/6 wild type or caspase 1−/− mice were immunized with two injections (5×10⁸ PFU per injection) of rVSV in each rear quadriceps, or were sham inoculated with sterile PBS. At 24 hours after infection mice (n = 2–3 mice per timepoint), were sacrificed and IL-1β in the draining popliteal LN (Panel A left) and quadriceps muscle (Panel A right) was quantitated via ELISA. The amount of IL-1β produced by wild type and caspase 1−/− mice was not significantly different in either organ. The comparison of IL-1β production by wild type and caspase 1−/− mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type and caspase 1−/− mice (n = 5 per group) after intramuscular challenge with 5×10⁸ PFU of rVSV. The comparison of WT and caspase 1−/− mice has been performed twice with consistent results. Caspase 1−/− mice lost significantly less weight than wild type controls on days 2 and 3 after challenge (P<0.05 via Mann Whitney test). Panel C shows average viral loads in the quadriceps muscle of infected mice (n = 6 per group). Data is compiled from two identically performed experiments. Viral loads in WT and caspase 1−/− mice were not significantly different. Panel D shows average serum neutralizing antibody titers for wild type and caspase 1−/− mice immunized with rVSV (n = 5 per group per timepoint). Error bars represent the upper and lower limits of the 95% confidence interval. Panel E shows average percent ± SEM of CD8 T cells in the blood binding to an MHC Class I tetramer recognizing an immunodominant epitope within VSV N. There were no significant differences in the antibody or CD8 T cell responses between the two groups at any time. The comparison of humoral and cellular immune responses in WT and caspase 1−/− mice has been performed twice with consistent results.</p

Wild type mice challenged with rVSV produce IL-1β locally and systemically.

<p>Groups of adult female C57BL/6 mice were immunized with two injections (5×10⁸ PFU per injection) of rVSV in each rear quadriceps, or were sham inoculated with sterile PBS. At 12 and 24 hours after infection mice (n = 4 per timepoint), were sacrificed and IL-1β in the blood (Panel A), draining popliteal LN (Panel B), and quadriceps muscle (Panel C) was quantitated via ELISA. Open and filled bars represent the 12 and 24 hour timepoints respectively. Dotted line on graphs shows the average amount of IL-1β detected in the blood and tissues of sham-inoculated mice (n = 4). This experiment has been performed twice with consistent results.</p

IL-1R−/− mice control VSV replication, make strong humoral and cellular immune responses, and are immune to rechallenge.

<p>Adult female wild type or IL-1R−/− (n = 3 per group) mice were immunized intramuscularly with a single injection of 5×10⁸ PFU rVSV in the rear quadriceps. Mice were sacrificed at 24 hours after infection and viral titers determined in the quadriceps muscle (Panel A). There was no significant difference in viral loads between the two groups. In Panels B–D, adult female wild type (n = 5) or IL-1R−/− (n = 4) mice were immunized intramuscularly with a single injection of 5×10⁸ PFU rVSV in the rear quadriceps. At the indicated timepoints after infection mice were bled and humoral and cellular immune responses were assayed. Panel B shows average anti-VSV neutralizing antibody responses by group as measured by microneutralization assay. Error bars represent the upper and lower limits of the 95% confidence interval. Panel C shows average percent CD8 T cells specific for the VSV N1 epitope as measured by MHC Class I tetramer. At 14 days after immunization, WT mice had significantly more (P = 0.03, Two-tailed T test) VSV N specific CD8 T cells than IL-1R−/− mice, but by day 28 the difference was no longer significant (P = 0.13). At eight weeks after the primary infection all mice were challenged intranasally with a semi-lethal dose of rVSV (1×10⁸ PFU). A cohort of naïve wild type mice (n = 7) was challenged at the same time. All pre-immune mice had robust immunity to rechallenge (Panel D) and did not lose weight or exhibit other signs of pathology. Two of the naïve mice succumbed to infection. Days on which naïve animals succumbed are indicated with an asterisk on the graph.</p