Infected Monocytes Research Articles

Temporal co-option of host cell trafficking by Yersinia pestis during bubonic plague. (P3140)

Abstract Bubonic plague, caused by the gram-negative bacteria Yersinia pestis, is characterized by pathologically swollen lymph nodes (LNs) or buboes. The mechanism by which these buboes form and the adaptive function they provide for the pathogen, if any, remains unknown. We hypothesize that bubo formation is a strategy for flea-borne Y. pestis to establish a foothold in the host for subsequent systemic infection. Our studies indicate that colonization of the draining lymph node (DLN) from the site of infection occurs through the trafficking of infected dendritic cells and monocytes in temporally distinct waves. This is effected by redundant chemotactic signals, including through the receptors CCR7 and CCR2, as evidenced by the upregulation of multiple chemoattractants in the DLN during Y. pestis infection and the improved survival of infected CCR7KO and CCR2KO mice compared to wild-type.

Tetramerization of SAMHD1 Is Required for Biological Activity and Inhibition of HIV Infection

SAMHD1 is a dGTP-activated dNTPase that has been implicated as a modulator of the innate immune response. In monocytes and their differentiated derivatives, as well as in quiescent cells, SAMHD1 strongly inhibits HIV-1 infection and, to a lesser extent, HIV-2 and simian immunodeficiency virus (SIV) because of their virion-associated virulence factor Vpx, which directs SAMHD1 for proteasomal degradation. Here, we used a combination of biochemical and virologic approaches to gain insights into the functional organization of human SAMHD1. We found that the catalytically active recombinant dNTPase is a dGTP-induced tetramer. Chemical cross-linking studies revealed SAMHD1 tetramers in human monocytic cells, in which it strongly restricts HIV-1 infection. The propensity of SAMHD1 to maintain the tetrameric state in vitro is regulated by its C terminus, located outside of the catalytic domain. Accordingly, we show that the C terminus is required for the full ability of SAMHD1 to deplete dNTP pools and to inhibit HIV-1 infection in U937 monocytes. Interestingly, the human SAMHD1 C terminus contains a docking site for HIV-2/SIVmac Vpx and is known to have evolved under positive selection. This evidence indicates that Vpx targets a functionally important element in SAMHD1. Together, our findings imply that SAMHD1 tetramers are the biologically active form of this dNTPase and provide new insights into the functional organization of SAMHD1.

Differential Cell Line Susceptibility to the Emerging Novel Human Betacoronavirus 2c EMC/2012: Implications for Disease Pathogenesis and Clinical Manifestation

The emerging novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) was recently isolated from patients with severe pneumonia and renal failure and was associated with an unexplained high crude fatality rate of 56%. We performed a cell line susceptibility study with 28 cell lines. HCoV-EMC was found to infect the human respiratory tract (polarized airway epithelium cell line Calu-3, embryonic fibroblast cell line HFL, and lung adenocarcinoma cell line A549), kidney (embryonic kidney cell line HEK), intestinal tract (colorectal adenocarcinoma cell line Caco-2), liver cells (hepatocellular carcinoma cell line Huh-7), and histiocytes (malignant histiocytoma cell line His-1), as evident by detection of high or increasing viral load in culture supernatants, detection of viral nucleoprotein expression by immunostaining, and/or detection of cytopathic effects. Although an infected human neuronal cell line (NT2) and infected monocyte and T lymphocyte cell lines (THP-1, U937, and H9) had increased viral loads, their relatively lower viral production corroborated with absent nucleoprotein expression and cytopathic effects. This range of human tissue tropism is broader than that for all other HCoVs, including SARS coronavirus, HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63, which may explain the high mortality associated with this disease. A recent cell line susceptibility study showed that HCoV-EMC can infect primate, porcine, and bat cells and therefore may jump interspecies barriers. We found that HCoV-EMC can also infect civet lung fibroblast and rabbit kidney cell lines. These findings have important implications for the diagnosis, pathogenesis, and transmission of HCoV-EMC.

Dipterinyl Calcium Pentahydrate Inhibits Intracellular Mycobacterial Growth in Human Monocytes via the C-C Chemokine MIP-1β and Nitric Oxide

Tuberculosis remains one of the top three leading causes of morbidity and mortality worldwide, complicated by the emergence of drug-resistant Mycobacterium tuberculosis strains and high rates of HIV coinfection. It is important to develop new antimycobacterial drugs and immunomodulatory therapeutics and compounds that enhance antituberculous immunity. Dipterinyl calcium pentahydrate (DCP), a calcium-complexed pterin compound, has previously been shown to inhibit human breast cancer cells and hepatitis B virus (HBV). DCP inhibitory effects were attributed to induction of apoptosis and/or increased production of interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study, we tested the ability of DCP to mediate inhibition of intracellular mycobacteria within human monocytes. DCP treatment of infected monocytes resulted in a significant reduction in viability of intracellular but not extracellular Mycobacterium bovis BCG. The antimicrobial activity of DCP was comparable to that of pyrazinamide (PZA), one of the first-line antituberculosis drugs currently used. DCP potentiated monocyte antimycobacterial activity by induction of the cysteine-cysteine (C-C) chemokine macrophage inflammatory protein 1β (MIP-1β) and inducible nitric oxide synthase 2. Addition of human anti-MIP-1β neutralizing antibody or a specific inhibitor of the l-arginase-nitric oxide pathway (N(G)-monomethyl l-arginine [l-NMMA] monoacetate) reversed the inhibitory effects of DCP on intracellular mycobacterial growth. These findings indicate that DCP induced mycobacterial killing via MIP-1β- and nitric oxide-dependent effects. Hence, DCP acts as an immunoregulatory compound enhancing the antimycobacterial activity of human monocytes.

Pharmacodynamic Evaluation of the Intracellular Activity of Antibiotics towards Pseudomonas aeruginosa PAO1 in a Model of THP-1 Human Monocytes

Pseudomonas aeruginosa invades epithelial and phagocytic cells, which may play an important role in the persistence of infection. We have developed a 24-h model of THP-1 monocyte infection with P. aeruginosa PAO1 in which bacteria are seen multiplying in vacuoles by electron microscopy. The model has been used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by using a pharmacodynamic approach (concentration-dependent experiments over a wide range of extracellular concentrations to calculate bacteriostatic concentrations [Cs] and maximal relative efficacies [Emax]; Hill-Langmuir equation). Using 16 antipseudomonal antibiotics (three aminoglycosides, nine β-lactams, three fluoroquinolones, and colistin), dose-response curves were found to be undistinguishable for antibiotics of the same pharmacological class if data were expressed as a function of the corresponding MICs. Extracellularly, all of the antibiotics reached a bacteriostatic effect at their MIC, and their Emax exceeded the limit of detection (-4.5 log(10) CFU compared to the initial inoculum). Intracellularly, Cs values remained unchanged for β-lactams, fluoroquinolones, and colistin but were approximately 10 times higher for aminoglycosides, whereas Emax values were markedly reduced (less negative), reaching -3 log(10) CFU for fluoroquinolones and only -1 to -1.5 log(10) CFU for all other antibiotics. The decrease in intracellular aminoglycoside potency (higher Cs) can be ascribed to the acid pH to which bacteria are exposed in vacuoles. The decrease in the Emax may reflect a reversible alteration of bacterial responsiveness to antibiotics in the intracellular milieu. The model may prove useful for comparison of antipseudomonal antibiotics to reduce the risk of persistence or relapse of pseudomonal infections.

Activation of transcription of immunoproteasome subunit genes in murine monocytes infected with different mycobacterial strains

Processing of mycobacterial antigens by immunoproteasome is necessary to control infection and to protect the organism from the development of active tuberculosis. We have investigated the activation of transcription of immunoproteasome subunit genes in peritoneal monocytes of C57Bl/6 mice infected with a vaccine M. bovis strain BCG and a virulent M. tuberculosis strain H37Rv. The level of transcription of LMP2, LMP7, and MECL1 subunits did not increase on the first and second days after a single infection. After two rounds of infection with M. bovis BCG, only the transcription of the LMP7 subunit gene was enhanced. However, the subsequent infection of monocytes, first with the vaccine strain, then with the virulent strain, resulted in a dramatic rise in the transcription of all immunoproteasome subunit genes. The transcription of the gene that encodes the PA28α subunit of the PA28 regulatory complex was activated only after a single infection of monocytes with M. bovis BCG. Thus, vaccination with M. bovis BCG promotes the efficient activation of immunoproteasomal genes in the case of subsequent contact with the virulent M. tuberculosis strain H37Rv.

Anatomically Restricted Synergistic Antiviral Activities of Innate and Adaptive Immune Cells in the Skin

Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci and numerous migratory dermal inflammatory monocytes that outlie the foci. We observed Ly6G(+) innate immune cells infiltrating and controlling foci, while CD8(+) T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8(+) T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8(+) T cells into foci, modestly reducing viral titers. Depletion of Ly6G(+) and CD8(+) cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.

The Myeloid Transcription Factor GATA-2 Regulates the Viral UL144 Gene during Human Cytomegalovirus Latency in an Isolate-Specific Manner

It is generally accepted that, following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in CD34(+) progenitor cells and other derivative cells of the myeloid lineage. In this study, we show that the viral UL144 gene is expressed during latent infection in two cell types of the myeloid lineage, CD34(+) and CD14(+) monocytes, and that the UL144 protein is functional in latently infected monocytes. However, this latency-associated expression of UL144 occurs only in certain isolates of HCMV and depends on the presence of functional GATA-2 transcription factor binding sites in the UL144 promoter, in contrast to the viral latency-associated gene LUNA, which we also show is regulated by GATA-2 but expressed uniformly during latent infection independent of the virus isolate. Taken together, these data suggest that the HCMV latency-associated transcriptome may be virus isolate specific and dependent on the repertoire of transcription factor binding sites in the promoters of latency-associated genes.

Ebola Virus Exploits a Monocyte Differentiation Program To Promote Its Entry

Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo. However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.

Granzyme A Produced by γ9δ2 T Cells Induces Human Macrophages to Inhibit Growth of an Intracellular Pathogen

Human γ9δ2 T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ9δ2 T cells produced soluble factors that could pass through 0.45 µm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ9δ2 T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ9δ2 T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ9δ2 T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ9δ2 T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ9δ2 T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections.

Активация транскрипции генов субъединиц иммунопротеасомы в моноцитах мышей при заражении различными штаммами микобактерий

Immunoproteasomal processing of mycobacterial antigens is necessary to control the infection and to protect the organism from development of active form of tuberculosis. Here we investigate the activation of immunoproteasome subunit genes transcription in peritoneal monocytes of C57Bl/6 mice infected with vaccine M. bovis BCG and virulent strain M. tuberculosis H37Rv. The level of transcription of LMP2, LMP7, MECL1 subunits didn't increase for one and two days after a single infection. Two rounds of infection with BCG strain M. bovis led to enhancement of the only LMP7 subunit gene transcription. However after subsequent infection of monocytes with vaccine followed by virulent strain infection the dramatic rise of all immunoproteasomal subunit genes transcription was observed. Activation of transcription of the gene coding the PA28alpha subunit of regulatory complex PA28 was observed only after a single infection of monocytes with strain M. bovis BCG. Thus, vaccination with strain M. bovis BCG promotes effective activation of immunoproteasomal genes in case of subsequent contact with virulent strain M. tuberculosis H37Rv.

Human immunodeficiency virus type 1 infection alters enzymatic and ultrastructural features of peripheral blood monocytes

INTRODUCTION:Human immunodeficiency virus-1 (HIV-1) infected monocytes are now believed to serve as a reservoir for HIV-1 infection, and to play a role in viral rebound phenomena in certain groups of patients who failed or stopped highly active antiretroviral therapy (HAART). Data characterizing the morphological changes of peripheral blood monocytes in HIV-1-infected individuals are limited.MATERIALS AND METHODS:In this study, we collected monocytes from 21 asymptomatic HIV-1-infected individuals with CD4 count more than 500 cells/mm3 and healthy individuals. The monocytes ultrastructural morphologic changes and α-naphthyl butyrate esterase (ANBE) activity were compared between the two groups.RESULTS:In monocytes from patients infected with HIV-1, activity of α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients. α-naphthyl butyrate esterase (ANBE) was markedly increased compared with normal monocytes. In both light microscopic and ultrastructural studies, the cytoplasm of monocytes from HIV-1-infected patients contained a haphazard appearing network of thin fibrils. Cell surface expression of the activation marker HLA-DR molecule was upregulated. There were no discernible differences between the cell surface expression of CD4, CD14, and CD16 molecules comparing normal monocytes to those from HIV-1-infected patients.CONCLUSIONS:Possibly, changes in the activity of ANBE along with a disrupted appearing cytoplasmic fibril network contribute to monocyte dysfunction in HIV-1-infected patients.

Pathology of Experimental Aerosol Zaire Ebolavirus Infection in Rhesus Macaques

There is limited knowledge of the pathogenesis of human ebolavirus infections and no reported human cases acquired by the aerosol route. There is a threat of ebolavirus as an aerosolized biological weapon, and this study evaluated the pathogenesis of aerosol infection in 18 rhesus macaques. Important and unique findings include early infection of the respiratory lymphoid tissues, early fibrin deposition in the splenic white pulp, and perivasculitis and vasculitis in superficial dermal blood vessels of haired skin with rash. Initial infection occurred in the respiratory lymphoid tissues, fibroblastic reticular cells, dendritic cells, alveolar macrophages, and blood monocytes. Virus spread to regional lymph nodes, where significant viral replication occurred. Virus secondarily infected many additional blood monocytes and spread from the respiratory tissues to multiple organs, including the liver and spleen. Viremia, increased temperature, lymphocytopenia, neutrophilia, thrombocytopenia, and increased alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, total bilirubin, serum urea nitrogen, creatinine, and hypoalbuminemia were measurable mid to late infection. Infection progressed rapidly with whole-body destruction of lymphoid tissues, hepatic necrosis, vasculitis, hemorrhage, and extravascular fibrin accumulation. Hypothermia and thrombocytopenia were noted in late stages with the development of disseminated intravascular coagulation and shock. This study provides unprecedented insight into pathogenesis of human aerosol Zaire ebolavirus infection and suggests development of a medical countermeasure to aerosol infection will be a great challenge due to massive early infection of respiratory lymphoid tissues. Rhesus macaques may be used as a model of aerosol infection that will allow the development of lifesaving medical countermeasures under the Food and Drug Administration's animal rule.

BET bromodomain inhibition as a novel strategy for reactivation of HIV-1

The persistence of latent HIV-1 remains a major challenge in therapeutic efforts to eradicate infection. We report the capacity for HIV reactivation by a selective small molecule inhibitor of BET family bromodomains, JQ1, a promising therapeutic agent with antioncogenic properties. JQ1 reactivated HIV transcription in models of latent T cell infection and latent monocyte infection. We also tested the effect of exposure to JQ1 to allow recovery of replication-competent HIV from pools of resting CD4(+) T cells isolated from HIV-infected, ART-treated patients. In one of three patients, JQ1 allowed recovery of virus at a frequency above unstimulated conditions. JQ1 potently suppressed T cell proliferation with minimal cytotoxic effect. Transcriptional profiling of T cells with JQ1 showed potent down-regulation of T cell activation genes, including CD3, CD28, and CXCR4, similar to HDAC inhibitors, but JQ1 also showed potent up-regulation of chromatin modification genes, including SIRT1, HDAC6, and multiple lysine demethylases (KDMs). Thus, JQ1 reactivates HIV-1 while suppressing T cell activation genes and up-regulating histone modification genes predicted to favor increased Tat activity. Thus, JQ1 may be useful in studies of potentially novel mechanisms for transcriptional control as well as in translational efforts to identify therapeutic molecules to achieve viral eradication.

The role of accessory proteins in the replication of feline infectious peritonitis virus in peripheral blood monocytes

The ability to productively infect monocytes/macrophages is the most important difference between the low virulent feline enteric coronavirus (FECV) and the lethal feline infectious peritonitis virus (FIPV). In vitro, the replication of FECV in peripheral blood monocytes always drops after 12h post inoculation, while FIPV sustains its replication in the monocytes from 45% of the cats. The accessory proteins of feline coronaviruses have been speculated to play a prominent role in virulence as deletions were found to be associated with attenuated viruses. Still, no functions have been ascribed to them. In order to investigate if the accessory proteins of FIPV are important for sustaining its replication in monocytes, replication kinetics were determined for FIPV 79-1146 and its deletion mutants, lacking either accessory protein open reading frame 3abc (FIPV-Δ3), 7ab (FIPV-Δ7) or both (FIPV-Δ3Δ7). Results showed that the deletion mutants FIPV-Δ7 and FIPV-Δ3Δ7 could not maintain their replication, which was in sharp contrast to wt-FIPV. FIPV-Δ3 could still sustain its replication, but the percentage of infected monocytes was always lower compared to wt-FIPV. In conclusion, this study showed that ORF7 is crucial for FIPV replication in monocytes/macrophages, giving an explanation for its importance in vivo, its role in the development of FIP and its conservation in field strains. The effect of an ORF3 deletion was less pronounced, indicating only a supportive role of ORF3 encoded proteins during the infection of the in vivo target cell by FIPVs.

Human cytomegalovirus induction of a unique signalsome during viral entry into monocytes mediates distinct functional changes: a strategy for viral dissemination

HCMV pathogenesis is a direct consequence of the hematogenous dissemination of the virus to multiple host organ sites. The presence of infected monocytes in the peripheral blood and organs of individuals exhibiting primary HCMV infection have long suggested that these blood sentinels are responsible for mediating viral spread. Despite monocytes being "at the right place at the right time", their short lifespan and the lack of productive viral infection in these cells complicate this scenario of a monocyte-driven approach to viral dissemination by HCMV. However, our laboratory has provided evidence that HCMV infection is able to induce a highly controlled polarization of monocytes toward a unique and long-lived proinflammatory macrophage, which we have demonstrated to be permissive for viral replication. These observations suggest that HCMV has evolved as a distinct mechanism to induce select proinflammatory characteristics that provide infected monocytes with the necessary tools to mediate viral spread following a primary infection. In the absence of viral gene products during the early stages of infection, the process by which HCMV "tunes" the inflammatory response in infected monocytes to promote viral spread and subsequently, viral persistence remains unclear. In this current review, we focus on the viral entry process of HCMV and the potential role of receptor-ligand interactions in modulating monocyte biology. Specifically, we examine the signaling pathways initiated by the distinct combination of cellular receptors simultaneously engaged and activated by HCMV during viral entry and how the acquisition of this distinct signalsome results in a nontraditional activation of monocytes leading to the induction of the unique, functional attributes observed in monocytes following HCMV infection.

60-OR

Aim Mycobacterium tuberculosis (Mtb) remains a world health threat, with 8.8 million cases reported in 2010, 65% of which were new and recurrent cases of tuberculosis. HLA mediated cellular immune responses are critical for controlling Mtb and recent studies have identified CD8+ T cells as important in controlling infection. Little is known about the minimal Mtb peptide ligands presented during an infection. The objective of this study is to biochemically purify ligands from the HLA of cells infected by Mtb to identify ligands distinct to infected cells. Methods 20 mg of soluble HLA-A ∗ 02:01 were harvested from Mtb infected THP-1 monocyte cells. Peptides were eluted from A ∗ 02:01, fractionated by HPLC, and comparatively analyzed using mass spectrometry (MS). Peptides unique to Mtb infected cells were identified, sequenced by tandem MS, and tested for immunogenicity in an IFNγ ELISOPT assay using PBMC from patients with acute and latent Mtb. Results Five novel Mtb derived ligands from five different Mtb open reading frames were presented by HLA-A ∗ 02:01 during infection. Patient T cells responded to four of the peptides. One peptide, LIL9 from a hypothetical Mtb protein, was dominant relative to the other four peptides, greater in both immunogenicity and in the number of Mtb patients with a response. A ligand from arsA, IMI10, only showed a measurable CD8 T-cell response in patients with latent tuberculosis infection. Conclusions We report the identification and characterization of 5 novel Mtb derived ligands, 4 of which were identified as CD8 T-cell epitopes. We further identify an epitope, who’s specific T-cells are only detectable in patients with latent tuberculosis infection. The reactive Mtb ligands identified not only represent novel epitopes, but also give us a glimpse of what Mtb proteins are processed and presented by class I HLA.

The diverse roles of monocytes in inflammation caused by protozoan parasitic diseases

The mononuclear phagocyte system (MPS), comprising monocytes, macrophages, and dendritic cells (DCs), plays an important role in the control of disease, but can also contribute to the establishment of persistent infections. Monocytes are derived from progenitors in the bone marrow. Once in circulation, they migrate into the tissues, differentiate into macrophages, and phagocytose microbes under inflammatory conditions. However, recent work shows they not only act as phagocytes, but are also precursors for particular types of DCs, inflammatory macrophages, and tissue macrophages. The role of monocytes during inflammation in models of bacterial and viral infections, cancer, atherosclerosis, and autoimmunity has been widely studied and reported. In this review we focus on the less understood role of monocytes in protozoan infections.

P102 Differential antiviral effect of porcine interferon alpha subtypes on classical swine fever virus infection of porcine monocytes

IntroductionClassical swine fever virus (CSFV) causes a serious disease of pigs that is a major threat to pork production industries worldwide [1]. The interferons (IFNs) elicit a variety of antiviral effects and recombinant IFNs have been shown to be prophylactically and therapeutically effective against virus infections [2,3]. Hence we are investigating the potential use of recombinant IFNs as a useful tool in the control of CSFV. We have previously demonstrated that pre-treating monocytes with recombinant porcine (Po) IFN-α subtype 1 and PoIFN-γ resulted in a reduction of CSFV infected monocytes [4]. As there are currently 17 different PoIFN-α subtypes, we investigated whether there are differences in anti-CSFV activity between the subtypes. MethodsHis-tagged recombinant PoIFN-α2–PoIFN-α16 proteins were expressed in Chinese hamster ovary (CHO) cells, affinity purified from culture supernatants and detected by western blot analysis. The activity of the purified proteins was determined with a vesicular stomatitis virus (VSV) bioassay [5] using PK-15 cells and a commercial PoIFN-α1 as a reference standard. CD14 positive porcine monocytes were pre-treated for 24h with standardised quantities of recombinant PoIFN-α subtypes, either alone or in combination. Cells were then cultured for a further 24h in the presence of CSFV and infection was assessed by flow cytometry [4]. ResultsPre-treatment of porcine monocytes with PoIFNα-2, 3, 4, 6, 8, 9, 10, 12, 13, 14, 16 and 17 was found to inhibit CSFV infection, with 20U/ml reducing infection of monocytes to less than 10%, compared to 55% infection in untreated cells. Treatment with poIFN-α15 reduced the percentage of cells infected, but was significantly less effective than the other 12 active subtypes. In contrast, treatment with poIFN-α5 and 7 did not result in any notable reduction in the percentage of monocytes infected by CSFV. A combination of PoIFN-α15 with PoIFN-γ or with PoIFN-α9 and -β enhanced the antiviral effect of PoIFNα15 against CSFV. ConclusionTreatment of monocytes with the majority of porcine IFN-α subtypes effectively induced protection against CSFV infection. Interestingly, two subtypes; poIFN-α5 and 7, had very little anti-CSFV activity, probably due to the virus blocking downstream signalling events of these IFNs. The limited activity of PoIFN-α15 was shown to be potentiated by combination with other type I or II IFNs. Ongoing studies are investigating which sub-type or subtype combination would be most suited to protection of pigs against CSFV infection.

Francisella tularensisregulates the expression of the amino acid transporter SLC1A5 in infected THP-1 human monocytes

Francisella tularensis, a Gram-negative bacterium that causes the disease tularemia in a large number of animal species, is thought to reside preferentially within macrophages in vivo. F. tularensis has developed mechanisms to rapidly escape from the phagosome into the cytoplasm of infected cells, a habitat with a rich supply of nutrients, ideal for multiplication. SLC1A5 is a neutral amino acid transporter expressed by human cells, which serves, along with SLC7A5 to equilibrate cytoplasmic amino acid pools. We herein analysed whether SLC1A5 was involved in F. tularensis intracellular multiplication. We demonstrate that expression of SLC1A5 is specifically upregulated by F. tularensis in infected THP-1 human monocytes. Furthermore, we show that SLC1A5 downregulation decreases intracellular bacterial multiplication, supporting the involvement of SLC1A5 in F. tularensis infection. Notably, after entry of F. tularensis into cells and during the whole infection, the highly glycosylated form of SLC1A5 was deglycosylated only by bacteria capable of cytosolic multiplication. These data suggest that intracellular replication of F. tularensis depends on the function of host cell SLC1A5. Our results are the first, which show that Francisella intracellular multiplication in human monocyte cytoplasm is associated with a post-translational modification of a eukaryotic amino acid transporter.