Recognition of HIV-1-infected fibrocytes lacking Nef-mediated HLA-B downregulation by HIV-1-specific T cells.

Fibrocytes were reported to be host cells for HIV-1, but the immunological recognition of HIV-1-infected fibrocytes has not been studied. Here, we investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific CD8+ T cells. CD8+ T cells specific for five HIV-1 epitopes (HLA-A*24:02-restricted, HLA-B*52:01-restricted, and HLA-C*12:02-restricted epitopes) produced IFN-γ and expressed CD107a after coculture with HIV-1-infected fibrocytes. HIV-1-infected fibrocytes were effectively killed by HIV-1-specific CD8+ T cells. Although it is well known that HIV-1 Nef-mediated downregulation of HLA-A and HLA-B critically affects the T cell recognition of HIV-1-infected CD4+ T cells and HIV-1-infected macrophages, Nef downregulated HLA-A, but not HLA-B, in HIV-1-infected fibrocytes. These findings suggested that HIV-1-specific CD8+ T cells could recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells or HIV-1-infected macrophages. HIV-1-infected fibrocytes were also recognized by HIV-1-specific HLA-DR-restricted T cells, indicating that HIV-1-infected fibrocytes can present HIV-1 epitopes to helper T cells. Collectively, these findings suggest that fibrocytes have an important role as antigen-presenting cells during HIV-1 infection. The present study demonstrates effective recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells and suggests possible roles of fibrocytes in the induction and maintenance of HIV-1-specific T cells. IMPORTANCE: Fibrocytes were identified as unique hematopoietic cells with the features of both macrophages and fibroblasts and were demonstrated to be host cells for HIV-1. However, T cell recognition of HIV-1-infected fibrocytes has not been studied. We investigated the recognition of HIV-1-infected fibrocytes by HIV-1-specific T cells. HIV-1-infected fibrocytes were effectively recognized and killed by CD8+ T cells specific for HIV-1 epitopes presented by HLA-A, HLA-B, or HLA-C and were recognized by HIV-1-specific HLA-DR-restricted CD4+ T cells. HIV-1 Nef-mediated downregulation of HLA-A and HLA-B was found in HIV-1-infected CD4+ T cells, whereas Nef did not downregulate HLA-B in HIV-1-infected fibrocytes. These results suggest that HIV-1-specific CD8+ T cells recognize HIV-1-infected fibrocytes more strongly than HIV-1-infected CD4+ T cells. The present study suggests the importance of fibrocytes in the induction and maintenance of HIV-1-specific T cells.

M-Sec induced by HTLV-1 mediates an efficient viral transmission.

Human T-cell leukemia virus type 1 (HTLV-1) infects target cells primarily through cell-to-cell routes. Here, we provide evidence that cellular protein M-Sec plays a critical role in this process. When purified and briefly cultured, CD4+ T cells of HTLV-1 carriers, but not of HTLV-1- individuals, expressed M-Sec. The viral protein Tax was revealed to mediate M-Sec induction. Knockdown or pharmacological inhibition of M-Sec reduced viral infection in multiple co-culture conditions. Furthermore, M-Sec knockdown reduced the number of proviral copies in the tissues of a mouse model of HTLV-1 infection. Phenotypically, M-Sec knockdown or inhibition reduced not only plasma membrane protrusions and migratory activity of cells, but also large clusters of Gag, a viral structural protein required for the formation of viral particles. Taken together, these results suggest that M-Sec induced by Tax mediates an efficient cell-to-cell viral infection, which is likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag.

M-Sec facilitates intercellular transmission of HIV-1 through multiple mechanisms.

BACKGROUND: HIV-1 promotes the formation of tunneling nanotubes (TNTs) that connect distant cells, aiding cell-to-cell viral transmission between macrophages. Our recent study suggests that the cellular protein M-Sec plays a role in these processes. However, the timing, mechanism, and to what extent M-Sec contributes to HIV-1 transmission is not fully understood, and the lack of a cell line model that mimics macrophages has hindered in-depth analysis. RESULTS: We found that HIV-1 increased the number, length and thickness of TNTs in a manner dependent on its pathogenic protein Nef and M-Sec in U87 cells, as observed in macrophages. In addition, we found that M-Sec was required not only for TNT formation but also motility of U87 cells, both of which are beneficial for viral transmission. In fact, M-Sec knockdown in U87 cells led to a significantly delayed viral production in both cellular and extracellular fractions. This inhibition was observed for wild-type virus, but not for a mutant virus lacking Nef, which is known to promote not only TNT formation but also migration of infected macrophages. CONCLUSIONS: By taking advantage of useful features of U87 cells, we provided evidence that M-Sec mediates a rapid and efficient cell-cell transmission of HIV-1 at an early phase of infection by enhancing both TNT formation and cell motility.

Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages.

Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.

Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages.

Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.

Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus.

UNLABELLED: Multiple host molecules are known to be involved in the cellular entry of filoviruses, including Ebola virus (EBOV); T-cell immunoglobulin and mucin domain 1 (TIM-1) and Niemann-Pick C1 (NPC1) have been identified as attachment and fusion receptors, respectively. However, the molecular mechanisms underlying the entry process have not been fully understood. We found that TIM-1 and NPC1 colocalized and interacted in the intracellular vesicles where EBOV glycoprotein (GP)-mediated membrane fusion occurred. Interestingly, a TIM-1-specific monoclonal antibody (MAb), M224/1, prevented GP-mediated membrane fusion and also interfered with the binding of TIM-1 to NPC1, suggesting that the interaction between TIM-1 and NPC1 is important for filovirus membrane fusion. Moreover, MAb M224/1 efficiently inhibited the cellular entry of viruses from all known filovirus species. These data suggest a novel mechanism underlying filovirus membrane fusion and provide a potential cellular target for antiviral compounds that can be universally used against filovirus infections. IMPORTANCE: Filoviruses, including Ebola and Marburg viruses, cause rapidly fatal diseases in humans and nonhuman primates. There are currently no approved vaccines or therapeutics for filovirus diseases. In general, the cellular entry step of viruses is one of the key mechanisms to develop antiviral strategies. However, the molecular mechanisms underlying the entry process of filoviruses have not been fully understood. In this study, we demonstrate that TIM-1 and NPC1, which serve as attachment and fusion receptors for filovirus entry, interact in the intracellular vesicles where Ebola virus GP-mediated membrane fusion occurs and that this interaction is important for filovirus infection. We found that filovirus infection and GP-mediated membrane fusion in cultured cells were remarkably suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and NPC1. Our data provide new insights for the development of antiviral compounds that can be universally used against filovirus infections.

A polymorphism of the TIM-1 IgV domain: implications for the susceptibility to filovirus infection.

Filoviruses, including Ebola and Marburg viruses, cause severe hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Human T-cell immunoglobulin and mucin domain 1 (TIM-1) is one of the host proteins that have been shown to promote filovirus entry into cells. In this study, we cloned TIM-1 genes from three different African green monkey kidney cell lines (Vero E6, COS-1, and BSC-1) and found that TIM-1 of Vero E6 had a 23-amino acid deletion and 6 amino acid substitutions compared with those of COS-1 and BSC-1. Interestingly, Vero E6 TIM-1 had a greater ability to promote the infectivity of vesicular stomatitis viruses pseudotyped with filovirus glycoproteins than COS-1-derived TIM-1. We further found that the increased ability of Vero E6 TIM-1 to promote virus infectivity was most likely due to a single amino acid difference between these TIM-1s. These results suggest that a polymorphism of the TIM-1 molecules is one of the factors that influence cell susceptibility to filovirus infection, providing a new insight into the molecular basis for the filovirus host range.

Suppression of Fas-mediated apoptosis via steric shielding by filovirus glycoproteins.

Apoptotic death of virus-infected cells is generally thought to be a defense mechanism to limit the spread of infectious virions by eliminating virus-producing cells in host animals. On the other hand, several viruses have been shown to have anti-apoptotic mechanisms to facilitate efficient viral replication and transmission. In this study, we found that the filovirus glycoprotein (GP) expressed on cell surfaces formed a steric shield over the Fas molecule and that GP-expressing cells showed resistance to cell death induced by a Fas agonistic antibody. These results suggest that filovirus GP-mediated steric shielding may interfere with the Fas-induced apoptotic signal transduction in infected cells and serve as an immune evasion mechanism for filoviruses.

Chimeric Newcastle Disease Virus Vectors Expressing Human IFN-γ Mediate Target Immune Responses and Enable Multifaceted Treatments.

The therapeutic potential of Newcastle disease virus (NDV) has been reported as both an oncolytic agent and a vaccine vector against many antigens. However, in the individuals already immunized with NDVs, second and subsequent administration does not provide substantial benefits. In this study, two types of recombinant chimeric NDVs using APMV-2 F and HN genes were generated. In rNDV-2HN, the wild-type NDV HN gene was replaced with the APMV-2 HN gene, and in rNDV-2F/2HN, both wild-type F and HN genes were replaced with APMV-2 F and HN genes, respectively. We enhanced the immune responses of these chimeric viruses by inserting the human IFN-γ gene. To examine the escape from NDV antiserum, each virus was treated with diluted NDV antiserum, and HEp-2 cells were infected with these virus particles. The two constructed chimeric viruses indicated notably lower virus-neutralizing titer compared to wild-type NDV and escaped the action of NDV antiserum. These two chimeric viruses infected both respiratory and colon cancer cell lines, indicating their potential as a cancer treatment tool. Chimeric viruses with enhanced immune responses can be considered a novel therapeutic strategy in cancer treatment that can be administered multiple times and used to enhance immune cells interaction.

An H5N1 highly pathogenic avian influenza virus that invaded Japan through waterfowl migration.

In 2010, an H5N1 highly pathogenic avian influenza virus (HPAIV) was isolated from feces of apparently healthy ducks migrating southward in Hokkaido, the northernmost prefecture of Japan. The H5N1 HPAIVs were subsequently detected in domestic and wild birds at multiple sites corresponding to the flyway of the waterfowl having stopovers in the Japanese archipelago. The Hokkaido isolate was genetically nearly identical to H5N1 HPAIVs isolated from swans in the spring of 2009 and 2010 in Mongolia, but less pathogenic in experimentally infected ducks than the 2009 Mongolian isolate. These findings suggest that H5N1 HPAIVs with relatively mild pathogenicity might be selected and harbored in the waterfowl population during the 2009-2010 migration seasons. Our data provide "early warning" signals for preparedness against the unprecedented situation in which the waterfowl reservoirs serve as perpetual sources and disseminators of HPAIVs.

Spontaneous and Immune Checkpoint Inhibitor-Induced Autoimmune Diseases: Analysis of Temporal Information by Using the Japanese Adverse Drug Event Report Database.

BACKGROUND AND OBJECTIVE: Immune checkpoint inhibitors (ICIs) such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitors have greatly improved cancer treatment. However, they are associated with immune-related adverse events, including autoimmune diseases (ADs) owing to their immune enhancement effect. As there are few comprehensive studies of ADs by ICIs, it is necessary to analyze the period information of drug-induced ADs. We also assumed that the temporal information may be useful to estimate the similarity of the pathogenic mechanism between spontaneous and ICI-induced ADs. METHODS: A period analysis including the Weibull analysis was performed on ICI-induced ADs using the Japanese Adverse Drug Event Report (JADER) database. For evaluating the similarity of spontaneous and ICI-induced ADs, a hierarchical cluster analysis was conducted to compare the different onset-time ranges. RESULTS: Type 1 diabetes mellitus, autoimmune colitis, and pemphigoid occurred earlier with CTLA-4 inhibitors (median: 46, 29.5 and 28 days, respectively) than with PD-1 inhibitors (> 130 days). Myasthenia gravis had a median time to onset of approximately 1 month, and the risk of onset would increase over time in ipilimumab combination therapy. This result reveals ADs that require attention. Using cluster analysis, we estimated six clusters with different patterns of onset times. Based on these results and a detailed previous research survey, the possible pathogenesis of drug-induced ADs was also discussed. CONCLUSIONS: This paper describes risk profiles with temporal information of ICI-induced ADs and proposes certain indicators for deciphering the mechanism of AD onset.

Tim4 recognizes carbon nanotubes and mediates phagocytosis leading to granuloma formation.

Macrophage recognition and phagocytosis of crystals is critical for the associated fibrosis and cancer. Of note, multi-walled carbon nanotubes (MWCNTs), the highly representative products of nanotechnology, induce macrophage NLRP3 inflammasome activation and cause asbestosis-like pathogenesis. However, it remains largely unknown how macrophages efficiently recognize MWCNTs on their cell surfaces. Here, we identify by a targeted screening of phagocyte receptors the phosphatidylserine receptors T cell immunoglobulin mucin 4 (Tim4) and Tim1 as the pattern-recognition receptors for carbon crystals. Docking simulation studies reveal spatiotemporally stable interfaces between aromatic residues in the extracellular IgV domain of Tim4 and one-dimensional carbon crystals. Further, CRISPR-Cas9-mediated deletion of Tim4 and Tim1 reveals that Tim4, but not Tim1, critically contributes to the recognition of MWCNTs by peritoneal macrophages and to granuloma development in a mouse model of direct mesothelium exposure to MWCNTs. These results suggest that Tim4 recognizes MWCNTs through aromatic interactions and mediates phagocytosis leading to granulomas.

C-type lectins do not act as functional receptors for filovirus entry into cells.

Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

Establishment of bone marrow-derived M-CSF receptor-dependent self-renewing macrophages.

Recent studies have revealed that tissue macrophages are derived from yolk sac precursors or fetal liver monocytes, in addition to bone marrow monocytes. The relative contribution of these cells to the tissue macrophage pool is not fully understood, but embryo-derived cells are supposed to be more important because of their capacity to self-renew. Here, we show the presence of adult bone marrow-derived macrophages that retain self-renewing capacity. The self-renewing macrophages were readily obtained by long-term culture of mouse bone marrow cells with macrophage colony-stimulating factor (M-CSF), a key cytokine for macrophage development. They were non-tumorigenic and proliferated in the presence of M-CSF in unlimited numbers. Despite several differences from non-proliferating macrophages, they retained many features of cells of the monocytic lineage, including the differentiation into dendritic cells or osteoclasts. Among the transcription factors involved in the self-renewal of embryonic stem cells, Krüppel-like factor 2 (KLF2) was strongly upregulated upon M-CSF stimulation in the self-renewing macrophages, which was accompanied by the downregulation of MafB, a transcription factor that suppresses KLF2 expression. Indeed, knockdown of KLF2 led to cell cycle arrest and diminished cell proliferation in the self-renewing macrophages. Our new cell model would be useful to unravel differences in phenotype, function, and molecular mechanism of proliferation among self-renewing macrophages with different origins.

Expression of IL-34 correlates with macrophage infiltration and prognosis of diffuse large B-cell lymphoma.

OBJECTIVES: Infiltration of macrophages through the tyrosine kinase receptor CSF1R is a poor prognosis factor in various solid tumors. Indeed, these tumors produce CSF1R ligand, macrophage colony-stimulating factor (M-CSF) or interleukin-34 (IL-34). However, the significance of these cytokines, particularly, the newly discovered IL-34 in haematological malignancies, is not fully understood. We therefore analysed the role of IL-34 in diffuse large B-cell lymphoma (DLBCL), the most common subtype of malignant lymphoma. METHODS: We analysed formalin-fixed paraffin-embedded lymphoma tissues of 135 DLBCL patients for the expression of IL-34 and the number of macrophages, and the survival of these patients. The expression of IL-34 in DLBCL cell lines and the activity of IL-34 to induce the migration of monocytic cells were also characterised. RESULTS: Several lymphoma tissues showed a clear IL-34 signal, and such signal was detectable in 36% of patients. DLBCL cell lines also expressed IL-34. Interestingly, the percentage of IL-34+ patients in the activated B-cell subtype was significantly higher than that in the germinal centre B-cell subtype. More interestingly, IL-34+ patients showed shorter survival periods and higher number of macrophages in lymphoma tissues. The recruitment of monocytes is likely the first step for the higher macrophage density in the IL-34+ lymphoma tissues. Indeed, IL-34 induced the migration of monocytic cells. CONCLUSION: Our results raise the possibility that IL-34 in lymphoma tissues of DLBCL patients recruits monocytes, leading to the higher number of macrophages in the tissues and poor prognosis of patients. IL-34 may be an additional therapeutic target of DLBCL.

Potential anti-lymphoma effect of M-CSFR inhibitor in adult T-cell leukemia/lymphoma.

The c-fms proto-oncogene is also known as macrophage colony stimulating factor receptor (M-CSFR) or colony-stimulating factor-1 receptor (CSF-1R), and is expressed on several types of malignant tumor cells and myeloid cells. In the present study, we found that overexpression of M-CSFR was present in adult T-cell leukemia/lymphoma (ATLL) cases. M-CSFR signaling was associated with lymphoma cell proliferation, and M-CSFR inhibition induced apoptosis in lymphoma cells. The ATLL cell line ATL-T expressed M-CSF/CSF-1 and interleukin (IL)-34, which are both M-CSFR ligands. M-CSF and IL-34 expression was seen in ATLL cases, and co-expression of these ligands was detected in 11 of 13 ATLL cases. M-CSFR inhibition suppressed programmed death-1 and -2 ligand in ATL-T cells and macrophages stimulated with conditioned medium from ATL-T cells. Thus, an M-CSFR inhibitor may be useful as additional therapy against ATLL due to direct and indirect mechanisms.

Discovery of an antibody for pan-ebolavirus therapy.

During the latest outbreak of Ebola virus disease in West Africa, monoclonal antibody therapy (e.g., ZMapp) was utilized to treat patients. However, due to the antigenic differences among the five ebolavirus species, the current therapeutic monoclonal antibodies are only effective against viruses of the species Zaire ebolavirus. Although this particular species has indeed caused the majority of human infections in Central and, recently, West Africa, other ebolavirus species (e.g., Sudan ebolavirus and Bundibugyo ebolavirus) have also repeatedly caused outbreaks in Central Africa and thus should not be neglected in the development of countermeasures against ebolaviruses. Here we report the generation of an ebolavirus glycoprotein-specific monoclonal antibody that effectively inhibits cellular entry of representative isolates of all known ebolavirus species in vitro and show its protective efficacy in mouse models of ebolavirus infections. This novel neutralizing monoclonal antibody targets a highly conserved internal fusion loop in the glycoprotein molecule and prevents membrane fusion of the viral envelope with cellular membranes. The discovery of this highly cross-neutralizing antibody provides a promising option for broad-acting ebolavirus antibody therapy and will accelerate the design of improved vaccines that can selectively elicit cross-neutralizing antibodies against multiple species of ebolaviruses.

Mapping of conserved and species-specific antibody epitopes on the Ebola virus nucleoprotein.

Filoviruses (viruses in the genus Ebolavirus and Marburgvirus in the family Filoviridae) cause severe haemorrhagic fever in humans and nonhuman primates. Rapid, highly sensitive, and reliable filovirus-specific assays are required for diagnostics and outbreak control. Characterisation of antigenic sites in viral proteins can aid in the development of viral antigen detection assays such immunochromatography-based rapid diagnosis. We generated a panel of mouse monoclonal antibodies (mAbs) to the nucleoprotein (NP) of Ebola virus belonging to the species Zaire ebolavirus. The mAbs were divided into seven groups based on the profiles of their specificity and cross-reactivity to other species in the Ebolavirus genus. Using synthetic peptides corresponding to the Ebola virus NP sequence, the mAb binding sites were mapped to seven antigenic regions in the C-terminal half of the NP, including two highly conserved regions among all five Ebolavirus species currently known. Furthermore, we successfully produced species-specific rabbit antisera to synthetic peptides predicted to represent unique filovirus B-cell epitopes. Our data provide useful information for the development of Ebola virus antigen detection assays.

Differential potential for envelope glycoprotein-mediated steric shielding of host cell surface proteins among filoviruses.

The viral envelope glycoprotein (GP) is thought to play important roles in the pathogenesis of filovirus infection. It is known that GP expressed on the cell surface forms a steric shield over host proteins such as major histocompatibility complex class I and integrin ß1, which may result in the disorder of cell-to-cell contacts and/or inhibition of the immune response. However, it is not clarified whether this phenomenon contributes to the pathogenicity of filoviruses. In this study, we found that the steric shielding efficiency differed among filovirus strains and was correlated with the difference in their relative pathogenicities. While the highly glycosylated mucin-like region of GP was indispensable, the differential shielding efficiency did not necessarily depend on the primary structure of the mucin-like region, suggesting the importance of the overall properties (e.g., flexibility and stability) of the GP molecule for efficient shielding of host proteins.