Interferon‐inducible protein,P56, inhibits HPV DNA replication by binding to the viral protein E1

Type I interferon (IFN) inhibits, by an unknown mechanism, the replication of human papillomaviruses (HPV), which are major human pathogens, Here, we present evidence that P56 (a protein), the expression of which is strongly induced by IFN, double‐stranded RNA and viruses, mediates the anti‐HPV effect of IFN. Ectopic expression of P56 inhibited HPV DNA replication and its ablation in IFN‐treated cells alleviated the inhibitory effect of IFN on HPV DNA replication. Protein–protein interaction and mutational analyses established that the antiviral effect of P56 was mediated by its direct interaction with the DNA replication origin‐binding protein E1 of several strains of HPV, through the tetratricopeptide repeat 2 in the N‐terminal region of P56 and the C‐terminal region of E1. In vivo, the interaction with P56, a cytoplasmic protein, caused translocation of E1 from the nucleus to the cytoplasm. In vitro, recombinant P56, or a small fragment derived from it, inhibited the DNA helicase activity of E1 and E1‐mediated HPV DNA replication. These observations delineate the molecular mechanism of IFN's antiviral action against HPV.     

Shp‐2 contributes to anti‐RSV activity in human pulmonary alveolar epithelial cells by interfering with the IFN‐α‐induced Jak/Stat1 pathway

Src homology phosphotyrosyl phosphatase 2 (Shp‐2) is a ubiquitously expressed protein that is involved in a variety of cellular processes, including antiviral interferon signalling pathways. In this study, we investigated the role of Shp‐2 in the host cell interactions of human respiratory syncytial virus (RSV). We report significant changes in the expression of Shp‐2 in human pulmonary alveolar epithelial cells (A549) upon RSV infection. We also report that blocking Shp‐2 does not affect viral replication or virus‐induced interferon‐alpha (IFN‐α) production. Interestingly, whereas A549 cells were activated by IFN‐α, the blocking of Shp‐2 resulted in increased viral replication that was associated with the reduced expression of the IFN‐stimulated genes of 2′,5′‐oligoadenylate synthetases and Mx1, and the concomitant inhibition of Stat1 tyrosine phosphorylation. Our findings suggest that Shp‐2 contributes to the control of RSV replication and progeny production in pulmonary alveolar epithelial cells by interfering with IFN‐α‐induced Jak/Stat1 pathway activation rather than by affecting the production of IFN‐α itself.     

Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1‐caspase‐1 axis

Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non‐structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11‐linked poly‐ubiquitin chains from caspase‐1 at Lys134, thus inhibiting the proteasomal degradation of caspase‐1. The enhanced stabilization of caspase‐1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future.     

Role of Autophagy in Innate Viral Recognition

Plasmacytoid dendritic cells (pDCs) detect viruses in the acidified endosomes via Toll-like receptors (TLRs) upon endocytosis of virions. Yet, pDC responses to certain single-stranded RNA viruses occur only following live viral infection. In our recent study, we presented evidence that the recognition of such viruses by TLR7 requires autophagy. We speculate that the requirement for autophagy in viral recognition reflects the necessity for transportation of cytosolic viral replication intermediates into the lysosome where TLR7 is activated. In addition, autophagy was found to be required for pDCs to produce type I interferon (IFN) in response to both ssRNA and dsDNA viruses. These results indicated that autophagy plays a key role in mediating virus detection and IFNα secretion in pDCs, and suggest that cytosolic replication intermediates of ssRNA viruses serve as pathogen signatures recognized by TLR7.

Addendum to:

Autophagy-Dependent Viral Recognition by Plasmacytoid Dendritic Cells

H.K. Lee, J.M. Lund, B. Ramanathan, N. Mizushima and A. Iwasaki

Science 2007; In press     

Impact of HIV on cell survival and antiviral activity of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are important mediators of innate immunity that act mainly through secretion of interferon (IFN)-alpha. Previous studies have found that these cells can suppress HIV in vitro; additionally, pDCs have been shown to be severely reduced in the peripheral blood of HIV-infected individuals. In the present study, we sought to determine the ability of pDCs to directly suppress viral replication ex vivo and to delineate the potential mechanisms whereby pDCs are depleted in HIV-infected individuals. We demonstrate that activated pDCs strongly suppress HIV replication in autologous CD4(+) T cells via a mechanism involving IFN-alpha as well as other antiviral factors. Of note, unstimulated pDCs from infected individuals who maintain low levels of plasma viremia without antiretroviral therapy were able to suppress HIV ex vivo via a mechanism requiring cell-to-cell contact. Our data also demonstrate that death of pDCs by both apoptosis and necrosis is induced by fusion of HIV with pDCs. Taken together, our data suggest that pDCs play an important role in the control of HIV replication and that high levels of viral replication in vivo are associated with pDC cell death via apoptosis and necrosis. Elucidation of the mechanism by which pDCs suppress HIV replication in vivo may have clinically relevant implications for future therapeutic strategies.     

Immature cell populations and an erythropoiesis gene-expression signature in systemic juvenile idiopathic arthritis: implications for pathogenesis

Introduction

Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IκB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs.

Methods

We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-α production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-α production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U.

Results

Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-α production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-α production, more strongly inhibited IFN-α production than TNF-α production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-α production by human PBMCs induced by the SLE serum and the in vivo serum IFN-α level induced by injecting mice with poly U.

Conclusions

These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.     

AKT isoforms modulate Th1‐like Treg generation and function in human autoimmune disease

Foxp3⁺ regulatory T cells (Tregs) exhibit plasticity, which dictates their function. Secretion of the inflammatory cytokine IFNγ, together with the acquisition of a T helper 1 (Th1)‐like effector phenotype as observed in cancer, infection, and autoimmune diseases, is associated with loss of Treg suppressor function through an unknown mechanism. Here, we describe the signaling events driving the generation of human Th1‐Tregs. Using a genome‐wide gene expression approach and pathway analysis, we identify the PI3K/AKT/Foxo1/3 signaling cascade as the major pathway involved in IFNγ secretion by human Tregs. Furthermore, we describe the opposing roles of AKT isoforms in Th1‐Treg generation ex vivo. Finally, we employ multiple sclerosis as an in vivo model with increased but functionally defective Th1‐Tregs. We show that the PI3K/AKT/Foxo1/3 pathway is activated in ex vivo‐isolated Tregs from untreated relapsing–remitting MS patients and that blockade of the pathway inhibits IFNγ secretion and restores the immune suppressive function of Tregs. These data define a fundamental pathway regulating the function of human Tregs and suggest a novel treatment paradigm for autoimmune diseases.     

Decreased TLR2 signal expression in peripheral blood mononuclear cell from patients with cryptococcal meningitis

Toll‐like receptors are the most important pattern recognition receptors that can recognize conserved molecular structures shared by large groups of pathogens. Here, the aim was to determine the expression and role of TLR2 in peripheral blood mononuclear cells (PBMCs) from patients with cryptococcal meningitis and healthy controls. TLR2 expression was measured using RT‐PCR and western blotting. The role of TLR2 in cytokine production by PBMCs after Cryptococcus neoformans exposure was assessed in healthy controls prior to incubation with anti‐TLR2. TLR2 mRNA and protein expression were both weaker in patients with cryptococcal meningitis than in healthy controls. Furthermore, pre‐incubation of PBMCs from healthy donors with anti‐TLR2 led to reduced expression of IFN‐γ and IL‐12p70, but not of IL‐4 and IL‐10, following C. neoformans stimulation. Our results suggest that impaired expression of TLR2 may be involved in defective host defense to C. neoformans through an attenuated Th1 response.     

Non‐invasive imaging of allogeneic transplanted skin graft by 131I‐anti‐TLR5 mAb

Although ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) uptake can be used for the non‐invasive detection and monitoring of allograft rejection by activated leucocytes, this non‐specific accumulation is easily impaired by immunosuppressants. Our aim was to evaluate a ¹³¹I‐radiolabelled anti‐Toll‐like receptor 5 (TLR5) mAb for non‐invasive in vivo graft visualization and quantification in allogeneic transplantation mice model, compared with the non‐specific radiotracer ¹⁸F‐FDG under using of immunosuppressant. Labelling, binding, and stability studies were performed. BALB/c mice transplanted with C57BL/6 skin grafts, with or without rapamycin treatment (named as allo‐treated group or allo‐rejection group), were injected with ¹³¹I‐anti‐TLR5 mAb, ¹⁸F‐FDG, or mouse isotype ¹³¹I‐IgG, respectively. Whole‐body phosphor‐autoradiography and ex vivo biodistribution studies were obtained. Whole‐body phosphor‐autoradiography showed ¹³¹I‐anti‐TLR5 mAb uptake into organs that were well perfused with blood at 1 hr and showed clear graft images from 12 hrs onwards. The ¹³¹I‐anti‐TLR5 mAb had significantly higher graft uptake and target‐to‐non‐target ratio in the allo‐treated group, as determined by semi‐quantification of phosphor‐autoradiography images; these results were consistent with ex vivo biodistribution studies. However, high ¹⁸F‐FDG uptake was not observed in the allo‐treated group. The highest allograft‐skin‐to‐native‐skin ratio (A:N) of ¹³¹I‐anti‐TLR5 mAb uptake was significantly higher than the ratio for ¹⁸F‐FDG (7.68 versus 1.16, respectively). ¹³¹I‐anti‐TLR5 mAb uptake in the grafts significantly correlated with TLR5 expression in the allograft area. The accumulation of ¹³¹I‐IgG was comparable in both groups. We conclude that radiolabelled anti‐TLR5 mAb is capable of detecting allograft with high target specificity after treatment with the immunosuppressive drug rapamycin.     

The activation of liver X receptors inhibits toll‐like receptor‐9‐induced foam cell formation

Toll‐like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG‐ODN, TLR9 agonist, and oxidized particles of LDL (Paz‐PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG‐ODN plus Paz‐PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)‐α, IFNβ, and IP‐10. The underlying mechanism by which TLR9 ligation influenced Paz‐PC in the FCF was NF‐κB‐ and IRF7‐dependent, as observed by higher levels of phosphorylated IκBα, increased nuclear translocation of the p65 subunit, lower levels of the total IKKα protein and higher release of interferon‐dependent cytokines, such as IP‐10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR‐dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG‐ODN plus Paz‐PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRα into the nucleus. TNF‐α, IP‐10, and IFNβ levels were reduced by the administration of GW3965 following CpG‐ODN and Paz‐PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF‐κB‐ and IRF7‐dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti‐atherosclerotic target. J. Cell. Physiol. 223: 158–167, 2010. © 2009 Wiley‐Liss, Inc.     

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4⁺ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4⁺ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-α/β cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.     

A G1‐like state allows HIV‐1 to bypass SAMHD1 restriction in macrophages

An unresolved question is how HIV‐1 achieves efficient replication in terminally differentiated macrophages despite the restriction factor SAMHD1. We reveal inducible changes in expression of cell cycle‐associated proteins including MCM2 and cyclins A, E, D1/D3 in macrophages, without evidence for DNA synthesis or mitosis. These changes are induced by activation of the Raf/MEK/ERK kinase cascade, culminating in upregulation of CDK1 with subsequent SAMHD1 T592 phosphorylation and deactivation of its antiviral activity. HIV infection is limited to these G1‐like phase macrophages at the single‐cell level. Depletion of SAMHD1 in macrophages decouples the association between infection and expression of cell cycle‐associated proteins, with terminally differentiated macrophages becoming highly susceptible to HIV‐1. We observe both embryo‐derived and monocyte‐derived tissue‐resident macrophages in a G1‐like phase at frequencies approaching 20%, suggesting how macrophages sustain HIV‐1 replication in vivo. Finally, we reveal a SAMHD1‐dependent antiretroviral activity of histone deacetylase inhibitors acting via p53 activation. These data provide a basis for host‐directed therapeutic approaches aimed at limiting HIV‐1 burden in macrophages that may contribute to curative interventions.     

Herpes simplex virus type 1 dysregulates anti‐fungal defenses preventing monocyte activation and downregulating toll‐like receptor‐2

We investigated the interplay occurring between pathogens in the course of dual infections, using an in vitro model in which the THP‐1 monocytic cell line is first infected with HSV‐1 and then exposed to Ca or Cn. These three pathogens share some pathogenic features: they cause opportunistic infections, target macrophages and are neurotropic. Here, we show that HSV‐1‐infected THP‐1 cells exhibited augmented phagocytosis against the two opportunistic fungi but reduced capability to counteract fungal infection: the better ingestion by monocytes was followed by facilitated fungal survival and replication. Reduced IL‐12 production was also observed. Cytofluorimetric analysis showed that HSV‐1‐infected monocytes exhibit: (i) downregulated TLR‐2 and TLR‐4, critical structures in fungal recognition; (ii) reduced expression of CD38 and CD69, known to be important markers of monocyte activation; and (iii) enhanced expression of apoptosis and necrosis markers, in the absence of altered cell proliferation. Overall, these findings imply that HSV‐1 infection prevents monocyte activation, thus leading to a significant dysfunction of the monocyte‐mediated anti‐Candida response; HSV‐1 induced apoptosis and necrosis of monocytes further contribute to this impairment.     

Inhibition of cytokine response to TLR stimulation and alleviation of collagen‐induced arthritis in mice by Schistosoma japonicum peptide SJMHE1

Helminth‐derived products have recently been shown to prevent the development of inflammatory diseases in mouse models. However, most identified immunomodulators from helminthes are mixtures or macromolecules with potentially immunogenic side effects. We previously identified an immunomodulatory peptide called SJMHE1 from the HSP60 protein of Schistosoma japonicum. In this study, we assessed the ability of SJMHE1 to affect murine splenocytes and human peripheral blood mononuclear cells (PBMCs) stimulated by toll‐like receptor (TLR) ligands in vitro and its treatment effect on mice with collagen‐induced arthritis (CIA). We show that SJMHE1 not only modulates the cytokine production of murine macrophage (MΦ) and dendritic cell but also affects cytokine production upon coculturing with allogeneic CD4⁺ T cell. SJMHE1 potently inhibits the cytokine response to TLR ligands lipopolysaccharide (LPS), CpG oligodeoxynucleotides (CpG) or resiquimod (R848) from mouse splenocytes, and human PBMCs stimulated by LPS. Furthermore, SJMHE1 suppressed clinical signs of CIA in mice and blocked joint erosion progression. This effect was mediated by downregulation of key cytokines involved in the pathogenesis of CIA, such as interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐6, IL‐17, and IL‐22 and up‐regulation of the inhibitory cytokine IL‐10, Tgf‐β1 mRNA, and CD4⁺CD25⁺Foxp3⁺ Tregs. This study provides new evidence that the peptide from S. japonicum, which is the ‘safe’ selective generation of small molecule peptide that has evolved during host–parasite interactions, is of great value in the search for novel anti‐inflammatory agents and therapeutic targets for autoimmune diseases.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.