HCMV vCXCL1 Binds Several Chemokine Receptors and Preferentially Attracts Neutrophils over NK Cells by Interacting with CXCR2

Summary: HCMV is a highly sophisticated virus that has developed various mechanisms for immune evasion and viral dissemination throughout the body (partially mediated by neutrophils). NK cells play an important role in elimination of HCMV-infected cells. Both neutrophils and NK cells utilize similar sets of chemokine receptors to traffic, to and from, various organs. However, the mechanisms by which HCMV attracts neutrophils and not NK cells are largely unknown. Here, we show a unique viral protein, vCXCL1, which targets three chemokine receptors: CXCR1 and CXCR2 expressed on neutrophils and CXCR1 and CX3CR1 expressed on NK cells. Although vCXCL1 attracted both cell types, neutrophils migrated faster and more efficiently than NK cells through the binding of CXCR2. Therefore, we propose that HCMV has developed vCXCL1 to orchestrate its rapid systemic dissemination through preferential attraction of neutrophils and uses alternative mechanisms to counteract the later attraction of NK cells. : Viral CXCL1 (vCXCL1) is a chemokine produced following infection with human cytomegalovirus. Yamin et al. show here that vCXCL1 binds to three chemokine receptors: CXCR1, CXCR2, and CX3CR1 and that neutrophils migrate faster and more efficiently than NK cells toward vCXCL1 through binding to CXCR2 expressed by neutrophils only

Neuraminidase-Mediated, NKp46-Dependent Immune-Evasion Mechanism of Influenza Viruses

Natural killer (NK) cells play an essential role in the defense against influenza virus, one of the deadliest respiratory viruses known today. The NKp46 receptor, expressed by NK cells, is critical for controlling influenza infections, as influenza-virus-infected cells are eliminated through the recognition of the viral hemagglutinin (HA) protein by NKp46. Here, we describe an immune-evasion mechanism of influenza viruses that is mediated by the neuraminidase (NA) protein. By using various NA blockers, we show that NA removes sialic acid residues from NKp46 and that this leads to reduced recognition of HA. Furthermore, we provide in vivo and in vitro evidence for the existence of this NA-mediated, NKp46-dependent immune-evasion mechanism and demonstrate that NA inhibitors, which are commonly used for the treatment of influenza infections, are useful not only as blockers of virus budding but also as boosters of NKp46 recognition

KRT14 haploinsufficiency results in increased susceptibility of keratinocytes to TNF-alpha-induced apoptosis and causes Naegeli-Franceschetti-Jadassohn syndrome

Naegeli-Franceschetti-Jadassohn syndrome (NFJS) is a rare autosomal dominant disorder characterized by loss of dermatoglyphics, reticulate hyperpigmentation of the skin, palmoplantar keratoderma, abnormal sweating, and other developmental anomalies of the teeth, hair, and skin. We recently demonstrated that NFJS is caused by heterozygous nonsense or frameshift mutations in the E1/V1-encoding region of KRT14, but the mechanisms for their deleterious effects in NFJS remain elusive. In this study, we further expand the spectrum of NFJS-causing mutations and demonstrate that these mutations result in haploinsufficiency for keratin 14 (K14). As increased apoptotic activity was observed in the epidermal basal cell layer in NFJS patients and as previous data suggested that type I keratins may confer resistance to tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in epithelial tissues, we assessed the effect of down-regulation of KRT14 expression on apoptotic activity in keratinocytes. Using a HaCaT cell-based assay, we found that decreased KRT14 expression is associated with increased susceptibility to TNF-alpha-induced apoptosis. This phenomenon was not observed when cells were cultured in the presence of doxycycline, a known negative regulator of TNF-alpha-dependant pro-apoptotic signaling. Collectively, our results indicate that NFJS results from haploinsufficiency for K14 and suggest that increased susceptibility of keratinocytes to pro-apoptotic signals may be involved in the pathogenesis of this ectodermal dysplasia syndrome

vMIP-II does not block the IL-8-mediated migration.

<p>(A) Freshly isolated naïve NK cells were double stained with IL-8-Ig and with anti-CD56. The percentages of the various populations are indicated in the figure. (B) CXCR1 expression on the transfectant 293T-CXCR1 cells (black open histogram) or on the 293T parental cells (filled grey histogram). (C) Binding of IL-8-Ig (left histogram) or vMIP-II-Ig (right histogram) to 293T-CXCR1 transfectant (black open histogram) or to the 293T parental cells (filled grey histogram). (D) 293T-CXCR1 cells were incubated with (black empty histogram) and without (dark gray empty histogram) rvMIP-II for 1 hour in 4°C and then stained with IL-8-Ig. The light gray filled histogram is the staining of IL-8-Ig on the 293T parental cells. (E) Freshly isolated naïve NK cells were incubated at 4°C for 1 hour with and without the proteins indicated in the x axis. RhIL-8 was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). *P<0.05. NS - not significant. Figure shows one representative experiment out of two performed.</p

vMIP-II-Ig mainly recognizes the naïve CD56^Dim CD16^Pos NK cell population which express the CX3CR1 and CXCR1 chemokine receptors.

<p>(A) Freshly isolated naïve NK cells were double stained with anti-CD56 mAb together with control-Ig or with vMIP-II-Ig. The CD56^Dim CD16^Pos and CD56^Bright CD16^Neg NK cell populations are indicated by an arrow. The percentages of the various populations are indicated. (B) Expression of various chemokine receptors on freshly isolated naïve NK cells. Staining was performed with anti-CD56 mAb together with specific antibodies against CCR1, CCR2, CCR3, CCR5 and CXCR4. The percentages of the various populations are indicated. (C) CCR5 expression varies between different individuals. Staining of various donors (indicated on top of the dot plots) was performed with anti-CD56 mAb together with specific antibodies against CCR5. (D and E) Chemokine receptors expressed primarily by the CD56^Dim CD16^Pos population. Freshly isolated naïve NK cells were double stained with anti-CD56 mAb together with anti-CXCR1 (D) or anti-CX3CR1 (E). The percentages of the various populations are indicated. Figure shows one representative experiment out of three performed.</p

vMIP-II binds activated NK cells that express CCR5.

<p>(A) vMIP-II-Ig binding to activated NK cells (Act-NK) (black open histogram). The filled grey histogram is the secondary antibody staining. (B) Activated NK cells were double stained with anti-CD56 together with anti-CX3CR1 (left dot plot) or with anti CXCR1 (right dot plot). Numbers represent percentages. (C) Activated NK cells were double stained with anti-CD56 mAb and antibodies against CCR1, CCR2, CCR3, CCR5 and CXCR4 (indicated in the X axis). Numbers represent percentages. (D and E) rhRANTES (D) or rvMIP-II (E) were placed in the bottom chamber of transwell plates and the migration of activated NK was quantified by FACS following a 3 hours incubation period at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). ***P<0.001. NS - not significant. Figure shows one representative experiment out of four performed.</p

vMIP-II blocks the migration of freshly isolated naïve NK cells to Fractalkine.

<p>(A) Freshly isolated naïve NK cells were double stained with Fck-Ig and with anti-CD56 mAb. The percentages of the various populations are indicated in the figure. (B) CX3CR1 expression on the transfectant 293T-CX3CR1 cells (black open histogram) or on 293T parental cells (filled grey histogram). (C) Binding of Fck-Ig (left histogram) or vMIP-II-Ig (right histogram) to 293T-CX3CR1 transfectant (black open histogram) or to the 293T parental cells (filled grey histogram). (D) 293T-CX3CR1 cells were incubated with (black empty histogram) and without (dark gray empty histogram) rvMIP-II for 1 hour in 4°C and then stained with Fck-Ig. The light gray filled histogram is the staining of Fck-Ig on the 293T parental cells. (E) Freshly isolated naïve NK cells were incubated at 4°C for 1 hour with and without the proteins indicated in the x axis. RhFck was placed in the bottom chamber and the numbers of migrated cells was determined by FACS following 3 hours incubation at 37°C. The migration of the NK cells without the appropriate chemokine was set as 1 and the results are presented as fold increase (FI). **P<0.01. NS - not significant. Figure shows one representative experiment out of four performed.</p

vMIP-II-Ig binds PBLs and naïve NK cells.

<p>Freshly isolated PBLs (A), freshly isolated naïve NK cells (B), Monocytes (C) and Neutrophils (D), (the various cell types are indicated in the right of the figure), were stained with different KSHV chemokines fused to human IgG1: vIL6-Ig, vMIP-I-Ig, vMIP-II-Ig or vMIP-III-Ig (X axis, indicated in the top of the figure). PBLs were double stained with the indicated chemokines fused to IgG and with anti-CD3. The percentages of various populations are indicated inside the dot plot. The Median Fluorescence Intensity (MFI) of the vMIP-II-Ig staining of CD3+ cells (A) and of NK cells (B) is indicated and is marked by an arrow. Figure shows one representative staining out of more than 3 performed.</p