Inhibitory KIR2DL2 receptor and HHV-8 in classic or endemic Kaposi sarcoma.

KIR2DL2, an inhibitory Killer cell Immunoglobulin-like Receptor (KIR), has been shown to predispose to the development of several herpesvirus-associated diseases by inhibiting the efficiency of Natural Killer (NK) cells against virus-infected cells. The aim of this observational study was to assess the prevalence of KIR2DL2 and Human Herpes Virus 8 (HHV8) in patients affected with classical and endemic Kaposi sarcoma (KS), as well as in controls. Blood samples collected from 17 Caucasian, HIV-negative, immunocompetent patients affected with classical KS (c-KS), 12 African, HIV-negative patients with endemic KS (e-KS), 83 healthy subjects and 26 psoriatic patients were processed for genotypization by PCR for two KIR alleles, such as KIR2DL2 and KIR2DL3 and analyzed for HHV-8 presence. The totality of both c-KS and e-KS patients presented HHV-8 infection, whereas HHV8 was found in 26.9% of psoriatic subjects and 19.3% of healthy subjects. KIR2DL2 was found in the 76.5% of c-KS subjects, while the receptor was found in 41.7% of the e-KS group, 34.6% of psoriatic patients and 43.4% of healthy controls (p < 0.0001). A significantly higher prevalence of KIR2DL2 in c-KS patients than in all the other subjects was also confirmed comparing age-matched groups. Based on these results, the inhibitory KIR2DL2 genotype appears to be a possible cofactor which increases the risk of developing c-KS in HHV8-positive, immunocompetent subjects, while it seems less relevant in e-KS pathogenesis.

The U94 Gene of Human Herpesvirus 6: A Narrative Review of Its Role and Potential Functions.

Human herpesvirus 6 (HHV-6) is a ß-herpesvirus that is highly prevalent in the human population. HHV-6 comprises two recognized species (HHV-6A and HHV-6B). Despite different cell tropism and disease association, HHV-6A/B show high genome homology and harbor the conserved U94 gene, which is limited to HHV-6 and absent in all the other human herpesviruses. U94 has key functions in the virus life cycle and associated diseases, having demonstrated or putative roles in virus replication, integration, and reactivation. During natural infection, U94 elicits an immune response, and the prevalence and extent of the anti-U94 response are associated with specific diseases. Notably, U94 can entirely reproduce some virus effects at the cell level, including inhibition of cell migration, induction of cytokines and HLA-G expression, and angiogenesis inhibition, supporting a direct U94 role in the development of HHV-6-associated diseases. Moreover, specific U94 properties, such as the ability to modulate angiogenesis pathways, have been exploited to counteract cancer development. Here, we review the information available on this key HHV-6 gene, highlighting its potential uses.

SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway.

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells' exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.

DNA Sensors' Signaling in NK Cells During HHV-6A, HHV-6B and HHV-7 Infection.

OBJECTIVES: The host DNA sensor proteins TLR9, STING, IFI16 are central signaling molecules that control the innate immune response to cytosolic nucleic acids. Here we propose to investigate how Natural killer (NK) cell infection by human herpesvirus (HHV)-6A, HHV-6B or HHV-7 is able to modify DNA sensor signaling in NK cells. METHODS: We infected the NK92 cell line and primary NK cells with cell-free inocula of HHV-6A, HHV-6B or HHV-7 and evaluated TLR9, STING, and IFI16 pathway expression by Real-Time PCR, Western Blot, immunofluorescence and flow cytometry for 1, 2, 3, and 6 days post-infection. We evaluated NK cell cytokine-producing by Real-Time PCR and enzyme immunosorbent assay. RESULTS: NK92 and primary NK cells were promptly infected by three viruses, as demonstrated by virus presence (DNA) and transcription (RNA) analysis. Our data show STING/STAT6 up-modulation in HHV-6A infected NK cells. NK cells infected with HHV-6B and HHV-7 up-regulated CCL3, IFN-alpha, TNF-alpha, IL-8 and IFN-gamma and slightly induced IL-4, and CCL4. HHV-6A infected NK cells up-regulated IL-4 and IL-13 and slightly induced IL-10, TNF-alpha, IFN-alpha, and IFN-gamma. CONCLUSION: For the first time, we demonstrate that HHV-6A, HHV-6B, and HHV-7 infections have a differential impact on intracellular DNA sensors. HHV-6B and HHV-7 mainly lead to the active control of in vivo viral spreading by pro-inflammatory cytokine secretion via TLR9. HHV-6A infected NK cells conversely induced STING/STAT6 pathway, as a mechanism of anti-viral activation, but they were characterized by a Th2 type response and a non-cytotoxic profile, suggesting a potential novel mechanism of HHV-6A-mediated immunosuppression.

Soluble HLA-G pre-transplant levels to identify the risk for development of infection in heart transplant recipients.

Infection is still a leading cause of death during the first year after heart transplantation. We evaluated the pre-transplant levels of HLA (Human Leukocyte antigen) - G molecules as a means of identifying heart recipients at risk of serious infections. We prospectively analyzed 122 adult heart transplant (HT) recipients. Serum samples were collected beforetransplantation and analyzed for sHLA-G levels by ELISA assay. The clinical follow-up period lasted 5 years. Clinical outcomes were bacterial infections requiring intravenous anti-microbial agents, cytomegalovirus (CMV) disease, and fungal infections requiring therapy. We found that 39 patients (32%) developed at least 1 serious bacterial infection. Higher pre-transplant sHLA-G levels were a risk factor for serious infection (above median value 5.4 ng/ml; relative risk 3.70; 95% confidence interval 1.03-12.64; p = 0.043). Patients with high levels of pre-transplant sHLA-G are also characterized by a lower overall survival at 5 years (p = 0.017), with microbial infections as major causes of death. No association was observed with the development rejection episode. Early monitoring of sHLA-G molecules proved useful for the identification of heart recipients who are at risk of serious infections.

HHV-6A infection induces amyloid-beta expression and activation of microglial cells.

BACKGROUND: The control of viral infections in the brain involves the activation of microglial cells, the macrophages of the brain that are constantly surveying the central nervous system, and the production of amyloid-beta (Aß) as an anti-microbial molecule. Recent findings suggest a possible implication of HHV-6A in AD. We evaluated the effect of HHV-6A infection on microglial cell expression Aß and the activation status, determined by TREM2, ApoE, cytokines, and tau expression. METHODS: We have infected microglial cells (HMC3, ATCC®CRL-3304), in monolayer and human peripheral blood monocyte-derived microglia (PBM-microglia) spheroid 3D model, with HHV-6A (strain U1102) cell-free virus inocula with 100 genome equivalents per 1 cell. We collected the cells 1, 3, 7, and 14 days post-infection (d.p.i.) and analyzed them for viral DNA and RNA, ApoE, Aß (1-40, 1-42), tau, and phospho-tau (Threonine 181) by real-time immunofluorescence and cytokines by immunoenzymatic assay. RESULTS: We observed a productive infection by HHV-6A. The expression of Aß 1-42 increased from 3 d.p.i., while no significant induction was observed for Aß 1-40. The HHV-6A infection induced the activation (TREM2, IL-1beta, ApoE) and migration of microglial cells. The secretion of tau started from 7 d.p.i., with an increasing percentage of the phosphorylated form. CONCLUSIONS: In conclusion, microglial cells are permissive to HHV-6A infection that induces the expression of Aß and an activation status. Meanwhile, we hypothesize a paracrine effect of HHV-6A infection that activates and induces microglia migration to the site of infection.

HHV-6A infection of endometrial epithelial cells affects immune profile and trophoblast invasion.

PROBLEM: We first reported human herpesvirus (HHV)-6A DNA presence in 43% of endometrial cells from women with idiopathic infertility, whereas no fertile control women harbored the virus. We investigated the effect of HHV-6A infection on the immunological status of the endometrium. METHOD OF STUDY: Endometrial biopsies, uterine flushing, and whole blood samples were collected from 67 idiopathic infertile women (mid-secretory phase). We analyzed the endometrial immunological status evaluating: (a) the effect of HHV-6A infection on endometrial immune profile analyzing the ratio of interleukin (IL)-15/ fibroblast growth factor-inducible 14 (Fn-14) and IL-18/ TNF-related weak inducer of apoptosis (TWEAK) mRNA as a biomarker of endometrial (e)natural killer activation/maturation, angiogenesis, and Th1/Th2 balance; (b) endometrial receptivity to trophoblasts in endometrial 3D in vitro model; (c) natural killer (NK) cells and T cells percentage and subpopulations by flow cytometry. RESULTS: We confirmed the presence of HHV-6A infection in a 40% of idiopathic infertile women, characterized by an immune profile reflecting eNK cell cytotoxic activation and a decrease in CD4+ CD25+ CD127dim/- regulatory T cells. The co-culture of endometrial epithelial cells with spheroids generated from the extravillous trophoblast-derived cell line JEG3 showed a twofold expansion of spheroids on endometrial epithelial-stromal cells (ESC) culture surface from HHV-6A negative women while no expansion was observed on the surface of ESC from HHV-6A positive women. CONCLUSION: The identification of an effect of HHV-6A infection on endometrial immune status opens new perspectives in idiopathic infertile women care management. In addition, it would be possible to select antiviral therapies as novel, non-hormonal therapeutic approaches to those idiopathic infertile women characterized by the presence of endometrial HHV-6A infection, to increase their pregnancy rate.

KIR2DS2/KIR2DL2/HLA-C1 Haplotype Is Associated with Alzheimer's Disease: Implication for the Role of Herpesvirus Infections.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, where neuroinflammation and immune cells are key pathological factors. Recently, it was suggested a possible association between AD and human herpesvirus 6 (HHV-6) infection. Since we recently observed that multiple sclerosis patients with KIR2DL2 expression on natural killer (NK) cells are more susceptible to herpesvirus infection, we tested the possible implication of KIR/HLA genetic for HHV-6A infection. We identified, for the first time, a possible implication of a specific KIR/HLA subset in AD. The combination KIR2DS2/KIR2DL2/C1 correlated with a lower MMSEDi score, representative of a severe AD status and an increased susceptibility to HHV-6A infection. Therefore, the results seem to converge on the hypothesis that herpesvirus infection might play a role in AD. If this hypothesis finds experimental confirmation, a new therapeutic strategy, modulating KIR2DL2 expression on NK cells, for AD might be envisaged.

Human Herpes simplex 1 virus infection of endometrial decidual tissue-derived MSC alters HLA-G expression and immunosuppressive functions.

OBJECTIVES: Mesenchymal stromal/stem cells have immunosuppressive functions. Our previous results demonstrated that one of the players of this immunomodulation can be ascribed to the Human Leukocyte Antigen-G. HLA-G, a non classical HLA class I antigen, is involved in immune tolerance during pregnancy, organ transplantation, autoimmune and inflammatory diseases. In this study we wanted to verify whether human endometrial decidual tissue derived (EDT)-MSC could express HLA-G. Additionally we assessed the permissivity to Human Herpesvirus infections, using HSV-1 as a model, and the possible effect on EDT-MSC immunosuppressive functions towards peripheral blood mononuclear cell (PBMC) proliferation. METHODS: We analyzed immune-inhibitory functions and HLA-G expression in human EDT-MSC before and after HSV-1 infection. RESULTS: We observed that EDT-MSC express HLA-G molecules, that partly are responsible for the immune-inhibitory functions of EDT-MSC towards PBMC proliferation. EDT-MSC are permissive for a productive infection by HSV-1, that decreases HLA-G expression and affects EDT-MSC immune-inhibitory functions. CONCLUSIONS: We demonstrate that EDT-MSC are susceptible to HSV-1 infection, that reduces HLA-G expression and their immune-inhibitory function. These data could have a clinical implication in the use of EDT-MSC as an immunosuppressant, in particular in steroid-refractory GvHD after allogeneic hematopoietic stem cell transplantation and in autoimmune diseases.

"Hemophagocytic Lymphohistiocytosis after EBV reactivation and ibrutinib treatment in relapsed/refractory Chronic Lymphocytic Leukemia".

Hemophagocytic Lymphohistiocytosis (HLH) is a rare syndrome characterized by ineffective T-cell and NK response. We report the clinical course of a patient with relapsed CLL who developed acute symptoms soon after starting ibrutinib. Hyperpyrexia, splenomegaly, hyperferritinemia, hypertriglyceridemia, cytopenias, and a typical cytokine pattern, i.e. high interleukin (IL)-6, IL10 and IL18, were consistent with a diagnosis of HLH. Coexistent Epstein Barr virus reactivation was documented. Ibrutinib-induced impairment of NK degranulation, associated with EBV reactivation and CLL-related immunodeficiency may have contributed to the development of HLH in our patient.

HHV-6A Infection of Endometrial Epithelial Cells Induces Increased Endometrial NK Cell-Mediated Cytotoxicity.

Background: We have recently reported the presence of Human herpesvirus-6A (HHV-6A) DNA in the 43% of endometrial epithelial cells from primary idiopathic infertile women, with no positivity in fertile women. To investigate the possible effect of HHV-6A infection in endometrial (e)NK cells functions, we examined activating/inhibitory receptors expressed by eNK cells and the corresponding ligands on endometrial cells during HHV-6A infection. Methods: Endometrial biopsies and uterine flushing samples during the secretory phase were obtained from 20 idiopathic infertile women and twenty fertile women. HHV-6A infection of endometrial epithelial cells was analyzed by Real-Time PCR, immunofluorescence and flow cytometry. eNKs receptors and endometrial ligands expression were evaluated by immunofluorescence and flow cytometry. Results: We observed the presence of HHV-6A infection (DNA, protein) of endometrial epithelial cells in the 40% of idiopathic infertile women. The eNK from all the subgroups expressed high levels of NKG2D and NKG2A receptors. Functional studies showed that NKG2D activating receptor and FasL are involved in the acquired cytotoxic function of eNK cells during HHV-6A infection of endometrial epithelial cells. In the presence of HHV-6A infection, eNK cells increased expression of CCR2, CXCR3 and CX3CR1 chemokine receptors (p = 0.01) and endometrial epithelial cells up-modulated the corresponding ligands: MCP1 (Monocyte chemotactic protein 1, CCL2), IP-10 (Interferon gamma-induced protein 10, CXCL10) and Eotaxin-3 (CCL26). Conclusion: Our results, for the first time, showed the implication of eNK cells in controlling HHV-6A endometrial infection and clarify the mechanisms that might be implicated in female idiopathic infertility.

The association between functional HLA-G 14bp insertion/deletion and +3142 C>G polymorphisms and susceptibility to multiple sclerosis.

We aimed to investigate two main polymorphisms in the 3' untranslated region (3'UTR) of the HLA-G gene [14bp insertion/deletion (INS/DEL) and +3142 C>G] and to assess their impact on the soluble HLA-G (sHLA-G) production in patients with multiple sclerosis (MS). This study included 60 patients with relasping-remitting (RR) MS and 112 healthy donors (HD). Mutations were identified by PCR and PCR-RFLP, and serum sHLA-G quantification was performed by ELISA. For the 14bp INS/DEL polymorphism, variants frequencies were similar in patients and controls, whereas a significant increased frequency of the +3142 G allele was found in MS patients compared to HD (63.4% vs 52.3%, p=0.04; OR=1.58, 95%CI=1.003-2.48). In addition, an association was found between MS susceptibility and the haplotypes regrouping both studied polymorphisms. Indeed, the 14bp DEL/+3142 G haplotype frequency was significantly increased in MS patients compared to HD (20.8% vs 12.5%, p=0.04, OR=1.84). On the other hand, no associations were detected between both polymorphisms and clinical parameters, except the lower age of disease onset (ADO) in patients with the +3142 C/C genotype. Moreover, our study doesn't show any significant variation of sHLA-G serum levels between patients and controls. Our findings showed that the +3142 C>G, but not the 14bp INS/DEL, polymorphism may constitute a genetic susceptibility factor to MS in the Tunisian population. However, no association was found between the two polymorphisms and sHLA-G serum levels.

Presence of HHV-6A in Endometrial Epithelial Cells from Women with Primary Unexplained Infertility.

To elucidate the roles of human herpesvirus (HHV)-6 primary unexplained infertile women, a prospective randomized study was conducted on a cohort of primary unexplained infertile women and a cohort of control women, with at least one successful pregnancy. HHV-6 DNA was analyzed and the percentage and immune-phenotype of resident endometrial Natural Killer (NK) cells, as the first line of defense towards viral infections, was evaluated in endometrial biopsies. Cytokine levels in uterine flushing samples were analyzed. HHV-6A DNA was found in 43% of endometrial biopsies from primary unexplained infertile women, but not in control women. On the contrary, HHV-6B DNA was absent in endometrial biopsies, but present in PBMCs of both cohorts. Endometrial NK cells presented a different distribution in infertile women with HHV6-A infection compared with infertile women without HHV6-A infection. Notably, we observed a lower percentage of endometrial specific CD56brightCD16- NK cells. We observed an enhanced HHV-6A-specific endometrial NK cell response in HHV-6A positive infertile women, with a marked increase in the number of endometrial NK cells activating towards HHV-6A infected cells. The analysis of uterine flushing samples showed an increase in IL-10 levels and a decrease of IFN-gamma concentrations in infertile women with HHV6-A infection. Our study indicates, for the first time, that HHV-6A infection might be an important factor in female unexplained infertility development, with a possible role in modifying endometrial NK cells immune profile and ability to sustain a successful pregnancy.

KIR2DL2 inhibitory pathway enhances Th17 cytokine secretion by NK cells in response to herpesvirus infection in multiple sclerosis patients.

We have previously demonstrated that multiple sclerosis (MS) patients with KIR2DL2 expression on Natural killer (NK) cells are more susceptible to herpes simplex virus 1 (HSV-1) infection. We explored cytokine expression by NK cells during HSV-1 infection in association with KIR2DL2 expression. MS KIR2DL2(+) NK cells failed to control HSV-1 infection and secreted high levels of Th17 cytokines, while MS KIR2DL2(-) NK cells released Th1 cytokines, mainly IFN-gamma. Our data showed, for the first time, a peculiar Th17 cytokine secretion by MS KIR2DL2(+) NK cells in the presence of HSV-1 infection, that could be implicated in MS pathogenesis.

Increase in Peripheral CD3-CD56brightCD16- Natural Killer Cells in Hashimoto's Thyroiditis Associated with HHV-6 Infection.

Hashimoto's thyroiditis (HT) is a very common autoimmune disease of the thyroid. In addition to genetic background, several viruses, including herpesviruses, have been suggested to play a role as possible environmental triggers of disease, but conclusive data are still lacking. Previous results showed that HT patients have an increased cellular immune response directed against the HHV-6 U94 protein and increased NK activity directed against HHV-6 infected thyrocytes.In this study, we characterized the antiviral antibody response and the NK cells activity and subtype in HHV-6 infected HT patients. The results showed that HT subjects have increased prevalence and titer of anti-U94 antibodies and a higher amount of CD3-CD56(bright)CD16(-)NK cell percentages compared to controls. Furthermore, the cell activation of CD3(-)CD56(bright) NK cells in HT patients significantly correlates with TPO and Tg Ab levels.The results suggest that HHV-6 might contribute to HT development, increasing NK cell secretion of inflammatory cytokines that could sustain the persistence of an inflammatory status in HT patients.

Implication of HLA-C and KIR alleles in human papillomavirus infection and associated cervical lesions.

Human papillomavirus (HPV) regulation of host immune response leads to cervical lesions. In particular, natural killer (NK) cells are crucial for HPV control. Since specific HLA-I/KIR interactions modify NK cell activation, we analyzed HLA-C and KIR alleles in HPV infection and lesion development in 150 controls, 33 condyloma acuminatum, and 111 invasive cervical cancer (ICC) patients. We showed an increase in HLA-C1/KIR2DL2 and HLA-C1/KIR2DL3 pairs in HPV high-risk infected patients (OR 3.05, 3.24) with ICC (OR 1.33, 3.68). These data suggest HLA-C and KIR typing as risk marker for HPV infection and lesion evolution.

Impact of HLA-G analysis in prevention, diagnosis and treatment of pathological conditions.

Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule that differs from classical HLA class I molecules by low polymorphism and tissue distribution. HLA-G is a tolerogenic molecule with an immune-modulatory and anti-inflammatory function on both innate and adaptative immunity. This peculiar characteristic of HLA-G has led to investigations of its role in pathological conditions in order to define possible uses in diagnosis, prevention and treatment. In recent years, HLA-G has been shown to have an important implication in different inflammatory and autoimmune diseases, pregnancy complications, tumor development and aggressiveness, and susceptibility to viral infections. In fact, HLA-G molecules have been reported to alternate at both genetic and protein level in different disease situations, supporting its crucial role in pathological conditions. Specific pathologies show altered levels of soluble (s)HLA-G and different HLA-G gene polymorphisms seem to correlate with disease. This review aims to update scientific knowledge on the contribution of HLA-G in managing pathological conditions.

Infection and HLA-G molecules in nasal polyposis.

Sinonasal polyposis (SNP) is a chronic inflammatory pathology with an unclear aetiopathogenesis. Human papillomavirus (HPV) infection is one candidate for the development of SNP for its epithelial cell trophism, hyperproliferative effect, and the induction of immune-modulatory molecules as HLA-G. We enrolled 10 patients with SNP without concomitant allergic diseases (SNP-WoAD), 10 patients with SNP and suffering from allergic diseases (SNP-WAD), and 10 control subjects who underwent rhinoplasty. We analyzed the presence of high- and low-risk HPV DNA and the expression of membrane HLA-G (mHLA-G) and IL-10 receptor (IL-10R) and of soluble HLA-G (sHLA-G) and IL-10 by polyp epithelial cells. The results showed the presence of HPV-11 in 50% of SNP-WoAD patients (OR:5.5), all characterized by a relapsing disease. HPV-11 infection was absent in nonrelapsing SNP-WoAD patients, in SNP-WAD patients and in controls, supporting the hypothesis that HPV-11 increases risk of relapsing disease. HPV-11 positive SNP-WoAD patients presented with mHLA-G and IL-10R on epithelial cells from nasal polyps and showed secretion of sHLA-G and IL-10 in culture supernatants. No HLA-G expression was observed in HPV negative polyps. These data highlight new aspects of polyposis aetiopathogenesis and suggest HPV-11 and HLA-G/IL-10 presence as prognostic markers in the follow-up of SNP-WoAD.