Prevalence of antibodies against Neospora caninum in dogs from urban areas in Central Poland

Neospora caninum is a protozoan parasite which causes abortion in cattle as well as reproduction problems and neurological disorders in dogs. To assess the prevalence of the parasite in urban dogs in the Mazovian Voivodeship, Central Poland, serum samples from 257 dogs were analyzed for the presence of specific IgG antibodies. The examined dogs visited three private veterinary clinics located in Warsaw due to control tests, vaccinations, or other reasons not directly connected with neosporosis. Using ELISA and Western blot, antibodies against the parasite were detected in 56 out of 257 dogs, giving a prevalence of 21.7%. A greater prevalence was observed in female dogs than in males, 28% and 17.3%, respectively, and the differences were statistically significant (p < 0.05). There were no significant differences in seroprevalence of Neospora infection within the age groups (p > 0.05). This study indicates the presence of N. caninum in the Mazovian Voivodeship, in dogs which live in urban areas and exposure of these dogs to the parasite. The fact that seropositive dogs had no contact with cattle confirms the important role of dogs in the parasite’s epidemiology

Rapid expansion and long-term persistence of elevated NK cell numbers in humans infected with hantavirus

Acute hantavirus infection in humans triggers a rapid expansion and long-term persistence of NK cells

NK Cell Activation in Human Hantavirus Infection Explained by Virus-Induced IL-15/IL15R alpha Expression

Clinical infection with hantaviruses cause two severe acute diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). These diseases are characterized by strong immune activation, increased vascular permeability, and up to 50% case-fatality rates. One prominent feature observed in clinical hantavirus infection is rapid expansion of natural killer (NK) cells in peripheral blood of affected individuals. We here describe an unusually high state of activation of such expanding NK cells in the acute phase of clinical Puumala hantavirus infection. Expanding NK cells expressed markedly increased levels of activating NK cell receptors and cytotoxic effector molecules. In search for possible mechanisms behind this NK cell activation, we observed virus-induced IL-15 and IL-15R alpha on infected endothelial and epithelial cells. Hantavirus-infected cells were shown to strongly activate NK cells in a cell-cell contact-dependent way, and this response was blocked with anti-IL-15 antibodies. Surprisingly, the strength of the IL-15-dependent NK cell response was such that it led to killing of uninfected endothelial cells despite expression of normal levels of HLA class I. In contrast, hantavirus-infected cells were resistant to NK cell lysis, due to a combination of virus-induced increase in HLA class I expression levels and hantavirus-mediated inhibition of apoptosis induction. In summary, we here describe a possible mechanism explaining the massive NK cell activation and proliferation observed in HFRS patients caused by Puumala hantavirus infection. The results add further insights into mechanisms behind the immunopathogenesis of hantavirus infections in humans and identify new possible targets for intervention

Increased HLA class I expression on hantavirus-infected cells inhibits NK cell effector functions.

<p>(A) Degranulation (CD107a) and cytokine production (TNF and IFN-γ) of resting, IL-15 and IFN-α pre-activated CD56^dim NK cells reactive against uninfected (white) or HTNV-infected (black) endothelial cells. Results from 2 independent experiments (n = 6) (*** p≤0.001, ** p≤0.01, * p≤0.05; paired <i>t</i>-test). (B) Representative staining of HLA class I and HLA-E expression on uninfected (white) or HTNV-infected (black) endothelial cells. Isotype control (grey). (C and D) CD56^dim NK cell responses against uninfected and HTNV-infected endothelial cells in the presence of anti-HLA class I or isotype control antibody. (C) Representative FACS analysis of the NK cell responses against uninfected and HTNV-infected endothelial cells in the presence of anti-HLA class I or isotype control antibody is depicted. (D) Frequency of CD56^dim NK cells expressing CD107a (n = 6), TNF (n = 3) and IFN-γ (n = 3) in response to uninfected (white) and HTNV-infected (black) endothelial cells in the presence of anti-HLA class I or isotype control antibody (*** p≤0.001, * p≤0.05; paired <i>t</i>-test).</p

Hantavirus-activated CD56^dim NK cells kill uninfected but not hantavirus-infected endothelial cells.

<p>(A) Experimental set-up: NK cells were incubated with uninfected or HTNV-infected cells for 24 h then transferred and incubated for another 5 h with uninfected or HTNV-infected cells, followed by assessment of NK cell degranulation and cytotoxicity. (B and C) Degranulation (CD107a) of CD56^dim NK cells pre-stimulated with uninfected or HTNV-infected endothelial cells against uninfected and HTNV-infected endothelial cells. (B) FACS analysis of one NK cell donor is shown. (C) CD107a expression on CD56^dim NK cells (n = 9) in response to uninfected and HTNV-infected endothelial cells after pre-stimulation with uninfected (white) or HTNV-infected (black) endothelial cells or medium alone (grey). Results from 3 independent experiments (*** p≤0.001; paired <i>t</i>-test). (D) Degranulation of CD56^dim NK cells (n = 4) against uninfected endothelial cells after pre-stimulation with uninfected (white) and HTNV-infected (black) endothelial cells. When indicated, IL-15 was blocked on endothelial cells during pre-stimulation. Results from 2 independent experiments (* p≤0.05; paired <i>t</i>-test). (E and F) Induction of apoptosis in endothelial cells exposed to NK cells pre-stimulated with uninfected or HTNV-infected endothelial cells. (E) One representative immunofluorescent staining is depicted: DAPI (blue), HTNV-nucleocapsid protein (green), TUNEL-positive cells (red). (F) Percentage of TUNEL-positive uninfected and HTNV-infected endothelial cells after exposure to NK cells (n = 6) pre-stimulated with uninfected (white) and HTNV-infected (black) endothelial cells. Results from 3 independent experiments (** p≤0.01; paired <i>t</i>-test). (G) NK cell-mediated specific lysis of uninfected endothelial cells after pre-stimulation with uninfected (white) or HTNV-infected (black) endothelial cells. Depicted are mean values (+/− SD) from 5 donors and 2 independent experiments (* p≤0.05).</p

CD56^dim NK cells are highly activated in HFRS patients.

<p>(A) PBMC from 16 PUUV-infected HFRS patients were collected in the early acute (median d6) and convalescence phase (d60). From 8 HFRS patients additional samples were collected at the indicated time-points and up to day 450. Analyses of NK cell phenotype shown in 1B and 1D–G were performed with samples from patients in the early acute (median d6) and early convalescent phase (d60) of HFRS. (B) Frequencies of CD69-positive CD56^bright and CD56^dim NK cells in early acute and convalescent (d60) HFRS infection (n = 8). (** p≤0.01, paired <i>t</i>-test). (C) Frequencies of CD69-positive CD56^dim NK cells from HFRS patients (n = 8) from early acute to convalescence phases are depicted. Values shown are mean (+/− SD). (D and E) Expression levels of activating NK cell receptors on CD56^bright and CD56^dim NK cells in acute and convalescent phases (D) Representative staining for NKG2D, 2B4, NKp30 and NKp46 on CD56^bright and CD56^dim NK cells during acute HFRS infection (black) and convalescence (white). Isotype (grey). (E) Expression levels (MFI) of NKG2D, 2B4, NKp30 and NKp46 on CD56^bright and CD56^dim NK cells in HFRS patients (n = 14–16) in acute (black) and convalescent phases (white). (*** p≤0.001, paired <i>t</i>-test). (F and G) Levels of intracellular cytotoxic effector molecules in CD56^bright and CD56^dim NK cells in acute and convalescent phases. (F) Representative intracellular staining for granzyme B and perforin in CD56^bright and CD56^dim NK cells in acute HFRS infection (black) and convalescence (white). Isotype (grey). (G) Expression levels (MFI) of intracellular granzyme B and perforin in CD56^bright and CD56^dim NK cells of HFRS patients (n = 14–16) in acute (black) and convalescent phases of infection (white). (*** p≤0.001, * p≤0.05, paired <i>t</i>-test).</p

CD56^dim NK cells acquire increased functional capacity after contact with hantavirus-infected cells.

<p>(A–C) Degranulation (CD107a) and intracellular cytokine production of CD56^dim NK cells reacting to K562 cells after pre-stimulation with uninfected or HTNV-infected endothelial and epithelial cells. (A) Representative FACS analysis of CD107a, IFN-γ and TNF expression in one NK cell donor is shown. (B and C) Summary of the CD56^dim NK cell responses against K562 cells (n = 9) after pre-stimulation with uninfected (white) or HTNV-infected (black) endothelial (B) and epithelial cells (C). (*** p≤0.001, ** p≤0.01; paired <i>t</i>-test). (D) NK cell-mediated specific lysis of K562 cells after pre-stimulation of NK cells with uninfected (white) and HTNV-infected (black) endothelial cells. Depicted are mean values (+/− SD) from 5 donors and 2 independent experiments (** p≤0.01, *p≤0.05; paired <i>t</i>-test).</p

Induced IL-15 and IL-15Rα expression in hantavirus-infected cells drives CD56^dim NK cell activation.

<p>(A and B) Kinetics of IL-15 and IL-15Rα mRNA-expression in HTNV-infected endothelial (A) and epithelial (B) cells analyzed with RT-qPCR. Fold change compared to the expression level in the respective uninfected cell is depicted. Results of one experiment run in triplicates are shown. (C and D) Expression of IL-15 and IL-15Rα protein in uninfected and HTNV-infected endothelial cells analyzed with flow cytometry after cell permeabilization. (C) One representative FACS analysis is shown. Uninfected cells (white), HTNV-infected cells (black) and isotype control (grey). (D) The expression levels (MFI) of IL-15 and IL-15Rα protein in uninfected (white) or HTNV-infected (black) endothelial cells (n = 3). (E–H) Surface expression of IL-15 and IL-15Rα on uninfected and HTNV-infected endothelial (E and F) and epithelial (G and H). (E and G) Representative FACS analysis of the surface expression of IL-15 and IL-15Rα on uninfected and HTNV-infected endothelial cells and epithelial cells. Uninfected cells (white), HTNV-infected cells (black) and isotype (grey). (F and H) The expression levels (MFI) of IL-15 and IL-15Rα on uninfected (white) or HTNV-infected (black) endothelial (n = 4) and epithelial cells (n = 4) are shown. (I–L) CD69 expression on CD56^dim NK cells after co-incubation with HTNV-infected endothelial cells (I and J) and epithelial cells (K and L) in the presence of anti-IL-15 or isotype control antibody. (I and K) Representative FACS analysis of CD69 expression on CD56^dim NK cells incubated with endothelial cells (I) and epithelial cells (K) in the presence of isotype control (black) or anti-IL-15 antibody (grey). (J and L) Expression levels (MFI) of CD69 on CD56^dim NK cells after co-incubation with endothelial cells (n = 8) (J) and epithelial cells (n = 6) (L). The dashed lines represent upper and lower SEM intervals for means of CD69 expression on CD56^dim NK cells after incubation with the respective uninfected cells.</p

Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

<div><p>Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.</p> </div