Neutrophil Activation in Acute Hemorrhagic Fever With Renal Syndrome Is Mediated by Hantavirus-Infected Microvascular Endothelial Cells

ORIGINAL RESEARCH published: 18 September 2018 doi: 10.3389/fimmu.2018.02098 Frontiers in Immunology | www.frontiersin.org 1September 2018 | Volume 9 | Article 2098 Edited by: Alan Chen-Yu Hsu, University of Newcastle, Australia Reviewed by: Erdong Cheng, University of Pittsburgh Cancer Institute, United States Albert Rizvanov, Kazan Federal University, Russia Tione Buranda, University of New Mexico, United States *Correspondence: Tomas Strandin [email protected] Specialty section: This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology Received: 31 March 2018 Accepted: 24 August 2018 Published: 18 September 2018 Citation: Strandin T, Mäkelä S, Mustonen J and Vaheri A (2018) Neutrophil Activation in Acute Hemorrhagic Fever With Renal Syndrome Is Mediated by Hantavirus-Infected Microvascular Endothelial Cells. Front. Immunol. 9:2098. doi: 10.3389/fimmu.2018.02098 Neutrophil Activation in Acute Hemorrhagic Fever With Renal Syndrome Is Mediated by Hantavirus-Infected Microvascular Endothelial Cells Tomas Strandin 1 *, Satu Mäkelä 2, Jukka Mustonen2and Antti Vaheri 1 1Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 2Department of Internal Medicine, Faculty of Medicine and Life Sciences, Tampere University Hospital, University of Tampere, Tampere, Finland Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) in humans. Both diseases are considered to be immunologically mediated but the exact pathological mechanisms are still poorly understood. Neutrophils are considered the first line of defense against invading microbes but little is still known of their role in virus infections. We wanted to study the role of neutrophils in HFRS using blood and tissue samples obtained from Puumala hantavirus (PUUV)-infected patients. We found that neutrophil activation products myeloperoxidase and neutrophil elastase, together with interleukin-8 (the major neutrophil chemotactic factor in humans), are strongly elevated in blood of acute PUUV-HFRS and positively correlate with kidney dysfunction, the hallmark clinical finding of HFRS. These markers localized mainly in the tubulointerstitial space in the kidneys of PUUV-HFRS patients suggesting neutrophil activation to be a likely component of the general immune response toward hantaviruses. We also observed increased levels of circulating extracellular histones at the acute stage of the disease supporting previous findings of neutrophil extracellular trap formation in PUUV-HFRS. Mechanistically, we did not find evidence for direct PUUV-mediated activation of neutrophils but instead primary blood microvascular endothelial cells acquired a pro-inflammatory phenotype and promoted neutrophil degranulation in response to PUUV infection in vitro. These results suggest that neutrophils are activated by hantavirus-infected endothelial cells and may contribute to the kidney pathology which determines the severity of HFRS. Keywords: hantavirus, HFRS, neutrophils, IL-8, endothelial cells, degranulation, NETs, NETosis INTRODUCTION Hantaviruses are the causative agents of two human diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Each hantavirus species is carried by its specific rodent host with no or minimal signs of disease but cause occasional human spillover infections with immune-mediated pathology (1). A hallmark of both hantavirus diseases is increased vascular permeability which mediates kidney and lung failure Strandin et al. Neutrophil Activation in HFRS associated with HFRS and HCPS, respectively (1). Endothelial cells lining the vasculature are also the prime target of viral replication in patients (2,3). Puumala hantavirus (PUUV) circulates in Northern Europe and Russia causing a relatively mild form of HFRS as compared to Hantaan (HTNV) or Dobrava hantavirus (DOBV)-caused HFRS and especially Andes (ANDV)- or Sin Nombre (SNV)-caused HCPS in which fatality rates can reach 40% (4). Typical laboratory findings in acute PUUV-caused HFRS are leukocytosis, thrombocytopenia, increased Creactive protein (CRP) level, and as signs of acute kidney injury (AKI), proteinuria, haematuria and elevated serum creatinine concentration (5). In addition to thrombocytopenia, hematological abnormalities include increased coagulation and fibrinolysis, complement activation, and elevated levels of proinflammatory cytokines which all have the potential to contribute to vascular permeability. Vascular permeability, in turn, could be the underlying cause of proteinuria which typically precedes AKI (6). The pathophysiology of PUUV-HFRS associated AKI is usually described as tubulointerstitial nephritis and infiltration of several immune cell types such as lymphocytes, monocytes, and polymorphonuclear leukocytes into kidneys have been observed (7,8). Neutrophils are the most abundant circulating leukocytes in humans and play a fundamental role in the innate immune response (9). Circulating neutrophils are the first type of immune cells recruited to sites of inflammation or infection. In humans, one of the most important chemotactic factors for neutrophils is interleukin (IL)-8, released at the site inflammation/infection (10). After receiving chemotactic signals neutrophils interact with endothelial cells lining the vasculature in order to traverse the endothelium and into the inflamed tissues (11). In vivo, the interactions between neutrophils and endothelial cells include initial rolling followed by firm adhesion and finally transendothelial migration. Firm adhesion is facilitated, among other factors, by endothelial intercellular adhesion molecule (ICAM)-1 expressed on the surface of endothelial cells and CD11b/CD18 integrin complex (also known as Mac-1, CR3 or αMβ2) on neutrophils (12). Once neutrophils reach the site of infection their primary role is to kill invading microbes by production of reactive oxygen species (ROS) and the release of antimicrobial proteins such as myeloperoxidase (MPO) and human neutrophil elastase (HNE) in a process of degranulation (13,14). In addition to ROS formation and degranulation, neutrophils are also able to release neutrophil extracellular traps (NETs), consisting of extracellular chromatin decorated with histones and granular proteins such as MPO and HNE, and with the potential to entrap and kill pathogens (15,16). The activation of neutrophils in several bacterial infections is well-described but their role in virus-mediated diseases has been neglected to a large extent (17). The involvement of neutrophils in the pathogenesis of hantavirus diseases is suggested by acutely increased serum levels of cytokines and chemokines with known functions in neutrophil chemotaxis, differentiation and mobilization (18–20). Increased numbers of circulating histones, cell-fee DNA and histone-DNA complexes have been described in the acute stage of PUUV infection (21–23), suggesting that neutrophils release NETs during HFRS. NETosis could potentially explain capillary leakage in HFRS (24). Interestingly, neutrophils are crucial for increased vascular permeability observed in response to HTNV infection in SCID mice (25). Furthermore, HTNV has also been shown to cause NETosis in vitro (21). In this study we wanted to elucidate the role of neutrophil activation in HFRS by determining markers of neutrophil activation (MPO, HNE, histones, and IL-8) in blood and tissues of patients suffering from acute PUUV-caused HFRS. In addition, to directly determine the role that hantavirus plays in mediating neutrophil responses in PUUV-HFRS, we investigated the potential of purified PUUV or PUUV-infected endothelial cells to activate neutrophils in vitro. Taken together, we found that the levels of circulating and tissue-localized MPO, HNE and IL-8 are elevated in acute PUUV-HFRS and correlate with kidney dysfunction, thereby corroborating the role of neutrophils in hantavirus pathogenesis. Mechanistically, our results do not support direct virus-mediated neutrophil activation but rather an indirect mechanism through infected endothelial cells. Finally, the antiviral function of neutrophils was pinpointed strongly to the release of proteases from neutrophils. MATERIALS AND METHODS Patient Samples The study was approved by the Ethics Committee of Tampere University Hospital (Nos. 99256 and R04180). All subjects gave written informed consent in accordance with the Declaration of Helsinki. The study consisted of plasma samples from patients treated for serologically confirmed acute PUUV infection at the Tampere University Hospital, Finland, during September 2000– March 2009. We assessed the extracellular circulating levels of MPO, HNE, histone H3 and IL-8 in plasma samples obtained from patients with acute PUUV-caused HFRS at 1st day of hospitalization (acute stage; median days after onset of fever 4 ±2), early recovery phase (15–30 days after hospitalization) and healthy controls. The study included Boiun-fixed, paraffin-embedded kidney biopsies obtained at Tampere University Hospital during 1985– 1987. The biopsies among patients with PUUV-HFRS were performed for clinical reasons at the time when there was no reliable serological test for PUUV infection available. All these biopsies were performed during the acute phase of the disease. The highest measured serum creatinine level of the patients ranged from 220 to 1,050 µmol/L. The biopsy findings were acute interstitial in two cases and acute tubulointerstitial nephritis in three, both findings being typical for PUUV-induced AKI (5). PUUV-negative cases served as controls. Indications for renal biopsies were AKI in one case, microscopic hematuria and/or proteinuria in four cases. The biopsy findings were normal morphology in two cases, acute tubulointerstitial nephritis, mesangial proliferative glomerulonephritis and IgMglomerulonephritis in one case each. In both groups of biopsy patients a positive (PUUV-HFRS cases) or negative (controls) Frontiers in Immunology | www.frontiersin.org 2September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS PUUV-serology was determined using stored serum samples obtained at the time of renal biopsy. Primary Antibodies Primary antibodies used in this study were rabbit polyclonal antibodies to MPO (Thermo scientific, #RB-373-A), Histone H3 (Abcam, #ab18521) or HNE (Abcam, #ab68672) and mouse monoclonal antibodies to IL-8 (RnD Systems; #Mab-208), CD18 (Millipore, #TS1-18) and ICAM-1 (RnD systems, #BBIG-I1) in addition to IgG1 isotype control (Immunotools). Rabbit polyclonal antibodies for PUUV nucleocapsid protein and glycoproteins have been described before (26). Histone Quantification Histones were measured by a dot-blot assay where 2 µl of patient or healthy control EDTA-plasma were pipetted on nitrocellulose membrane, air-dried and probed with Histone H3 antibody in blocking buffer [1.5% milk in Tris–EDTA–NaCl– Tween (TENT)]. After washing with TENT, the primary antibody was detected with IR800-conjugated anti-rabbit antibody (Li-cor) in blocking buffer. After additional washing, signal intensity was determined by Odyssey instrumentation (Li-cor). Recombinant Histone H3 (New England Biolabs) was used for standard preparation. Enzyme-Linked Immunosorbent Assays (ELISAs) The levels of MPO, HNE, and IL-8 were measured from patient or control plasma using ELISA kits provided by Abcam (for MPO, HNE, and IL-8) or Immunotools (for IL-8). Immunohistochemistry (IHC) IHC was performed on kidney sections after heat-mediated antigen retrieval using the protocol provided in Vectastain ABC Elite HRP kit from Vector labs. Avidin/biotin blocking kit, biotinylated goat anti-mouse IgG or anti-rabbit secondary antibodies and DAB substrate were used as suggested by the vendor (Vector labs). The mean percentage of DAB positive area was counted from four images taken (1.5 ×1.5 mm) from each individual section using Fiji Image J software. Cultures of Primary Blood Microvascular Endothelial Cells (BECs) BECs were obtained from Lonza and maintained in endothelial basal medium (EBM-2) supplemented with SingleQuotsTM Kit containing 5% fetal bovine serum (FBS), human endothelial growth factor, hydrocortisone, vascular endothelial growth factor, human fibroblast growth factor-basic, ascorbic acid, R3insulin like growth factor-1, gentamicin and amphotericin-B (Lonza). For experiments the cells were used at passages 7–10. Virus Propagation and Titration PUUV Kazan strain was propagated in Vero E6 cells (green monkey kidney epithelial cell line; ATCC no. CRL-1586) grown in Minimum Essential Medium containing 10% FCS, penicillin and streptomycin (cMEM). For experiments, PUUV was purified from Vero E6 cell supernatant through a 30% sucrose cushion by ultracentrifugation. Virus titers were measured by incubating diluted virus stocks on Vero E6 cells for 24 h at 37◦C and subsequently staining acetone-fixed cells with a polyclonal antibody specific for PUUV nucleocapsid protein and AlexaFluor488-conjugated secondary antibody. Focus-forming units (FFU)/ml were counted under an UV-microscope. Infection of BECs Confluent BECs were infected for 1 h at 37◦C using purified PUUV diluted in BEC growth medium at multiplicity of infection (MOI) of 10. For inactivation of PUUV, virus stocks were kept under UV light for 30 min. Immunofluorescence Immunofluorescence of either mock-, UV-PUUV or live PUUV-infected BECs was performed on cells grown on black, glass-bottomed cell culture plates. After fixation with 4% formaldehyde, cells were permeabilized (3% BSA, 0.3% TritonX-100) for 10 min and incubated with primary antibodies to MPO, HNE, ICAM-1 or viral nucleocapsid protein followed by appropriate Alexafluor488or Alexafluor594conjugated secondary antibodies (Thermo Scientific). To stain the nuclei, cells were incubated with Hoechst 33258. After washing, fluorescence intensity was counted using Hidex sense microplate reader (Hidex) and images taken using Leica TCS SP8 X confocal microscope (Biomedicum Imaging Unit, Biomedicum, University of Helsinki, Helsinki, Finland). Neutrophil activation was quantitated by the percentage of decondensed polymorphonuclear cells with simultaneous MPO relocalization in 4 immunofluorescence images (500 ×500 µm) taken randomly from each well by confocal microscope. PMN Cultures Fresh blood from healthy volunteers was drawn into EDTAtubes and PMNs immediately isolated using Polymorphoprep separation medium (Axis-Shield) according to manufacturer’s protocol. PMN purity and viability were assessed by phenotypic polymorphonuclear characterization using Hoechst 33258 fluorescence microscopy and trypan blue exclusion test, respectively (both were routinely found to be over 90%). Isolated PMNs were diluted into BEC growth medium (106cells/ml) and incubated with mock-, UV-PUUV or live PUUV-infected BECs for 1 or 3 h at 37◦C (10 times excess PMNs over BECs). Cells were washed and subjected directly to immunofluorescence staining as described above for determination of PMN binding. When PMN-BEC co-cultures were incubated in the presence of 10 µg/ml neutralizing antibodies to IL-8, CD18 or viral glycoproteins, PMNs were pre-treated with FcR blocking reagent (Immunostep) for 10 min. Neutrophil Activation Assays Purified virus or virus-containing Vero E6 cell culture supernatants were incubated with freshly isolated PMNs (MOI 1) in cMEM for 3 h at 37◦C. The incubation time was chosen based on the optimal time needed to detect adequate levels of PMN activation marker expression with low concurrent spontaneous activation due to culturing. After fixation with Frontiers in Immunology | www.frontiersin.org 3September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS 2% formaldehyde, extracellular DNA was quantitated with the fluorescent, cell-impermeable Pico-Green dsDNA binding reagent (Thermo Scientific) using Hidex Sense microplate reader. Peroxidase activity was assessed from supernatants of pelleted PMNs (400 ×g 5 min) by the chromogenic peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB). PUUV Viability Assay Purified PUUV was incubated for 3 h with freshly isolated PMNs (in a 1:1 ratio) in assay buffer (10 mM Hepes pH 7,4; 150 mM NaCl). Neutrophil activator phorbol myristate acetate (PMA; 1µg/ml) and one of the following inhibitors NaN3(0.01 or 0.002%), PMSF (0.2 or 1 mM), EDTA (2 mM) or DNAse I (10 U/ml) were added where indicated. After incubation, PMNs were pelleted by centrifugation (400 ×g 5 min) and one third of the supernatant used for PUUV titer measurement as described above. The rest of the sample was used for immunoblotting by standard procedures for PUUV structural proteins (27) using polyclonal antibodies specific for Gn, Gc or N followed by IRD800-conjugated secondary antibody (Li-Cor) and detection by Odyssey. Statistics Significant differences between groups of normally distributed data was assessed by student’s T-test and non-normally distributed data by Mann-Whitney U-test or Kruskal-Wallis H-test for multiple populations. The normality of data was estimated by Shapiro-Wilk test. Correlations between parameters were assessed using Spearman’s rank correlation test. All analyses were done with SPSS software version 24 (SPSS Inc., Chicago, IL, USA) or GraphPad prism (La Jolla, CA, United States). RESULTS Circulating Levels of MPO, HNE, Histones, and IL-8 Are Elevated in Acute PUUV-Caused HFRS Circulating levels of MPO, HNE, histones, and IL-8 were all significantly higher in the acute stage of the disease as compared to the recovery stage or healthy controls (Figure 1). The levels of all the measured markers remained elevated also in the recovery stage as compared to controls but this difference was not statistically significant. Strongly elevated circulating levels of MPO and HNE suggest that neutrophils are activated in acute PUUV-HFRS. Furthermore, increased numbers of extracellular histones imply that neutrophil activation could, at least in part, be due to NETosis (21). Interestingly, in the present study, neutrophil activation was accompanied by elevated levels of IL-8, a chemotactic and priming factor for neutrophils. To get further insight to the role of neutrophil activation in the pathogenesis of PUUV-HFRS by we correlated the acute plasma levels of MPO, HNE, histone H3, and IL-8 to variables reflecting severity of AKI (maximum plasma creatinine level measured during the hospital stay), and to hematological variables (minimum blood platelet count, maximum blood leukocyte count, and plasma tissue plasminogen activator (tPA) level) (Table 1). We found that HNE and histone H3 positively correlated with the severity of AKI. In addition, MPO and HNE correlated with all the tested hematological variables. Not surprisingly, given the likely neutrophil origin of MPO and HNE, they also correlated strongly with each other. The acute IL-8 levels correlated significantly with low platelets and exceptionally strongly with MPO, HNE and histones (p<0.005 for all) suggesting that IL-8 could play a role in neutrophil activation. Localization of MPO, HNE, and IL-8 in the Kidneys of Acute PUUV-HFRS In order to investigate whether markers of neutrophil activation could also be detected in tissues of patients suffering from acute PUUV-HFRS, we made use of archival biopsies to detect the expression of MPO, HNE and IL-8 in patient kidneys by immunohistochemistry. We did not include histones in this analysis since we expected that differentiating NET-associated histones from viable cells would be challenging. Elevated expression of MPO, HNE and IL-8 could be detected more readily in acute PUUV-HFRS patients than PUUV-negative control patient samples (Figure 2A). By counting the tissue area staining positive for MPO, HNE, or IL-8 we could observe a statistically significant difference in MPO expression between PUUV positive and negative cases (n=5; p=0.04; Figure 2B). In the case of HNE and IL-8 higher expression was found in 2 and 3 PUUV cases, respectively, as compared to controls but this was not enough to reach statistical significance (n=5; p=0.08 and p=0.15, respectively). The most prominent localization of MPO, HNE and IL8 in HFRS patients was the tubulointerstitial space, in line with the diagnosis of tubulointerstitial nephritis. Therein we could observe both cell-associated (arrowheads) and extracellular expression (filled arrows) of MPO and HNE suggesting both neutrophil infiltration and activation, respectively. Interestingly, HNE and IL-8 localized also to in the tubular epithelial cells in two HFRS patients (empty arrows). IL-8 could also occasionally be observed in the tubular cells of PUUV-negative patients albeit with a lower intensity and frequency as compared to HFRS patients (Figures 2A,B). Taken together, these results show that MPO is a component of the inflammatory response toward PUUV in the kidneys of acute HFRS, possibly accompanied by HNE and IL-8, and suggest that neutrophils (and their activation products) infiltrate kidneys through the capillary endothelium. In addition, the current findings together with our previous observations of elevated IL-8 levels in urine of acute PUUVHFRS (28) suggest that IL-8 is produced locally by kidney epithelial cells in PUUV-HFRS and likely acts as a chemotactic factor inviting neutrophil recruitment and extravasation in the kidney. Live PUUV Does Not Activate Neutrophils in vitro It has been shown that HTNV can directly bind CD18 integrin on neutrophils and induce NETosis (21). We wanted to determine whether PUUV can also induce NETosis which could explain our findings of neutrophil activation in acute PUUV-HFRS. We did this by incubating mockor PUUV-infected Vero E6 cell culture supernatants or purified PUUV (with the same infectious titer) with freshly isolated PMNs and subsequently quantified the release of extracellular DNA from PMNs. NETosis was Frontiers in Immunology | www.frontiersin.org 4September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS FIGURE 1 | Circulating levels of MPO, HNE, histone H3 and IL-8 in PUUV-HFRS. Plots of the acute (median days after onset of fever 4 ±2) and recovery stage (15–30 days post onset) levels of MPO (A), HNE (B), histones (C) or IL-8 (D) in PUUV-infected patient plasma samples (n=32, 30, 53 and 36, respectively) as compared to healthy controls (n=5, 5, 5, and 8, respectively) distributions across groups were compared by Kruskal-Wallis H test and statistically significant differences indicated as either *p<0.05 or **p<0.01. clearly induced above mock control by PUUV-containing Vero E6 supernatant but not with purified PUUV (Figure 3A). To elaborate on the finding that live PUUV does not cause NETosis, UV-inactivation of PUUV-infected Vero E6 supernatant did not affect its ability to induce NETosis (Figure 3B). These findings indicate that live PUUV alone is not capable of inducing NETosis but instead involves other factor/s released from hantavirusinfected Vero E6 cells supernatants which might act alone or synergistically with the inactivated virus. In order to analyze whether purified hantaviruses could induce PMN degranulation we incubated fresh PMNs with purified PUUV, TULV, HTNV, or PMA (as a positive control) and analyzed peroxidase activity in cell supernatants (Figure 3B). MPO activity was induced by PMA but not with any of the hantaviruses tested, suggesting that hantavirus do not induce significant PMN degranulation. These results imply that neutrophil activation in HFRS is not caused by direct contact with hantavirus but instead requires additional factors. Thus, we wanted to analyze whether PUUV-infected endothelial cells could play a role in neutrophil activation during HFRS. PUUV Induces a Pro-Inflammatory Phenotype in BECs Endothelial cells are the prime target of hantavirus infection in vivo (2,3). Thus, we hypothesized that PUUV could mediate neutrophil activation through infected endothelial cells. To test this, we either mock-infected or infected primary blood endothelial cells (BECs) with purified live PUUV or UVinactivated PUUV (UV-PUUV). By visualizing the expression of viral nucleocapsid protein N in the cells by immunofluorescence we observed that BECs were close to 100% infected at 3 days post infection (dpi), followed by significant drop in infectivity levels at 6 dpi (Figure 4A). The downregulation of PUUV infection in BECs is type I interferon-mediated, as reported previously (29), and is probably due to MxA protein sequestering the viral N protein (30) We analyzed the supernatants of infected BECs for the presence of IL-8 by ELISA and observed that BECs infected with live PUUV upregulate the secretion of IL-8 at 3 dpi as compared to mockor UV-PUUV infections (Figure 4B). However, longer culturing of mockor UV-PUUV infected BECs also upregulated IL-8 in the cell supernatants which finally led to Frontiers in Immunology | www.frontiersin.org 5September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS TABLE 1 | Correlation between different clinical variables reflecting HFRS disease severity and markers of neutrophil activation. Creatinine (max) Platelets (min) Leukocyte count (max) tPA MPO HNE Histones IL-8 MPO r0.215 −0.582** 0.405** 0.571* 1.000 0.363** 0.329* 0.659** Sig. 0.123 0.000 0.003 0.021 0.008 0.016 0.000 n53 52 52 16 53 53 53 36 HNE r0.364** −0.420** 0.296* 0.636** 0.363** 1.000 0.130 0.474** Sig. 0.007 0.002 0.033 0.008 0.008 0.352 0.004 n53 52 52 16 53 53 53 36 Histones r0.228* −0.260 0.273 −0.101 0.329* 0.130 1.000 0.464** Sig. 0.036 0.063 0.050 0.632 0.016 0.352 0.004 n53 52 52 16 53 53 53 36 IL-8 r0.138 −0.379* 0.292 0.536 0.659** 0.474** 0.464** 1.000 Sig. 0.423 0.025 0.089 0.089 0.000 0.004 0.004 n36 35 35 11 36 36 36 36 The clinical variables creatinine, platelet and leukocyte counts are maximum or minimum values from the course of hospital stay whereas tPA and markers of neutrophil activation are from 1st day of hospitalization. r, Spearman’s rho correlation coefficient; Sig., Significance of correlation (p); n, number of patients; MPO, Myeloperoxidase; HNE, Neutrophil elastase; IL-8, Interleukin-8; tPA, Tissue plasminogen activator; *p<0.05, **p<0.01. comparable, high levels of IL-8 irrespective of PUUV infection at 6 dpi. Since IL-8 could act as a general marker of inflammation (10), we wanted to see whether PUUV-infected BECs would upregulate also other inflammatory factors potentially important for neutrophil recruitment. Thus we assessed the expression of ICAM-1, a ligand for neutrophil-expressed CD11b/CD18 integrin complex, on the plasma membrane of BECs by immunofluorescence-based imaging and quantification assays. We found elevated levels ICAM-1 on the surface of PUUVinfected BECs as compared to mockor UV-PUUV infected BECs at 3 dpi (Figure 4C). The elevated ICAM-1 levels remained only slightly lower as compared to TNF-αtreated BECs, used as a positive control. At 6 dpi, ICAM-1 expression returned to baseline levels in PUUV-infected BECs concomitantly with the reduction of viral replication. As expected, TNF-αwas found to be a robust inducer of ICAM-1 in BECs and thus we hypothesized that TNF-αcould induce ICAM-1 also in PUUVinfected BECs. However, we did not find any evidence for TNF-α in PUUV-infected BEC supernatants by ELISA suggesting that the induction of ICAM-1 in PUUV-infected BECs is independent of this cytokine (data not shown). PMNs Adhere to PUUV-Infected BECs Next, we wanted to determine whether freshly isolated PMNs (containing mainly neutrophils) could adhere to PUUV-infected BECs which express elevated levels of IL-8 and ICAM-1. Fresh PMNs were incubated with mock-, UV-PUUV or live PUUVinfected BECs for 1 h, non-bound PMNs removed by washing and remaining numbers of BEC-bound PMNs determined by immunofluorescence-based imaging and quantitation of MPO (used as a marker of PMNs). In addition to MPO, bound PMNs were differentiated from BECs based on their segmented nuclear morphology by DNA staining. We found that PMN binding to PUUV-infected BECs was significantly elevated as compared to mockor UV-PUUV infected BECs at 3 dpi, but not at 6 dpi (Figures 5A,B), consistent with the elevated expression of ICAM-1 and higher virus replication in PUUVinfected BECs at 3 dpi. As positive and negative controls, we used TNF-αtreated BECs and PUUV infected-BECs without PMN incubation, respectively. Based on these results we consistently allowed PMNs to bind the 3-day BEC cultures for 1 h in the following experiments assessing PMN-BEC interaction as shown in Figure 5. We wanted to analyze further whether the binding of PMNs to PUUV-infected BECs is mediated by chemotactic signals driven by IL-8 and/or is dependent of the neutrophil-expressed CD11b/CD18 binding to ICAM-1 on BECs. We did this by incubating PMNs with either BECs infected with either live PUUV or UV-PUUV as the negative control for infection in the presence of neutralizing antibodies to IL-8 or CD18. We could determine that binding to PUUV-infected BECs was dependent on CD11b/CD18 on neutrophils but not on the presence of IL-8 (Figures 5A,C). Not surprisingly, CD11b/CD18 seemed to mediate the adhesion of PMNs also to TNF-αtreated BECs, although less dramatically (Figure 5D). The fact that CD18 neutralizing antibody was not as efficient in blocking PMN adhesion to TNF-αtreated BECs could be due to the excess level of ICAM-1 upregulated by TNF-αas compared to PUUV infection (Figure 4C). Given that HTNV has been shown to bind CD18 integrin (21) we also tested the effect of viral glycoprotein (Gn and Gc) neutralizing antibodies (31) on PMN-BEC interaction. However, we could not observe any significant effects (Figure 5E) by Gn or Gc neutralizing antibodies suggesting that PMN adhesion to infected BECs is mediated by host-derived inflammatory factors but not viral proteins. Unfortunately, we could not reliably determine the role of ICAM-1 in PMN-BEC interaction since the presence of ICAM-1 specific antibodies resulted in further elevated binding of PMNs to BECs regardless of BECs being infected with PUUV or not (data not shown). We hypothesize that this phenomenon could be due to antibody-mediated cross-linking of neutrophiland BEC-associated ICAM-1 proteins. In addition, ICAM-1 antibodies are known to activate cross-linking and activation Frontiers in Immunology | www.frontiersin.org 6September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS FIGURE 2 | Immunohistochemical analysis of MPO, HNE and IL-8 in kidney sections from patients with acute PUUV-HFRS. Kidney sections from acute PUUV-HFRS or unrelated kidney diseases (n=5 for both) were analyzed for the presence of MPO, HNE or IL-8 by standard immunohistochemical techniques and visualized by DAB staining. (A) Images from three cases with acute PUUV-HFRS and one PUUV-negative control section is shown. (B) The tissue area positive for MPO, HNE or IL-8 was evaluated as percentages in all sections and mean values ±standard deviation reported for acute PUUV-HFRS and PUUV-negative sections. Significant differences were assessed by student’s T-test and statistically significant differences indicated as *p<0.05. of ECs on cell surfaces in some conditions (32) which could potentially explain this finding. PUUV-Infected BECs Induce PMN Degranulation Next, we wanted to determine whether more extensive coculturing of PMNs and PUUV-infected BECs could result in PMN activation (degranulation or NETosis) which could explain our findings of increased levels of extracellular neutrophil markers in PUUV-HFRS patients (Figures 1,2). We observed a time-dependent morphological change in PMNs bound to PUUV-infected BECs but not in mockor UV-PUUV infected BECs which was visible after 3 but not 1 h of co-culture (Figures 6A,B). This was evident by increased nuclear swelling of PMNs together with altered localization of MPO from diffuse to plasma membrane-associated staining. Furthermore, MPO could be observed also extracellularly, which strongly suggests that PMN degranulation was taking place. The fluorescent phenotype of PMNs bound to PUUV-infected BECs was strikingly similar although less pronounced as observed for PMNs bound to TNF-αtreated cells after a 3 h of co-culture. We did not find any evidence for NETosis in PMNs bound either on PUUV-infected or TNF-αtreated cells or by PUUV-infected BEC supernatants (data not shown) in these experimental conditions. Antiviral Effect of PMNs Is Mediated by Protease Release in vitro Finally, we wanted to determine whether neutrophils possess antiviral function and if so, by which mechanism. We incubated purified PUUV with freshly isolated PMNs, which were either non-activated or activated with PMA. Furthermore, incubations Frontiers in Immunology | www.frontiersin.org 7September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS FIGURE 3 | Live PUUV does not induce neutrophil activation. (A) Mockor PUUV-containing Vero E6 supernatants (Sup) or purified PUUV were incubated with freshly isolated PMNs for 3 h (MOI 1) (n=2). The release of extracellular DNA was assessed by an impermeable DNA binding fluorescent dye. (B) Mock-, live PUUVor UV-inactivated PUUV-containing Vero E6 supernatants were incubated with PMNs and assessed for DNA release as in (A) (n=3). (C) Purified PUUV, TULV or HTNV were incubated with fresh PMNs for 3 h (MOI 1) and peroxidase activity assessed from cell culture supernatants by TMB (n=3). PMA-treated PMNs were used as a positive controls. Difference between groups were assessed by one-way ANOVA +Dunnett’s multiple comparisons test and statistically significant differences indicated as either *p<0.05 or **p<0.01. FIGURE 4 | PUUV infection causes pro-inflammatory responses in BECs. (A) BECs were either mock-infected or infected with live or UV-inactivated PUUV (UV-PUUV) and assessed for viral nucleocapsid protein expression at 3 and 6 days post infection by immunofluorescence (red). The nuclei of BECs were visualized with Hoechst 33420 (blue). (B) IL-8 was measured from the respective supernatants of mock, UV-PUUV or PUUV-infected BECs by ELISA. (C) ICAM-1 expression in TNF-αtreated or mock-, UV-PUUV or PUUV-infected BECs was visualized by immunofluorescence (green) and shown as an overlay with Hoechst 33258 staining (blue). Fluorescence intensity of ICAM-1 expression on BECs was quantified and reported as fold change to mock-infected cells. Differences between groups were assessed by one-way ANOVA with Dunnett’s multiple comparisons test and statistically significant differences indicated as **p<0.01. n=2 in all panels. Results shown are representatives of three independent experiments. Frontiers in Immunology | www.frontiersin.org 8September 2018 | Volume 9 | Article 2098 Strandin et al. Neutrophil Activation in HFRS FIGURE 5 | Binding of quiescent PMNs to PUUV-infected BECs. Freshly isolated PMNs were allowed to bind to mock-, UV-PUUV or live PUUV-infected BECs for 1 h at 3 or 6 days post infection (dpi). (A) PMNs bound to either TNF-αtreated, UV-PUUV or PUUV-infected BECs were visualized at 3 dpi by immunofluorescence using MPO-specific antibody. The nuclei were stained with Hoechst 33420. (B) BEC-bound PMNs were quantified at 3 and 6 dpi as fold change to mock-infected cells. (C) PMNs were allowed to bind to PUUV-infected BECs at 3 days post infection in the presence of neutralizing antibodies to IL-8, CD18 or an isotype control and quantified as above. (D) PMNs were allowed to bind to BECs treated with TNF-αfor 3 days in the presence or absence of CD18 antibody and quantified as above. (E) PMNs were allowed to bind at 3 days post infection to PUUV-infected BECs treated with Gnor Gc-specific neutralizing antibodies and quantified as above. Statistically significant differences between groups were assessed either by student’s T-test (B,D) or one-way ANOVA with Dunnett’s multiple comparison test (C,E) and indicated as **p<0.01. n=2 in all panels. Results shown are representatives of three independent experiments. of virus with PMA-activated PMNs were performed in the presence of one of the following inhibitors: NaN3(an inhibitor of MPO activity), phenylmethylsulfonylfluoride (PMSF; inhibitor of serine proteases), EDTA (inhibitor of metalloproteinases) and DNAse (degradation of NETs). After incubation, samples were subjected to immunoblotting in order to detect degradation of Frontiers in Immunology | www.frontiersin.org 9September 2018 | Volume 9 | Article 2098