Acute clearance of human metapneumovirus occurs independently of natural killer cells

Acute Clearance of Human Metapneumovirus Occurs Independentlyof Natural Killer Cells

Sherry C. Wen,a Sharon J. Tollefson,b Monika Johnson,b Pavlo Gilchuk,a Kelli L. Boyd,a Bryan Shepherd,c Sebastian Joyce,a

John V. Williamsa,b

Department of Pathology, Microbiology, and Immunology,a Department of Pediatrics,b and Department of Biostatistics,c Vanderbilt University School of Medicine,Nashville, Tennessee

Human metapneumovirus (HMPV) is a major cause of respiratory disease. The role of NK cells in protection against HMPV isunclear. We show that while HMPV-infected C57BL/6 mice had higher numbers of functional lung NK cells than mock-treatedmice, comparing NK cell-depleted and control mice did not reveal differences in lung viral titers, histopathology, cytokine levels,or T cell numbers or function. These data indicate that NK cells are not required for host control of HMPV.

Human metapneumovirus (HMPV) is a major cause of lowerrespiratory illness (1, 2). No vaccine is currently available,

and HMPV causes recurrent infections, even in the immunocom-petent. The importance of natural killer (NK) cells for immunityagainst large DNA viruses is established (3–7); however, whetherNK cells contribute to RNA virus immunity is controversial.While one study reported that NK cells decreased the influenzavirus titer in mice (8), others found that NK cells increased lunginflammation associated with respiratory syncytial virus (RSV)and influenza (9, 10). The influence of NK cells on the adaptiveimmune response to respiratory viruses is also unclear and has notbeen addressed for HMPV. We sought to test the hypothesis thatNK cells are required to clear HMPV infection.

Lung NK cell numbers increase during HMPV infection andare functionally competent. We examined NK cells by flow cy-tometry in C57BL/6 (B6) mice (Jackson Laboratory) infected in-tranasally with 6 � 105 PFU HMPV (A2 clinical strain TN/94-49)(11) (Fig. 1A and B). Lung NK cell numbers in HMPV-infectedanimals increased significantly by day 1 postinfection (p.i.) andpeaked on day 3; however, there was no increase of NK cells inmock-treated mice (inoculated with cell lysate) or in mice inocu-lated with UV-inactivated HMPV (Fig. 1C). To measure NK cellfunctionality, we performed intracellular cytokine staining forgamma interferon (IFN-�) and surface staining for CD107a, amarker for cytotoxic-granule release (12, 13). There was a signif-icantly higher number of reactive NK cells in HMPV-infectedmice than in the mock-treated and UV-inactivated-HMPVgroups, indicating that NK cells in infected animals were func-tional (Fig. 1D). Surface expression of CD69, an inducible activa-tion marker (14), significantly increased on NK cells in infectedmice on days 1 and 3 p.i. but not in mice in the mock-treated andUV-inactivated-HMPV groups (Fig. 1E) (data not shown). Therewas also a higher number of CD3� T cells in the HMPV-infectedgroup than in the mock-treated and UV-inactivated-HMPVgroups (data not shown). Together, these results suggest thatHMPV replication results in increases in both NK cell number andfunctionality.

Lung viral titers and cytokines remain unchanged with NKcell depletion. To test whether NK cells are required to clearHMPV, we intraperitoneally injected B6 mice with either anti-NK1.1 or an isotype control antibody (BioXCell) 5 days beforeinfection, on the day of infection, and on day 5 p.i. for longer

experiments (Fig. 2A). This protocol depleted �95% of NK cellsin lungs and spleens, confirmed by flow cytometry on both organs(data not shown). Isotype control mice did not lose weight, as waspreviously described for other HMPV models (15); NK cell-de-pleted mice did not differ significantly from isotype control micein weight (Fig. 2B). We quantified viral titers by plaque titration asdescribed previously (11). In agreement with reports on influenzavirus (16, 17), NK cells did not affect viral titer, as NK cell-depletedmice had a peak viral load (day 5) and clearance kinetics equal tothose of the controls (Fig. 2C). Thus, in contrast to the criticalfunction of NK cells during infection by herpesviruses (3, 5), NKcells are nonessential for HMPV clearance.

A prior report by Alvarez et al. (18) suggested that NK cells hada role in limiting HMPV replication, as NK cell-depleted BALB/cmice had higher lung titers; however, that model described bipha-sic viral kinetics and long-term persistence not confirmed by oth-ers (13, 19–26). The discrepancy could be due to different mouseand virus strains or different depletion antibodies or protocols.

Since NK1.1 is expressed on both NK and natural killer T(NKT) cells, the anti-NK1.1 antibody could potentially depleteboth cell types (27); however, using anti-NK1.1 antibody is thecurrent preferred method of depleting NK cells (28–31), as anti-asialo-GM1 also affects basophils (32) and monocytes (33). Toaddress the issue of NK cell depletion, we used CD1d�/� mice (agift from Luc Van Kaer, Vanderbilt University), which lackNK1.1-positive (NK1.1�) NKT cells but have normal NK cellnumbers (34). HMPV-infected CD1d�/� mice had weights andviral titers similar to those of NK cell-depleted and isotype controlB6 mice (Fig. 2D) (data not shown). These results indicate thatneither NK nor NKT cells help clear HMPV. As NK and NKT cellsproduce a variety of cytokines (35, 36) and can affect cytokinesecretion by other leukocytes (12), we examined lung cytokinelevels in HMPV-infected control, NK cell-depleted, and CD1d�/�

Received 29 May 2014 Accepted 17 June 2014

Published ahead of print 25 June 2014

Editor: D. S. Lyles

Address correspondence to John V. Williams, [email protected].

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01558-14

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animals by real-time reverse transcription (RT)-PCR (13). Over-all, there were similar levels of cytokine gene expression in allgroups on days 5 and 7 p.i. (Fig. 2E to H) (data not shown), withmodest differences in interleukin-10 (IL-10) mRNA on day 7.IFN-� protein production was verified by enzyme-linked immu-nosorbent assay (ELISA) (Fig. 2I). These results indicate that theabsence of neither NK nor NKT cells substantially affects cytokineproduction during HMPV infection.

NK cells do not modulate lung histopathology. Since NK cellscontributed to RSV-associated inflammation (13), we examinedwhether NK cells affected lung inflammation post-HMPV infec-tion. Histology samples were analyzed qualitatively and quantita-tively in a blind manner by an expert pathologist (13, 37). Incontrast to the effect of NK cells in RSV infection (13, 38), thedegree of lung inflammatory-cell infiltration post-HMPV infec-tion was not altered by NK cell depletion (Fig. 3A to C). As NK

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FIG 1 Lung NK cell numbers increase after HMPV infection and are functionally competent. B6 mice were inoculated intranasally with mock-treated cell lysate,UV-inactivated HMPV, or HMPV, and the lung NK cell response was assessed. (A) Cells were stained with fluorescently labeled antibodies as indicated on theaxes. NK cells were defined as the DX5� CD3� population; experiments were confirmed with NKp46 as the NK cell marker (not shown). (B) Samples werestimulated with PMA and ionomycin. (C and D) The total number of NK cells (C) and NK cells producing IFN-� or CD107a� (indicating degranulation) (D)were quantified. Each symbol represents the mean of the results of two independent experiments with three individual mice per group for each time point andexperiment � the standard error of the mean (SEM). Dotted lines represent the numbers at baseline in uninfected mice. (E) CD69-expressing NK cells werequantified as a percentage of total NK cells on day 1 postinfection. For all graphs shown in panels C and D, the values for the HMPV group are significantly higherthan for the other two groups, with a P value of �0.005 when the entire trajectory curves are compared. A multiple linear model, including experiment, daypostinfection, and group (mock-treated, UV-inactivated, or HMPV), was fitted. The association between day and outcome was modeled using a quadratic term(i.e., day � day2) and an interaction between group and day (group � day group � day2). To test the overall effect of the group on outcome trajectories, weperformed a likelihood ratio test, where the model was fitted with the exclusion of group. Pairwise analyses were performed (i.e., by comparing the trajectoriesfor the mock-treated versus UV-inactivated group, the mock-treated versus HMPV group, and the UV-inactivated versus HMPV group). For panel E, the data(�SEM) from three independent experiments with three mice per group per experiment are combined. *, P � 0.05; two-tailed Student’s t test. SSC-A, side-scatterarea; FSC-A, forward-scatter area.

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cells were reported to regulate macrophage numbers (35, 39), lungsections were analyzed for macrophages by F4/80 staining; no dif-ferences were found (Fig. 3D). In both isotype control and NKcell-depleted groups, inflammation was predominately perivascu-lar on days 3 and 5 (Fig. 3E) (data not shown), although there wassome peribronchiolar inflammation on day 7 (Fig. 3F). These re-sults indicate that NK cells do not affect the degree of lung inflam-mation during acute HMPV infection.

CD4� (TCD4) and CD8� (TCD8) T cell numbers and functionsare preserved in the absence of NK cells. Since our results indi-cated that NK cells do not affect the host innate immune re-sponse to HMPV, we wondered whether NK cell depletionwould alter the adaptive immune response. TCD8 cells are im-

portant for HMPV clearance during primary infection (40);furthermore, the absence of NK cells alters the number of TCD8

cells responding to other viruses (41–46). To study the TCD8

cell response, we used an HLA B7.2 transgenic (B7tg) mousemodel (47) in which the M195-203 (M195) peptide is the immu-nodominant HMPV epitope (13).

We quantified lung TCD8 cells by tetramer staining on day 10p.i., the peak of the TCD8 response to HMPV (13). We found nosignificant differences in the total numbers of lymphocytes, TCD8

cells, or HMPV M195-specific TCD8 cells (Fig. 4A to C). There wasa higher percentage of TCD8 cells in the NK cell-depleted groupthan in the isotype control group (Fig. 4D); however, the absolutenumbers of TCD8 cells were not different.

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FIG 2 Lung viral titers and cytokines remain unchanged with NK cell depletion. (A) B6 mice were injected with either isotype (control) antibody or anti-NK1.1antibody and infected i.n. with HMPV. (B) Mice were weighed daily from the day of infection until euthanasia. (C) Viral titers were quantified in PFU per gramof lung tissue. (D) Additional experiments were performed to compare viral titers in B6 mice and CD1d�/� mice. The dotted line represents the limit of viraldetection. (E to H) Mice were infected with HMPV, and lung RNA was extracted for quantification of cytokine gene expression using RT-PCR. Cytokine levelswere normalized to the value for the housekeeping gene Hprt, and relative gene expression was expressed as the fold change compared to the average value forthe isotype control. (I) ELISA was performed to quantitate IFN-� protein production, expressed as picograms per gram of lung tissue. The data in panel B arecombined from five independent experiments, the data in panel C are combined from two to five independent experiments, and the data in panel D are combinedfrom two independent experiments with at least three mice per group for each time point and experiment �SEM. The data in panels E to I are combined fromtwo to four independent experiments with three to five individual mice per group for each time point and experiment �SEM. *, P � 0.05; two-tailed Student’st test.

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Programmed death-1 (PD-1), an inhibitory receptor, medi-ates TCD8 impairment during acute and chronic infections (13,48, 49). Since NK cell depletion reduced the expression of TCD8

exhaustion markers during lymphocytic choriomeningitis vi-rus (LCMV) infection (41), we tested whether NK cell deple-tion would affect TCD8 PD-1 expression during HMPV infec-tion. We found no significant differences in the percentages ornumbers of total TCD8 or M195-specific TCD8 cells expressingPD-1 (Fig. 4E to G). To quantify TCD8 cell functionality, wemeasured IFN-� and CD107a using lung lymphocytes from thesame mice used for analysis by tetramer staining. Consistentwith TCD8 cell impairment previously reported (13), only afraction of the HMPV-specific TCD8 cells produced IFN-� anddegranulated (Fig. 4H). However, comparing the NK cell-de-pleted and isotype groups revealed no significant differences inthe percentages or numbers of TCD8 cells degranulating or pro-ducing IFN-� or in the IFN-� mean fluorescence intensity(MFI) (Fig. 4H) (data not shown).

To determine whether NK cell depletion affected TCD4 cells, westained for CD4 and quantified IFN-� by intracellular cytokinestaining following stimulation with phorbol myristate acetate(PMA)-ionomycin. We found no significant differences betweenthe NK cell-depleted and isotype groups in percentages or num-bers of total TCD4 cells, IFN-�-producing TCD4 cells, or IFN-�MFIs, indicating that TCD4 cell function is preserved during NKcell depletion (Fig. 4I and J) (data not shown). Together, thesedata suggest that in the context of HMPV infection, NK cell de-pletion does not have a significant effect on TCD4 or TCD8 cellfunctionality.

In summary, the present work indicates that acute HMPV in-fection can be cleared independently of NK cells. Our results agreewith previous reports that found certain components of the im-mune system to be dispensable in some situations but essential inothers (50). Taken together, these findings suggest that aspects ofthe immune response to HMPV infection are different from thoseto RSV, consistent with prior findings with humans (51). Addi-

FIG 3 NK cells do not modulate lung histopathology. Mice were infected with HMPV, and whole lungs were excised for histology. Slides were stained withhematoxylin and eosin (H&E) (A to C) or F4/80 (D). Representative images are shown for day 5 (�400 magnification) (A), day 7 (�100 magnification)(B), day 7 (�400 magnification) (C), and day 7 (�400 magnification) (D). Arrows point to areas of inflammation. Inflammation was scored in a blindmanner by an expert pathologist on a scale from 0 to 4, with 4 being the maximum level of inflammation, on day 5 (E) and day 7 (F). Error bars indicatestandard deviation.

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FIG 4 CD4� and CD8� T cell numbers and function are preserved in the absence of NK cells. B7tg mice were infected with HMPV, and lung lymphocytes wereisolated on day 10 postinfection for analysis by flow cytometry. The total numbers of lymphocytes (A), CD8� T cells (B), and M195 tetramer-specific CD8� Tcells (C) are shown. (D) Lung CD8� T cell population expressed as a percentage of total lymphocytes in isotype (control) and NK cell-depleted animals. The totalnumber of PD-1� CD8� T cells (E) and the number of PD-1� M195 epitope-specific CD8� T cells (F) are also shown. (G) Representative flow cytometry plotsafter staining with PD-1 and M195 tetramer labeled with allophycocyanin (APC) (left), PD-1 and an irrelevant vaccinia virus tetramer (middle), and isotypecontrol antibody for PD-1 and M195 tetramer (right). The number in each quadrant represents the subset population as a percentage of CD8� T cells. In parallelwith tetramer staining, lymphocytes were stimulated in vitro with M195 peptide and stained for CD3, CD8, IFN-�, and CD107a. (H) Percent M195 tetramer-positive and percent functional M195-specific CD8� T cells. In other samples, lymphocytes were stimulated with PMA and ionomycin and stained for CD3, CD4,and IFN-�. (I) Total number of CD4� T cells. (J) Total number of IFN-�-producing CD4� T cells. Eight thousand total CD4� or CD8� T cells per sample werecollected by flow cytometry. Data are combined from three independent experiments with four mice per group per experiment �SEM. *, P � 0.05; two-tailedStudent’s t test.

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tional research will further our understanding of the pathophysi-ology associated with HMPV infection.

ACKNOWLEDGMENTS

We thank D. Flaherty, B. Matlock, and C. Warren at the Vanderbilt FlowCytometry Shared Resource. We also thank A. Sette, F. Lemonnier, and L.Van Kaer for providing mice.

The VMC Flow Cytometry Shared Resource is supported by the Van-derbilt Ingram Cancer Center (P30 CA68485) and the Vanderbilt Diges-tive Disease Research Center (DK058404). This work was supported byR01 AI85062 (to J. V. Williams) and by T32 GM07347 from the NationalInstitute of General Medical Studies for the Vanderbilt Medical ScientistTraining Program.

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