The role of M2-2 PDZ-binding motifs in pulmonary innate immune responses to human metapneumovirus

2.1 rHMPV preparation and titration

WT-rHMPV or recombinant mutants with mutations in the motif 29-DEMI-32 (Mut-1) or the motif 39-KEALSDGI-46 (Mut-2), were generated and propagated in LLC-MK2 cells (ATCC, Manassas, VA), as previously described.^{21, 23, 24} Viral titers of purified recombinant viruses or viruses from the infected mouse lungs were determined by immunostaining in LLC-MK2 cells, as described previously.^{21, 25}

2.2 Mice

Six- to eight- week-old female BALB/c mice were purchased from The Jackson Laboratory and maintained under specific-pathogen-free conditions at the University of Texas Medical Branch (UTMB) at Galveston. To investigate the innate response of mice to the primary infection, mice were anesthetized and administered intranasally with 5 × 10⁶ pfu of rHMPV, diluted in Dulbecco's PBS (D-PBS) (Invitrogen, Carlsbad, CA), or with an equivalent volume of sucrose diluted in D-PBS (mock-infection), as previously described.²⁶ To study whether mice with primary inoculation of rHMPV develop protection, mice were challenged with 10⁷ pfu of HMPV/CAN/97/83/A2 on day 28 postprimary infection. Lungs at day 5 postprimary infection or post-challenge were harvested to determine the HMPV titers in the lungs. All of the studies were approved by the UTMB Institutional Animal Care and Use Committee.

2.3 Bronchoalveolar lavage analysis

Bronchoalveolar lavage (BAL) fluid was collected by washing the lungs two times with 1 mL D-PBS at different days postinfection (p.i.). After centrifuging at 1500 rpm for 5 minutes at 4°C, the supernatants were used for quantification of cytokines/chemokines using Luminex-based Bio-Plex Pro Mouse Cytokine 23-plex Assay (Cat #M60009RDPD; Bio-Rad Laboratory, Hercules, CA) according to the manufacturer's instructions. The examined cytokines include Eotaxin, G-CSFm GM-CSF, interferon (IFN)-γ, interleukin (IL)-1α, IL-1β, IL-2, IL-3, Il-4, Il-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and tumor necrosis factor-α. The detection limit for all cytokines by the Bioplex assay is 3 pg/mL. The IFN-β was quantified by enzyme-linked immunosorbent assay (ELISA) (PBL Biomedical Laboratories, Piscataway, NJ) with the detection limit of 50 pg/mL.

2.4 Airway hyperresponsiveness

Airway hyperresponsiveness (AHR) was assessed in unrestrained conscious mice by noninvasive nonventilated enhanced pause (Penh) measurement, as described previously.^{26, 27} Freely moving mice were placed in whole-body barometric plethysmography (Buxco Electronics, Troy, NY) and respiratory activity was recorded for 4 minutes to measure baseline Penh values.

2.5 Fluorescence-activated cell sorting analysis of immune cells

Lung samples of mice, mock-inoculated or inoculated with WT-rHMPV or mutants, were harvested at days 5 and 7 p.i. Total lung cells were prepared, as previously described.^{25, 26} Isolated cells were incubated with anti-FcγRII/RcγRIIImAb (24G2; BD Biosciences, San Jose, CA) followed by cell-surface marker staining. For cell-surface marker staining, isolated cells were stained with the following antibodies: anti-CD11c in combination with anti-CD80, anti-CD86, or anti-MHCII (all from BD Pharmingen, San Jose, CA) for DCs and anti-CD4 or anti-CD8 in combination with anti-CD3 (all from BD Pharmingen) for T cells. Samples were stained at 4°C in PBS with 1% fetal bovine serum and analyzed with a BD FACSCanto flow cytometer equipped with BD FACSDiva software (both from Becton Dickinson Immunocytometry Systems, Franklin Lakes, NJ). The analysis was performed using Cyflogic (Cyflo Ltd, Turku, Finland).

2.6 Statistical analysis

Statistical significance was determined by the Mann-Whitney U test. Values of P < .05 were considered significant. Values for viral replication, cell numbers, and cytokine/chemokine production experiments were presented as mean ± standard error (SEM) of the mean.

3.1 M2-2 PDZ-binding motifs are important to pulmonary HMPV replication and the modulation of pulmonary cytokine/chemokine induction

To determine whether the motifs 29-DEMI-32 and 39-KEALSDGI-46 are important in mediating pulmonary innate responses to HMPV, we first compared pulmonary viral loads in WT-, Mut-1-, or Mut-2-infected mice, at day 5 p.i., the replication peak of primary HMPV infection.^{26, 27} In brief, BALB/c mice were intranasally inoculated with WT, Mut-1, or Mut-2 at 5 × 10⁶ pfu/mice, followed by lung harvesting and viral titration. The mock-inoculation was used as a negative control. As shown in Figure 1A, mice infected with Mut-1 and Mut-2 exhibited more than a log reduction in lung viral titer than those infected with WT, indicating the replication attenuation of mutants. To confirm the mutations after infection, we isolated total RNAs from infected lungs at day 5 p.i. The DNA sequencing of reverse transcription PCR products demonstrated that mutations of each motif were kept intact (data not shown), supporting the importance of motifs in the regulation of HMPV replication. The sequencing was done in the Molecular Genomics Core at UTMB.

We then investigated the pulmonary cytokine/chemokine induction by infection, WT or mutants. In brief, we inoculated mice with WT or mutants, using mock-inoculation as a control, and measured the abundance of cytokines/chemokines in pulmonary BAL fluid at day 1 and 5 p.i by Bio-Plex Pro Mouse Cytokine 23-plex Assay (Bio-Rad). Generally speaking, the induction of most cytokines/chemokines by HMPV infection peaks at day 1 p.i., and goes back to the basal level at day 5 p.i.²⁸ Herein, Figure 1B,C show the induction at day 1 p.i. Mediators with the induction >5000 pg/mL are listed in Figure 1B, while the mediators with the induction <2500 pg/mL are shown in Figure 1C. Compared with the cytokine/chemokine induction by WT-rHMPV, the induction of these molecules was significantly enhanced by Mut-1 or Mut-2 infection. Pulmonary IFN-β induction was comparable between WT- and Mut-2 infected lungs. At day 5 p.i., the abundance of pulmonary cytokines/chemokines became significantly less in all groups, compared with that at day 1 p.i. The ones detected are shown in Figure 1D. Our Bio-plex assay did not detect any induction of Th2 cytokines including IL-4, IL5, and IL-13 in all experimental groups. Overall, these results demonstrated that both motifs 29-DEMI-32 and 39-KEALSDGI-46 of M2-2 suppress the induction of pulmonary innate immune mediators after HMPV infection.

3.2 Mutant infection of mice provides protection against viral challenge with a clinical isolate

To investigate the impact of motifs on the lung function in response to HMPV infection, we compared the AHR in WT- and mutant-infected mice by Penh measurement, a method using a noninvasive and nonventilated unrestrained whole-body plethysmography to correlate changes in airway physiology by the infection.^29-31 Mock-inoculation was used as a negative control. The basal Penh in response to HMPV infection usually peaks between day 5 and day 7 p.i.²⁷ Therefore, we chose day 6 p.i. for the Penh investigation. We found that, at day 6 p.i., there were no significant Penh differences between WT- and Mut-2-infected mice, while Mut-1 induced moderately higher Penh than WT and Mut-2 (Figure 2A). At day 8 p.i., the Penh in response to WT, Mut-1, and Mut-2 infections became comparable among the groups.

Innate inflammatory cytokines and viral loads are important determinants of lung pathogenesis. Both are generally suggested to be associated with the disease severity of HMPV and respiratory syncytial virus (RSV).^32-40 Although the attenuation of mutant replication (Figure 1A) and enhanced induction of protective immune mediators, such as IL12-p40,³³ could prevent severe lung injury from possible adverse effects of mutant-enhanced inflammatory cytokines, there are three recent publications from different groups, who supported that higher innate cytokines triggered by an essential amount of infectious particles are necessary to prevent severe RSV diseases.^41-43 Viruses with a low inoculation indeed can cause more severe disease because of their failure to induce sufficient innate immune responses, demonstrating complicated balance mechanisms underlying disease severity.^41-43 Although no such reports are available for HMPV infection, and we do not know whether mutant-enhanced cytokine/chemokine induction is beneficial or deleterious to lung functions, our Penh results did not show uncontrollable pathogenesis by mutant infections, compared with WT infection. No animal death was observed after the primary infection of WT or mutants either.

Next, to evaluate the protective capacity of rHMPV mutants, we challenged mice with HMPV/CAN/97/83/A2 at 10⁷ pfu/mice on day 28 post-initial inoculation with WT or mutant rHMPV. The mock-inoculation was used as a negative control. The lungs were harvested at day 5 post-challenge for viral titration. No pulmonary viral particles could be detected in WT- and mutant rHMPV-inoculated mice, while mock-inoculated mice had significant virus replication (Figure 2B), suggesting that mutants could generate protection. We also did not observe the difference in Penh at day 5 post-challenge among mice which were immunized with WT, Mut-1, and Mut-2 (Figure 2C). No animal death and detectable Th2 cytokines (IL-4, IL-5, and IL-13) were observed after the challenge. The Th2 cytokine quantification was done by ELISA kits (R&D Systems, Minneapolis, MN).

3.3 The motifs inhibit lung DC maturation in response to HMPV

It has been previously shown that HMPV infection leads to the pulmonary recruitment of plasmacytoid DCs (pDCs) and conventional DCs (cDCs) with different kinetics after the primary infection of mice.⁴⁴ Recently, we also demonstrated that monocyte-derived DCs (moDCs), an appropriate model for lung cDCs,^{45, 46} are regulated by the M2-2 protein in response to HMPV infection.²⁰ Herein, we investigated whether M2-2 uses its putative PDZ-binding motifs to regulate the DCs in rHMPV-infected lungs. Fluorescence-activated cell sorting (FACS) analysis revealed that the number of lung pDCs and cDCs was increased after mutant inoculation compared with WT inoculation and this increase was most significant in Mut-2-infected mice on day 7 p.i. (Figure 3A). In addition to enhanced cDC infiltration, we also found the cDC maturation was affected by the motifs. In brief, lung cells isolated from WT- or mutant-infected mice were stained with DC maturation markers of CD80, CD86, or MHCII and followed by FACS analysis. At day 7 p.i., CD11c⁺ lung cells from Mut-1- and Mut-2-infected mice had a significant increase in the expression of CD80 and MHCII (Figure 3B,C) and Mut-1 had more impact on CD86 expression than WT-infected lungs. These results supported that M2-2 PDZ-binding motifs have an inhibitory effect on DC maturation and recruitment in HMPV infection.

3.4 The motifs suppress pulmonary T cell infiltration and activation in response to HMPV

DCs play an essential role in bridging innate and adaptive immunity.⁴⁷ DC maturation identified by costimulatory molecules including CD80 and CD86 is important for T cell function.^{48, 49} Improved DC maturation by mutant infection, therefore, likely leads to enhanced T cell responses. To test this hypothesis, pulmonary T cells were assessed at day 7 p.i. in mice infected with WT or mutants through FACS analysis. Compared with WT infection, the number of lung CD4 and CD8 cells was increased by Mut-1 infection (Figure 4A). Mut-2 infection also led to significant enhancement in CD4 cells, but not in CD8 cells. As shown in Figure 4B, the percentage of both CD4 and CD8 T cells was significantly enhanced by mutants, compared with pulmonary T cells of WT-infected lungs. Collectively, these results suggest that M2-2 PDZ-binding motifs are important in regulating T cell responses to HMPV infection.