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. 2024 May 8;15(5):e0055024.
doi: 10.1128/mbio.00550-24. Epub 2024 Mar 26.

IFN-λ drives distinct lung immune landscape changes and antiviral responses in human metapneumovirus infection

Jorna Sojati  1 , Olivia B Parks  1 , Yu Zhang  1 , Sara Walters  1 , Jie Lan  1 , Taylor Eddens  1 , Dequan Lou  2 , Li Fan  2 , Kong Chen  2 , Tim D Oury  3 , John V Williams  1   4   5
Affiliations

IFN-λ drives distinct lung immune landscape changes and antiviral responses in human metapneumovirus infection

Jorna Sojati et al. mBio. .

Abstract

Human metapneumovirus (HMPV) is a primary cause of acute respiratory infection, yet there are no approved vaccines or antiviral therapies for HMPV. Early host responses to HMPV are poorly characterized, and further understanding could identify important antiviral pathways. Type III interferon (IFN-λ) displays potent antiviral activity against respiratory viruses and is being investigated for therapeutic use. However, its role in HMPV infection remains largely unknown. Here, we show that IFN-λ is highly upregulated during HMPV infection in vitro in human and mouse airway epithelial cells and in vivo in mice. We found through several immunological and molecular assays that type II alveolar cells are the primary producers of IFN-λ. Using mouse models, we show that IFN-λ limits lung HMPV replication and restricts virus spread from upper to lower airways but does not contribute to clinical disease. Moreover, we show that IFN-λ signaling is predominantly mediated by CD45- non-immune cells. Mice lacking IFN-λ signaling showed diminished loss of ciliated epithelial cells and decreased recruitment of lung macrophages in early HMPV infection along with higher inflammatory cytokine and interferon-stimulated gene expression, suggesting that IFN-λ may maintain immunomodulatory responses. Administration of IFN-λ for prophylaxis or post-infection treatment in mice reduced viral load without inflammation-driven weight loss or clinical disease. These data offer clinical promise for IFN-λ in HMPV treatment.

Importance: Human metapneumovirus (HMPV) is a common respiratory pathogen and often contributes to severe disease, particularly in children, immunocompromised people, and the elderly. There are currently no licensed HMPV antiviral treatments or vaccines. Here, we report novel roles of host factor IFN-λ in HMPV disease that highlight therapeutic potential. We show that IFN-λ promotes lung antiviral responses by restricting lung HMPV replication and spread from upper to lower airways but does so without inducing lung immunopathology. Our data uncover recruitment of lung macrophages, regulation of ciliated epithelial cells, and modulation of inflammatory cytokines and interferon-stimulated genes as likely contributors. Moreover, we found these roles to be distinct and non-redundant, as they are not observed with knockout of, or treatment with, type I IFN. These data elucidate unique antiviral functions of IFN-λ and suggest IFN-λ augmentation as a promising therapeutic for treating HMPV disease and promoting effective vaccine responses.

Keywords: host-pathogen immunity; human metapneumovirus; interferon; respiratory infection.

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Conflict of interest statement

J.V.W. previously served on the Scientific Advisory Board of Quidel and an Independent Data Monitoring Committee for GlaxoSmithKline, neither activity involved with the work under consideration. All other authors declare no conflicts of interest.

Figures

Fig 1
Fig 1
IFN-λ is highly upregulated during HMPV infection. Mice were infected with 5 × 105 PFU of HMPV strains TN/94-49 or C2-202 or mock cell lysate and IFN-λ2/3, IFN-β, and IFN-γ were measured in lung homogenate day 1 post-infection by (A) Luminex; (B) ELISA; or (C) qPCR. Data in C were normalized to HPRT1 gene and mock-infected mice by the 2−∆∆Ct method. (D) Mice were infected with HMPV C2-202 and IFN-λ2/3, IFN-β and IFN-γ were measured in lung homogenate days 1, 5, and 7 post-infection by Luminex. (E) BEAS-2B cells were infected with TN/94-49 or C2-202 and IFN-λ1 and IFN-λ2 measured in supernatant days 0–3 post-infection by Luminex. (F) C10 cells were infected with TN/94-49 or C2-202 and IFN-λ1 and IFN-λ2 measured in supernatant days 0–3 post-infection by Luminex. Cells were infected at multiplicity of infection (MOI) of 0.05. Limit of detection noted by dashed line. Data are shown as mean ± standard deviation. Analyses by student’s one-way or two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 2
Fig 2
Alveolar type II epithelial cells are primary IFN-λ inducers during early HMPV infection. Mice were infected with 5 × 105 PFU of HMPV strain C2-202 or mock cell lysate and IFN-λ expression in mouse lungs was measured by flow cytometry day 1 post-infection. (A) Schematic diagram of flow cytometric IFN-λ staining. (B) Gating for IFN-λ expression on type II alveolar epithelial cells. Fluorescence-minus-one control and isotype control (irrelevant T-cell marker RORγT conjugated to the same fluorophore) were used. For C–G, IFN-λ expression was assessed in lung epithelial and myeloid populations of mock- or C2-202 HMPV-infected mice day 1 post-infection. (C) Frequency of IFN-λ2/3+ cells in CD45+ (immune) vs CD45− (non-immune) populations. (D) Frequency of IFN-λ2/3+ cells in alveolar vs airway epithelial cell types. (E) Frequency of IFN-λ2/3+cells in alveolar cell subpopulations in lungs of mice, showing upregulation of IFN-λ in type II alveolar epithelial cells with HMPV infection. (F) Frequency of IFN-λ2/3+ cells in parent populations of macrophages vs dendritic cells. (G) Frequency of IFN-λ2/3+ cells in dendritic cell subpopulations. Data are shown as mean ± standard deviation. Analyses were done by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 3
Fig 3
Epithelial cells respond to IFN-λ and internalize HMPV in early infection. Single-cell RNA sequencing (scRNA-seq) was performed on cells isolated from lungs of mice infected with 5 × 105 PFU C2-202 HMPV and harvested day 1 post-infection. (A) Type I IFN receptor expression was measured. Gene expression of subunits Ifnar1 (right) and Ifnar2 (left) stratified by lung cell type. (B) IFN-λ receptor expression was measured. Gene expression of subunits Ifnlr1 (right) and IL10Rb (left) stratified by lung cell type. (C) Expression levels of HMPV transcripts was analyzed and stratified by cell type. (D) qPCR of HMPV expression was done for FACS-sorted populations from lungs of C2-202-infected mice harvested day 1 post-infection. Data are normalized to the HPRT1 gene and the null condition of mock-infected mouse lung homogenate by the 2−∆∆Ct method. Analysis was done by one-way ANOVA. **P < 0.01, ***P < 0.001.
Fig 4
Fig 4
IFN-λ limits HMPV lung replication without contributing to inflammatory disease. C57BL/6 (B6) mice (shown in black), Ifnar1−/− (called IFNAR−/−, shown in green), and Ifnlr1−/− (called IFNLR−/−, shown in purple) were infected with 5 × 105 PFU C2-202 HMPV and disease assessed by measuring body weight (A) and clinical severity scores (B) to day 7 post-infection. Weight represented as % of day 0. Clinical severity scores were measured by assigning 1 point out of 5 for each of the following: hunching, huddling, fur ruffling, rapid breathing, and lethargy. Analyses were done by two-way ANOVA. ***P < 0.001, ****P < 0.0001 for IFNAR−/− vs both WT and IFNLR−/−. (C) Lung histology was performed for 5 × 105 C2-202 HMPV-infected mice euthanized day 5 or day 7 post-infection. Scoring criteria per field included: 0: no inflammation; 1: <25% inflammation; 2: 25%–50% inflammation; 3: 50%–75% inflammation; 4: >75% inflammation. Score for each sample was added and divided by total number of fields analyzed. Representative lung histology images shown on right. (C, D) HMPV titer (PFU/g) was measured in lung homogenates (E) or nasal turbinates (F) of 5 × 105 C2-202 HMPV-infected mice day 5 post-infection. Limit of detection noted by dashed line. Analyses were done by two-way ANOVA, ***P < 0.001, ****P < 0.0001.
Fig 5
Fig 5
IFN-λ promotes differential lung innate cell recruitment. WT (shown in black), Ifnar1−/− (labeled IFNAR−/−, shown in green), and Ifnlr1−/− (labeled IFNLR−/−, shown in purple) mice were infected with 5 × 105 PFU C2-202 HMPV, and lung myeloid and epithelial cell populations quantified day 1 post-infection by flow cytometry. (A–D) (Left) Frequency of lung innate immune cell populations, including M2 macrophages (A), CD11b+CD11c+ interstitial macrophages (B), inflammatory monocytes (C), and plasmacytoid dendritic cells (D) day 1 post-infection. (Right) Frequency of lung immune cell populations of C2-202 HMPV-infected mice from days 1–5 post-infection. (E, F) (Left) Frequency of lung epithelial cell populations, including ciliated (E) or basal (F) epithelial cells day 1 post-infection. (Right) Frequency of lung epithelial cell populations of C2-202 HMPV-infected mice from days 1–5 post-infection. Data are shown as mean ± standard deviation. Analyses were done by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 6
Fig 6
IFN-λ limits HMPV spread from upper to lower airways. (A) C57BL/6 (B6) mice were infected intranasally with 5 × 105 PFU TN/94-49 HMPV in 10-, 15-, 20-, and 50 µL volumes (n = 10 mice per group). Mice were euthanized 5 min post anesthesia recovery. Lung and nasal turbinate viral titers (plaque-forming units/g) were quantified by plaque assay. (B) Schematic diagram of upper respiratory tract (URT)-restricted HMPV infection. (C, D) WT (shown in black), Ifnar1−/− (labeled IFNAR−/−, shown in green), and Ifnlr1−/− (labeled IFNLR−/−, shown in purple) mice were infected with the URT-restricted infection model of 10 µL of 5 × 105 PFU TN/94-49 HMPV intranasally, and titer (PFU/g) was measured in lung homogenates (D) or nasal turbinates (C) by plaque assay. (E) Data from 6C and 6D graphically depicted to highlight lower airway HMPV spread on day 5 post-infection. Limit of detection was noted by dashed line. Analyses were done by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 7
Fig 7
Inflammatory cytokine expression is altered by loss of IFN-λ signaling. WT (shown in black), Ifnar1−/− (labeled IFNAR−/−, shown in green), and Ifnlr1−/− (labeled IFNLR−/−, shown in purple) mice were infected with 5 × 105 PFU C2-202 HMPV. (A) Protein expression levels by Luminex assay (ng/mL) of inflammatory cytokines in lung homogenates collected on day 1 post-infection from 5 × 105 PFU C2-202 HMPV-infected mice graphically represented by heat map. (B) Protein expression levels by Luminex of IFN-λ2/3, IFN-β, IFN-γ, IL-6, MIP-1α, and MCP-1 cytokines in lung homogenate from WT, Ifnlr1−/− (labeled IFNLR−/−), and Ifnar1−/− (labeled IFNAR−/−) mice infected with 5 × 105 PFU C2-202 HMPV and harvested day 1 post-infection. Data are shown in 7A and 7B from one representative Luminex plate (n = 3 mice per group) with similar findings on repeat assay. (C) qPCR analysis of interferon-stimulated genes was done for lung homogenate of mice described in 7B. Data are shown as mean ± standard deviation. Analyses were done by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 8
Fig 8
IFN-λ prophylaxis and treatment reduce HMPV burden without inflammation. C57BL/6 (B6) mice were infected with 5 × 105 PFU C2-202 HMPV. (A) In a prophylaxis model (schematic diagram shown), recombinant IFN-λ (1 µg) or IFN-β (5 µg) was administered intranasally 1 days prior to infection. Mock prophylaxis included intranasal delivery of an equal volume of 0.1% bovine serum albumin (BSA). Mice receiving mock prophylaxis (shown in black), IFN-β prophylaxis (shown in green), and IFN-λ prophylaxis (shown in purple) were characterized (B–F). Disease was assessed by measuring body weight (B) and clinical severity scores (C) to day 7 post-infection. Weight represented as % of day 0. Clinical severity scores were measured by assigning 1 point out of 5 for each of the following criteria: hunching, huddling, fur ruffling, rapid breathing, and lethargy. Analyses were done by two-way ANOVA. ### P < 0.001, #### P < 0.0001 for mock vs both IFN-β and IFN-λ. **P < 0.01, ***P < 0.001, ****P < 0.0001 for IFN-λ vs IFN-β. (D, E) HMPV titer (PFU/g) was measured in lung homogenates (D) or nasal turbinates (E) of mice receiving IFN prophylaxis. (F) Frequency of lung innate immune and epithelial cell populations in mice receiving IFN prophylaxis euthanized day 1 post-infection by flow cytometry. (G) In a treatment model (schematic diagram shown), recombinant IFN-λ (1 µg) or IFN-β (5 µg) was administered intranasally 2 days post-infection. Mice receiving mock treatment (shown in black), IFN-β treatment (shown in green), and IFN-λ treatment (shown in purple) were characterized (G–K). Disease was assessed by measuring body weight (H) and clinical severity scores (I) to day 7 post-infection. Mock treatment included intranasal delivery of an equal volume of 0.1% BSA. (J, K) HMPV titer (PFU/g) was measured in lung homogenates (J) or nasal turbinates (K) of mice receiving treatment. Limit of detection noted by dashed line. Analyses were done by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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