Lair1^−/− mice were generated on the C57BL/6 background by Taconic Artemis as described (13). BALB/c mice were procured from Harlan (Horst, the Netherlands). All animal studies were approved by the Institutional Animal Care and Use Committee and carried out in accordance with the national and institutional guidelines.

Eight to Twelve-week-old female C57BL/6 Lair1^−/− mice or their wild-type littermates were intranasally infected with 1 × 10⁷ PFU of RSV-A2 in 50 μl PBS. RSV-A2 preparation, quantitative assay for RSV-A2 titration and RSV-A2 infection of mice, including, intranasal inoculation, termination, and sample collection, was performed as described previously (14, 15). Mice were sacrificed on day 2 or 5 post-infection.

For LAIR-1 blocking experiments, recombinant mouse LAIR-1 fused with the Fc portion of mouse IgG2a was produced in-house. The Fc tail was mutated to prevent binding to Fc receptors and complement as described previously (16).

Eight to twelve-week-old female BALB/c wild-type mice were injected intraperitoneally with 200 or 400 μg of mouse LAIR-1-Fc chimeric protein in 100 μl PBS or PBS alone as control, 1 day before and 2 days after RSV infection. Intranasal RSV infection was performed as described above.

Bronchoalveolar lavage (BAL) fluid collection was performed by flushing the lungs 2 times with 1.0 ml of ice-cold PBS. BAL fluid was centrifuged; supernatants were stored at −80°C for further analyses. Total cell counts in BAL were determined using a Bürker-Türk hemocytometer. BAL cells were analyzed by flow cytometry or examined by light microscopy.

For differential cell analysis by light microscopy, at least 200 cells were counted to assign relative quantities of macrophages, lymphocytes, and neutrophils based on morphology after May–Grünwald–Giemsa stain.

RSV-A2 concentrations in BAL fluid supernatants were analyzed by real-time PCR as described previously (15). Mouse CXCL1 (KC) and IL-6 levels in BAL fluid supernatants were measured by ELISA (Peprotech, London, UK) according to the manufacturer's instructions.

Male, 8–12 weeks old, wild-type and Lair1^−/− C57BL/6 mice were randomly assigned to undergo cigarette smoke (CS) or control air exposure. Mice were exposed to whole body mainstream CS generated from standard research cigarettes (3R4F; 9.4 mg tar/0.726 mg nicotine, University of Kentucky) using a Watson-Marlow roller pump (323 E series, speed 35 RPM; Watson Marlow, Rotterdam, The Netherlands) that directed the CS into the exposure chamber (25 L of air volume). Carbon monoxide and oxygen levels in the exposure chamber were measured using a gas analyzer from Bacharach (PCA-3 series; Bacharach, New Kensington, PA, USA) carbon monoxide concentrations were held at 150–300 ppm and oxygen levels were kept at 20%. Mice were exposed to CS twice daily for a maximum of 30 min with a 5-h smoke-free interval, for 10 consecutive days. On the first day of CS exposure, mice were exposed to CS from 2 pairs of cigarettes, followed by CS of two times 3 cigarettes on the second day, CS of 4 and 5 cigarettes on the third day, CS of 5 and 6 cigarettes on the fourth day, and CS of two times 6 cigarettes on fifth and remaining days. Mice were weighed daily. On the final day, mice were sacrificed and BAL fluid was collected.

Eight to Sixteen week-old male and female Lair1^−/− mice (on a C57BL/6 background) or their wild-type littermates were randomly assigned to be instilled with 0.1 μg or 0.5 μg CXCL1 (R&D Systems, Minneapolis, MN, USA) in 50 μl PBS or PBS alone. After 4 h, mice were sacrificed and BAL fluid was collected. Cells were counted and analyzed by flow cytometry as described below.

Spleen and lymph nodes were mechanically dissociated and filtered sequentially through 100 and 70 μm cell strainers (BD Biosciences, San Jose, CA, USA). Bone marrow was flushed from femurs and tibiae and filtered through 70 μm cell strainers. Lungs were mechanically dissociated using the gentleMACS Dissociator (Miltenyi Biotec, Leiden, the Netherlands) as recommended by the manufacturer, and were enzymatically digested for 30 min at 37°C with 0.13 Wünch U/ml of LiberaseTM (Roche, Basel, Switzerland) and 200 μg/ml DNAse I (Roche, Basel, Switzerland) in RPMI-1640. Subsequently, the digested lung cell suspension was passed through 70 μm cell strainers. Red blood cells were lysed by 10 min incubation at 4°C in ammonium chloride carbonate buffer containing 155 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA. Single-cell suspensions were analyzed by flow cytometry.

Single-cell suspensions from tissues and cells from BAL fluid were stained for surface markers for 30 min at 4°C in PBS containing 0.01% (m/v) sodium azide and 1% (m/v) BSA. Propidium iodide (0.3 μg/mL, Sigma, St. Louis, MO, USA) in PBS was used to distinguish vital cells. Samples were treated with rat anti-mouse CD16/CD32 antibody (clone 2.4G2, BD Biosciences, Basel, Switzerland) to block FcR-mediated non-specific antibody binding prior to incubation with fluorochrome-conjugated antibodies. Acquisitions were made with a BD Canto II or LSR Fortessa and analyzed using FlowJo software (version 10.0.7, Treestar).

The statistical significance of differences between two groups was calculated with the unpaired Mann-Whitney or Student's t test where appropriate and more than two groups were compared with the 2-way ANOVA or Kruskal-Wallis test as mentioned in the legends. Statistical analyses were performed using Prism 6 (GraphPad) software. P-values < 0.05 were considered significant and are marked in the graphs where applicable. All unmarked differences are non-significant.

We recently demonstrated that LAIR-1 is a functional inhibitory receptor on airway-infiltrated neutrophils of RSV infection-induced bronchiolitis patients in an ex-vivo setting (10). We therefore hypothesized that LAIR-1 regulates the neutrophil response in vivo during viral bronchiolitis. To test this hypothesis, wild-type and Lair1^−/− C57BL/6 mice were intranasally inoculated with the RSV-A2 strain. Mice were sacrificed on day 2 or 5 post-infection and BAL was performed to assess the cellular airway infiltrate, cytokine levels, and viral load.

Total leukocyte influx into the airways was notably increased in LAIR-1-deficient compared with wild-type mice at both the time points (Figure 1A). Early in infection, the increase in cell numbers was mostly contributed by enhanced neutrophil recruitment in LAIR-1-deficient mice, while at day 5 post-infection lymphocytes mainly constituted the infiltrating population (Figures 1B,C). There were no differences in macrophage recruitment between genotypes (Figure 1D). Thus, LAIR-1 limits neutrophil and lymphocyte recruitment during RSV infection in vivo.

Despite the enhanced leukocyte recruitment, concentrations of the major neutrophil chemoattractant CXCL1 and the inflammatory cytokine IL-6 were not increased in the BAL fluid of RSV-infected Lair1^−/− mice compared with wild-type mice (Figures 1E,F), nor were there differences in the viral load (Figure 1G). Thus, the data suggest that LAIR-1 negatively regulates neutrophil and lymphocyte recruitment during RSV infection without directly affecting the local inflammatory milieu or viral replication.

To rule out basal differences, we performed immunophenotyping of unchallenged Lair1^−/− C57BL/6 mice and confirmed that there were little to no differences in the composition of immune cell populations (Supplemental Figures S1, S2) as described before (13, 17). Moreover, we demonstrate that unchallenged wild-type and Lair1^−/− mice did not differ in neutrophil numbers and activation state—indicated by the CD11b, CD62L, and CD182 markers—in either blood or bone marrow (Figure 2A). In unchallenged wild-type mice, circulating neutrophils did not express LAIR-1, whereas tissue-infiltrated neutrophils did (Figure 2B). However, upon infection with RSV, circulating neutrophils started to express LAIR-1 (Figure 2C). In line with the observation regarding tissue infiltrated neutrophils in unchallenged mice, airway-infiltrated neutrophils also expressed LAIR-1 and were highly activated—indicated by upregulation of CD11b and shedding of CD62L (Figure 2C). Thus, RSV infection induced LAIR-1 expression on circulating and airway-infiltrated neutrophils which may directly regulate the function/adhesion of neutrophils.

To further rule out developmental differences in Lair1^−/− mice as cause of the observed phenotype, the interaction between endogenous LAIR-1 and its ligands was blocked during RSV infection by injecting wild-type mice with LAIR-1-Fc chimeric protein. Here, BALB/c mice rather than C57BL/6 mice were used as the former are more sensitive to RSV infection (18, 19). The results obtained with RSV-infected BALB/c mice in which endogenous LAIR-1-collagen interactions were blocked, mimic those of the RSV-infected Lair1^−/− C57BL/6 mice, when compared to their respective vehicle-treated or wild-type mice. The total cell count in the BAL of RSV-infected LAIR-1-Fc chimeric protein-treated mice was increased (Figure 3A). This was due to enhanced neutrophil and lymphocyte recruitment, whereas macrophage numbers remained unaffected (Figures 3B–D). Concentrations of CXCL1 and IL-6 as well as viral load were comparable between vehicle- and LAIR-1-Fc-treated mice (Figures 3E–G). The data further confirm that LAIR-1 negatively controls neutrophil and lymphocyte recruitment during RSV infection with no direct effect on the local inflammatory milieu or viral replication.

Despite the enhanced neutrophil recruitment during RSV infection in mice that lack LAIR-1 signaling, there was no effect on disease severity as measured by weight loss (data not shown). However, RSV pathophysiology in humans and mice is notably different. While neutrophils dominate the cellular inflammatory response in RSV bronchiolitis patients, with neutrophils comprising ≥80% of infiltrating leukocytes (20–22), lymphocytes are more prominent in the airways of mice during experimental RSV infection (Figures 1A–D, 3A–D). We hypothesized that in a genuine neutrophil-driven disease model the loss of LAIR-1-mediated immune regulation and the corresponding increase in neutrophil infiltration would exacerbate disease severity. Therefore, we employed another model of neutrophilic airway inflammation and exposed wild-type and Lair1^−/− C57BL/6 mice to cigarette smoke. Smoke-exposed Lair1^−/− mice lost more body weight and showed delayed and significantly attenuated recovery as compared to their wild-type counterparts (Figure 4A). In line with this, BAL fluid analysis revealed that Lair1^−/− mice had significantly higher lung immune infiltration in response to smoke-exposure as compared to the wild-type (Figure 4B). This increase in total cell influx was contributed by increased recruitment of neutrophils, macrophages and lymphocytes to Lair1^−/− lungs (Figures 4C–E). The increase in BAL cell counts in Lair1^−/− mice was highest for neutrophils (~3 fold) followed by macrophages (~2 fold), while the increase in lymphocytes was modest and failed to reach statistical significance. We confirmed that airway-infiltrated neutrophils of smoke-exposed mice expressed LAIR-1 and were highly activated (Supplemental Figure S2). Thus, LAIR-1 limits neutrophil recruitment to the airways during cigarette smoke-induced lung inflammation and regulates disease severity.

The local inflammatory milieu in the lung, as reflected by CXCL1 and IL-6 production, was not directly regulated by LAIR-1 (Figures 1E,F, 3E,F). Since the lung is enriched in LAIR-1 ligands such as collagen, SP-D and airway-infiltrated neutrophils in both the RSV infection and the smoke exposure model express LAIR-1 (Figure 2C; Supplemental Figure S3), we hypothesized that LAIR-1 can directly limit the migratory capacity of neutrophils. To examine this hypothesis, the lungs of wild-type and Lair1^−/− C57BL/6 mice were instilled with the neutrophil chemoattractant CXCL1 by intranasal administration and analyzed for the BAL fluid cellular infiltrates. We observed significantly higher total BAL cell influx in Lair1^−/− mice as compared to wild-type mice in response to 0.5 μg of CXCL1 (Figure 5A). Flow cytometry confirmed that CXCL1 specifically attracted neutrophils, as they constitute ~88% of total cell population in BAL fluid (Figure 5B), which were highly activated and expressed LAIR-1 (in wild-type mice) (Figure 5C). These data demonstrate that LAIR-1 intrinsically limits neutrophil infiltration of the airways, thereby controlling neutrophilic airway inflammation.

LB and LM have regular interaction with pharmaceutical and other industrial partners. They have not received personal fees or other personal benefits. LB's institute has received major funding (>€100,000 per industrial partner) for investigator-initiated studies from AbbVie, MedImmune, Janssen, the Bill and Melinda Gates Foundation and MeMed Diagnostics. LB's institute has received minor funding participation in trials by Regeneron and Janssen since 2015 (total annual estimate <€20,000). LB received minor funding for consultation and invited lectures by AbbVie, MedImmune, Ablynx, Bavaria Nordic, MabXience, Novavax, Janssen (total annual estimate <€20,000). LM's institute has received funding for investigator-initiated studies from Nextcure, Boehringer Ingelheim, Ono Pharmaceuticals, Ablynx and Janssen. LM received minor funding for consultation from Novo Nordisk, Biogen and Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.