This study was conducted according to the guidelines outlined by National Science Foundation Animal Welfare Requirements and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. This project was approved by the Institutional Animal Care and Use Committee at Wakayama Medical University (approved number: 882).

P2431, a serotype 6A S. pneumoniae (Sp.) strain resistant to streptomycin was used in all experiments. It is already known that transmission ratio varies depending on serotype (Zafar et al., 2016). To investigate the promoting effect on transmission by CSE, we selected serotype 6A which is unlikely to transmit comparing to other serotypes. The bacteria were grown in tryptic soy (TS) broth (Becton Dickinson (BD), Franklin Lakes, NJ) to mid-exponential phase at 37°C. When the bacterial culture reached the desired optical density (OD) at 600 nm, bacteria were washed and diluted in sterile phosphate-buffered saline (PBS) for inoculation. Quantitative culture was performed by plating 10-fold serial dilutions in triplicate on TS agar plates containing streptomycin (200 µg/ml) and catalase (6,300 U/plate) (Worthington Biochemical Corporation, Lakewood, NJ). Plates were incubated overnight at 37°C + 5% CO₂. Bacterial stocks were stored in 20% glycerol at -80°C.

Pregnant female wildtype C57BL/6 mice were obtained from Charles River Laboratories Japan, INC (Yokohama, Japan). Mice were maintained in a conventional animal facility and pups remained with their dam for the course of the experiment. Pups inoculated intranasally with chemical or infectious agents were monitored throughout the duration of the experiments and appeared healthy and gained weight similar to untreated animals.

CSE was obtained from CMIC Pharma Science Co., Ltd (Yamanashi, Japan). Briefly, CSE is prepared by bubbling a stream of smoke from 50 of Hi-Light cigarettes (Japan Tobacco Inc., Tokyo) through 50ml of saline (Mizutani et al., 2009). For standardization, CSE was diluted 100-fold, and an OD at 267 nm was used as the indicator of concentration; the standardized concentration was adjusted to ~1.308. Undiluted CSE was used for all experiments similarly as previous reports (Mizutani et al., 2009; Ueha et al., 2016; Ueha et al., 2017). CSE was stored at -80°C until use.

From day 4-7 of life, pups were given CSE (3µl/mouse), intranasally, twice a day without anesthesia; pups in the control group received 3µl PBS. On day 8 of life, pups were inoculated intranasally with 8,000 CFU of S. pneumoniae suspended in 3µl of PBS, without anesthesia.

The pneumococcal nasal colonization levels were measured by enumerating colonies in a nasal wash or a nasal tissue. Pups were euthanized with isoflurane and the upper respiratory tract was lavaged with 200µl of sterile PBS from a 25G needle inserted in the trachea; lavages were collected from the nares. After washing nasal cavity, the nasal tissue was homogenized in 1ml of sterile PBS. The nasal lavages or homogenized tissues were serially diluted and plated on TS agar plates containing streptomycin (200 µg/ml) and catalase (6,300 U/plate) and incubated overnight at 37°C + 5% CO₂. The pneumococcal colonies were counted to determine the colonization in each mouse. The limit of detection was 666 CFU/ml.

To assess pneumococcal shedding, from day 1 to 4 p.i., daily nasal secretions were collected by gently tapping (10 times) the nares onto TS agar plates containing streptomycin (200 µg/ml) and catalase (6,300 U/plate) as reported previously (Richard et al., 2014; Kono et al., 2016; Zafar et al., 2016; Zafar et al., 2017). The sample was then evenly spread across the plate using a sterile swab and incubated overnight at 37°C + 5% CO₂ for quantitative culture.

The transmission model was modified for the current study (Richard et al., 2014; Kono et al., 2016). First, all pups were treated intranasally with 3µl of CSE (“index-and-contact CSE” group) or 3µl of PBS (“index-and-contact PBS” group) from days 4-7 of life as described above. Then half of the pups in a litter were intranasally inoculated with Sp. (8,000 CFU) at day 8 of life (‘index mice’). The pups were then returned to the dam and their uninfected littermates (‘contact mice’). At day 4 p.i. (day 12 of life), all pups were euthanized by isoflurane and the nasal lavages were collected. A transmission event was defined as the presence of the pneumococcus in the nasal wash of contact mice.

In other transmission experiments, only index or only contact pups received CSE (“index-CSE” and “contact-CSE” groups). In index-CSE groups, index mice received intranasal CSE and the contact mice received PBS; in contact-CSE groups, contact mice received intranasal CSE and the index mice received PBS. The litters were infected with Sp. and transmission was assessed as described above.

After the pre-treatment with CSE or PBS on days 4-7 of life, pups were inoculated intranasally with a low dose of S. pneumoniae (~1,000 CFU) on day 8 of life. All pups were euthanized 7 or 24 hours after infection; nasal lavage was collected and pneumococcal colonization was determined as described as above.

Pups were pre-treated with CSE or PBS and infected with Sp. as described above. On day 2 p.i. (day 10 of life), all pups were euthanized. The heads were removed and fixed in 4% paraformaldehyde for 2 days, then decalcified by ethylenediaminetetraacetic acid (EDTA) for 2 weeks. After decalcification, the tissue was dehydrated and embedded into paraffin. Histological analysis was performed with coronal sections. Tissues were sectioned with a thickness of 3µm at the nasal cavity and stained with hematoxylin and eosin (HE). Tissue samples for immunohistochemistry (IHC) double staining of type 6A pneumococcal capsule polysaccharide and Alcian blue for mucus were sectioned with a thickness of 4µm. After the activation of antigen for 20 min at 95°C, samples were incubated with rabbit antisera against type 6A pneumococcal polysaccharide (Statens Serum Institut, Denmark, 1:5,000 diluted in Dako REAL Antibody Diluent (Dako, Santa Clara, CA)) for 30 min at room temperature, and then incubated with un-diluted anti-rabbit secondary antibody (Histofine Simple Stain MAX PO (R), Nichirei Bioscience Inc., Tokyo, Japan) for 30 min at room temperature. In order to develop the color, samples were incubated with diaminobenzidine tetrahydrochloride (DAB) for 5 min at room temperature. The sections were then stained with Alcian blue solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 30 min at room temperature and nuclei were stained by kernechtrot (MUTO PURE CHEMICALS CO., LTD., Tokyo, Japan) for 5 min at room temperature. Images were captured using KEYENCE BZ-X810 All-in-One Fluorescene Microscope (KEYENCE Japan, Osaka, Japan).

Neutrophils in nasal lavages were stained as previously described (Kono et al., 2016; Zafar et al., 2017). Pups exposed to CSE or PBS were pre-treated and infected with 8,000 CFU Sp. as described above. Pups were euthanized on day 2 or 4 p.i. and nasal lavages (200 µl) were collected. Pellets of nasal lavages were resuspended with PBS + 1% bovine serum albumin. Cells were stained with a 1:150 dilution of the following antibodies: anti-CD11b-V450 (BD), anti-Ly6G-PerCP-Cy (BD), and anti-CD45-APC-Cy7 (BD) for 30 min on ice in the dark after FcR blocking with a 1:200 dilution of anti-CD16/32 (BioLegend) for 15 min on ice. Cells were then fixed with 4% paraformaldehyde until analysis on FACS Verse (BD). Neutrophils were detected as CD11b+, Ly-6G+, CD45+ events.

After the pre-treatment of CSE or PBS, pups were intranasally inoculated with ~1,000 CFU and euthanized 24 hours post-infection. Nasal lavage samples (200µl) were 3-fold serially diluted and 300µl of each dilution was applied to a nitrocellulose membrane (Amersham Protran NC 0.2, Cytiva, Tokyo, Japan) with a slot-blot vacuum apparatus. The membranes were blocked with PBS containing 1% BSA, then probed with 1:500 dilution of the biotin-labeled Maackia Amurensis Lectin II (MAL II) (Vector Laboratories, Burlingame, CA) and then probed with 1:100,000 dilution of streptavidin conjugated to horseradish peroxidase (Abcam plc, Cambridge, UK) for 1 hour each, at room temperature. Immuno-reactive bands were visualized by chemiluminescence (ECL Prime Western Blotting Detection Reagent, Cytiva) and detected by imaging apparatus (LuminoGraph II, ATTO, Tokyo, Japan). The relative intensities of the bands on the immunoblot were quantified by measuring the integrated pixel density (IPD) using Adobe Photoshop (21.1.0 Release). For the purpose of analysis, the color of the blot was inverted so the background was black and the bands were white. An ellipse was drawn to encompass a band and the measurement was recorded. The same ellipse was used to measure all bands on the blot. A blank ellipse on the blot was measured to provide a background value that was subtracted from all other band IPD values.

Mann-Whitney U test or Fisher’s exact test was used for comparisons between two groups using GraphPad Prism 7 (GraphPad Software Inc., SanDiego, CA). Differences with P values of < 0.05 were considered statistically significant.

To determine how cigarette smoke exposure affects pneumococcal colonization of the nasopharynx, neonatal mice were daily inoculated intranasally with 3µl of CSE or 3µl of phosphate buffered saline (PBS) from day 4-7 of life and on day 8 of life challenged with 8,000 CFU of Sp. Type 6A; pneumococcal colonization in the nasopharynx was assessed on day 10 or 12 of life. The experimental schematic is shown in Figure 1A . The density of nasal colonization was significantly higher in the CSE than the PBS control group 4 days post-infection (p=0.0079), although there was no difference between the groups on day 2 p.i. ( Figure 1B ). We also examined pneumococcal colonization in nasal tissue, but there were not any significant differences between the CSE and PBS groups (data not shown). These results suggest that passive smoking increases colonization density or the number of bacteria attaching to the epithelial surface rather than contributing to an increased number of bacteria penetrating the nasal subepithelial tissue.

As the number of bacteria shed by colonized pups is one of critical factors impacting transmission, the effect of cigarette smoke exposure on pneumococcal shedding was evaluated. After pre-treatment of with CSE or PBS on days 4-7 of life and infection with Sp. P2431 on day 8 of life, pneumococcal shedding was monitored daily from day 1-4 p.i. ( Figure 1A ). Shedding values were significantly higher on day 2 p.i. in CSE-treated pups compared to PBS-treated pups ( Figure 2A , p=0.030). Comparing the total number of shed Sp. from days 1-4 p.i. shows a significant increase in the number of Sp. shed in the mice treated with CSE over those which received PBS ( Figure 2B , p=0.0041). Based on the previous report showing that over 300 CFU/20 taps of shedding were required for effective pneumococcal transmission (Zafar et al., 2016), we evaluated the number of events from days 1-4 p.i. that shedding were over 150 CFU/10 taps and the ratio of positive events was significantly higher in CSE-treated group by Fisher’s exact test (p=0.031). As previous reports have shown suggestions of a positive correlation between pneumococcal shedding and local neutrophil recruitment in the nasal cavity (Richard et al., 2014; Kono et al., 2016; Zafar et al., 2016), we performed flow cytometry with the nasal lavages obtained on day 2 p.i. from CSE or PBS pre-treated pups as this was the day when the bacterial shedding was significantly different between both groups ( Figure 2C ). There were significantly higher numbers of neutrophils in the nasal cavity of the CSE group on day 2 p.i. (p=0.0014). Together, these results suggest that passive smoking increases pneumococcal shedding early in infection by inducing increased inflammation in the URT mucosa and a higher density of colonizing bacteria.

To evaluate the impact of passive smoking on pneumococcal transmission, we compared transmission of Sp. among index and contact-CSE group (both index and contact pups were CSE-treated) to transmission within PBS-treated litters (control group). The density of Sp. colonization among contact pups in the index and contact-CSE group was significantly higher than the control group (p=0.021; Figure 3A ) and the transmission rate among the CSE-treated group (55%) was also significantly higher compared to the PBS-treated litters (7%) by Fisher’s exact test (p=0.021; Table 1 ). Next, to evaluate whether shedding or acquisition is the factor more influenced by cigarette smoke exposure, we assessed transmission within litters in which only the index pups received CSE (index-CSE group) compared to transmission in litters in which only the contact pups received CSE (contact-CSE group; Figure 3B and Table 1 ). There were no transmission events observed in the index-CSE litters, and in the contact-CSE groups the transmission rate was 25%; however, there were not any significant differences compared to the control group (in which both index and contact mice received PBS; Table 1 ). These results suggest that exposure of CSE in both the donor (index) and recipient (contact) pups increases rates of pneumococcal transmission.

To investigate how passive smoking influences initial pneumococcal colonization, we examined upper respiratory tract colonization density at early points after intranasal infection. For this experiment, we infected pups with a lower dose of Sp. to more closely model natural acquisition during transmission experiments. We chose a bacterial dose of >1,000 CFU as the total Sp. shed from a single colonized mouse in the CSE-treated group over 4 days was estimated at ~1,000 CFU ( Figure 2B ). In pups given 1000 CFU intranasally after CSE treatment, there were significantly more bacteria at 7 hours (p=0.0065) and 24 hours (p=0.029) post-infection ( Figures 4A, B ) compared to pups that were pre-treated with PBS. On the other hand, there could not find statistical difference in the bacterial density in the nasal cavity at day 4 post-infection (data not shown). This suggests that passive smoking enhances susceptibility to an initial step of colonization by pneumococcus in a new host.

At day 2 p.i., when the shedding of the CSE-treated group was significantly higher than the PBS group ( Figure 2A ), we performed a histological examination of the nasal tissue from CSE and PBS pre-treated pups. Representative images are shown in Figure 5 . Following staining of nasopharyngeal tissue sections with hematoxylin and eosin (HE), we observed more damaged tissue and vascular leakage (hemorrhage and inflammatory cells) in the nasal cavity of the mice pre-treated with CSE than mice treated with PBS ( Figures 5A, B compared to 5G, H ). In sections which were stained for the pneumococcal capsule by anti-polysaccharide-specific sera and for mucus by Alcian blue, we identified clusters of Sp. surrounded by mucus ( Figures 5E, F ) and disruption of the mucosal membrane, as well as adhesion of the pneumococcus to exposed submucosa in the CSE-treated pups ( Figures 5C, D ). In contrast, the disruption of the mucosal membrane and luminal bacteria were fewer in the PBS group, although the bacteria were still observed along the undisrupted surfaces of the nasal cavity ( Figures 5I–L ). These findings suggest CSE causes tissue damage in upper respiratory tract which appears to facilitate attachment of the pneumococcus to the host mucosal surface.

To assess the effect of CSE on mucus production in the upper respiratory tract, we quantified sialic acid levels in the nasal lavages by immunoblotting, probing with Maackia Amurensis Lectin II (MAL II), which binds α-2,3-linked sialic acids. We wanted to assess the relationship between CSE treatment and mucus secretion under “acquisition model” conditions (infecting with a low-dose of Sp.). After pre-treatment with CSE or PBS ( Figure 1A ), pups were infected with ~1,000 CFU Sp.; nasal lavages were obtained 24 hours p.i. The levels of sialic acid were significantly increased in the URT lavages obtained from the CSE-treated group compared to the PBS-treated group ( Figures 6A, B ), suggesting that mucus secretion is elevated by passive smoking in the setting of pneumococcal acquisition in the early phase of infection.