β_2-adrenergic signaling controls neonatal respiratory syncytial virus infection by promoting viral clearance and airway protection

Domain: neuroscience
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
Respiratory syncytial virus (RSV) is the most common cause of acute bronchiolitis and pneumonia in children under 5 years of age. RSV infection is characterized by lung inflammation, viral expansion, and airway barrier destruction. The crosstalk between neurons and immune and non-immune cells develops from early life onwards. The lungs and airways are densely innervated by sympathetic neurons where they secrete the neurotransmitter noradrenaline (NA) at homeostasis and following injury in adult mice. NA, once secreted into the lungs, can act on immune and non-immune cells through β-adrenergic receptors (β-ARs). However, it is unknown whether NA-β-AR signaling axis regulates antiviral immunity during early-life infections. In a mouse model of neonatal RSV infection, we find that stimulation of adrenergic signaling via β2-AR agonist increases viral clearance, anti-inflammatory responses, and disease recovery. By contrast, loss of sympathetic neurons and β2-AR increases the RSV viral load and immunopathology in neonates. Stimulating β2-AR signaling has several downstream effects, including reduced airway mucus secretion and protection from endothelial barrier disruption, which together prevent airway blockade, respiratory failure, and facilitate control of RSV infection. Collectively, our studies demonstrate that sympathetic neurons and β2-AR signaling protect neonates from viral expansion and lung barrier breakdown, promoting antiviral defense.

Introduction
Respiratory viral infections are the most common cause of morbidity and mortality among children under 5 years old, affecting millions of lives globally1,2. Respiratory syncytial virus (RSV), a non-segmented single-stranded RNA virus, is the most common cause of bronchiolitis and viral pneumonia in infants and young children3,4. In 2019, 33 million RSV-associated cases and >100,000 RSV-associated deaths were estimated worldwide3. RSV infection causes illnesses ranging from mild to severe diseases caused by viral factors and host responses5. Upon infection, RSV first binds to epithelium of the upper respiratory tract. During later stages of infection, RSV can cause severe lung damage and obstruction of the airways due to barrier destruction, uncontrolled mucus production, and immune infiltration6–9. RSV infection severely affects lower respiratory tract in newborns and cause viral pneumonia. Current clinical approaches include antibody-based prophylaxis such as Palivizumab and Nirsevimab, or supportive antiviral treatments post-infection10,11. Recently approved maternal vaccine provide an active immunization in pregnant mothers and protection to infants. However, all available vaccines provide only short-term protection to infants and children and cannot prevent from reinfections. Thus, there is a need of better understanding of the pathogenesis of RSV infection and development of therapies for infected newborn and infant patients.

The airways and lungs are densely innervated by sympathetic neurons, which mediate stress signaling and bronchomodulation. These neurons secrete the neurotransmitter noradrenaline (NA) from nerve terminals, which has pleiotropic effects on multiple lung cell-types during homeostasis, inflammation and injury12–16. NA can act on β-adrenergic receptors (β-ARs) expressed on airway smooth muscle cells to drive bronchodilation, and on immune cells to suppress cytokine production17. While such studies have been mainly conducted in adult animals, it is unknown whether the sympathetic neurons and signaling to β-AR modulate lung inflammation in neonates and impact the host response to RSV infection.

A key aspect of successful respiratory defense to infection is induction of host tolerance mechanisms to prevent immunopathology and barrier destruction. One of the first host responses after respiratory RSV infection is viral recognition by TLR4/7/8, RIG-I and other recognition pathways that mediate the antiviral responses, which leads to induction of interferon (IFN) production and RSV-specific CD8+ T cells to promote viral clearance9,18. However, RSV-infection can also drive increased pulmonary neutrophils and increased levels of TNF-α, IL6, and CXCL-1 that cause immunopathology and mortality6,9,19. Thus, an effective antiviral host response against RSV requires both promotion of viral clearance and regulation of harmful immunopathology and barrier destruction. Tissue-reparative macrophages, antiviral monocytes, anti-inflammatory factors (e.g., IL-10), and functional endothelial cells (ECs) can promote tolerance and barrier protection9.

Alveolar ECs & lung-resident macrophages (alveolar and interstitial macrophages) express high levels of β2-adernergic receptor20,21. We hypothesized that activation of β2-adernergic receptors on these cells might provide lung protection from endothelial barrier disruption and immunopathology during RSV infection. Upon activation, β2-AR signals via Gs-based induction of cAMP and PKA to drive immunomodulation by suppressing cytokine levels and other transcriptional changes. The β2-agonists (albuterol, salmeterol) are FDA approved drugs and have been used for the treatment of asthma and COPD by enhancing bronchodilation and easing breathing22. Previous work has shown that β2-agonists in adult mice suppress type 2 inflammation and anti-helminth immunity via suppression of type 2 innate lymphoid cell responses in the lung23. However, it is less understood if β2-agonists can alter the lung damage and host defense against viral infection. It is also unknown if β-AR signaling promotes antiviral cytokine production (e.g., IFN pathways) and viral clearance, nor barrier protection and disease recovery in the setting of RSV-infection in neonates.

While the revised 2014 American Academy of Pediatrics guidelines suggest β2-agonist treatment can provide transient breathing relieve in infants with RSV-induced bronchiolitis like it does for asthma and COPD patients, the updated guidance recommends against its use because of no clear benefit in recovery and length of hospital stay among infected patients in clinical studies24,25. However, few clinical studies also suggest that β2-agonist use might be helpful in some cases, and further in-depth studies are needed to determine the subgroups among children by considering factors such as age group, family history, immune parameters, previous exposure to disease, and/or time extent of bronchodilator use25–27. Importantly, there is a knowledge gap in clinical studies whether β2 agonist can alter lung immune cell dynamics, airway inflammation, and viral clearance during RSV infection. Our study is significant as it aims to fill these gaps. We here found that the local β2-AR agonist (albuterol) treatment in RSV-infected neonatal mice decreased morbidity and lung pathology. By contrast, depletion of sympathetic neurons and β2-AR deficiency resulted in enhanced immunopathology and RSV load. Mechanistically, β2-AR signaling increases interstitial macrophage abundance, type III interferon (IFN-λ2) production, barrier maintenance, and viral clearance. Thus, our data indicate a potential beneficial role of sympathetic neurons and β2-AR signaling in promoting airway protection to RSV infection.

Results
Local administration of β2-adrenergic receptor agonist limits RSV-induced morbidity in neonates
We first investigated whether treating neonatal mice with a β2-agonist alters their host response against RSV. To address this question, we first established a non-lethal neonatal RSV infection model in postnatal day 7 (P7) pups of C57BL/6J mice28. In this model, P7 pups were intranasally infected with 1.6 × 10^6 TCID50/10 µL (5 µL per nostril) of RSV or with vehicle (mock inoculation), followed by daily measurements of body weight of pups until 14 days post-infection (dpi). To determine the therapeutic efficacy of β2-agonist, we intranasally inoculated two doses of albuterol (5 µg/10 µL/neonate) or PBS at 2 and 5 dpi (Fig. 1A). While all neonatal groups gained weight as previously shown28,29, RSV-infected neonates showed a mark reduction in body weight gain starting from 1 dpi until 14 dpi as compared to the percentage weight gain in mock-inoculated group (the average difference reached >35% at 7 dpi and >56% at 14 dpi) (Fig. 1B, C). We observed a significant increase in percentage weight gain in albuterol-treated group as compared to percentage weight gain in PBS-treated group at 3-9 dpi and 13-14 dpi, suggesting that the local albuterol treatment is reducing the morbidity of RSV-infected neonates (Fig. 1C). From 3-7 dpi, the increase in body weight gain in albuterol-treated RSV-infected neonates compared to the percentage body weight gain in PBS-treated infected mice was observed to be >10% on average. By contrast, there were no significant differences in body weight gain between mock-inoculated neonates treated with PBS and albuterol at any time point between 1-14 days post-inoculation, suggesting albuterol itself is not having an impact on the overall physiology of the neonates (Fig. 1B). Further, we did not find a significant difference in actual measured body weight (g) between PBS- and albuterol-treated P7 neonates at day 0 of inoculation for both mock- and RSV-inoculated groups (Supplementary Fig. 1), indicating enhanced recovery of RSV-infected neonates with β2-agonist treatment was not due to increased body weight before inoculation of β2-agonist and RSV. Upon UV-inactivated RSV virus challenge (1.6 × 10^6 TCID50 per neonate), there was no difference in body weight gain between PBS- and albuterol-treated neonates at any time point up to 14 days post-challenge (Fig. 1D), suggesting the β2-agonist-mediated host protection might involve RSV replication control mechanism in the infected lungs. Also, we did not find a difference in actual measured body weight (g) among P7 PBS- and albuterol-treated neonates at day 0 of UV-inactivated RSV challenge (Supplementary Fig. 1). In sum, our data suggest that the intranasal treatment of β2-agonist restricts disease morbidity in RSV-infected neonates.

β2-agonist treatment protects neonates from RSV-induced immunopathology
Previous studies have shown that RSV infection causes lung damage that is associated with peribronchial and perivascular inflammatory cellular infiltration6,8,30. We next asked if β2-agonist treatment reduces RSV-induced immunopathology in neonates and contributes to disease recovery of infected mice. To assess lung pathology, we performed morphometric analyses of Hematoxylin and Eosin (H&E)-stained lung sections of albuterol- and PBS-treated neonates after days 7 RSV or mock inoculation, a time point where we observed significant difference in body weight gain. In mock-inoculated neonates, we did not observe differences in lung tissues, alveolar structures and cellular infiltration between albuterol-treated and PBS-treated groups (Fig. 2A–C, Supplementary Fig. 2). RSV-inoculated PBS-treated mice showed significant lung damage as judged by increased cellular influx, airway collapse, and red blood cell (RBC) accumulation (Fig. 2A, B, Supplementary Fig. 2), confirming that the RSV infection causes immunopathology in neonates. By contrast, we observed a significant amelioration of lung damage in RSV-infected neonates treated with β2-agonist (albuterol) by histopathological analyses of lung sections and histology scoring (Fig. 2B, C, Supplementary Fig. 2). In particular, RSV-induced RBC accumulation in the air spaces and airway collapse were found to be significantly lower in the lungs of albuterol-treated neonates when compared to the lungs of PBS-treated neonates (Fig. 2B, C). These data indicate that the β2-AR stimulation has a beneficial impact in limiting lung inflammation and airway barrier disruption in RSV-infected neonates.

Another hallmark of lung pathology during RSV infection is increased pro-inflammatory cytokine and chemokine production7,9,31. Previous studies have demonstrated a positive correlation of disease severity with elevated levels of inflammatory cytokines in RSV infections31–33. We next examined expression of pro-inflammatory cytokines and chemokines with and without β2-agonist treatment. We found a significant reduction in the transcript levels of Il6, Cxcl1 and Il1b mRNA at 7 dpi in the lungs of albuterol-treated mice when compared to the levels in the lungs of PBS-treated mice (Fig. 3A). Baseline expression levels of Il6, Cxcl1 and Il1b in PBS- and albuterol-treated lungs did not show any significant differences in the mock groups (Fig. 3A). Using a LEGENDPlex inflammatory cytokine panel to detect protein levels of cytokines, we found that β2-agonist treatment suppressed the levels of IL-1β, IL-6, and CXCL-1 in RSV-infected mice (Fig. 3B). We also examined the levels of Th2 cytokines (IL-4, IL-5 and IL-13) in lung homogenates from RSV-infected neonates. A significant reduction in the level of IL-13 in albuterol-treated neonates at 7 dpi was found as compared to the level in PBS-treated group, but no differences were observed in the levels of IL-4 and IL-5 (Supplementary Fig. 3). These results suggest that there could be some impact of β2-agonist treatment in dampening type 2 inflammation in RSV-infected neonates especially the production of IL-13 cytokine.

Of note, RNAseq data from the Tabula Muris consortium34 and ImmGen database (www.immgen.org) show that lung macrophages and monocytes show high expression of both β1-AR (Adrb1) and β2-AR (Adrb2). We found that Adrb1 and Adrb2 are both present in lung homogenates from neonatal mice, and that albuterol treatment does not impact the expression level of either β-AR in the presence and absence of RSV infection (Supplementary Fig. 4). Next, we aimed to determine if β-AR signaling can promote modulation of cytokine production in macrophages exposed to a virus-like trigger. R848 (Resiquimod) is a synthetic ligand for TLR7/8 receptors, which also play key roles in recognizing RSV-derived RNA7,9. We used R848 to stimulate bone marrow-derived macrophages (BMDMs) in the presence of PBS, albuterol or xamoterol (a β1-agonist). We observed a significant decrease in the levels of pro-inflammatory cytokine (TNF-α) and chemokine (CXCL-1) by R848-treated BMDMs co-treated with albuterol (1 µM), but not by BMDMs co-treated with xamoterol (1 µM) (Fig. 3C), suggesting that β2-AR but not β1-AR signaling can significantly suppress pro-inflammatory cytokine production in macrophages. We found similar suppression of cytokine production by β2-agonist treatment of BMDMs that were derived from P14 neonates (Supplementary Fig. 5). Furthermore, levels of anti-inflammatory cytokine (IL-10) were robustly increased in R848-treated adult and neonatal BMDM cultures co-treated with β2-agonist, but not with β1-agonist (xamoterol) (Fig. 3C and Supplementary Fig. 5). Importantly, the immunosuppressive effects of albuterol were abolished when BMDMs were derived from Adrb2−⁄− mice (Fig. 3D). Interestingly, TNF-α and CXCL-1 levels were enhanced in Adrb2−⁄− BMDMs compared to wild-type BMDMs (Fig. 3D), indicating lack of ADRB2 leads to enhanced pro-inflammatory responses in macrophages. Together, our data indicate that β2-adrenergic signaling promotes anti-inflammatory responses in macrophages, and this may contribute to immunomodulation during RSV infection.

One of the major characteristic features of severe RSV infection in children is RSV-induced goblet cell hyperplasia and mucus hypersecretion that can cause airway obstruction and respiratory failure35,36. However, the underlying mechanism of RSV-induced mucus secretion is not fully understood. Previous studies have demonstrated that TLR3, NF-κB, and p38/JNK-AP-1 signaling pathways induce mucins including MUC1 and MUC5AC35,37–39. Moreover, pro-inflammatory cytokines (such as IL-1 and TNF-α) promote NF-κB and the MAPK signaling to drive mucus overproduction38–40. To determine whether the host protective role of β2-AR signaling involves suppression of mucus production, we analyzed mucus production in infected lungs of neonates with and without β2-agonist treatment by performing Periodic acid-Schiff (PAS) staining. We found that numbers of mucus-producing goblet cells (dark pink cells) were markedly increased in lungs after 7 dpi with RSV as compared to mock-inoculated mice (Fig. 3E). Albuterol treatment at 2 and 5 dpi robustly decreased this goblet cell hyperplasia and mucus production (Fig. 3E). In mock-inoculated neonates, albuterol treatment did not alter the goblet cell numbers in the lungs (Fig. 3E), suggesting decrease in goblet cell numbers in RSV-infected neonates with albuterol treatment is not due to off-target effects on mucus producing cells.

To confirm further the PAS staining results, we assessed mRNA levels of Muc5ac and Gob5 genes. MUC5AC is a major mucin in the outer gel layer in respiratory mucus35. GOB5, a calcium-activated chloride channel expressed in secretory granules of goblet cells, has been positively associated with RSV-induced mucus secretion and disease severity41,42. We observed ∼2.3-fold increase in both Muc5ac and Gob5 mRNA levels in PBS-treated lung homogenates of RSV-infected neonates when compared to the levels in mock-inoculated neonates (Fig. 3F). In RSV-infected neonates with albuterol treatment, we observed a significant reduction of Muc5ac and Gob5 mRNA levels (Fig. 3F).

Early life RSV infection and RSV-induced bronchiolitis can cause asthma-like symptoms at a later age. To determine the impact of neonatal β2-AR agonist treatment in development of asthma-like symptoms in later life, we performed Flexivent measurements of lung mechanics to assess pulmonary hyperresponsiveness in RSV-infected neonates at 12-15 weeks of age. As in other studies43–46, we used methacholine (6.25 mg/mL) as a positive control to assess the airway hyperreactivity (Resistance, Rrs; Elastance, Ers; Newtonian resistance, Rn; and tissue damping, G). As in previous studies with lung injury/infection43,47, all four measured airway hyperreactivity parameters were increased with methacholine (6.25 mg/mL) treatment as compared to without treatment (0 mg/mL) condition for both mock-inoculated and RSV-infected mice (Supplementary Fig. 6). However, the differences were found more significant among infected mice (Supplementary Fig. 6B). We did not find any difference in airway hyperreactivity between PBS- and albuterol-treated mice that were RSV-infected when they were 7 days old (Supplementary Fig. 6B), suggesting that albuterol treatment in RSV-infected neonates has no impact in development of RSV-induced pulmonary hyperresponsiveness during adulthood (12-15 weeks old).

All together, these findings suggest that stimulating β2-AR signaling via agonist treatment dampens pathological lung inflammation and mucus secretion during RSV infection in neonates but has no impact on pulmonary hyperresponsiveness when infected neonates reach adulthood.

β2-agonist treatment limits endothelial barrier disruption during neonatal RSV infection
Another aspect of β2-adrenergic-mediated anti-RSV response could be via protection of vascular integrity. β2-AR gene (Adrb2) is expressed at high levels by lung ECs as seen in violin plots of single-cell RNAseq (scRNAseq) analyses from the Tabula Muris Consortium34, suggesting that agonist may signal to these cells (Fig. 4A). Lung histology data also showed that albuterol treatment decreased airway collapse and vascular leakiness of RBCs (Fig. 2). We hypothesized that β2-AR signaling may protect neonatal lungs from endothelial barrier disruption during RSV infection. To examine this, we employed various methods to determine the impact of β2-agonist treatment in endothelial barrier protection. We quantified the presence of protein, albumin, and RBCs in bronchoalveolar lavage (BAL) fluid at 7 dpi in RSV-infected neonates (Fig. 4B). Albuterol treatment decreased levels of total BAL protein compared to the levels in PBS-treated RSV-infected neonates (Fig. 4C). At baseline, we did not observe differences in total BAL protein levels between albuterol- and PBS-treated neonates (Supplementary Fig. 7A), suggesting β2-agonist treatment does not impact the airway barrier function at homeostasis. We also observed marked reduction in BAL albumin levels and the number of total RBCs (CD45-Ter119+ cells) in the BAL fluid (BALF) from albuterol-treated mice than that in PBS-treated mice at 7 dpi (Fig. 4D, E, Supplementary Fig. 7B).

Previous study has shown decreased expression of tight junction proteins in the lungs of RSV-infected mice48. Thus, we hypothesized that albuterol may help maintain barrier function and tight junction protein expression during RSV infection. We performed gene expression analyses of tight junction proteins and adhesion molecules in RSV-infected lungs with and without albuterol treatment. The mRNA expression levels of tight junction proteins including Claudin-1, Claudin-4 and Claudin-18 and Occludin, and adhesion molecule (E-Cadherin) were decreased in RSV-infected neonates compared to mock-inoculated mice and in albuterol treatment increased these levels (Fig. 4F). We also observed an effect of albuterol treatment in baseline induction of tight junction and adhesion molecules in mock-inoculated neonates (Fig. 4F). Moreover, albuterol treatment robustly increased the mRNA expression of cell proliferation marker Ki-67 in RSV-infected lungs when compared to the expression in PBS-treated infected mice, supporting the idea of increased cellular regeneration and barrier repair with β2-agonist treatment (Fig. 4F).

We next sought to examine if sympathetic neurons are in close vicinity with lung endothelium to allow direct signaling to lung ECs. To determine this possibility, we performed immunohistochemistry of the lung sections from P14 neonates to examine co-localization of neurons and lung ECs. We stained the lung sections with EC marker (CD31) together with sympathetic neuronal marker (Tyrosine hydroxylase, TH). We observed sympathetic neurons (TH+ neurons), and ECs (CD31+) are situated in proximity in the airways as well as in lung parenchyma (Fig. 4G and Supplementary video 1-3). These results suggest that the lung endothelium and sympathetic neurons co-develop from an early stage of life and that there could be neuroendothelial signaling at play in our model. In sum, our data suggest that β2-AR stimulation via albuterol treatment increases the lung endothelial barrier function during RSV infection.

Loss of β2-adrenergic signaling increases RSV-induced morbidity and lung pathology in neonates
Since we observed decreased RSV-induced morbidity and immunopathology with β2-AR stimulation, we next determined whether β2-AR deficient mice may have worsened morbidity and lung pathology during RSV infection. To determine these, we infected P7 neonatal β2-AR-sufficient (Adrb2+/+) and β2-AR-deficient (Adrb2-/-) mice with RSV, followed by analysis of disease morbidity (Fig. 5A) and lung damage. As we demonstrated in Fig. 1, the percentage of body weight gain was markedly higher in mock-inoculated neonates as compared to the RSV-infected groups (Fig. 5B, C). We observed a marked decrease in percentage weight gain in RSV-infected Adrb2−/− neonates at 10-12 dpi when compared to percentage weight gain in RSV-infected Adrb2+/+ neonates, suggesting loss of β2-AR shows a negative impact in controlling RSV infection and disease severity (Fig. 5C). The decrease in body weight among Adrb2−/− neonates was observed to be >6.5% on average when compared to Adrb2+/+ neonates during 6-13 dpi and >9.5% on average during 10-12 dpi (Fig. 5C). Consistent with weight gain data among mock-inoculated neonates, we did not observe difference in body weight in Adrb2−/− vs. Adrb2+/+ neonates up to 4-6 dpi in RSV-inoculated groups (Fig. 5C), suggesting the effect of β2-adrenergic signaling blockade in the morbidity of RSV-infected neonates does not develop rapidly following RSV infection. Furthermore, we did not find a significant difference in actual body weight (g) due to β2-AR deficiency at day 0 of inoculation for the P7 neonates in Fig. 5B, C except a modest increase in weight of the RSV-infected Adrb2–/– neonates when compared to the weight of RSV-infected Adrb2+/+ neonates (Supplementary Fig. 8). These RSV-infected P7 Adrb2–/– neonates also showed increased body weight when compared to mock-inoculated P7 Adrb2–/– and Adrb2+/+ neonates, suggesting a developmental and maternal nursing effect for the increased body weight for this neonatal group at day 0 of inoculation.

The increased morbidity of RSV-infected Adrb2−/− neonates was consistent with augmented lung damage and barrier disruption as observed by H&E analyses of lung sections of Adrb2−/− neonates at 7 dpi (Fig. 6A, B). While there were no differences in baseline alveolar/perivascular structures and inflammation of the lungs between Adrb2−/− and Adrb2+/+ neonates with mock inoculation, Adrb2−/− neonates showed a marked increased airway cellular influx and damage compared to Adrb2+/+ neonates at 7 dpi with RSV (Fig. 6B, C). Of note, we also observed an increase in airway RBCs in Adrb2−/− mice at the baseline level, indicating the importance of β2-AR in baseline barrier maintenance (Fig. 6C).

Sympathetic neurons represent the major cell type that drive β2-AR stimulation via releasing NA in the lungs and have been found to modulate both LPS-induced and type 2 inflammation in the lung23,49. To determine their role in RSV infection, we depleted sympathetic neurons in P4 neonates via treatment with 6-Hydroxydopamine (6-OHDA), which leads to chemical sympathectomy. The 6-OHDA- vs. vehicle-treated mice were then intranasally infected with RSV at P12. Consistent with histopathology results in Adrb2−/− and Adrb2+/+ neonates, we observed increased airway inflammation and alveolar damage in 6-OHDA-treated neonates at 7 dpi as compared to the inflammation and damage in vehicle-treated neonates (Fig. 6D-F). Mock-inoculated 6-OHDA- vs. vehicle-treated mice did not show major baseline differences in lung inflammation or immune cell influx due to neuronal ablation (Fig. 6D-F).

Collectively, these data suggest that the endogenous blockade of β2-AR signaling dampens the neonatal host defense to RSV by increasing morbidity and lung pathology in neonates.

β2-adrenergic signaling increases monocyte recruitment and regulates the abundance of interstitial macrophages during RSV infection
Like other respiratory viral infections, RSV infection leads to increase in leukocyte numbers in the lungs, mainly the numbers of neutrophils, monocytes, macrophages and lymphocytes50–52. Tissue-resident immune cells in the lung (such as alveolar macrophages, AMs) and airway epithelium engulf RSV viral particles or RSV-infected cellular debris, and secrete cytokines and chemokines that regulate the entry of myeloid and lymphoid cells into the lung, and enhance viral clearance52,53. Lung-infiltrating T cells play an indispensable role in killing of virus-infected cells and preventing viral replication in the lung9,53. To delineate the role of β2-adrenergic signaling on composition of immune cell infiltrates at the site of infection, we performed immune subset analysis of recruited and non-recruited cells in the airways in albuterol-treated versus PBS-treated neonates (Supplementary Fig. 9A). After RSV infection, albuterol-treated neonates showed similar numbers of total BAL cells as in PBS-treated neonates at 7 dpi (Fig. 7A), suggesting albuterol treatment does not have an impact on airway recruitment of total cells after RSV infection. Similar to previous findings28,52, we observed significant increase in total BAL and CD45+ cells in RSV-infected neonatal lungs as compared to the numbers in mock-inoculated neonates (Fig. 7A, B). At mock-inoculated groups of mice, we did not find difference in baseline total BAL cells and leukocytes (CD45+ cells) (Fig. 7A, B), suggesting no effect of albuterol treatment in baseline immune cell influx in airways. Immune subset analyses in BALF at 7 dpi showed a reduction trend in total numbers of B cells (CD45+CD11b− B220+ cells) and CD4+ T cells and significant increase in total number of interstitial macrophages (IMs, CD45+CD11b+ Ly6G− F4/80+ Siglec-F− cells) in albuterol-treated neonates as compared to the numbers in PBS-treated neonates (Supplementary Fig. 9B-C and Fig. 7C). Furthermore, we did not observe significant differences in absolute numbers of AMs, neutrophils, CD8+ T cells, NK cells and non-T/B/NK cells (Supplementary Fig. 9B-C) and the levels of IgA in the BALF (Supplementary Fig. 9D) between PBS- and albuterol-treated neonates, suggesting a minor or no role of adaptive immune cell numbers in β2-AR-mediated control of RSV infection.

We also quantified myeloid and lymphoid subsets in whole lungs from RSV-infected neonates after albuterol or PBS treatment by flow cytometry (Supplementary Fig. 10A). Similar to results in BALF immunophenotyping, albuterol- vs. PBS-treated neonates did not show major differences in leukocyte numbers (Supplementary Fig. 10B), but showed an increased percentage and number of IMs (CD45+CD11b+ Ly6G− Siglec-F− F4/80+ cells) with albuterol treatment (Fig. 7D). Also, the numbers of Ly6chi monocytes (CD45+CD11b+ Ly6G− F4/80− Ly6chi cells) and Ly6clo monocytes (CD45+CD11b+ Ly6G− F4/80− Ly6clo cells) were increased with albuterol treatment as compared to the numbers in PBS-treated neonates (Fig. 7E). There were no differences in CD4+ T and B cell numbers in the lungs in both groups (Fig. 7F, Supplementary Fig. 11). At mock-inoculated groups of neonates at 7 days post-inoculation, we did not find difference in baseline numbers of total IMs in BALF and whole lungs (Fig. 7C, D), suggesting albuterol-mediated promotion of increase in IM numbers occur during RSV infection only. Consistent to BAL immune cell number data, we did not observe significant differences in the numbers of AMs, CD8+ T cells, NK cells and non-T/B/NK cells in RSV-infected lungs with and without albuterol treatment (Fig. 7E, F). Also, the total number of RSV-specific CD8+ T cells did not change in albuterol- and PBS-treated neonates at 7 dpi (Fig. 7G, Supplementary Fig. 11), suggesting β2-adrenergic control of RSV infection does not involve the increased recruitment of RSV-specific CD8+ T cells.

Altogether, our immunophenotyping results from BALF and lungs suggest a link of β2-adrenergic signaling pathway in recruitment and maintenance of IMs in anti-RSV host response.

β2-adrenergic signaling pathway enhances viral clearance via interferon-λ production during neonatal RSV infection
While myeloid cells (such as macrophages, DC, monocytes) were found to secrete type I IFNs (IFN-α, IFN-β) during RSV infection30,52, respiratory epithelial cells were demonstrated to produce IFN-λ during influenza virus infection and confers host protection in mice against influenza virus challenge54. Myeloid and epithelial cells mediate potent antiviral effects to viral infection via multiple mechanisms, involving suppression of viral mRNA synthesis, translation of viral particles and assembly of virus particles9. We thus asked whether β2-adrenergic-mediated protection against RSV infection involves enhanced RSV viral clearance and IFN production. To investigate this possibility, we quantified mRNA levels of both RSV and IFNs in the lung homogenates after RSV infection.

To determine RSV multiplication, we quantified the RSV L (RNA-dependent RNA polymerase) and RSV N (nucleoprotein) viral copies per µg of mRNA by qPCR analyses of RSV-infected lungs. We also performed qPCR analyses to quantify the mRNA levels of IFNs (IFN-α, IFN-β, and IFN-λ) in the lung homogenates. First, we performed time-course analyses of viral load and IFN production at 2, 3, 4, 5, and 7 dpi. The viral copies of RSV N were detected starting from 2 dpi up to 7 dpi (Supplementary Fig. 12A). To quantify the live infectious viral titers and assess active RSV replication in infected lungs, we performed a plaque-forming assay as published in other studies55,56. Similar to qPCR viral load results, we detected infectious viral titers (as plaque-forming units, PFUs) from 2 to 7 dpi, with a maximal titer at 3 and 5 dpi (Supplementary Fig. 12B). We found significantly increased viral titers at 5 dpi compared to 2 dpi, an indication of active viral replication in infected neonates (Supplementary Fig. 12B). The production of IFN-β (Ifnb1 mRNA expression) peaked at 3 and 4 dpi, followed by at 7-dpi (Supplementary Fig. 12C). In contrast, production levels of IFN-λ (Ifnl2 mRNA expression) was found to peak at 5-7 dpi (Supplementary Fig. 12C). The early IFN-β production was paralleled with suppressed viral load, whereas IFN-λ production at latter phase of infection was found to be associated with suppressed viral load at 7 dpi (Supplementary Fig. 12A and 12C). To determine the full efficacy of two doses of β2-agonist treatment (at 2 and 5 dpi), we then analyzed the impact of albuterol treatment in viral load and IFN production at 7 dpi. While there were no detectable viral copies of RSV L and N genes in mock-inoculated lungs of albuterol- and PBS-treated neonates at 7 days post-inoculation, we observed robust suppression of viral copy numbers of RSV N and L genes in albuterol-treated neonates at 7 dpi as compared to the levels in PBS-treated group (Fig. 8A). We also found a significant reduction in PFU numbers in the lung homogenates from albuterol-treated neonates when compared to the numbers in PBS-treated mice (Fig. 8B), suggestive of reduction of infectious viral titers in the lungs with local albuterol treatment. To complement our results regarding increased RSV clearance in β2-agonist-treated neonates, we also performed immunohistochemical staining of RSV (all antigens) in the lung tissue sections collected from PBS- and albuterol-treated neonates at 7 dpi. We found that the lung sections from albuterol-treated neonates showed a small fraction of the airway epithelium positive for RSV (Supplementary Fig. 13). In contrast, in PBS-treated neonates, RSV was distributed throughout the airway epithelium (Supplementary Fig. 13). Further, we found visibly more airway damage and epithelial sloughing (shown by white arrows) in PBS-treated RSV-infected neonates than the airway damage in albuterol-treated infected neonates (Supplementary Fig. 13).

Moreover, we observed an increased mRNA levels of IFN-λ (∼1.9 fold) in the lung homogenates between mock- and RSV-inoculated neonates at day 7 post-inoculation, confirming that the RSV infection increases IFN-λ production (Fig. 8C). With albuterol treatment at 2 and 5 dpi, we found a higher levels of IFN-λ mRNA in RSV-infected neonates at 7 dpi (Fig. 8C). When quantified in protein levels, we observed an increase in IFN-λ protein in the lung homogenates from albuterol-treated neonates when compared to IFN-λ levels in PBS-treated neonates at 7 dpi (Supplementary Fig. 14). We did not observe significant changes in expression levels of Ifna1 and Ifnb1 between albuterol- and PBS-treated mice at 7 dpi, indicating β2-agonist treatment has a minor impact in production of type I IFNs during RSV infection (Fig. 8C). Also, we did not observe increase in expression levels of interferon-stimulated genes (ISGs) with albuterol treatment (Supplementary Fig. 15), implying a minor role of ISG upregulation in neighboring cells for antiviral responses in β2-agonist-treated neonates.

To further confirm the antiviral role of β2-adrenergic signaling in RSV infection, we pursued to investigate the viral load and IFN production in Adrb2+/+ and Adrb2−/− neonates at 7 dpi. Consistent with the results with β2-AR agonism, we found an increase of viral copies of RSV N and L genes in the lungs of Adrb2−/− neonates as compared to the levels in Adrb2+/+ neonates (Fig. 8D). These results are well correlated with marked decrease in Ifnl2 expression levels in Adrb2−/− neonates (Fig. 8E). Decrease in RSV clearance and IFN-λ production in Adrb2−/− neonates was also observed in sympathetic neuron-ablated [6-Hydroxydopamine (6-OHDA)-treated] neonates when compared to vehicle-treated neonates (Fig. 8F, G). Finally, we confirmed the antiviral effect of IFN-λ in neonatal RSV infection by intranasal administration of two doses of recombinant IFN-λ2 in RSV-infected neonates and analyzing the RSV load in the lungs at 7 dpi (Fig. 8H, I). We found significant suppression of viral replication as judged by viral copy numbers of RSV L and N genes in IFN-λ2-treated neonates when compared with viral replication in vehicle-treated neonates (Fig. 8I).

It is possible that β2-agonist treatment enhances the phagocytosis of viral particles and RSV-infected cells for viral clearance. To determine the extent to which β2-agonist impacts the phagocytic capacity of monocytes and macrophages during RSV infection, we assessed the impact of albuterol treatment on RSV binding/phagocytosis in neonatal BMDMs by examining the expression levels of phagocytosis receptors and the extent of RSV surface binding and phagocytosis in BMDMs. We first challenged neonatal BMDMs with different doses of live RSV to validate macrophages responding to live RSV infection. As expected, we found a dose-dependent increase in the expression of IL-6 mRNA with a multiplicity of infection (MOI) of 1 to 10 (Supplementary Fig. 16A). The increase in cytokine response is consistent with increased RSV binding and phagocytosis, as judged by a dose-dependent increase in RSV viral load in macrophages that were washed with PBS before analyzing the viral load (Supplementary Fig. 16B). In albuterol-treated BMDMs, we found robust suppression of Tnfa mRNA levels (Supplementary Fig. 16C), consistent with our in vitro cytokine data showing albuterol suppresses TNF-α production when stimulated with R848 (Supplementary Fig. 5). These albuterol-treated RSV-infected BMDMs (MOI of 5) also exhibited increased viral load, as indicated by increased RSV N gene copy numbers from BMDM lysates (Supplementary Fig. 16B). Furthermore, mRNA level analyses of phagocytosis markers showed increased expression of Mrc1 (code for CD206), Tlr4, and Cd14 in RSV-infected macrophages treated with albuterol as compared to the expression levels in PBS-treated RSV-infected macrophages (Supplementary Fig. 16D). Interestingly, albuterol robustly increased the baseline expression of Cd14 and Tlr4, but not the baseline expression of Mrc1 and Fcgr1 (Supplementary Fig. 16D). These results suggest that albuterol treatment enhances the expression of phagocytosis receptors, contributing to the enhanced phagocytosis of RSV viral particles and/or RSV-infected cell debris.

In summary, we demonstrate a critical role of β2-AR signaling axis in controlling RSV replication in neonates via promoting type III IFN production and phagocytosis.

Methods
Approval
All animal experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University (KSU), with approval numbers 4571 and 5021. The use of the pathogen was approved by the Institutional Biosafety Committee of KSU (approval numbers: 1546, 1766). Seven-day-old (P7) and twelve-day-old (P12) neonatal mice, as well as RSV-infected adult mice, were used in the experiments.

Mice
C57BL/6J and Adrb1tm1Bkk Adrb2tm1Bkk /J (described here as Adrb1–/– Adrb2–/–) mice were originally purchased from Jackson Laboratories. Adrb1–/– Adrb2–/– mice were first bred with C57BL/6J mice to generate heterozygous mice. The Adrb2–/– mice used in the experiments were generated after multiple backcrosses in the C57BL/6J background. Adult C57BL/6J and Adrb2–/– mice were housed and bred in a pathogen-free animal facility at KSU. Before a week of delivery of pups, pregnant females were separated and transferred to the ABSL-II facility at KSU. Pregnant females were housed in a micro isolator with 12-hour light and dark cycle, maintaining 30-70% humidity, and monitored daily for their litters until birth. Infection was carried out in postnatal day 7 (P7) pups, and the experiment was ended at the indicated time points before or at 14 dpi. For the infection in 6-OHDA-treated pups, infection was performed at P12 age. All P7 or P12 neonates from each litter were used only for 1 experiment and considered as one independent experiment. Litter of size <3 pups was not used for the experiment. Both male and female neonates in the litter were included in the study design, and data were not separated or reported based on sex. Sample collection and processing were performed in an aseptic environment. For Adrb2–/– experiments, in-house C57BL/6J pups were used as receptor-sufficient (Adrb2+/+) groups.

Virus and intranasal inoculation
Human respiratory syncytial virus (RSV-A2 strain, Cat# VR-1540) used for in vivo and in vitro infection experiments was obtained from ATCC. Once the virus was received from ATCC, it was aliquoted and stored at −80°C for infection. On the day of infection, the virus was transported to an ABSL-II facility, where it was maintained at a cold temperature (in dry ice) and then thawed immediately before intranasal inoculation. As described in our previous study28, RSV-A2 (1.6 × 10^6 TCID50/10 µL) was inoculated via the intranasal route into isoflurane-induced pre-anesthetized P7 or P12 neonates. Mock group neonates were intranasally inoculated with 10 µL of sterile 1X PBS. The RSV aliquots were exposed to UV for 120 seconds and were used for UV-inactivated RSV challenge experiments. The UV inactivation of RSV was confirmed by the absence of plaques in a plaque-forming assay using UV-exposed virus. Infected neonates were kept with their moms in a micro-isolator and monitored twice a day till the endpoint of the experiments.

Compound treatment and inoculation
For ablation of sympathetic neurons, neonates were intraperitoneally injected with 6-Hydroxydopamine hydrochloride (6-OHDA, Cat# H4381, Sigma-Aldrich) in 20 μL total volume (100 mg/kg in 0.1% ascorbic acid/PBS) for 4 consecutive days starting from P4 till P7. The control group of mice received vehicle (0.1% ascorbic acid/PBS) only. At the P12 stage, RSV was intranasally inoculated as described above, and the experiment was ended after 7 dpi. For the treatments of albuterol (Cat# 1012600, Sigma-Aldrich) or IFN-λ2 (Cat# 250-33, Peprotech), RSV-infected neonates were intranasally inoculated with albuterol (5 μg/10 μL/neonate) or IFN-λ2 (1 μg/10 μL/neonate) at 2 dpi and 5 dpi. Neonates were monitored for survival and body weight until 14 dpi or 7 dpi for other experiments.

Collection of BALF and Whole lung tissue
For BALF collection, neonates were first euthanized with CO2 overdose, followed by an incision of the neck area using sterile forceps to expose the trachea and a cannula (22G) catheter was inserted into the trachea. Next, the lung cavity was flushed twice with 250 μL of BAL buffer containing heparin (20 U/mL, Sigma-Aldrich), dextrose (1 mg/mL, Fisher Chemicals) and 1X PBS. A total volume of ~0.4 mL of BALF was collected in 1.5 mL sterile microcentrifuge tube. For the whole lung collection, the chest cavity was opened aseptically, and the lung lobes were collected in a 1.5 mL sterile microcentrifuge tube and processed for gene and protein expression level analyses or plaque assay. All samples were kept on ice until further processing or immediately stored at −80°C.

Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining
After 7 dpi, RSV- and mock-inoculated neonates were euthanized with CO2 exposure. The intact whole lung together with heart and trachea was isolated and kept in 10 mL of neutral buffered formalin (10%) (Sigma-Aldrich) solution at room temperature for 24 hours. The formalin fixed tissue samples were sent to Veterinary Diagnostic Laboratory, KSU for sectioning, staining, and slide scanning. Digitally scanned images of H&E and PAS-stained lung sections were processed using Aperio ImageScope software. H&E images were scored by a trained person who was blinded with samples following the previous publications with minor modifications29,81. The scored data were generated for each lobe after examining 3-4 different area of the lobe with higher magnifications (20X) images and then the scores were averaged for each mouse and the average score per mouse was plotted for graphical representation. H&E images were scored using the following criteria: Score of 0-1 (Normal/healthy), 1-2 (Mild/minimal), 2-3 (Moderate), and 3-4 (Severe) damage as described in the supplementary table 2.

BALF processing and flow cytometry analyses
After collection, BALF was centrifuged at 490g for 5 minutes at 4°C and the pellet was resuspended in 1 mL PBS and 250 µL cell suspension (1/4th) was used for RBC analysis. The rest of the cells suspension was re-centrifuged and resuspended in RBC lysis buffer (Thermo Fisher) for 7 minutes at room temperature. Then, cells were washed with Flow buffer [1X PBS, 2% FBS (Hyclone) and 0.2 mM EDTA (RPI Research)] and centrifuged at 490g for 5 minutes at 4°C. Cell pellets were then resuspended with a 200 µL Flow buffer. Next, resuspended cell suspension (10 µL) were mixed in 1:20 dilution of trypan blue (0.04%) (Sigma-Aldrich) and viable cells were counted using a hemocytometer. For flow cytometry analyses, the cells suspensions were first transferred to the V-bottom plate, treated with FcR-blocking solution (Miltenyi Biotec), followed by dark incubation with antibody cocktails prepared in Flow staining buffer (1X PBS and 2% FBS) for 25 minutes on ice. Next, antibody-stained cells were centrifuged at 490g for 5 minutes at 4°C and then washed with Flow buffer and re-centrifuged at 490g for 5 minutes/4°C. Finally, stained cell pellets were resuspended with 200 µL fixative buffer (0.5 mM EDTA, 2% paraformaldehyde and 1X PBS) and were kept on ice for 25 minutes. Antibodies used for RBC experiment are listed in the Supplementary Table 3. Antibodies used for immune cell characterization are listed in the Supplementary Table 4. Positive immune cells were gated using fluorescence minus one (FMO) as a negative staining control. To exclude dead cells, Zombie Yellow (Biolegend) was used. Data was acquired in LSRII Fortessa (BD) or Northern Lights (Cytek) flow cytometer. Then, exported FCS data files were analyzed with FlowJo (Treestar, version 10.10) and the table was created for quantification of immune cells or RBCs.

BAL protein and albumin measurement
After collection, BALF was centrifuged at 490g for 5 minutes at 4°C and the supernatant was transferred to new sterile tubes. Next, protease and phosphatase inhibitor (1%, Thermo Scientific) was added to the supernatant, then aliquoted and stored at −80°C until further processing. The levels of total protein in BALF were measured using BCA assay (PierceTM BCA protein assay kit) and the levels of BALF albumin were measured by albumin assay (BCG Albumin assay kit, Sigma), following manufacturer’s instructions. Furthermore, BALF IgA levels were measured using IgA ELISA (Mouse IgA ELISA Antibody Pair kit, STEMCELL Technologies) following manufacturer’s instructions.

Whole lung single cell preparation and flow cytometry analyses
After whole lung collection, tissue was chopped into small pieces in RPMI and then incubated at 37°C with 250 rpm shaking in a digestion buffer (RPMI, 2% FBS) containing 30 µg/mL DNase I (Roche) and 1.5 mg/mL Collagenase IV (Sigma-Aldrich) for 50 minutes. Cells and smaller tissue chunks were then passed through a 28G syringe 3X times and filtered through a 70-µm cell strainer into a prefilled RPMI tube. Next, cells were centrifuged at 490g for 5 minutes at 4°C and the pellet was resuspended with 1X RBC lysis buffer (Thermo Fisher) for 10-12 minutes at room temperature. Cells were then washed with Flow buffer and centrifuged at 490g for 5 minutes at 4°C. Cell pellets were then resuspended with 1 mL Flow buffer, followed by dilution of cell suspension with trypan blue (0.04%) (Sigma-Aldrich) and viable cells were counted using a hemocytometer. For flow cytometry analyses, antibody cocktails for myeloid and lymphoid panels were prepared 10-15 minutes advance in Flow staining buffer in the separate tubes. Next, 2×10^6 cells/well were transferred to the V-bottom plate and treated with FcR-blocking solution (Miltenyi Biotec), followed by incubation of cells with antibody cocktails for 25 minutes in ice in dark. After incubation, antibody-stained cells were centrifuged at 490g for 5 minutes at 4°C and then washed with Flow buffer and re-centrifuged at 490g for 5 minutes/4°C. Finally, stained cell pellets were resuspended with 300 µL fixative buffer (0.5 mM EDTA, 4% paraformaldehyde and PBS) and were kept in ice in dark until data was acquired in the flow cytometer. For RSV-specific CD8 T+ cell staining, PE-conjugated RSV M2 tetramer was used (received from NIH Tetramer Core Facility, Emory University, Atlanta). Antibodies used for staining lung immune cells are listed in the supplementary table 5 & 6. Positive immune cell populations were determined by using FMO as a negative staining control. To exclude dead cells, LIVE/DEAD fixable aqua (Invitrogen) or Zombie Yellow (Biolegend) was used. Flow cytometry was conducted on LSRII Fortessa flow cytometer (BD) and data files were analyzed using FlowJo (Treestar, version 10.10) software.

Measurement of cytokine and chemokine levels in lung tissues
Immediately after the whole lung collection, lung tissues were weighed, homogenized with BB beads (Daisy Outdoor Products) in 1 mL PBS and centrifuged to collect the whole lung supernatant. On the collected supernatant, protease and phosphatase inhibitor (1%) (Thermo Scientific) was added and then aliquoted and stored at −80°C until further processing for total protein and cytokine measurements. Cytokines/chemokines from whole lung homogenates were measured by ELISA (BioLegend & Proteintech) or LEGENDPlex assay (BioLegend), following the manufacturer’s instructions. Total lung protein was measured using the BCA assay (PierceTM BCA Protein Assay Kit) according to the manufacturer’s protocol, and cytokine/chemokine levels were normalized to tissue weight or total protein. ELISA data were obtained from the Epoch ELISA plate reader using Gen5 software, and LEGENDPlex data were obtained from the Northern Lights (Cytek) instrument and processed and analyzed using LEGENDPlex online Data Analysis software (BioLegend).

Measurement of live infectious viral titers by plaque-forming assay
A plaque-forming assay was performed to measure infectious viral titers (PFU) in RSV-infected neonatal lungs82. Immediately after the whole lung collection, lung tissues were weighed, homogenized with BB beads in DMEM, and centrifuged to collect the supernatant. The collected supernatant was freshly used in the assay. The supernatant was diluted, and then the diluted samples were placed over pre-cultured PBS-washed Hep2 cells (ATCC# CCL-23) in 12-well plates. After 2 hours of incubation for viral adsorption, the preheated microcrystalline overlay media was added onto the Hep2 culture and incubated for 7 days with 5% CO2 at 37°C without disturbing the plates. On the final day, the media was gently removed from each well, washed with PBS, and the cells were fixed using Crystal Violet, Formaldehyde, and alcohol solution for 15 minutes. The fixed cells were washed with water multiple times and then dried. Plaques were counted, and representative wells were imaged with iBright FL1500 imager (Thermo Fisher) using a default iBA analysis software with universal mode and the visible crystal violet setting enabled. Mock-inoculated mice were used as a negative control (zero plaque) for the plaque assay.

Immunohistochemistry (IHC) and confocal microscopy of lung lobes
At the indicated time points, neonates were first euthanized with CO2 overdose. Neonatal lungs were then inflated with 4% PFA/PBS, then collected after closing the trachea with suture thread, and kept at 4°C in 4% PFA/PBS overnight. Next, lungs were washed twice in 1X PBS and transferred to a 30% sucrose (Fisher Chemicals) solution and kept in 4°C until the tissue reached to the bottom of the tube. Lobes were separated out and tissue were embedded in optimal cutting temperature compound (Sakura Fintek) and blocks were stored at −80°C until sectioning in Cryostat (Leica Biosystems). The lobes were cut into 20-μm or 60-μM thick sections and kept in slides at −80°C until staining. On the first day of staining, sections were blocked in 5% normal donkey serum (NDS) and 5% normal goat serum (NGS) or 10% NDS only (for RSV staining) for 5 hours at room temperature. Then, for neuron staining, sections were incubated with primary rabbit anti-mouse TH (EMD Millipore, Catalog# EMD-AB152) and rat anti-mouse CD31 (Dianova, Catalog# DIA-310) antibodies overnight at 4°C in plate rocker. For RSV staining, sections were incubated overnight at 4°C in a plate rocker with primary goat anti-RSV (Meridian Biosciences, Cat# B65860G), rat anti-F4/80 (Abcam, Cat# Ab90247), and rabbit anti-EpCAM (Abcam, Cat# Ab282457) antibodies. After incubation, sections were washed with PBST (PBS, 0.3% Triton X-100) and incubated with appropriate secondary antibodies [donkey anti-rabbit Alexa Fluor 647 (Thermo Fisher), goat anti-rat Alexa Fluor 594 (Abcam), donkey anti-goat Alexa Fluor 633 (Thermo Fisher), donkey anti-rabbit Alexa Fluor plus 555 (Thermo Fisher), and donkey anti-rat Alexa Fluor 488 (Thermo Fisher)], for 3 hours at room temperature. After the incubation, sections were washed in 1X PBS and mounted on slides using VectaShield (Vector laboratories) containing DAPI (2 µg/mL, Sigma). All antibodies used in IHC are listed in the supplementary table 7. Stained sections in slides were kept in dark and stored at 4°C until microscopy. A laser scanning microscope (LSM880 Airyscan, Zeiss) was used to capture images of the sections with Z-stacks and tiling, or Z-stacks only for RSV staining. Once the images were acquired, they were processed in Zen software (Zeiss) for maximum intensity projection and addition of scale bars. For creating 3D movies, FIJI software was used.

Isolation and culture of adult and neonatal bone marrow-derived macrophages (BMDMs)
Adult (8-week-old) and neonatal (P14) mice were first euthanized by using CO2 overdose. Then, muscles were separated to get the limb bones (tibias and femurs) and washed with sterile 1X PBS containing 2% FBS. Bones were then crushed using a sterile mortar and pestle with sterile 1X PBS containing 2% FBS to release bone marrow (BM) cells. The BM cells suspension was passed through a 70-μM cell strainer (Cell Treat Scientific) and cells were incubated with 1X RBC lysis buffer at room temperature for 8-11 minutes, followed by washing of cells twice with 1X PBS containing 2% FBS and centrifuging at 490g for 5 minutes/4°C to get the cell pellet. Next, the cell pellets were suspended in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. Approximately 10×10^6 BM cells from adult mice and 5×10^6 BM cells from P14 mice were cultured at 37°C with 5% CO2 in Petri dishes with 10 mL of conditioned DMEM (cDMEM) media containing 20% FBS, 30% L929 cell culture supernatant and 1% antibiotic-antimycotic solution (Gibco). On day three of incubation, an additional 10 mL cDMEM was added to the cultured cells and incubated at 37°C. On days six or seven, supernatant was removed from the dishes and differentiated BMDMs were dislodged with ice-cold 2.5 mM EDTA/PBS solution and cells were collected in DMEM containing 10% FBS. Cell suspension was centrifuged at 490g for 5 minutes at 4°C and cell pellets were resuspended with 2 mL of DMEM/10% FBS for cell counting and were used for seeding into 96-well plates.

In vitro stimulation of BMDMs and cytokine measurement
For cytokine measurement, 1.5×10^5 adult or neonatal BMDMs were first seeded into flat-bottomed 96-well plates in DMEM containing 10% FBS and incubated at 37°C overnight with 5% CO2. Cells were then washed with warm 1X PBS and co-treated with R848 (Cat# tlrl-r848-1, Invivogen) together with PBS, albuterol or xamoterol (Cat# 0950, Tocris) for 8 hours. Cells were centrifuged at 490g for 5 minutes/4°C and supernatant was stored at −80°C until the cytokine measurement. The levels of cytokines and chemokines were measured by ELISA (BioLegend) as per manufacturer’s instructions.

For the measurement of expression levels of phagocytosis receptors, 2 × 10^5 neonatal BMDMs were first seeded into flat-bottomed 96-well plates in DMEM containing 10% FBS and incubated with 5% CO2 at 37°C overnight. Cells were then washed with warm sterile 1X PBS and infected with live RSV-A2 (Cat# VR1540-P, ATCC) together with PBS or albuterol for 4 hours. Cells were then washed with warm PBS and lysed directly in the wells using Trizol, and lysates were stored at −80°C until the RNA was isolated.

Gene expression and viral load measurement
Whole lung samples were homogenized with sterile BB beads (Daisy Outdoor) in 1 mL Trizol reagent (Invitrogen) using TissueLyser II (Qiagen). RNA was isolated using PureLinkTM RNeasy Mini Kit (Thermo Fisher Scientific). To minimize DNAse contamination, in-column PureLinkTM DNAse (Invitrogen) treatment protocol was followed as recommended by the manufacturer. Then, RNA was reverse-transcribed to cDNA using SuperScript II cDNA Synthesis Kit (Invitrogen) after treatment with RNAse OUT (Invitrogen). The relative gene expression was determined by quantitative PCR on ABI 7000 System (Applied Biosystems) using PowerUpTM SYBR Green Master Mix (Applied Biosystems). Primers used for gene expression analysis were used in previous studies and are listed in supplementary table 8. The mRNA level of each gene was normalized to the expression of Gapdh. The normalized mRNA expression levels were presented as a fold change difference using the ΔΔCt method. For heatmap generation, we used mean of the fold changes of the group. For viral load measurement, the levels of RSV N & RSV L viral RNA were quantified using a standard curve that determines the absolute copy number of viral RNA. A standard curve was generated using quantitative RSV genomic RNA (VR-1540DQ, ATCC). The sample was serially diluted 2-fold and 10-fold. The detected Ct values were plotted to create a standard curve. Undetermined values were considered as absence of copy number and were used as zero in analyses.

For phagocytosis receptor gene expression analyses, BMDM RNA was isolated using PureLinkTM RNeasy Mini Kit (Thermo Fisher Scientific) and quantified. Then, RNA was reverse-transcribed to cDNA using the SuperScript II cDNA Synthesis Kit (Invitrogen). The relative gene expression was determined using quantitative PCR with the QuantStudio-3 (Thermo Fisher) and PowerUp SYBR Green Master Mix (Applied Biosystems). The primers used in this experiment were previously used and are listed in supplementary table 8. The mRNA level of each gene was normalized to the expression of Gapdh. For viral RNA measurement, the sample was serially diluted 10-fold. The levels of RSV N viral RNA were quantified using a standard curve method to determine the absolute copy number of viral RNA as discussed above.

Pulmonary hyperresponsiveness measurement
Neonatal mice were inoculated with RSV or mock at seven days of their birth, followed by albuterol or PBS treatment intranasally at 2 and 5 days post-inoculation. Lung mechanics were measured to determine pulmonary hyperresponsiveness in all cohorts of mice (12-15 weeks old) using a Flexivent (SCIREQ). For this, each mouse was first anesthetized by inhalation of 3-4% isoflurane, followed by a tracheostomy using a 22G cannula and connection to the Flexivent. All mice were ventilated at 150 Hz, with a tidal volume of 10 mL/Kg, and a positive end-expiratory pressure level of 300 mmH2O. The nebulizer was used for 10 seconds to administer saline (0.9% NaCl) or methacholine chloride (MCh, Methapharm). For each mouse, the baseline (0 mg/mL MCh) measurement was first performed using saline only, followed by measurement with exposure of 6.25 mg/mL MCh (dissolved in 0.9% NaCl). The following lung mechanical parameters were measured: Rrs, Ers, Rn, and G. Rrs measurements provide the information of total airway constriction, whereas Ers provide information about total airway stiffness, Rn about central airway constriction, and G about small and alveolar tissue constriction. Data files were acquired with the Flexiware software (Version 8), and all data were then analyzed and graphed in Prism.

Statistical analysis
Sample size and statistical details are described in figure legends and source data file. All statistical analyses were performed using GraphPad Prism 10 software. Data were tested for normality and differences in variance was used to apply appropriate statistical test (parametric or non-parametric). Mean differences of two experimental groups were compared using either a one-tailed, two-tailed unpaired t-test or Mann-Whitney test. Means of more than two groups were analyzed using One-way ANOVA, Brown-Forsythe and Welch ANOVA, Two-way repeated measure ANOVA and Kruskal-Wallis test. Fisher’s test, uncorrected Dunn’s test, Dunn’s multiple comparisons test, and Sidak’s test for post hoc analysis were performed as indicated in each figure legend. Mean differences between experimental groups were represented as follow: not significant (ns), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Data representation and visualization
For heatmaps, data generated from RT-qPCR experiments were normalized and averaged. All averaged data were plotted to generate a heatmap using Morpheus software (Broad Institute). BioRender (http://biorender.com) was used to create schematic. All generated representations, graphs and schematics from PRISM, Morpheus, FlowJo and BioRender were aligned and finalized for figures in Adobe Illustrator (Adobe Inc.).

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
All data needed to evaluate the conclusions are included in the article, Supplementary Information or available upon request. The raw numbers for charts and graphs are available in the Source Data file whenever possible. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Dear Editor, dear Authors\nRSV is one of the most important pathogens for infants and elderly world-wide. It is merely since very recently that\nvaccination using either viral antigens or virus neutralizing antibodies have become available at least in some regions.\nAlbeit these prophylactic interventions will most likely have a major impact, therapeutic interventions are still mainly\nrestricted to supportive care. Hence, there is still a huge need for efficient therapies to treat disease in RSV infected infants\nand adults. The findings presented in the submitted manuscript by Adhikari et. al., provide new avenues for treatment of RSV\ninfections in neonatal and possible other viral infections. The authors demonstrate that treatment with a b2-AR agonist\novercomes the reduced gain of weight in RSV infected neonatal mice and that this is associated with a reduces lung\npathology including less inflammation and less disruption of the lung/blood barrier. This is nicely mirrored by the enhanced\nloss of weight gain and pathology in mice lacking the b2-adrenergic receptor. Detailed analysis, indicated that the protective\nactivity of b2-AR signaling might be attributed to increased IL-10 production, infiltration of monocytes and interstitial\nmacrophages that contribute to viral clearance. The presented research is well designed well-analyzed and clearly\npresented. The observations might be instrumental to use a drug (that is already approved to treat the symptoms of long term\nconsequences of severe viral lung infection at young age) also during the infection itself. There are however several issues\nthat need to handled before publication.\n1. My major concern is that lack of evidence that actual viral replication is involved in the observed responses. Mice are only\nweakly permissive for RSV infections, with C57BL/6 mice being even far less so then BALB/C mice. Moreover, at least in\nBALB/c mice RSV replicates less efficient in the lungs of neonatal mice as compared to adult mice (Ruckwardt et al 2011,\nPlos pathogens). The presented manuscript does not report on the levels of replicating RSV that can be isolated in the lungs\nof the investigated mice. So it is unclear if any viral replication in these mice is ongoing and involved in the observed\nresponses. Figure S8 illustrates that the levels of viral mRNA (as analyzed by QPCR) are highest at day 2, the first time point\nof investigation after challenge. This does not indicate viral replication, and might rather argue for the lack of measurable\nreplication. In this way it cannot be excluded that the observed responses merely result from a non-infectious inoculate in the\nneonatal respiratory tract. I think the best way to test if viral replication is ongoing an involved in the observed responses is to\ninclude as control neonatal mice ( in both PBS treated and albuterol treated groups) that were either treated with an RSV\nneutralizing antibody (Palivizumab or Nirsevimab) before infection or neonatal mice from which the mother was treated with\nan RSV neutralizing antibody. Quantifying the level of replicating RSV in the lungs would demonstrate to which extend viral\nreplication is ongoing in this model. If only poor or no viral replication is observed then the impact of the neutralizing\nantibody on the observed immune and lung pathology will indicate to which extend viral replication is involved in the\nobserved responses. Ideally one would use an antibody lacking effector functions (eg LALAPG Fc variant or Fab, nanobody,\n….) to also rule out a possible impact of effector function triggering by the used antibodies. Such an approach would also\nnicely relate to the prophylactic RSV vaccination (passive and active) that currently rolled out. Alternatively, one can use UV-\ninactivated RSV as a control group. Also staining of viral antigen would be informative to clarify if viral replication co-locates\nwith the observed lung pathology.\n2. Neonatal mice are of especial interest to study the long-term consequences of RSV infections and the Th2 driven immune\npathology. RSV infection in neonatal mice results in a Th2 skewed immune responses and primes of asthma-like symptoms\nupon secondary infection (or pulmonary immune triggering) at later age (Culley et al, J Exp Med, 2002). In children\nprophylaxis using monoclonal antibodies overcomes the development of pulmonary hyperresponsiveness with asthma-like\nsymptoms at later age. Therefore it would be very interesting to test if also b2-AR agonist- treatment might overcome this.\nThis is merely a suggestion but it would be already very informative to test and report on the levels of Th2 cytokines (IL-4, IL-\n5 and IL-13) in the lung samples (or BMDMs) in the current manuscript.\n3. The authors illustrate in Fig 4A that also myeloid cells including monocytes express high level of the B2-A Receptor and\nsuggest that the b2-AR agonist albuterol enhances viral clearance. Next to the increased recruitment of monocytes and the\nincrease in the numbers of interstitial macrophages by albuterol it would be very interesting to investigate to which extend\nalbuterol also impacts the phagocytic capacity of these cells that account for clearance.\n4. Figure S3 illustrates that albuterol robustly increases IL-10 levels in BMDM of d14 mice. This seems far less the case for\nde d7 neonates (Fig 3C). please clarify and adapt in the text.", "reviewer_2": "In this manuscript, Adhikari et al evaluate the role of β2-adrenergic signaling on RSV infection and pathogenesis in the lung\nof neonatal mice by either dosing mice with albuterol or by using Adrb2 deficient mice. The concept that sympathetic\nneurons may regulate RSV replication and its course of disease in the lung is interesting. The conclusion of the authors is\nthat neonatal RSV infection induces lung inflammation, mucus production, barrier breakdown, and reduced IFN-lambda\nproduction and that treatment with a β2-adrenergic agonist (albuterol) can reduce morbidity by decreasing virus replication,\nlung damage, and barrier disruption and increasing IFN-lambda production. The significance of these findings is uncertain\ngiven clinical data showing no benefit of giving albuterol treatment to hospitalized RSV infected infants in regard to reducing\ndisease severity and recovery time.\nMajor Comments:\nThe authors should discuss their results in context with current AAP guidelines which recommend against using the β2-\nadrenergic agonist albuterol in hospitalized RSV-infected infants because it does not decrease the severity of disease or\nimprove recovery times. Are the benefits of albuterol being observed specific to the model that they are using?\nMouse body weights are presented at the %Original Weight and there is a significant difference in the %Original Weight\nbetween RSV infected mice administered PBS vs albuterol and between RSV infected Adrb2+/+ mice vs Adrb2-/- mice. The\nauthors should also show and statistically compare the measured weights of the mice over time as it is difficult to ascertain\nhow meaningful the difference between groups are when comparing %Original Weight. Also, it appears that the %Original\nWeight were already lower in the group of RSV infected mice administered PBS vs the group of mice administered in\nAlbuterol prior to the administration of albuterol (Figure 1B, right panel)\nThe viral load of RSV in the lungs of mice is quantified by PCR and represented as the fold change. It is not clear what is\nbeing compared to calculate a fold change. The reference for this method reports the data as the copy number relative to hki.\nThere also appears to be a lot of variability in the fold change in RSV N and RSV L levels between experiments in the RSV\ninfected/PBS treated at 7 days post infection (Figure 8 A, C, E, H). Do the decreases in fold change of RSV N and RSV L\nRNA correspond to decreases in infectious viral titers and/or number of RSV infected cells in lung tissue via histology?\nIn Figure 6C, there do not appear to be any significant differences in histology scores between mock infected Adrb2 +/+ vs\nRSV infected Adrb2 +/+. However, there are significant differences in the histology scores for airway RBS, alveolar RBC,\nairway inflammation, alveolar integrity, and interstitial inflammation between mock infected and RSV infected mice in Figure\n2C. In general, the histology scores appear lower in the RSV infected mice in Figure 6C compared to RSV infected mice\nFigure 2C.\nIn Figure 3B, the cytokines levels the lungs of PBS and albuterol treated RSV infected mice are represented as the log2 fold\nchange over mock. The colorimetric scale is from -1 to 2.5. It is difficult to assess how meaningful these differences are\nbased on how the data is presented. The authors should graph and compare the measured value of the cytokines\n(normalized by lung weight) for each mouse/group.\nIn Figure 8B, D, and F, the same colorimetric scale should be used to when representing the relative expression level of\nIfna1, Ifnb1, and Ifnl2 in the heat plots. The variability in the scale can exaggerate differences between groups. Also, does\nthe increase in Ifnl2 expression following albuterol treatment correspond to significant increase in IFN-lamda2 protein levels\nin the lung?\nThe manuscripts text states, “We did not observe any major impact of albuterol treatment in the baseline expression of tight\njunction and adhesion molecules (Fig. 4F)” (line 236-237). However, the heatmap would suggest that there are differences\nin Caludin-3, Claudin-4, Claudin-18, Occludin, and E-cadherin expression in mock infected mice administered albuterol vs\nPBS.\nMinor comments.\nFigure 3A shows that the levels of IL1b transcripts were reduced in the lungs of albuterol-treated mice at 7 days post-RSV\ninfection. Were the levels of IL1b measured in the inflammatory cytokine panel and if so, were they significantly lower in\nalbuterol-treated animals?\nThe manuscript text states that the virus load was peaked at 2 dpi, followed by at 4 and 7 dpi (Fig S8A) (lines 336-337). The\npeak viral load in the time course was at 2 dpi. There appears to be a small amount of variability at later time points,\npotentially because the levels of virus are low.\nIn the legends for Figure 2, the black arrows indicating RBC accumulation are shown in panel A and not in panel B."}, "author_response": "Point by point response\nWe thank the reviewers and editors for their careful reading of our manuscript and constructive\nfeedback and comments. We have performed additional experiments and have revised our manuscript\nto address each point raised by the reviewers. We provide a point-by-point response to each comment\nbelow and have created rebuttal figures (Rebuttal Figure R1, R2, R3, etc.) to address each of the\ncomments. We also refer to the figures in the revised manuscript, where the new data is presented,\nand the pages where the changes are made. Text revisions are written in red in revised manuscript.\nWe believe our manuscript is significantly strengthened, and we hope the reviewers will find it\nacceptable for publication.\nReviewer #1:\nRSV is one of the most important pathogens for infants and elderly world-wide. It is merely since very\nrecently that vaccination using either viral antigens or virus neutralizing antibodies have become\navailable at least in some regions. Albeit these prophylactic interventions will most likely have a major\nimpact, therapeutic interventions are still mainly restricted to supportive care. Hence, there is still a\nhuge need for efficient therapies to treat disease in RSV infected infants and adults. The findings\npresented in the submitted manuscript by Adhikari et. al., provide new avenues for treatment of RSV\ninfections in neonatal and possible other viral infections. The authors demonstrate that treatment with\na b2-AR agonist overcomes the reduced gain of weight in RSV infected neonatal mice and that this is\nassociated with a reduces lung pathology including less inflammation and less disruption of the\nlung/blood barrier. This is nicely mirrored by the enhanced loss of weight gain and pathology in mice\nlacking the b2-adrenergic receptor. Detailed analysis, indicated that the protective activity of b2-AR\nsignaling might be attributed to increased IL-10 production, infiltration of monocytes and interstitial\nmacrophages that contribute to viral clearance. The presented research is well designed well-analyzed\nand clearly presented. The observations might be instrumental to use a drug (that is already approved\nto treat the symptoms of long term consequences of severe viral lung infection at young age) also\nduring the infection itself. There are however several issues that need to handled before publication.\nResponse: We thank the reviewer for their feedback and insightful comments, which have led to the\ngeneration of important new datasets that strengthen the conclusions and rigor of the study.\nReviewer #1 Major Comments:\n1. My major concern is that lack of evidence that actual viral replication is involved in the observed\nresponses. Mice are only weakly permissive for RSV infections, with C57BL/6 mice being even far less\nso then BALB/C mice. Moreover, at least in BALB/c mice RSV replicates less efficient in the lungs of\nneonatal mice as compared to adult mice (Ruckwardt et al 2011, Plos pathogens). The presented\nmanuscript does not report on the levels of replicating RSV that can be isolated in the lungs of the\ninvestigated mice. So it is unclear if any viral replication in these mice is ongoing and involved in the\nobserved responses. Figure S8 illustrates that the levels of viral mRNA (as analyzed by QPCR) are\nhighest at day 2, the first time point of investigation after challenge. This does not indicate viral\nreplication, and might rather argue for the lack of measurable replication. In this way it cannot be\nexcluded that the observed responses merely result from a non-infectious inoculate in the neonatal\nrespiratory tract. I think the best way to test if viral replication is ongoing an involved in the observed\nresponses is to include as control neonatal mice ( in both PBS treated and albuterol treated groups)\nthat were either treated with an RSV neutralizing antibody (Palivizumab or Nirsevimab) before infection\nor neonatal mice from which the mother was treated with an RSV neutralizing antibody. Quantifying the\nlevel of replicating RSV in the lungs would demonstrate to which extend viral replication is ongoing in\nthis model. If only poor or no viral replication is observed then the impact of the neutralizing antibody\non the observed immune and lung pathology will indicate to which extend viral replication is involved in\nthe observed responses. Ideally one would use an antibody lacking effector functions (eg LALAPG Fc\nvariant or Fab, nanobody,….) to also rule out a possible impact of effector function triggering by the\nused antibodies. Such an approach would also nicely relate to the prophylactic RSV vaccination\n(passive and active) that currently rolled out. Alternatively, one can use UV-inactivated RSV as a control\ngroup. Also staining of viral antigen would be informative to clarify if viral replication co-locates with the\nobserved lung pathology.\nviral replication in the infected neonates. To quantify the live infectious viral titer and assess the RSV\nreplication in infected lungs, we performed the plaque forming assay (PFU), as published in other\nstudies1,2. We observed infectious viral titers from 2-7 days post-infection (dpi), with maximal titer at 3\nand 5 dpi (Rebuttal Fig R1). We found significantly increased viral titers at 5dpi compared to 2dpi,\nsuggestive of active viral replication in infected neonates (Rebuttal Fig R1B). Furthermore, we also\nperformed PFU assay from the lung homogenates from PBS- and albuterol-treated RSV-infected\nneonates at 7 dpi. Similar to our original qPCR findings of viral load (Previously in Fig 8A), these results\nshowed a significant reduction in PFU numbers in albuterol-treated neonates compared to the numbers\nin PBS-treated mice (Rebuttal Fig. R2A). These new data are now added in revised manuscript (Fig.\n8B,Supplemental Fig S12B) and have mentioned in the manuscript (page 9; lines 374-380, 388-391).\nTo evaluate our finding of increased RSV clearance in 2-agonist-treated neonates, we also performed\nimmunohistochemical staining of RSV (all antigens, Meridian Biosciences, Cat# B65860G) in the lung\ntissue sections collected from PBS- and albuterol-treated neonates at 7 dpi. We found that the lung\nsections from albuterol-treated neonates showed a small fraction of the airway epithelium positive for\nRSV (Rebuttal Fig R3). In contrast, in PBS-treated neonates, RSV was distributed throughout the\nairway epithelium (Rebuttal Fig R3). Further, we found visibly more airway damage and epithelial\nsloughing (shown by white arrows) in PBS-treated RSV-infected neonates (Rebuttal Fig R3). On the\nother hand, albuterol-treated RSV-infected lung showed intact airway epithelium (Rebuttal Fig R3).\nWe would also like to refer to Rebuttal Fig 11 where we also did not observe such epithelial cell damage\nin mock-inoculated neonates. Overall, these results indicate that RSV antigens co-localize with lung\npathology and that 2-agonist treatment protects infected neonates from airway epithelial damage\nRebuttal Figure R1. Time kinetics of infectious\nviral titers in the lungs after neonatal RSV\ninfection\nThe whole lungs were collected at indicated time\npoints after RSV infection (1.6 x 106 TCID per\n50\nneonate) in P7 neonates of C57BL/6 mice and\nhomogenized in 1X DMEM. The visible plaques\n(as plaque-forming units, PFUs) developed in 12-\nwell plate format after 7 days culture of Hep2 cells\nthat were treated with lung homogenates for viral\nadsorption were counted. Details about plaque\nforming assay are provided in Method section. (A)\nThe representative images of plaques developed\nafter 7 days culture of Hep2 cells are shown. (B)\nThe counted PFUs for each time point were\nnormalized to lung weight and represented as\nPFU per gram. Undetected (UD) plaques (no\nplaques) were considered as absence of\ninfectious viral titer in the sample and were used\nas zero in the graph plot. n = 3-4 mice/group were\nused, and the data are shown as mean ± SEM.\nOne-way ANOVA with uncorrected Fisher’s LSD\ntest for multiple comparisons was used (*P <\n0.05, ns = not significant).\nFinally, as reviewer suggested, we also performed morbidity study using UV-inactivated RSV as a\ncontrol to evaluate the efficacy of 2-agonist treatment in live infectious viral clearance and subsequent\nreduction in lung pathology and morbidity. The UV inactivation of RSV was confirmed by the complete\nabsence of plaques in a plaque-forming assay. The % body weight gain among PBS- and albuterol-\ntreated neonates did not significantly change at any time point up to 14 days post-challenge of UV-\ninactivated RSV (1.6 x 106 TCID /neonate) (Rebuttal Fig R2B). Our results demonstrate that live RSV\n50\ninfection is critical to induce morbidity and that the 2 agonist-mediated host defense involves live viral\nclearance. These new data are added in revised manuscript (Fig. 1D, Supplemental Fig S13) and\nhave mentioned in page 4, lines 152-156 and page 9, lines 391-398.\nAltogether, our new results have now greatly strengthened our conclusion regarding the actual viral\ninfectivity in RSV-infected neonates in our model, and that the 2-AR agonist treatment increases live\n2\nviral clearance to mediate host protection against neonatal RSV infection.\nRebuttal Figure R2. Infectious viral titers at 7dpi with and without β-agonist treatment & morbidity analyses of neonates after\nTCID per neonate) and homogenized in 1X DMEM. The representative images of plaques (from 3 mice/group) developed after 7 days\n50\nculture of Hep2 cells in 12 well plates that were treated with RSV-infected lung homogenates (7 dpi) from PBS- and albuterol-treated\nneonates (left). The visible plaques were counted and quantified and data were normalized to lung weight and represented as PFU per\ngram (right). Mock-inoculated mice were used as a negative control (zero plaque) for the plaque assay. RSV-infected, n=9-12 mice/group\nand mock-inoculated, n=4 mice. The Mann-Whitney test was used to compare two infected groups, and the data are shown as mean ±\nSEM. (B) The percent original weight change difference between PBS and albuterol-treated neonates after UV-inactivated RSV challenge\n(1.6 x 106 TCID per neonate) over 14 days post-challenge period (till P21). n=7-9 mice (males and females combined) per group were\n50\nused. Two-way repeated measure ANOVA with uncorrected Fisher’s LSD test for multiple comparisons was used (ns = not significant).\nRebuttal Figure R3.\nTreatment of RSV-\ninfected neonates with\nβ-agonist reduces the\n2\nnumber of RSV positive\nairway epithelial cells\nin the lung.\nRepresentative confocal\nimages of RSV-infected\nlung sections (from 7 dpi)\nfrom PBS- and albuterol-\ntreated neonates after\nimmunofluorescence\nstaining for RSV,\nepithelial cells and\nmacrophages. White\narrow indicate epithelial\nsloughing. Total 3\nimages per mouse were\ntaken from n=3 mice per\ngroup. RSV is in green;\nEpCAM, epithelial cell\nmarker in yellow; F4/80,\nmacrophage marker in\nred; and DAPI, nuclei\nmarker in blue. The\nimage on the left is a 20X\nmagnification, followed\nby 40X optical\nmagnification images on\nthe right, from the\nselected area (in a\nrectangle) of the left\nimage with a Z-stack.\nScale bars, 50 µm (left)\nand 20 µm (right).\n3\n2. Neonatal mice are of especial interest to study the long-term consequences of RSV infections and\nthe Th2 driven immune pathology. RSV infection in neonatal mice results in a Th2 skewed immune\nresponses and primes of asthma-like symptoms upon secondary infection (or pulmonary immune\ntriggering) at later age (Culley et al, J Exp Med, 2002). In children prophylaxis using monoclonal\nantibodies overcomes the development of pulmonary hyperresponsiveness with asthma-like symptoms\nat later age. Therefore it would be very interesting to test if also b2-AR agonist- treatment might\novercome this. This is merely a suggestion but it would be already very informative to test and report\non the levels of Th2 cytokines (IL-4, IL-5 and IL-13) in the lung samples (or BMDMs) in the current\nmanuscript.\nResponse: We thank the reviewer for this important question about the impact of neonatal 2-AR\nagonist treatment in the development of pulmonary hyperresponsiveness at later age of life. As reviewer\nsuggested, we performed additional experiments using Flexivent system to assess the pulmonary\nhyperreactivity when RSV-infected neonates reached to adulthood (12-15 weeks old). To set up these\nexperiments, we first purchased the Flexivent system (SCIREQ) and optimized the parameters for\naccurate measurements of pulmonary hyperresponsiveness. As in other studies 3-6, we used\nmethacholine (6.25 mg/mL) as a positive control to assess the pulmonary/airway hyperreactivity\n(Resistance, R ; elastance, E ; Newtonian resistance, R ; and tissue damping, G). R measurements\nrs rs n rs\nprovide information about total airway constriction, whereas E provide information for total airway\nrs\nstiffness, R about central airway constriction and G about small and alveolar tissue constriction. As\nn\nreported in previous studies with lung injury/infection3,7, all four measured airway hyperreactivity\nRebuttal Figure R4. Lung mechanics measurements in adult mice after neonatal β2-agonist treatment in RSV-infected\nneonates\n12 to 15 weeks old C57Bl/6J mice that were mock-inoculated (A) or RSV-infected (B) at P7 stage were first exposed to saline (0.9%\nNaCl) only for the baseline (methacholine, MCh, 0 mg/mL) measurements of lung mechanics in Flexivent, followed by 6.25 mg/mL MCh\n(dissolved in saline) exposure and Flexivent measurement. Details of Flexivent measurement are provided in Method section. Lung\nresistance (Rrs), elastance (Ers), Newtonian resistance (Rn), and tissue damping (G) were measured. One-way ANOVA with Fisher’s\nLSD test for multiple comparisons was used. n = 4-5 mice/group (A) and n = 10-12 mice/group (B) were used, and the data are shown\nas mean ± SEM. ns = not significant, *P < 0.05, ** P < 0.001, *** P < 0.0001\n4\nparameters were increased with methacholine treatment as compared to without treatment (0 mg/mL)\ncondition for both mock-inoculated and RSV-infected mice (Rebuttal Fig. R4A-B), suggesting our\nFlexivent setup providing reliable and accurate measurements. However, the differences were found\nmore significant among RSV-infected mice (Rebuttal Fig. R4B). We did not find any difference in\nairway hyperreactivity between PBS-and albuterol-treated mice that were RSV infected when they were\n7 days old (Rebuttal Fig. R4B). This data suggests that the albuterol treatment among RSV-infected\nneonates has no impact in RSV-induced pulmonary hyperresponsiveness development during 12-15\nweeks old age. While we did not find any obvious differences in pulmonary hyperresponsiveness\nbetween PBS and albuterol-treated RSV-infected mice at later stage of age, we cannot completely rule\nout the possibility of 2-agonist in altering RSV-induced pulmonary hyperresponsiveness during\nadulthood. Given that albuterol is short-acting 2 agonist and we only analyzed the lung mechanics\nwhen mice reached the age of 12-15 weeks, we are not sure if the effect will also be same at early\nstages of adult age (<12 weeks old) and if we use the long-acting agonist (e.g., Salmeterol). Further\nstudy is needed to fully determine the impact of 2-agonist administration in RSV-infected neonates in\nreducing pulmonary hyperresponsiveness development during adulthood. We have now added these\nnew data in Fig. S6 and has described about it in page 6, lines 230-242 of the revised manuscript.\nRebuttal Figure R5. Treatment of RSV-infected neonates with β-agonist suppresses IL-13 but not IL-4 and IL-5 cytokine levels.\nThe levels of cytokines were determined by ELISA (IL-4 & IL-5: BioLegend) & (IL-13: Proteintech) according to the manufacturer’s\nprotocol. The Mann-Whitney test was used with n = 10-12 mice per group (IL-4 & IL-5), n = 9 mice per group (IL-13), data from 1-2\nexperiments. Data are presented as mean ± SEM (ns = not significant, ** P < 0.001).\nAs reviewer suggested, we also examined the levels of Th2 cytokines (IL-4, IL-5 and IL-13) in lung\nhomogenates from RSV-infected neonates treated with PBS and albuterol. We found a significant\nreduction in the levels of IL-13 in albuterol-treated neonates at 7dpi compared to the levels in PBS-\ntreated group, but no differences were found for IL-4 and IL-5 (Rebuttal Fig. R5A-C). These results\nsuggest that there could be some impact of 2 agonist treatment in dampening type 2 inflammation in\nRSV-infected neonates especially the production of IL-13 cytokine. Further experiments with Th2\ncytokine analyses at different time points are necessary to more specifically delineate the impact of 2\nagonist treatment in affecting the type 2 inflammation in neonates with RSV infection. We have now\nadded Th2 ELISA data in the revised manuscript (Fig. S3) and have mentioned the role of 2 agonist\nin type 2 inflammation (page 5, lines 184-188).\n3. The authors illustrate in Fig 4A that also myeloid cells including monocytes express high level of the\nB2-A Receptor and suggest that the b2-AR agonist albuterol enhances viral clearance. Next to the\nincreased recruitment of monocytes and the increase in the numbers of interstitial macrophages by\nalbuterol it would be very interesting to investigate to which extend albuterol also impacts the phagocytic\ncapacity of these cells that account for clearance.\nResponse: We fully agree with the reviewer that it would be interesting to examine up to which extent\nalbuterol impacts the phagocytic capacity of monocytes and macrophages during RSV infection. We\n5\nnow provide experimental data regarding the impact of 2 agonist treatment in RSV\nbinding/phagocytosis in macrophages. As reported in literature 8-10. we did in vitro neonatal bone\nmarrow-derived macrophage (BMDM) culture for the infection with live RSV with and without albuterol\ntreatment condition, followed by examination of expression levels of phagocytosis markers and the\nextent of RSV surface binding and phagocytosis in macrophages. We first challenged BMDMs with\ndifferent doses of live RSV to validate that macrophage respond to live RSV infection. As expected, we\nfound a dose dependent increase in expression of IL-6 mRNA with multiplicity of infection (MOI) of 1 to\n10 (Rebuttal Fig. R6A). Increase in cytokine response is consistent with increased RSV binding and\nphagocytosis as we observed dose dependent increase in RSV viral load by macrophages that were\nwashed with PBS before analyzing the viral load (Rebuttal Fig. R6B). In albuterol-treated neonatal\nBMDMs, we found robust suppression of Tnfa mRNA levels, consistent with our original in vitro data\n(Fig. 3C) showing albuterol suppresses TNF- production when stimulated with R848 (Rebuttal Fig.\nR6C). These albuterol-treated RSV-infected BMDMs (MOI of 5) also showed increased viral load\nbased on RSV N gene copy numbers recovered from BMDM lysates (Rebuttal Fig. R6B).\nFurthermore, mRNA level analyses of phagocytosis markers showed increased expression of Mrc1\n(code for CD206), Tlr4 and Cd14 in RSV-infected macrophages treated with albuterol as compared to\nthe expression levels in PBS-treated RSV-infected macrophages (Rebuttal Fig. R6D-G). These results\nsuggest that the albuterol treatment enhances the expression of phagocytosis markers which might\nRebuttal Figure R6. Treatment of RSV-infected neonatal macrophages with β-agonist enhances expression of phagocytosis\n2\nreceptors\n(A) Expression level of Il6 by neonatal BMDM cultures after 4h infection with live RSV of multiplicity of infection (MOI) of 1 to 10. Control\n= uninfected negative controls. (B) The standard curve generated for absolute quantification of viral copy number of RSV N gene by\nqPCR using pre-quantified genomic RSV RNA with known copy numbers (left). The RSV mRNA was transcribed to cDNA first and 10-\nfold diluted, and then average Ct values were determined by qPCR using RSV N primers. Absolute quantification of viral copies of RSV\nN gene expression after 4 hours infection of neonatal BMDMs with RSV of MOI of 1 to 10 (right). Undetermined (UD) values were\nconsidered as absence of RSV N copy number in the samples and were used as zero copy number in the graph plot. (C-G) Expression\nlevels of Tnfα, Mrc1, Tlr4, Cd14 and Fcgr1 by neonatal BMDMs after 4h of infection with RSV (MOI 5) in the presence and absence of\nalbuterol (1µM). Mock-treated BMDMs received sterile PBS only. One-way ANOVA with Tukey test for multiple comparisons was used\nexcept for Tlr4 statistics, which used Fisher’s LSD test for multiple comparisons. n =4, data are shown as mean ± SEM (ns = not\nsignificant, *P < 0.05, **P < 0.01, ***P < 0.001 & ****P < 0.0001).\ncontribute to increased binding and internalization of RSV viral particles. Interestingly, albuterol\nrobustly increased the baseline Cd14 and Tlr4 expression (Rebuttal Fig. R6E-F). Further, albuterol\ntreatment also markedly increases the expression of Mrc1 during RSV infection but not the baseline\nexpression (Rebuttal Fig. R6D). Collectively, our new data support the idea that albuterol can enhance\nthe expression of phagocytosis receptors for the enhanced phagocytosis of RSV viral particles and/or\nRSV-infected neonatal cells. These new data are added in Supplemental Fig. S16 of the revised\nmanuscript and the results are mentioned in page 10, lines 420-439.\n6\n4. Figure S3 illustrates that albuterol robustly increases IL-10 levels in BMDM of d14 mice. This seems\nfar less the case for de d7 neonates (Fig 3C). please clarify and adapt in the text.\nResponse: We thank reviewer for pointing out the different results of IL-10 levels in BMDMs versus\nlung homogenates. There could be multiple possibilities for such discrepancy in results. One would be\nthe different nature of stimuli (R848 for BMDMs vs. live RSV for lung infection). Also, BMDMs represent\nsingle cell type and might contribute to robust increase in IL-10 production when stimulated with R848\ntogether with albuterol. On the other hand, lung has diverse types of immune and non-immune cells\nand these cell types might not be responding similarly with 2 AR agonism for IL-10 production during\nRSV infection (7dpi). There could also be possibility that non-macrophage cell type in lung suppressing\nIL-10 production after albuterol treatment. The time-course in vivo experiments are required to fully\ndelineate the role of lung cell type in IL-10 production in response to RSV during 2 agonist treatment.\nThis caveat has now been mentioned in the revised manuscript (page 11, lines 491-499).\nReviewer #2:\nIn this manuscript, Adhikari et al evaluate the role of β2-adrenergic signaling on RSV infection and\npathogenesis in the lung of neonatal mice by either dosing mice with albuterol or by using Adrb2\ndeficient mice. The concept that sympathetic neurons may regulate RSV replication and its course of\ndisease in the lung is interesting. The conclusion of the authors is that neonatal RSV infection induces\nlung inflammation, mucus production, barrier breakdown, and reduced IFN-lambda production and that\ntreatment with a β2-adrenergic agonist (albuterol) can reduce morbidity by decreasing virus replication,\nlung damage, and barrier disruption and increasing IFN-lambda production. The significance of these\nfindings is uncertain given clinical data showing no benefit of giving albuterol treatment to hospitalized\nRSV infected infants in regard to reducing disease severity and recovery time.\nResponse: We thank the reviewer for their positive and constructive feedback including highlighting\nour findings. We agree with reviewer regarding the caveat that our results contradict with the clinical\ndata that had shown no benefit of albuterol treatment in hospitalized RSV-infected infants 11,12. Few\nclinical studies also suggest that albuterol use might be helpful in some cases and it requires further in-\ndepth studies to determine the subgroups among children by considering factors such as age group,\nfamily history, previous exposure to disease and/or time extent of bronchodilator use13-17. After careful\nexamining the published literature regarding the efficacy of albuterol in RSV-induced morbidity in\nhospitalized infants/children, we believe there could be multiple reasons for the contradictory results\nwe observed with clinical data. First, in most of the clinical studies, use of albuterol was started after\nthe patients reaching at certain stages of disease severity (i.e. after RSV-induced bronchiolitis and\nhospitalizations) 11. But in our model, we administered the albuterol in RSV-infected neonates starting\nat 2 dpi, time where infected neonates do not or minimally exhibit clinical signs. There could be different\nimpact on RSV clearance and thus host response with early vs. late treatment of albuterol in RSV-\ninfected patients. Second, the dose and route of albuterol administration might be critical in anti-RSV\nhost response as we delivered the agonist (5 µg/neonate) intranasal route. Our method of\nadministration might help the agonist reaching more to the distal alveolar region of the lung as\ncompared to nebulization (i.e. inhalational) route as reported in published clinical studies 18-20. To this\nsupport, some studies have suggested that the 2 agonist may not consistently reach to the deep\nalveolar region with nebulization route of delivery19,20. Since our results involve increase in interstitial\nmacrophage (IM) and monocyte numbers and reduction in lung inflammation and mucus production in\n -agonist-treated neonates, it might be logical to say that the anti-RSV host response following\n2\nalbuterol treatment increases with the delivery route that helps deliver the agonist reaching to deep\nairway region of the lung where IMs and monocytes are located. Third, we used two doses of albuterol\ntreatment in our neonatal RSV infection model and this might explain at least 2 doses of albuterol\ntreatment (at 2 and 5 dpi) is critical for inducing anti-RSV defense in neonatal mice. It is less likely that\nalbuterol-treated infants in clinical studies received such combination of treatment with similar doses.\nWe believe that this combination of agonist treatment is essential for suppressing pathologic airway\nand interstitial inflammation in RSV-infected neonates while maintaining the endothelial barrier function\nand IFN-λ induction21,22. In sum, while our study could not explain fully why our results do not align with\n7\nclinical studies regarding the efficacy of albuterol in RSV-infected infants, there are however multiple\nfactors that might explain the discordant results and further investigation is needed to fully determine\nthe underlying confounding factors. This study is significant as it aims to fill the knowledge gaps\nregarding the role of 2-adrenergic signaling pathway in RSV infection. We have now explained this\ncaveat in the revised manuscript (page 3-4, lines 116-124; page 12, lines 545-56).\nReviewer #2 Major Comments:\nThe authors should discuss their results in context with current AAP guidelines which recommend\nagainst using the β2-adrenergic agonist albuterol in hospitalized RSV-infected infants because it does\nnot decrease the severity of disease or improve recovery times. Are the benefits of albuterol being\nobserved specific to the model that they are using?\nResponse: We thank the reviewer for this excellent point, and we apologize for not discussing the\nresults with current AAP guidelines in previous submission. While the revised 2014 AAP guidelines\nsuggest the albuterol treatment can provide transient breathing relieve in infants with RSV-induced\nbronchiolitis like it does for asthma and COPD patients, the updated guidance recommends against its\nuse because of no clear benefit in infected patients 23. We now carefully revised our manuscript and\nthe statements are refrained regarding the efficacy of albuterol in improving clinical signs and recovery\nand has now reasoned in the context with AAP guidelines in the revised manuscript (page 3-4, lines\n116-124). Regarding the question about the benefits of albuterol treatment is specific to our model or it\ncould be applied more broadly to other scenario, we acknowledge future study would be needed to fully\nunderstand the generalizability of albuterol efficacy in anti-RSV response. We also believe that our\nstudy is different in many ways with clinical studies and the results we observed might generate proof\nof concept idea and be critical for the future study to understand antiviral role of β2 adrenergic signaling\npathway. We have now mentioned this in the discussion of revised manuscript (page 12, lines 545-\n564; page 12-13, lines 568-572).\nMouse body weights are presented at the %Original Weight and there is a significant difference in the\n%Original Weight between RSV infected mice administered PBS vs albuterol and between RSV\ninfected Adrb2+/+ mice vs Adrb2-/- mice. The authors should also show and statistically compare the\nmeasured weights of the mice over time as it is difficult to ascertain how meaningful the difference\nbetween groups are when comparing %Original Weight. Also, it appears that the %Original Weight\nwere already lower in the group of RSV infected mice administered PBS vs the group of mice\nadministered in Albuterol prior to the administration of albuterol (Figure 1B, right panel).\nResponse: We appreciate the reviewer’s comments about the %Original Weight vs. measured weight\n(g) and the interpretation of these results. Given that mouse pups at the time of infection (day 7 of birth)\nare in a stage of gaining weight and the body weight gain of the P7 pups depends a lot on maternal\nand nursing behaviors of dams. Because of these caveats, there are always some differences in actual\nbody weight of pups between litters/cohorts with or without infection. The time course studies of\n%Original body weight in neonatal respiratory viral infection in mice have also been reported previously\nby other groups24-27. As reviewer suggested, we also converted the plots based on actual measured\nbody weight (in gram). As expected, there are differences in body weight gain pattern between\nlitters/cohorts (Rebuttal Fig R7A-B). However, we did not find a significant difference in measured\nbody weight (g) at day 0 of infection for the groups in the figure plots except small increased weight of\nthe pups of RSV-infected Adrb2–/– mice at day 0 compared to the weight of Adrb2+/+ pups (Rebuttal\nFig. R8A-B). These RSV-infected Adrb2–/– neonates also showed increased body weight when we\ncompared to mock-inoculated Adrb2–/– and Adrb2+/+ neonates at day 0 of infection (Rebuttal Fig. R8B),\nsuggesting developmental effect for the increased body weight for this particular neonatal group at day\n0 of infection. Overall, these actual body weight data at day 0 of infection help ruling out the possibility\nof differences in body weight in mock and RSV-inoculated neonates prior albuterol treatment and\nbecause of β2-AR deficiency in Adrb2–/– neonates. The measured body weight (g) data at day 0 of\ninfection for the morbidity experiments are now presented in Supplemental Fig S1 & S8 and has\n8\nmentioned about these results in page 4, lines 149-152 & page 7, lines 295-301.\nRebuttal Figure R7. Actual\nbody weight (in gram) of mock-\nand RSV-inoculated neonates\nwith and without β-agonist\n2\ntreatment and blockade of β-\n2\nadrenergic receptor\n(A) Body weight change\ndifference between PBS and\nalbuterol-treated neonates in\ngram after mock and RSV\ninoculation (From Figure 1B and\n1C). n=9-14 mice (males and\nfemales) per group, pooled from\n1-3 independent experiments.\n(B) Body weight change\ndifferences (in gram) between\nAdrb2+/+ and Adrb2−⁄− mice after\nmock and RSV inoculation for 14-\nday post-inoculation period (till\nP21) (From Figure 5B-C). Sterile\nPBS was used for the mock\ninoculation. n=6-12 mice (males and\nfemales) per group, pooled from\n2 independent experiments.\nTwo-way repeated measure\nANOVA with uncorrected Fisher’s\nLSD test for multiple comparisons\nwas used. All data are shown as\nmean ± SEM (*P<0.05,\n***P<0.001, ns- not significant).\nRebuttal Figure R8. Body weight of P7 neonates at day 0 of inoculation (From Figure 1B-D and Figure 5B-C)\n(A) Actual body weight (in gram) of mock-, live RSV- and UV-inactivated RSV-inoculated P7 neonates at day 0 of inoculation (From\nFigure 1B-D). n=9-14 mice (males and females) per group, pooled from 1-3 independent experiments. (B) Actual body weight (in gram)\nof mock-and RSV-inoculated P7 Adrb2+/+ and Adrb2−⁄− neonates at day 0 of inoculation (From Figure 5B-C). n=6-12 mice (males and\nfemales) were used per group, pooled from two independent experiments. Ordinary one-way ANOVA with Tukey multiple comparisons\ntest was used. All data are shown as mean ± SEM (*P<0.05, ns- not significant).\nThe viral load of RSV in the lungs of mice is quantified by PCR and represented as the fold change. It\nis not clear what is being compared to calculate a fold change. The reference for this method reports\nthe data as the copy number relative to hki. There also appears to be a lot of variability in the fold\nchange in RSV N and RSV L levels between experiments in the RSV infected/PBS treated at 7 days\npost infection (Figure 8 A, C, E, H). Do the decreases in fold change of RSV N and RSV L RNA\ncorrespond to decreases in infectious viral titers and/or number of RSV infected cells in lung tissue via\nhistology?\nbetter clarity, we have now converted our PCR data into viral copies/µg RNA, as reported by others\nfor the viral load measurements of RSV 28-31 and SARS-Cov232. For this, we first bought VR-1540 DQ\n(Quantitative genomic RNA of RSV-A2) from ATCC and synthesized cDNA, followed by qPCR using\nsame procedure used for viral load measurements from whole lung lysates to generate standard curves\nof RSV A2 Large Polymerase (L) and RSV A2 Nucleoprotein (N) gene expression (Rebuttal Fig R9A).\nWe then converted all our viral load graphs (previously Fig 8A, 8C, 8E, 8H & S8A) to viral copies/µg\nRNA from the Ct values of RSV L and RSV N using equation from the generated standard curves\n(Rebuttal Fig R9A)32. The revised results (Rebuttal Fig R9B-F) are consistent with our previous data\nRebuttal Figure R9. Viral copy measurements for absolute viral load quantification (Related to Fig 8A, D, F, I & S8A)\n(A) The standard curves generated for absolute quantification of viral copy numbers of RSV N and RSV L genes by qPCR using pre-\nquantified genomic RSV RNA with known copy numbers. The RSV mRNA was first transcribed to cDNA, diluted 2- and 10-fold, and then\naverage Ct values were determined by qPCR using RSV N and RSV L primers. (B) The absolute viral copy numbers of RSV L and RSV\nN were quantified by qPCR in the lung lysates from RSV-or mock-inoculated neonates that were treated with albuterol or PBS at 2 and\n5 days of post-inoculation. Kruskal-Wallis statistics with Dunn’s multiple comparisons post-tests were used. Sample size: mock (PBS)\nn=5, mock (Albuterol) n=5, RSV (PBS) n=5 and RSV (Albuterol) n=7. (C) The absolute viral copy numbers of RSV L and RSV N were\nquantified by qPCR in the lung lysates from RSV-infected whole lungs of Adrb2+/+ and Adrb2–/– mice at 7 dpi. The Unpaired t-test with\nWelch’s correction was used. Sample size: Adrb2+/+ (RSV), n=9 mice; Adrb2–/– (RSV), n=9 mice. (D) The absolute viral copy numbers of RSV L and RSV\nN in the lung lysates from RSV-infected whole lungs of vehicle-and 6-OHDA-treated mice at 7 dpi. Sample size: vehicle-\ntreated (RSV), n=6 mice & 6-OHDA-treated (RSV), n=7 mice. (E) Absolute quantification of viral copy number by qPCR in RSV-infected\nwhole lungs of PBS- and IFN-λ2-treated neonates. The Mann-Whitney test was used. Sample size: PBS (RSV) n=8 mice and IFN-λ2\n(RSV) n=10 mice. (F) Viral copy number was determined at 2, 3, 4, 5 and 7 dpi in the lung homogenates from RSV-infected neonates\nby qPCR analyses of RSV-N gene. Mock-inoculated mice are uninfected control mice. n=3 mice per group. Undetermined values were\nconsidered as absence of RSV copy number in the samples and were used as zero. Data are shown in mean ± SEM. *P < 0.05, **P <\n0.01, ***P < 0.001.\n10\nshowing fold expression relative to hki. We have now added more details regarding the method of viral\nload quantification in the revised manuscript (page 17, lines 799-803).\nWe agree with the reviewer’s comment regarding a variability in RSV N and RSV L levels between\nexperiments at 7dpi (previously Fig. 8A, 8C, 8E). In particular, the viral load data in RSV-infected PBS\ntreated group in Rebuttal Fig R9D is in lower magnitude as compared to other data (Rebuttal FigR9B).\nWhile there could be several reasons for this, but neonates used for Rebuttal Fig R9D were infected at\nP12, not at P7, after 4 (P4 to P7) consecutive treatment of PBS or 6-OHDA. There could be an impact\non viral infectivity because of difference in initial body weight of neonates in P7 vs P12 (Rebuttal Fig\nR10) at the day of infection. We have mentioned about this in the discussion of the revised manuscript\n(page 13, lines 597-599). To quantify the live infectious viral titers as reviewer suggested, we\nperformed plaque-forming assay of whole-lung homogenates from PBS- and albuterol-treated\nneonates at 7 dpi, also suggested by the reviewer 1. We found a significant reduction in PFU numbers\nin albuterol-treated neonates compared to the numbers in PBS-treated mice (Rebuttal Fig R2A),\nsuggesting that albuterol treatment reduces the infectious viral titer in the lungs. This new data is added\nin revised manuscript (Fig 8B).\nRebuttal Figure R10. Body\nweight differences in P7 & P12\nFollowing the suggestion of the\nneonates (From Fig 8A and 8F)\nreviewer, we also performed Actual body weight in gram of P7\nimmunofluorescence of the RSV- and P12 neonates used for two\ndifferent experiments. P7\ninfected lung sections to visualize the neonates were from RSV-infected\nRSV-infected cells at 7dpi using PBS group of Fig 8A and P12\nneonates were from PBS-treated\nantibody that can react with RSV\ngroup of Fig 8F). Unpaired t-test\nantigens (Meridian, BiosciencesTM was used to compare the group\nCat# B65860G)33. We also co-stained mean. Sample size: n=5-6\nmice/group. Data is shown in\nmacrophages (using anti-F4/80\nmean ± SEM (****P < 0.0001).\nantibody) and epithelial cells (using\nanti-EpCAM antibody). First, we\ninvestigated the level of infection in the lung regions and found that RSV immunoreactivity was mainly\nabundant at large as well as small/medium airway epithelial cells (Rebuttal Fig R3 and R11),\nsuggesting RSV infection can reach to distal alveolar regions of the lungs in our model and that\nepithelial cells are the primary sites of RSV infection. We also observed that the macrophages (F4/80+\ncells) are sparsely distributed in PBS-treated neonates whereas their number increased in albuterol-\ntreated neonates (Rebuttal Fig R3). In albuterol-treated neonates, we found the RSV-infected epithelial\ncells were markedly less in numbers than in PBS-treated RSV-infected neonates (Rebuttal Fig R3).\nAltogether, our additional results strengthen our conclusion regarding the impact of albuterol treatment\nin reducing the infectious viral titers in RSV-infected neonates at 7dpi. These new data are now added\nin Supplemental Fig. 13 of the revised manuscript and has mentioned in page 9, lines 391-398.\nRepresentative confocal images of mock-inoculated and RSV-infected lung sections from P14 neonates after immunofluorescence\nstaining for RSV, epithelial cells and macrophages. RSV antigens are in green; EpCAM, epithelial cell marker in yellow; F4/80,\nmacrophage marker in red; DAPI, nuclei marker in blue. White arrows indicate epithelial sloughing. Total 3 images per mouse were taken\nfrom n=3 mice per group. The image on the left is a 20X magnification, followed by 63X optical magnification images on the right, from\nthe selected area (in a rectangle) of the left image with a Z-stack. Scale bars, 50 µm (left) and 10 µm (right).\nIn Figure 6C, there do not appear to be any significant differences in histology scores between mock\ninfected Adrb2 +/+ vs RSV infected Adrb2 +/+. However, there are significant differences in the\nhistology scores for airway RBC, alveolar RBC, airway inflammation, alveolar integrity, and interstitial\ninflammation between mock infected and RSV infected mice in Figure 2C. In general, the histology\nscores appear lower in the RSV infected mice in Figure 6C compared to RSV infected mice Figure 2C.\nResponse: We agree with the reviewer regarding the lower histology scores in RSV-infected neonates\nas shown in Figure 6C compared to RSV-infected neonates in Figure 2C.The differences in disease\nseverity in neonates after RSV infection could potentially be affected by the animal strain, maternal\n12\nnursing behavior, size of litter, and microbiota 34,35. Further, there could be an impact of repeated post-\ninfection intranasal inoculations (i.e. PBS/albuterol treatment at 2 and 5 dpi) in increasing RSV-induced\nlung damage in Figure 2C when compared to no post-infection inoculation in Figure 6C. There could\nbe a possibility of reaching more live RSV and RSV-infected cell debri to deep alveolar region with\nrepeated post-infection inoculations that could increase the lung damage and infection severity.\nAdditionally, we noticed that the experiments performed in Figure 2C and Figure 6C involved RSV viral\ntiters from different lots of ATCC and there could be some variation in infectivity/pathogenesis because\nof different lots even we used the same dose of RSV (1.6 x 106 TCID /neonate) for all experiments.\nexperiments performed to generate data for the Figures (Rebuttal Table 1). We have also now\nmentioned this caveat in discussion as limitation of the study (page 13, lines 595-597). We believe\nexperimental groups should be compared to their corresponding internal control group to account for\nany differences between experiments and litters.\nRebuttal Table 1. RSV-A2 lots and corresponding experiments\nRSV-A2 Experimental Figures Lot#\n(Cat#)\nVR-1540 1(B & C), 2, 3E, 7 (D-G) 70040896\nVR-1540 1(D), 3(A, B & F), 4, 5, 6, 7 (A-C), 8, S3, S4, S6, S12, S13, S14, S15 70053997\nVR-1540 DQ Standard curve 70060095\nVR-1540P S16 70072445\nIn Figure 3B, the cytokines levels the lungs of PBS and albuterol treated RSV infected mice are\nrepresented as the log2 fold change over mock. The colorimetric scale is from -1 to 2.5. It is difficult to\nassess how meaningful these differences are based on how the data is presented. The authors should\ngraph and compare the measured value of the cytokines (normalized by lung weight) for each\nmouse/group.\nResponse: We thank the reviewer for these comments. We previously plotted the heatmaps\nfollowing a previous publication 36. As reviewer suggested, we have now plotted the graphs and\ncompared values of the cytokines after normalizing by lung protein (Fig. 3B).\nIn Figure 8B, D, and F, the same colorimetric scale should be used to when representing the relative\nexpression level of Ifna1, Ifnb1, and Ifnl2 in the heat plots. The variability in the scale can exaggerate\ndifferences between groups. Also, does the increase in Ifnl2 expression following albuterol treatment\ncorrespond to significant increase in IFN-lamda2 protein levels in the lung?\nResponse: We appreciate this suggestion from reviewer. As suggested, we have now normalized all\nthe heatmaps in Figure 8C,8E and 8G (previously Fig8B, 8D and 8F) to a single scale ranging from\n0.25 to 4.85 (Rebuttal Fig R12). Furthermore, as reviewer suggested, we ran IFN-lamda2/3 ELISA\n(R&D systems, cat# DY1789B-05) to determine if albuterol treatment increase the IFN-λ2 protein levels\nin the RSV-infected lungs. Our results show an increase in protein levels of IFN-λ2/3 in the lungs from\nalbuterol-treated neonates as compared to the levels in PBS-treated neonates at 7 dpi (Rebuttal Fig\nR13). We have now included this data (in Supplemental Fig. S14) and mentioned about this result in\nrevised manuscript (page 9, lines 402-404).\n(A) The mRNA levels of IFNs (Ifna1, Ifnb1, and Ifnl2), were quantified by qPCR in the lung homogenates from RSV-or mock-inoculated\nneonates after albuterol or PBS treatment at 2 and 5 days of post-inoculation. Sample size: mock (PBS) n=7-11, mock (Albuterol) n=5-\n9, RSV (PBS) n=12 and RSV (Albuterol) n=12-14 mice. (B) Quantification of mRNA levels of IFNs (Ifna1, Ifnb1, and Ifnl2), in RSV-\ninfected whole lungs of Adrb2+/+ and Adrb2–/– mice at 7 dpi. Sample size: Adrb2+/+ (RSV), n=8-10 mice; Adrb2–/– (RSV), n=9-10 mice. (C)\nQuantification of mRNA levels of IFNs, in RSV-infected whole lungs of vehicle-and 6-OHDA-treated mice at 7 dpi. Sample size: vehicle-\ntreated (RSV), n=6-7 mice; 6-OHDA-treated (RSV), n=7-9 mice. The relative IFN gene expression is shown in averages on the heatmaps\nin a scale range of 0.25 to 4.65\nThe manuscripts text states, “We did not observe any major\nimpact of albuterol treatment in the baseline expression of tight\njunction and adhesion molecules (Fig. 4F)” (line 236-237).\nHowever, the heatmap would suggest that there are differences\nin Caludin-3, Claudin-4, Claudin-18, Occludin, and E-cadherin\nexpression in mock infected mice administered albuterol vs\nPBS.\nResponse: We apologize for not being clear in our statements\nin previous submission regarding the baseline expression of\ntight junction and adhesion molecules. We have now revised the\nmanuscript text about these statements to indicate there are\nsome changes in the baseline expression because of albuterol\ntreatment (page 6, line 267-268).\nReviewer #2 Minor Comments: Rebuttal Figure R13. β 2 -agonist treatment\nenhances lung interferon lambda\nproduction in RSV-infected neonates\nFigure 3A shows that the levels of IL1b transcripts were reduced Quantification of whole lung homogenate IFN-\nλ levels in PBS- or albuterol-treated RSV-\nin the lungs of albuterol-treated mice at 7 days post-RSV\ninfected neonates (7 dpi) using ELISA (R&D\ninfection. Were the levels of IL1b measured in the inflammatory Systems). One-tailed unpaired t-test was\ncytokine panel and if so, were they significantly lower in used. Sample size: n=4 mice/group. Data is\nshown in mean ± SEM (*P < 0.05).\nalbuterol-treated animals?\nResponse: We thank the reviewer for this question. Yes, we did observe the reduction of IL-1β protein\nlevels in our inflammatory cytokine panel analyses in the lung homogenates from albuterol-treated\nneonates compared to the levels in PBS-treated mice at 7dpi (Fig. 3B).\nThe manuscript text states that the virus load was peaked at 2 dpi, followed by at 4 and 7 dpi (Fig\nS8A) (lines 336-337). The peak viral load in the time course was at 2 dpi. There appears to be a small\namount of variability at later time points, potentially because the levels of virus are low.\nResponse: We appreciate reviewer’s comment regarding the time kinetics of viral load during neonatal\nRSV infection. We agree with the reviewer about the time kinetic variability due to the levels of virus.\n(Rebuttal Fig. R1A-B). Both datasets suggest that the viral load are detectable in the lungs between\n2-7 dpi and we have now revised our statements about these results in the revised manuscript (page\n9, lines 375-378).\nIn the legends for Figure 2, the black arrows indicating RBC accumulation are shown in panel A and\nnot in panel B.\nResponse: We apologize for not showing arrows in panel B in the previous submission. We have now\nadded arrows in panel B in revised Figure 2B.\nRebuttal references\nassay for human respiratory syncytial virus (HRSV). J Immunol Methods 446, 15-20 (2017).\nhttps://doi.org:10.1016/j.jim.2017.03.020\nfusion protein inhibitor. Nat Commun 15, 1173 (2024). https://doi.org:10.1038/s41467-024-\n45241-y\nNature\n631, 601-609 (2024). https://doi.org:10.1038/s41586-024-07608-5\nby ERK signaling in brain stem cholinergic neurons. Cell Rep 11, 934-943 (2015).\nhttps://doi.org:10.1016/j.celrep.2015.04.012\nAllergy 75, 603-615 (2020). https://doi.org:10.1111/all.14039\noscillation and forced expiration measurements in mice for the assessment of airway\nhyperresponsiveness. Respir Res 11, 82 (2010). https://doi.org:10.1186/1465-9921-11-82\nalters lung function in adult mice. Sci Rep 6, 22751 (2016). https://doi.org:10.1038/srep22751\nmacrophages challenged with respiratory syncytial virus. BMC Genomics 14, 190 (2013).\nhttps://doi.org:10.1186/1471-2164-14-190\nvariable inflammatory responses in association with altered expression of cell surface dectin-\n1. J Immunol Methods 329, 157-166 (2008). https://doi.org:10.1016/j.jim.2007.10.003\npersistence in macrophages upregulates Fcgamma receptors expression. Viruses 6, 624-639\n(2014). https://doi.org:10.3390/v6020624\nHospitalized with Acute RSV Bronchiolitis. Interdiscip Perspect Infect Dis 2012, 585901\n(2012). https://doi.org:10.1155/2012/585901\nRev 35, 519-530 (2014). https://doi.org:10.1542/pir.35-12-519\nprescription of albuterol in infants hospitalized for viral bronchiolitis. Allergol Immunopathol\n(Madr) 48, 469-474 (2020). https://doi.org:10.1016/j.aller.2019.10.007\nBronchiolitis. West J Emerg Med 19, 475-483 (2018).\nhttps://doi.org:10.5811/westjem.2018.3.35837\nFinnish hospitals. Acta Paediatr 106, 1512-1518 (2017). https://doi.org:10.1111/apa.13931\n15\nappropriate to use albuterol, at least on a therapeutic trial basis? Allergol Immunopathol\n(Madr) 49, 153-158 (2021). https://doi.org:10.15586/aei.v49i1.12\nbronchiolitis phenotyping: Is it time to consider phenotype-specific responses to individualize\npharmacological management? Paediatr Respir Rev 34, 53-58 (2020).\nhttps://doi.org:10.1016/j.prrv.2019.04.003\nthan 24 months of age. Pediatrics 132, e341-348 (2013). https://doi.org:10.1542/peds.2013-\n0303\ninfants. J Nucl Med 43, 487-491 (2002).\neffectiveness of nebulized therapy with epinephrine compared with albuterol and saline in\ninfants hospitalized for acute viral bronchiolitis. J Pediatr 141, 818-824 (2002).\nhttps://doi.org:10.1067/mpd.2002.129844\ninfection. Science 369, 712-717 (2020). https://doi.org:10.1126/science.abc2061\nand Type III Interferons. Immunity 50, 907-923 (2019).\nhttps://doi.org:10.1016/j.immuni.2019.03.025\nbronchiolitis. Pediatrics 134, e1474-1502 (2014). https://doi.org:10.1542/peds.2014-2742\nInfluenza Infection that Are Associated with Type 2 Immunity. Immunity 49, 531-544 e536\n(2018). https://doi.org:10.1016/j.immuni.2018.07.011\nthe enhancement of respiratory syncytial virus infection severity during adult reinfection of\nneonatally sensitized mice. J Virol 82, 4115-4124 (2008). https://doi.org:10.1128/JVI.02313-07\nsequelae of neonatal respiratory syncytial virus infection are dependent on cells of the innate\nimmune system. J Virol 88, 604-611 (2014). https://doi.org:10.1128/JVI.02620-13\ngamma in a novel neonatal mouse model of respiratory syncytial virus infection. PLoS One 7,\ne40499 (2012). https://doi.org:10.1371/journal.pone.0040499\nto RSV through recruitment of antiviral monocytes. J Exp Med 212, 699-714 (2015).\nhttps://doi.org:10.1084/jem.20140825\ninflammation and disease via IL-1alpha and TNF-alpha. Mucosal Immunol 17, 1184-1198\n(2024). https://doi.org:10.1016/j.mucimm.2024.08.002\ninfection. Nat Commun 9, 3999 (2018). https://doi.org:10.1038/s41467-018-06508-3\ninfection through a GPR43-type 1 interferon response. Nat Commun 10, 3273 (2019).\nhttps://doi.org:10.1038/s41467-019-11152-6\nthe lung for early control of SARS-CoV-2. Nat Commun 14, 8229 (2023).\nhttps://doi.org:10.1038/s41467-023-43447-0\na pathogenic pulmonary innate immune response. JCI Insight 2 (2017).\nhttps://doi.org:10.1172/jci.insight.94605\nExp Med 218 (2021). https://doi.org:10.1084/jem.20210235\n16\nthe persistence of symptoms in children. Cell Rep Med 5, 101836 (2024).\nhttps://doi.org:10.1016/j.xcrm.2024.101836\nCoV-2 variants including Omicron. Cell Rep 39, 110799 (2022).\nhttps://doi.org:10.1016/j.celrep.2022.110799\n17"}, {"round": 2, "reviewer_comments": {"reviewer_1": "As a response to my comments the authors performed additional experiments. These were performed in a correct way and provided additional insight in the mechanism by which a B2-AR agonist restricts RSV infection. I now consider the manuscript eligible for publication. kind regards", "reviewer_3": "The response to earlier reviewers comments are reasonable. Two edits/changes to consider and one comment.\n1. In intro, lines 76-77, the authors state, “In 2019, 33 million RSV cases were registered and 118,000 RSV-associated deaths reported worldwide3.” These are estimated and not registered or reported cases and deaths.\n2. Line 82, “prophylaxis” is correct/better term than “therapy”\n3. There is as yet no truly effective treatment for clinical RSV infection with the exception of human challenge studies when treatment is given very early in the infection, e.g. first PCR positive specimen with daily testing post challenge. Treatment later in the infection at the time of presentation no treatment has proven effective. It would be of interest, for later studies, to look at different treatment at different time points after challenge."}, "author_response": "Point by point response\nReviewer #1:\nAs a response to my comments the authors performed additional experiments. These were performed\nin a correct way and provided additional insight in the mechanism by which a B2-AR agonist restricts\nRSV infection. I now consider the manuscript eligible for publication. kind regards\nResponse: We thank the reviewer for their enthusiasm to our research and constructive comments\nwhich allowed us to strengthen our manuscript.\nReviewer #3:\nThe response to earlier reviewers comments are reasonable. Two edits/changes to consider and one comment.\nResponse: We thank the reviewer for their feedback and comment.\n1. In intro, lines 76-77, the authors state, “In 2019, 33 million RSV cases were registered and 118,000\nRSV-associated deaths reported worldwide3.” These are estimated and not registered or reported\ncases and deaths.\nResponse: We apologize for an inaccurate statement. We have now revised the statement as reviewer\nsuggested in the revised manuscript.\n2. Line 82, “prophylaxis” is correct/better term than “therapy”\nResponse: We thank the reviewer for the suggestion. We have now replaced “therapy” with\n“prophylaxis” in revised manuscript.\n3. There is as yet no truly effective treatment for clinical RSV infection with the exception of human\nchallenge studies when treatment is given very early in the infection, e.g. first PCR positive specimen\nwith daily testing post challenge. Treatment later in the infection at the time of presentation no treatment\nhas proven effective. It would be of interest, for later studies, to look at different treatment at different\ntime points after challenge.\n.\nResponse: We fully agree with this excellent comment from the reviewer regarding follow-up\nexperiment in the future. While our study found that the local β -AR agonist (albuterol) treatment in\npathology, we have to yet determine the clinical beneficial role of albuterol at later phase of infection\n(after 14 dpi) and also cumulative efficacy of the compound when administer more than two doses at\nearly and later phase of infection where clinical signs would be more obvious. We’re also pursuing to\nexamine the full spectrum of clinical benefit of β -AR agonist treatment in RSV-infected neonatal mice\n2\nusing Salmeterol, a long-acting β agonist.\n2\n1"}]

Decision letter