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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Ann Anat. 2023 Jan 20;247:152048. doi: 10.1016/j.aanat.2023.152048

Intravenous Surfactant Protein D inhibits lipopolysaccharide-induced systemic inflammation

Sarah K Mierke a, Kelsey L Rapier a, Anna M Method a, Brooke A King a, Paul S Kingma a,b,c
PMCID: PMC9992088  NIHMSID: NIHMS1867208  PMID: 36690045

Abstract

Background:

Surfactant protein D (SP-D) is an innate host defense protein that clears infectious pathogens from the lung and regulates pulmonary host defense cells. SP-D is also detected in lower concentrations in plasma and many other non-pulmonary tissues. Plasma levels of SP-D increase during infection and other proinflammatory states; however, the source and functions of SP-D in the systemic circulation are largely unknown. We hypothesized that systemic SP-D may clear infectious pathogens and regulate host defense cells in extrapulmonary systems.

Methods:

To determine if SP-D inhibited inflammation induced by systemic lipopolysaccharide (LPS), E.coli LPS was administered to mice via tail vein injection with and without SP-D and the inflammatory response was measured.

Results:

Systemic SP-D has a circulating half-life of 6 hours. Systemic IL-6 levels in mice lacking the SP-D gene were similar to wild type mice at baseline but were significantly higher than wild type mice following LPS treatment (38,000 vs 29,900ng/ml for 20mg/kg LPS and 100,700 vs 73,700ng/ml for 40mg/kg LPS). In addition, treating wild type mice with purified intravenous SP-D inhibited LPS induced secretion of IL-6 and TNFα in a concentration dependent manner. Inhibition of LPS induced inflammation by SP-D correlated with SP-D LPS binding suggesting SP-D mediated inhibition of systemic LPS requires direct SP-D LPS interactions.

Conclusions:

Taken together, the above results suggest that circulating SP-D decreases systemic inflammation and raise the possibility that a physiological purpose of increasing systemic SP-D levels during infection is to scavenge systemic infectious pathogens and limit inflammation-induced tissue injury.

Keywords: Collectin, Lung, Sepsis, Endotoxin shock, Inflammation, Lipopolysaccharide

1. INTRODUCTION

Surfactant protein D (SP-D) is a member of the collectin family of innate host defense proteins. SP-D binds a broad range of viruses, Gram-negative bacteria, Gram-positive bacteria, fungus, and mite extracts and facilitates the clearance of these pathogens from the respiratory system by host defense cells (Arroyo and Kingma, 2021; Sorensen, 2018; Watson et al., 2020). Although binding infectious organisms is a critical feature of SP-D physiology, targeted deletion of the SP-D gene (Sftpd) in transgenic mice revealed more complex roles of SP-D in pulmonary immune cell regulation. Sftpd−/− mice survived normally but developed progressively worsening pulmonary inflammation that is characterized by increased numbers of alveolar macrophages that are enlarged, lipid laden, and release metalloproteinases and reactive oxygen species (Korfhagen et al., 1998; Wert et al., 2000a; Wert et al., 2000b). Moreover, oxygen radical release and pulmonary production of the proinflammatory mediators, tumor necrosis factor (TNF)α interleukin (IL)-1, and IL-6, are increased in Sftpd−/− mice when challenged with either a bacterial or viral infection (Kingma et al., 2006; LeVine et al., 2004; LeVine et al., 2000). A similar increase in pulmonary inflammation in Sftpd−/− mice was also observed when indirect lung injury was induced in these mice via a systemic sepsis model (King and Kingma, 2011). Therefore, SP-D promotes a controlled immune response in the lung that effectively clears invading pathogens while also limiting the potential damaging effects of immune cell mediated inflammation and microbial invasion in the lung parenchyma.

SP-D is secreted in highest concentration in the lung, but low levels of SP-D are also detected by immunostaining and mRNA expression in other tissues, including vascular endothelium (Madsen et al., 2000; Snyder et al., 2008; Sorensen et al., 2006; Stahlman et al., 2002). Studies suggest that during acute respiratory distress syndrome and other forms of lung injury, damage to the alveolar-capillary barrier leads to leakage of pulmonary SP-D into the vascular compartment and several studies have demonstrated that blood levels of SP-D increase during sepsis and other pro-inflammatory states (Greene et al., 2002; Greene et al., 1999; Kuroki et al., 1998; Takahashi et al., 2006; Wu et al., 2009). However, the functions of SP-D in the systemic circulation are largely unknown. Since pulmonary SP-D is critical for maintaining a controlled immune response in the lung that limits tissue damage resulting from inflammation, we hypothesized that systemic SP-D may limit systemic inflammation and tissue damage in a manner similar to the pulmonary immune system. Therefore, to investigate the physiologic function of systemic SP-D and to assess the potential clinical application of intravenous/systemic SP-D in treating septic shock, the effect of SP-D on lipopolysaccharide (LPS) induced systemic inflammation was determined. Results indicate that SP-D inhibits inflammation induced by systemic LPS and suggest that SP-D may have similar functions in both the pulmonary and extrapulmonary organ systems.

2. MATERIALS AND METHODS

2.1. Animal Husbandry-

Mice were handled in accordance with approved protocols through the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Medical Center. All mice had been maintained in the vivarium in barrier containment facilities. Sentinel mice in the colony were serologically negative for common murine pathogens. Studies were performed on 6- to 10-week old Sftpd−/− and age-matched Sftpd+/+ litter mate wild-type controls generated as previously described. For experiments that did not utilize the Sftpd−/− transgenic mice, the C57BL/6N mouse model was utilized.

2.2. Preparation of SP-D-

Recombinant human SP-D (rhSP-D) was made in transfected Chinese hamster ovary cells expressing a complementary DNA encoding full-length human SP-D and purified, as previously described (Ikegami et al., 2006). All preparations of SP-D were purified to a single band on silver stained gel and with less than 0.1 endotoxin units per microgram protein. The rhSP-D was diluted to a concentration of 0.5 mg/mL in a buffer containing 20 mmol/L Tris, 200 mmol/L NaCl, and 1 mmol/L ethylenediaminetetraacetic acid at pH 7.4. Control mice were instilled with the same volume of dilution buffer.

2.3. Systemic SP-D clearance-

Wild type (C57BL/6N) mice were temporarily anesthetized with isoflurane and treated with rhSP-D (150 μg/kg) via tail vein injection. Blood was harvested at the indicated time intervals and SP-D levels were determined by enzyme-linked immunosorbent assay (ELISA) as previously described (Kingma et al., 2006). All samples were diluted to keep SP-D levels within the acceptable concentration range of the ELISA. Assays utilized a mouse anti-SP-D monoclonal antibody that was developed by exposing Sftpd−/− mice to purified, full length, human SP-D and selecting cell lines that demonstrated a high affinity for SP-D and minimal cross-reactivity with SP-A (Seven Hills Bioreagents, Cincinnati, OH).

2.4. Inflammation in the absence of SP-D-

Wild type (Sftpd+/+) and Sftpd−/− mice were temporarily anesthetized with isoflurane and treated with control buffer or LPS (serotype Escherichia coli O111:B4, 20 mg/kg or 40 mg/kg) via intraperitoneal injection. Blood was harvested 6 hours after injection and plasma IL-6 levels were determined.

2.5. Inflammation in presence of exogenous SP-D-

Wild type (C57BL/6N) mice were treated with LPS (serotypes Escherichia coli O111:B4 and 0127:B8, 5 μg/kg) or with LPS and increasing amounts of purified SP-D (25–150 μg/kg) via tail vein injection. To facilitate more consistent and fewer injections, LPS was combined with SP-D where indicated and given as part of a single injection. Blood was harvested 2 hours after injection and plasma cytokine levels were measured. Mice were euthanized with intraperitoneal injection of pentobarbital. Blood samples were obtained by cardiac puncture at the time of sacrifice and stored at −20°C until cytokine levels were determined. IL-6 (M600B, R&D Systems, Minneapolis, MN) and TNFα (MTA00B, R&D Systems, Minneapolis, MN) were quantified in plasma with a murine ELISA using a commercially available kits. Chromogenic limulus amebocyte lysate test was used to quantify endotoxin (Cambrex BioScience, Walkersville, MD).

2.6. SP-D LPS Binding Assay-

Sample wells of a 96 well plate were coated with LPS at 10ng/ml and incubated at 4°C for 3 days. Wells were blocked for an hour prior to the addition of increasing amounts of SP-D (0, 10, 25, 50, 75, 100, 200, 400, 600, 1000ng/ml) with 100mM CaCl2 and incubated for 2 hours. Samples were washed prior to incubation with a polyclonal rabbit anti-SP-D antibody (1:750) (Seven Hills Bioreagents, Cincinnati, OH) for 1 hour. Samples were washed and incubated with donkey anti-rabbit IgG (1:10,000) for 1 hour before adding TMB substrate. Reaction was stopped using 2M H2SO4 and samples absorbance was measured at 450nm using a BioTek Synergy 2.

2.7. LPS clearance assay-

Wild type (C57BL/6N) mice were temporarily anesthetized with isoflurane and treated with rhSP-D (150 μg/kg) via tail vein injection. Blood was harvested at the indicated time intervals and LPS levels were determined by chromogenic limulus amebocyte lysate test.

2.8. Statistical Analysis-

All results are given as the mean ± standard error of the mean. The groups were compared using two-tailed Student’s t-test. Differences of p < 0.05 were considered significant.

3. RESULTS

To evaluate if systemic SP-D is immediately cleared from the systemic circulation, the clearance of intravenously administered rhSP-D was measured (Figure 1). Results indicate that rhSP-D was not immediately cleared and remained in plasma with a half-life of ~6-hours. In the absence of inflammation or lung injury, mouse endogenous SP-D levels in blood were 25±6 ng/ml, while in the presence of lung injury induced by 20 mcg/kg intratracheal LPS injection SP-D levels in blood were 132±56 ng/ml (data not shown).

Figure 1.

Figure 1.

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rhSP-D (150 μg/kg) was administered to wildtype mice (n=5) via tail vein injection. Blood was collected 15 minutes after injection (t=0) and at the indicated time points. Plasma SP-D levels were determined by ELISA. Error bars represent SEM.

3.1. Systemic inflammation in the absence of SP-D-

Endotoxemia was induced in wild type and Sftpd−/− mice by intraperitoneal injection of E.coli 0111:B4 LPS (20 mg/kg and 40 mg/kg) and systemic inflammation was evaluated by measuring plasma IL-6 levels 6 hours after injection. When compared to wild type mice, systemic IL-6 levels were not elevated in Sftpd−/− mice in the absence of LPS induced inflammation (Figure 2). However, following challenge with intraperitoneal LPS, plasma IL-6 levels in Sftpd−/− mice were significantly higher than IL-6 levels in wild type mice.

Figure 2.

Figure 2.

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Wildtype (open bar) and Sftpd−/− (closed bar) mice (n=10 for each group) were treated with control buffer or LPS via intraperitoneal injection. Blood was harvested 6 hours after injection and plasma IL-6 levels were determined by ELISA. Blood IL-6 levels were significantly higher in Sftpd−/− mice treated with 40 mg/kg LPS when compared to wildtype mice (p=0.32 for 20 mg/kg LPS and p=0.04 for 40 mg/kg LPS). Error bars represent SEM.

3.2. Purified intravenous SP-D inhibits LPS induced inflammation-

To determine if the increase in systemic inflammation in Sftpd−/− mice was directly related to the absence of SP-D versus secondary changes to the mouse immune system and to determine if administering exogenous rhSP-D to healthy wild type mice would also inhibit LPS induced systemic inflammation, LPS (5 μg/kg) was administered to wild type mice with control buffer or increasing concentrations of rhSP-D and the cytokine response was measured in the plasma 2 hours after injection. A much lower dose of LPS was utilized to minimize the downstream effects of LPS (eg. acute respiratory distress syndrome and multi-organ system failure) that might confound our results. rhSP-D significantly decreased levels of IL-6 and TNFα in a concentration dependent manner with 150 μg/kg rhSP-D producing a maximum reduction of 40% and 50% in IL-6 and TNFα levels, respectively (Figure 3).

Figure 3.

Figure 3.

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Wildtype mice (n=5 for each group, 6–8 weeks old) were treated with LPS or with LPS and increasing amounts of rhSP-D via tail vein injection. Blood was harvested 2 hours after injection and plasma cytokine levels were measured by ELISA. Top panel: IL-6 levels were lower in mice treated with SP-D (p=0.13, p=0.01, p<0.002 for 25, 75, and 150 mg/kg SP-D, respectively). Bottom panel: TNF-α levels were lower in mice treated with SP-D (p=0.14, p=0.13, p=0.006 for 25, 75, and 150 mg/kg SP-D, respectively). Error bars represent SEM.

Because the previous experiments combined LPS with rhSP-D prior to a single intravenous injection, these experiments represented optimum conditions for evaluating the effect of SP-D on LPS induced systemic inflammation. To assess the potential of systemic rhSP-D to locate and inhibit LPS circulating in the blood, rhSP-D was administered via tail vein injection 30 minutes before or after LPS injection and the cytokine response was measured in plasma 2 hours after LPS injection (Figure 4). Systemic IL-6 levels were significantly reduced when rhSP-D was administered 30 minutes before or with LPS injection. IL-6 levels were also lower when rhSP-D was administered 30 minutes after LPS, but the results did not reach statistical significance.

Figure 4.

Figure 4.

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Wildtype mice (n=5 for each group, 6–8 weeks old) were treated with LPS (5 μg/kg) via tail vein injection without rhSP-D or with rhSP-D (150 μg/kg) that was administered 30 minutes before (t=−30), with (t=0), or 30 minutes after (t=+30) the LPS dose. Blood was harvested 2 hours after the LPS dose and plasma cytokine levels were measured by ELISA. Systemic IL-6 levels were significantly reduced when rhSP-D was administered 30 minutes before (p=0.04) or with (p=0.03) LPS injection. Error bars represent SEM. IL-6 levels were also lower when rhSP-D was administered 30 minutes after LPS, but the results did not reach statistical significance (p=0.25).

3.3. SP-D-mediated inhibition of LPS induced inflammation correlates with SP-D LPS binding affinity-

To determine if SP-D inhibits LPS induced systemic inflammation through pathways that are dependent or independent of LPS binding, we compared the effect of SP-D on inflammation induced by a low and high SP-D affinity LPS serotype. Using an ELISA based SP-D LPS binding assay, the binding affinity of rhSP-D for LPS from several E.coli strains was measured and one with a high binding affinity (E.coli 0111:B4) and one with a low binding affinity (E.coli 0127:B8) were identified (Figure 5A). The effect of rhSP-D on systemic IL-6 levels 2 hours following tail vein injection of either the low or high binding LPS was measured. rhSP-D significantly decreased inflammation induced by the high binding LPS strain, but rhSP-D did not reduce plasma IL-6 levels induced by the low binding LPS strain (Figure 5B).

Figure 5.

Figure 5.

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Left Panel. An ELISA based binding assay was used to quantify rhSP-D binding to E.coli 0111:B4 or 0127:B8 LPS. rhSP-D binding to 0111:B4 LPS was significantly higher. Right Panel. E.coli 0111:B4 and 0127:B8 LPS (50,000 EU/kg) was administered via tail vein injection to wildtype mice (n=5 for each group) with (+) and without (−) rhSP-D (150 mg/kg). Blood was harvested at 2 hours and IL-6 levels were determined by ELISA. IL-6 levels in mice treated with LPS without SP-D were set to 100% and compared to mice treated with SP-D and 0111:B4 (p=0.96) or 0127:B8 (p=0.12) LPS. Error bars represent SEM.

3.4. SP-D does not increase systemic LPS clearance-

To determine if SP-D inhibits systemic LPS induced inflammation by increasing systemic LPS clearance, mice were treated with LPS via tail vein injection with or without rhSP-D and plasma LPS concentrations were measured by Limulus assay. LPS clearance rates were similar with and without rhSP-D, suggesting the SP-D does not inhibit LPS induced systemic inflammation by enhancing systemic LPS clearance (Figure 6).

Figure 6.

Figure 6.

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Wildtype mice (n=5 for each group, 6–8 weeks old) were treated with LPS (5 μg/kg) via tail vein injection without rhSP-D or with rhSP-D (150 μg/kg) and plasma LPS concentrations were measured by Limulus assay. Error bars represent SEM.

4. DISCUSSION

Sftpd−/− mice develop progressive pulmonary inflammation that is exacerbated by intratracheal challenge with infectious particles or toxicants indicating that SP-D is essential for regulating respiratory host defense cells during lung infection or injury (LeVine et al., 2004; LeVine et al., 2000; LeVine et al., 2001; Wert et al., 2000a; Wert et al., 2000b). Although SP-D is present in only low amounts outside the lung, systemic levels of SP-D increase significantly during sepsis and other forms of lung injury (Greene et al., 2002; Greene et al., 1999; Kuroki et al., 1998; Takahashi et al., 2006). This increase in systemic levels of SP-D has been hypothesized to represent a leakage of SP-D from the alveoli through the alveolar capillary barrier and into the systemic circulation (Determann et al., 2010; Eisner et al., 2003; Lomas et al., 2009; Lopez-Cano et al., 2017). Importantly, it is unknown whether circulating SP-D has a biological role or if it is merely a byproduct of alveolar injury. We hypothesized that systemic SP-D may limit systemic inflammation and help clear systemic pathogens in a manner comparable to the role of pulmonary SP-D in the pulmonary innate immune system.

Similar to prior studies evaluating pulmonary infection and injury, systemic inflammation was also increased in Sftpd−/− mice when systemic injury was induced by intraperitoneal injection of LPS. Although the magnitude of the exaggerated systemic inflammatory response in Sftpd−/− mice was less than what has been observed in models of pulmonary inflammation and injury in Sftpd−/− mice, the increase in systemic inflammation was still significant when compared to wildtype mice. The reason for the slightly subdued systemic inflammatory response in Sftpd−/− mice is unclear, but it may be due to compensatory actions of the other circulating collectins such as mannose binding protein that are not present in the lung.

Studies evaluating Sftpd−/− mice have demonstrated several abnormalities in the innate immune system in the absence of SP-D therefore the elevated systemic inflammation observed in Sftpd−/− mice maybe due to the downstream effects of Sftpd deletion rather than reflecting a direct role of SP-D in systemic inflammation (LeVine et al., 2004; LeVine et al., 2000; LeVine et al., 2001; Wert et al., 2000a; Wert et al., 2000b). To address this potential explanation, we examined the ability of purified SP-D to inhibit LPS induced systemic inflammation. We observed a consistent SP-D-mediated reduction in pro inflammatory cytokines following treatment with intravenous LPS. The maximum reduction in inflammatory cytokines that we observed with intravenous SP-D was approximately 40%. Although SP-D treatment was unable to eliminate the inflammatory response induced by LPS, the reduction in inflammation was consistent and concentration dependent. In addition, to clear infectious pathogens while also limiting inflammatory tissue injury, one would expect an immune regulatory protein to control but not completely abolish the systemic inflammatory response in the setting of systemic infection.

To maximize the consistency of our LPS+SP-D experiments, we simultaneously injected LPS with SP-D. This approach potentially created a biased scenario where the SP-D was potentially able to bind and inhibit the LPS prior to injection. Therefore, we performed a subset of experiments where we injected the SP-D either 30 minutes before or 30 minutes after the LPS. SP-D mediated inhibition of LPS was similar whether the SP-D was injected at the same time or 30 minutes prior to the LPS suggesting that SP-D is capable of locating and inhibiting LPS in the systemic circulation and that the effects seen in other experiments were not an artifact of the simultaneous injection. We observed a small reduction in inflammation in the animals that received SP-D 30 minutes after LPS injection, but this trend was not significant. LPS has a very quick mechanism of action and it is likely that the activation of inflammatory pathways had already occurred prior to the injection of SP-D 30 minutes after the LPS. Therefore, while systemic SP-D may not be able to reverse the effects of LPS that are initiated before systemic levels of SP-D reach physiologically relevant concentrations, these results would suggest that during an ongoing infection, once SP-D is present in blood it can locate LPS and block subsequent LPS induced inflammation.

LPS activates immune cells via CD14 and Toll-like receptor (TLR) signaling pathways that stimulate the release of TNFα IL-1, IL-6, IL-8, IL-12, and several other inflammatory mediators (Chow et al., 1999; Diks et al., 2004; Freudenberg and Galanos, 1990; Raetz et al., 1991). These mediators recruit and activate surrounding host defense cells causing a cascade of inflammation, which if left unchecked, can damage the surrounding tissue. In vitro studies suggest that SP-D may influence several steps in LPS signaling pathways including direct LPS binding, CD14 inhibition, and TLR4 binding (Bufler et al., 2003; Kuroki et al., 2007; Liu et al., 2010; Nie et al., 2008; Ohya et al., 2006; Sano et al., 2000; Senft et al., 2005; Yamazoe et al., 2008). Although SP-D has been implicated in several immune modulatory pathways, unfortunately the precise mechanism of action of SP-D in these pathways is not well understood. SP-D also has a high affinity for the core oligosaccharides of LPS, but the relative affinity varies depending on the strain of bacterial LPS utilized. In contrast, SP-D binding of CD14 and TLR4 occurs independently of SP-D LPS interactions. Our results suggest that with our model of inflammation, inhibition of systemic LPS requires LPS binding by SP-D and is not mediated through receptor mediated interactions that occur independent of LPS binding.

SP-D is known to enhance the clearance of several microbial pathogens from the lung (Hartshorn, 2010; LeVine et al., 2004; LeVine et al., 2000; LeVine et al., 2001). In addition, SP-D binds aerosolized LPS immediately as it enters the lung and SP-D bound to LPS is cleared from the lung 1.7-fold faster than SP-D alone (Ikegami et al., 2007; van Rozendaal et al., 1999; Van Rozendaal et al., 1997). Since our results suggest that SP-D mediated inhibition of systemic inflammation requires LPS binding we hypothesize that SP-D may inhibit LPS induced inflammation by enhancing LPS clearance. However, when we examined the effect of SP-D on LPS clearance we found that the half-life of systemic LPS was similar with or without SP-D suggesting that SP-D does not inhibit LPS induced inflammation by accelerating its clearance from the systemic circulation.

Several studies have evaluated SP-D outside of the lung. A few groups have examined the function of SP-D in extrapulmonary tissues using cell culture and/or animal models in disease models of the eye, pancreas, kidney, intestines, and large arteries (Du et al., 2016; Heimer et al., 2013; Hu et al., 2016; Khamri et al., 2007; Liu et al., 2015a; Liu et al., 2015b; Mun et al., 2009; Murray et al., 2002; Oberley et al., 2004; Salminen et al., 2012; Zhang et al., 2015). Work by Sorensen et al evaluated the association between circulating SP-D and atherosclerosis and found that intravenous treatment with a fragment of human SP-D resulted in a reduction in HDL cholesterol, LDL cholesterol, and total cholesterol (Sorensen et al., 2006). Several other studies have examined circulating SP-D levels following various forms of lung injury but mostly limited their evaluation to the potential of circulating SP-D as a marker of lung injury (Greene et al., 2002; Greene et al., 1999; Kuroki et al., 1998; Takahashi et al., 2006; Wu et al., 2009).

5. CONCLUSIONS

Our data suggests that if full size SP-D is present in the circulatory system, SP-D regulates systemic host defense cells and decreases systemic inflammation. Although further studies are needed to determine the precise systemic inflammatory pathways regulated by SP-D, our results raise the possibility that a physiological purpose of increasing systemic SP-D levels during infection is to utilize the large pulmonary supply of SP-D to scavenge systemic infectious pathogens and limit inflammation-induced tissue injury. These results also suggest that patient outcomes may be improved by supplementing with exogenous intravenous SP-D during diseases such as bacterial, viral or fungal infections or other diseases characterized by excessive systemic inflammation.

Acknowledgements

This work was supported by National Institutes of Health grant HL089505 (PSK)

Abbreviations:

SP-D

surfactant protein D

LPS

lipopolysaccharide

TNF

tumor necrosis factor

IL

interleukin

rhSP-D

recombinant human Surfactant Protein D

TLR

toll like receptor

ELISA

enzyme-linked immunosorbent assay

Sftpd

surfactant protein d gene

Footnotes

Declaration of Interests:

Paul Kingma was a consultant from 2011–21 for Airway Therapeutics Inc. which is developing SP-D as a human therapeutic agent. Paul Kingma has no current relationship with Airway Therapeutics Inc.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics Statement

The work described has been carried out in accordance with http://www.wma.net/en/30publications/10policies/b3/index.html; EU Directive 2010/63/EU for all animal experiments. Animals were handled in accordance with approved protocols through the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Medical Center.

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