Post-burn Lung Inflammation Is Associated With Induction of Pulmonary Cathelicidin-related Antimicrobial Peptide and S100a8 in Mice

Abstract

Burn trauma triggers dysregulated systemic inflammation, leading to multiorgan dysfunction. Respiratory failure often follows burn injury, resulting in morbidity and mortality, in part, because of excessive and prolonged release of local and systemic pro-inflammatory mediators. One class of important mediators of inflammation at mucosal surfaces are antimicrobial peptides (AMPs), and their expression is notably altered in inflammation. We sought to determine whether pulmonary AMPs are induced in inflammatory lung after burn. C57BL/6 male mice were given a 12%–15% full-thickness total body surface area dorsal scald burn or sham injury. Survival rate and pulmonary function of the mice were assessed at 24 h. Histopathological examination and quantification of pro-inflammatory mediators, IL-6 and CXCL1, in the lungs at 24 h after burn were performed. mRNA expression of a subset of prominent lung AMPs in whole lung, alveolar macrophages (AMs), and primary lung epithelial cells were measured. Our data showed decreased survival and impaired respiratory function after burn injury. Moreover, hematoxylin and eosin-stained lung sections of burned mice showed pulmonary edema and congestion, and pulmonary IL-6 and CXCL1 were elevated. AMP analysis revealed that burn triggered a dramatic rise in lung Camp and S100a8 above that of sham mice. To our surprise, lung epithelial cells, and not AMs, were the cellular source of burn-induced Camp and S100a8 in this murine model of burn injury. Taken together, these data reveal for the first time that lung inflammation post-burn involves a rise in AMPs, Camp and S100a8, from lung epithelial cells.

INTRODUCTION

Burn trauma remains a critical healthcare challenge in the United States with more than 29 000 people admitted to the hospital annually.¹ While more patients are surviving due to advances in acute medical care, pulmonary complications present a major obstacle to recovery from burn injury, even in the absence of inhalation injury.^1,2

Burn-related pulmonary complications, such as acute respiratory distress syndrome (ARDS) and pneumonia, can result in respiratory failure.^1,3 Clinical and experimental studies from our lab and others have shown that burn injury leads to pulmonary inflammation,^4–10 characterized by extracellular fluid accumulation in lung interstitium and inflammatory cell infiltration, features associated with ARDS.⁹ This post-burn pulmonary inflammation leads to rigid lungs, shallow breathing, and hypoxemia, contributing to morbidity and mortality after burn.^9,11

Post-burn lung inflammation also involves activation of the innate immune system, which includes macrophages, neutrophils, and lung epithelial cells.^3,12 Mediators produced by these cells, such as interleukin-6 (IL-6) and chemokine (C-X-C motif) ligand 1 (CXCL1), further activate and recruit immune cells, amplifying inflammation. Antimicrobial peptides (AMPs) are important mediators of inflammation at mucosal surfaces. Studies have reported that pulmonary inflammation can upregulate lung AMPs, cathelicidins, defensins, and calprotectin (a complex of subunits S100A8 and S100A9).^13–17 Cathelicidins are a family of cationic endogenous AMPs, which can be synthesized and stored in neutrophil granules,^18,19 and are also induced in macrophages and lung epithelial cells.^13,20 The only known human cathelicidin is called human cationic antimicrobial peptide 18 (hCAP-18),²¹ and its mature form is called LL-37.²¹ The murine ortholog of LL-37 is cathelicidin-related antimicrobial peptide (CRAMP), which is encoded by the gene Camp. Defensins are another family of endogenous cationic AMPs, categorized into α-and β-defensins.¹⁴ α-defensin 1(Defa1)^22,23 and β-defensins 1 through 3 (Defb1, Defb2, Defb3) have been implicated in lung inflammation.^24–26 These defensins can originate from granules of neutrophils or expressed by monocytes, macrophages, or epithelial cells of respiratory tract.^27,28 S100A8, S100A9, and calprotectin, a heterodimer protein of S100A8 and S100A9, are danger-associated molecular patterns (DAMPs) that can act as AMPs in lungs.^17,29–33 Similar to cathelicidins, S100A8 and S100A9 can be produced by macrophages, neutrophils, and bronchial and alveolar epithelial cells.¹⁷ While initially discovered for their direct antimicrobial activities against infectious organisms,¹⁵ studies have shown that AMPs can have other pro-inflammatory effects, such as leukocyte recruitment and stimulating chemokine and cytokine secretion from activated cells, such as neutrophils, macrophages, and epithelial cells.^14,15,30,34

It is currently unknown whether AMP expression is induced in the lung after remote burn trauma in young mice. Herein, we report upregulation of pulmonary AMPs, Camp and S100a8, and this parallels impaired respiratory function, heightened pulmonary inflammatory histopathological changes (ie, tissue cellularity, alveolar wall thickness, alveolar congestion), and increased lung inflammatory mediators, IL-6 and CXCL1, at 24 h after burn injury. Moreover, our data revealed that lung epithelial cells, not alveolar macrophages (AMs), were responsible for burn-induced pulmonary expression of Camp and S100a8 at 24 h in mice.

METHODS

Animals

Male C57BL/6 mice (Jackson Laboratories) were housed at the University of Colorado Anschutz Medical Campus under specific pathogen-free conditions for 2 weeks prior to studies. Animals were between 10 and 12 weeks old and weighed between 27 and 32 g at the time of the experiment. All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus (protocol number 00087). Animals were housed in a temperature- and humidity-controlled room with a 14-h light cycle and 10-h dark cycle.

Murine model of dorsal scald burn injury

We used our well-established clinically relevant murine model of burn injury,³⁵ with modification.^36–38 Mice were anesthetized (25 mg/kg of ketamine and 2.5 mg/kg of xylazine with constant isoflurane) (Webster Veterinary) and their dorsa was shaved. Anesthetized mice were shaved and subjected to a 12%–15% total body surface area scald burn injury by using a template and exposing the shaved dorsum to a 92 °C to 93 °C water bath for 8 s, resulting in a full-thickness skin injury.³⁹ Sham-injured animals were treated comparably and exposed to room-temperature water. Both sham and burn-injured mice received fluid resuscitation (1 mL of saline intraperitoneally, i.p.) immediately post-burn in addition to pain medication (1.0 mg/kg buprenorphine SR-LAB; Zoo Pharm).^38,40 Additional saline was given i.p. at 10 μL/g of body weight at 8 h post-burn. Experiments were performed between the hours of 8 and 10 am to minimize confounding effects of circadian variation in corticosterone and other hormones, which can influence inflammatory and immune responses. Surviving mice were euthanized at 24 h post-injury.

Survival analysis

Data are presented as percent survival of mice from sham and burn groups at 24 h post-injury, which was combined from 2 independent experiments, with n = 13 for sham and n = 22 for burn groups.

Whole body plethysmography

Pulmonary function was assessed in conscious mice at 24 h post-injury using unrestrained whole-body barometric plethysmography (Buxco Research Systems), as previously described.³⁶ Briefly, mice were acclimated to the environment in the chamber for 5 min before lung function parameters were recorded for 10 minutes on a breath-by-breath basis by the manufacturer’s software (Buxco FinePointe). Enhanced pause (Penh), breath frequency (f), tidal volume (TVb), and minute volume (MVb) were analyzed. Mean values for each parameter per mouse were used for analysis.

Histopathology of lungs

The left lung was inflated with 10% formalin and fixed overnight, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E), as previously described.^6,36 Lung sections were evaluated using a BX46 light microscope and CellSens software (Olympus), and histology images were taken in the peripheral lung, in a nonbiased fashion, at 400× magnification. The pathologist was blinded to the treatment groups while reviewing the lung sections. Inflammation in the images was examined by assessing for pathological changes, such as alveolar wall thickening, exudates in alveolar spaces, and pulmonary congestion.^36,37 These histopathological changes were quantified, also in a blinded manner, using ImageJ, as previously described by our laboratory.³⁷ Ten high-power field (400×) images of peripheral lung were taken per animal in a nonbiased fashion. Images were converted into binary to differentiate lung tissue from airspace and then analyzed for percent area covered by lung tissue in each field of view as described previously.^36,37

Alveolar macrophage isolation

Mouse primary AMs were isolated by bronchoalveolar lavage (BAL), as previously described.⁴¹ Lungs were flushed 10 times with 0.8 mL cold PBS, and lavage cells were then examined by flow cytometric analysis for assessment of purity as described by our laboratory.⁴² Cell count and viability were assessed by trypan blue exclusion.⁴¹ Directly conjugated antibodies against the following surface molecules were used: CD11b APC (Clone M1/70; BD BioSciences), Ly6G APC-Cy7(Clone 1A8; BD BioSciences), F480 PerCP-Cy5-5 (Clone BM8; BD BioSciences), and SiglecF BV421 (Clone E50-2440; BD BioSciences). Multiparameter flow cytometry was performed using an LSR-II instrument (BD Biosciences) compensated with single fluorochromes and analyzed using FacsDiva (BD Biosciences) and FlowJo software.

Lung epithelial cell isolation

Lung lobes were dissociated into a single cell suspension using the Miltenyi Lung Dissociation Kit (Cat. #:130-095-927; Miltenyi Biotec, Inc.), as described by our laboratory.⁴¹ Cell count and viability were assessed by trypan blue exclusion.⁴¹ Primary lung epithelial cells were isolated from the single-cell suspension via magnetic column separation, as previously described.⁴³ Cells were stained with anti-CD45 microbead (Cat. #: 130-052-301; Miltenyi Biotec, Inc.) and anti-Epithelial Cell Adhesion Marker (EpCAM) microbead (Cat. #: 130-105-958; Miltenyi Biotec, Inc.) and then separated by LS MACS column (Cat. #: 130-042-401 Miltenyi Biotec, Inc.) on a MACS separator magnet (Cat. #: 130-091-051; Miltenyi Biotec, Inc.). Lung epithelial cells were characterized as CD45 negative (CD45⁻) and EpCAM positive (EpCAM⁺), since EpCAM is not expressed on leukocytes.⁴⁴ Column isolated CD45⁻ EpCAM⁺ mouse lung epithelial cells were then examined by flow cytometric analysis for assessment of purity using the following antibodies: CD45 BV510 (Clone 30-F11; BD Biosciences) and EpCAM eFluor450 (Clone G8.8; Thermo Fisher Scientific).

Cytokine/chemokine levels in the lung

Flash frozen lung tissue was homogenized and ELISA’s for IL-6 and CXCL1 were performed as previously described.^36,45 The results were normalized to total protein, quantified by Pierce BCA Protein Assay (Cat. #: 23225; ThermoFisher).

Quantitative RT-PCR

For mRNA expression, lung tissue or isolated cells were homogenized in Qiagen RLT buffer (Cat. #: 74106; Qiagen Inc., Valencia, CA), as previously described.⁶ Quantitative RT-PCR was run with primers for Camp (Mm00438285_m1, ThermoFisher), S100a8 (Mm00496696_g1, ThermoFisher), Defa1 (Mm02524428_g1, ThermoFisher), Defb1 (Mm0043 2803_m1, ThermoFisher), Defb2 (Mm00657074_m1, ThermoFisher), or Defb3 (Mm04214158_sThermoFisher), and Gapdh as endogenous control (4352339E, ThermoFisher). Ct values from each sample were measured by QuantStudio 3 Real-Time PCR System (Applied Biosystems). Relative expression was calculated using 2^−ΔCt algorithm, previously described.^38,46

Western blot analysis

Protein levels of CRAMP were measured in whole lung tissue by western blot analysis, as previously described.⁴⁷ Briefly, western blots were probed with either CRAMP antibody (cat# 12009-1-AP, ThermoFisher) at 1:200 dilution or β-actin primary antibody (Cat# 4970L, ThermoFisher) at 1:1000 dilution for 1.5 h at room temperature, followed by secondary immunoblotting with antirabbit IgG (Cat. #: 7074, Cell Signaling) for 1 h, and Clarity Western chemilum-inescence solution (Cat. #: 1705061, BioRad, Hercules) was added for color development. ImageLab (BioRad, Hercules) was used to quantify band intensity, and densitometry was performed using β-actin normalization.

Statistical analysis

Statistical comparisons between sham and burn injury groups were made by unpaired 2-tailed Student’s t-test for parametric data and Mann–Whitney U-test for nonparametric data. One-way ANOVA with Tukey’s post hoc test was used to compare Camp and S100a8 expression in whole lung tissue and AMs from sham and burn groups. Significant differences are reported when P < .05. Data are reported as mean values ± the standard error of the mean (SEM). For survival assessment, comparison between the 2 groups was made by log-rank test from Kaplan–Meir survival analysis. Each data set is representative of 2 independent experiments with n = 4–5 for the sham group and 4–12 for the burn group. Data were analyzed with Graph Pad Prism software for macOS version 10.4.0 (GraphPad Software).

RESULTS

Burn injury, survival, and lung function in mice

First, we determined the percent survival of mice at 24 h post-injury. There were no deaths in the sham group (n = 13/13) and 73% survived at 24 h in the burn group (n = 16/22), combined from 2 experiments (P < .05). In surviving mice, lung function was assessed 1 h prior to euthanasia at 24 h post-injury.^36,38 Measurements included breath frequency, tidal volume, minute volume and enhanced pause (Penh),^36,38,48 an indicator for control of breathing (Figure 1). Breath frequency of sham-injured mice was 330 ± 9.0 breaths/minute, while that of burned mice was reduced by 68% (Figure 1A, P < .05). Tidal volume in sham mice was 0.37 ± 0.05 mL and was comparably decreased after burn injury by 63% (Figure 1B, P < .05). Additionally, when compared to sham mice, minute volume in burn injury was reduced by 87% (Figure 1C, P < .05). Lastly, we evaluated Penh and found that sham mice had average Penh of 0.84 ± 0.05 and burn injury raised this value to 11 ± 2.0 (Figure 1D, P < .05). Together, these data demonstrate that relative to sham-injured mice, at 24 h post-burn, injured mice display abnormal breathing patterns, indicated by dampened breath frequency, tidal volume, and minute volume and elevated Penh, contributing to overall respiratory dysfunction.

Figure 1.

Lung Function After Burn Injury. Twenty-four hour post-injury the following lung parameters were measured by whole-body plethysmography: (A) breath frequency; (B) tidal volume; (C) minute volume; (D) Penh or enhanced pause. Data are reported as mean values of each parameter per mouse ± SEM. *P < .05 compared to sham group by Student’s t-test. n =5 (sham group) and 10 (burn group) per experiment. Representative of 2 independent experiments in which similar results were obtained.

Burn induces inflammatory lung histopathology

Consistent with our prior publications,^6,37,45 we observed that mice who sustained burn injury had pulmonary inflammation. Sham mice had minimal to no lung inflammation at 24 h after injury (Figure 2A and B) and, in contrast, the lungs of burn-injured mice had increased alveolar wall thickening, exudate in alveolar space, and pulmonary congestion (Figure 2C and D). To quantify these changes, ImageJ was used,³⁷ and we found that compared to sham, burned mice had a 1.4-fold increase in percent tissue area, which corresponds to reduced alveolar airspace (Figure 2E, P < .05). Our laboratory has previously reported that in inflation-fixed lungs, an increase in alveolar wall thickening and exudate in alveolar space correlate with reduced alveolar airspace and greater pulmonary congestion.^6,36,37,45 Therefore, these data confirm our earlier findings that burn trauma results in inflammatory lung histopathology.⁶

Figure 2.

Pulmonary Inflammation After Burn Trauma. (A and C) Lung sections from sham and burn groups were stained with hematoxylin and eosin (H&E) and examined for histopathological changes 24 h post-injury. Representative micrographs are shown from lung sections from 2 independent experiments. (B and D) Enlarged insets from representative high-power fields are shown. All images are at 400× optical magnification. (E) Image J software was used to assess pulmonary congestion in 10 high-power fields per mouse and reported as mean percentage of tissue area covered by lung tissue in 10 field of views ± SEM. *P < .05 compared to sham by Student’s t-test. n = 9 from sham group and 12 from burn group, combined from 2 independent experiments.

Heightened pulmonary IL-6 and CXCL1 after burn

Similar to our previous studies,^6,37 burn injury raised levels of inflammatory mediators, IL-6 and CXCL1, in the lung, compared to sham-injured animals. Sham-injured mice had IL-6 levels of 10.2 ± 0.88 pg/mg of total protein and, after burn, there was a 2.2-fold increase in pulmonary IL-6 (Figure 3A, P < .05). Similarly, sham-injured mice had CXCL1 levels that were 9.7 ± 1.6 pg/mg of total protein, and this was raised by 11.7 fold at 24 h post-injury (Figure 3B, P < .05). Together, these data demonstrate that burn injury results in lung inflammation at 24 h in mice.

Figure 3.

Increased Pro-Inflammatory Mediators, IL-6 and CXCL1, After Burn Injury. Lung tissue was collected from sham and burn injured and levels of (A) IL-6 and (B) CXCL1 were measured by ELISA. Data are shown as mean pg/mg of total protein ± SEM. *P < .05 compared to sham by Student’s t-test. n = 4 (sham group) and 5 (burn group) per experiment. Representative of 2 independent experiments in which similar results were obtained.

Burn injury induces lung Camp and S100a8

Studies have shown that burn injury leads to immune dysregulation,^49–56 and inflammatory lung diseases can increase pulmonary AMPs.^15,16,57,58 Therefore, we sought to determine whether remote burn trauma-induced expression of lung AMPs at 24 h post-burn. There was minimal expression of Camp mRNA in whole lung tissue from sham mice (Table 1) and a remarkable 550-fold elevation in Camp at 24 h after burn injury (P < .05). There was also a corresponding induction in CRAMP protein level in lungs from burned mice, as compared with whole lung from sham mice (Figure 4, P < .05). Similarly, burn trauma also induced expression of S100a8 (Table 1). There was a low mRNA level of S100a8 from sham mice; however, burn injury increased lung expression by 10-fold (P < .05). While there was some expression of Defa1 in lungs of sham and burn-injured mice, there was no significant difference between the groups (Table 1). Moreover, Defb1, Defb2, and Defb3 were not detectable, regardless of injury (Table 1). Together, these data show that burn trauma leads to an increased AMP response in the lung by inducing CRAMP and S100a8 in mice.

Table 1.

Pulmonary AMP Expression from Sham and Burn-Injured Mice at 24 h

	Sham	Burn	P-value
Camp	0.0003 ± 0.0002	0.05 ± 0.009	.001
S100a8	0.3 ± 0.1	2 ± 0.2	.0009
Defa1	0.02 ± 0.007	0.03 ± 0.005	.3
Defb1	N.D.	N.D.	N/A
Defb2	N.D.	N.D.	N/A
Defb3	N.D.	N.D.	N/A

mRNA levels were measured by quantitative RT-PCR in whole lung tissue. The mean ± SEM of 2^(−ΔCT) between target gene and internal control (Gapdh) are listed. The p-values were calculated using Student’s t-test. n = 3–4 (sham group) and 5–10 (burn group) per experiment. Representative of 2 experiments. Abbreviations: N/A, not applicable; N.D., not detectable.

Figure 4.

Pulmonary CRAMP Protein After Sham and Burn Injury in Mice. Whole lung tissue was collected from mice 24 h post-injury. (A) Representative western blot of CRAMP protein in lungs from sham (n = 3) and burn (n = 3) and (B) Quantitation of CRAMP protein band density relative to β-actin protein. Data are shown as relative density ± SEM. *P < .05 compared to sham by Student’s t-test. n = 6 (sham group) and 7 (burn group), combined from 2 independent experiments.

Cellular source of lung Camp and S100a8 after burn injury

To begin to explore possible cellular sources of burn-induced pulmonary Camp and S100a8, we first examined expression of Camp and S100a8 mRNA in isolated primary AMs. Using BAL, we obtained AMs, which were >95% of all recovered BAL cells (Figure 5A), regardless of injury, which is consistent with prior publications from our laboratory.⁴¹ To our surprise, AMs from both sham and burn-injured mice failed to express Camp (Figure 5B). While there was low expression of S100a8 in AMs from sham and burn mice, there were no difference in expression between the groups (Figure 5C). Next, we isolated primary lung epithelial cells, which were CD45⁻ EpCAM⁺ with 90% purity (Figure 6A). In contrast to AMs, we found Camp expression in lung epithelial cells from burn-injured mice and observed that expression of Camp mRNA was significantly greater (an impressive 550-fold elevation) in primary lung epithelial cells from burn-injured mice relative to sham-injured animals (Figure 6B, P < .05). Similarly, S100a8 expression was found to be 204-fold greater in pulmonary epithelial cells from burn-injured mice compared to sham mice (Figure 6C, P < .05). These data show that lung epithelial cells, and not AMs, are the cellular source of burn-induced lung Camp and S100a8.

Figure 5.

Whole Lung and AMs Expression of Camp or S100a8 24 h After Burn Injury. (A) Representative gating strategies of Ly6g⁻ CD11b⁺ SiglecF⁺ F4/80⁺ AMs from BAL. (B and C) Camp or S100a8 mRNA expression in whole lung tissue and AMs from sham and burn groups. n = 4 (sham group) and 4–6 (burn group) per experiment. *P < .05 compared to all other groups by One-way ANOVA with Tukey’s post hoc test. Representative of 2 independent experiments in which similar results were obtained. Abbreviation: n.d., not detected.

Figure 6.

Lung Epithelial Cell Expression of Camp and S100a8 After Burn Injury. (A) Representative gating strategies of isolated CD45⁻ EpCAM⁺ lung epithelial cells. (B and C) Camp and S100a8 mRNA expression in lung epithelial cells. n = 6 (sham group) and 8–9 (burn group). *P < .05 compared to sham by Mann–Whitney U-test. Data are combined from 2 independent experiments.

DISCUSSION

The post-burn inflammatory responses are excessive, involving multiple major organs,^{49–56,59,60} and lungs are amongst the first organs to fail following injury.¹ Burn patients often develops ARDS,⁹ characterized by rapid decline in pulmonary function as a result of increased capillary permeability, lung infiltration by inflammatory cells, and hypoxia.⁶¹ Consistent with our prior studies,³⁶ at 24 h post-injury, in our murine burn model we observed impaired lung function after burn, demonstrated by decreased breathing rates and increased Penh. We chose to characterize the breathing patterns in mice using a non-invasive, unrestrained whole-body plethysmography as we were able to assess lung function without anesthesia or surgical intervention. Thus, this method allowed us to measure effects of the burn injury on respiratory parameters without additional stress. Other tools for characterizing lung function, including lung resistance, requires anesthesia, surgical intervention, and mechanical ventilation. While there is some evidence that lung resistance more accurately reflects pulmonary function,⁶² the invasive nature of the procedure and mechanical ventilation increases BAL cell numbers.⁶³ In noninvasive plethosmography, Penh is a better indicator of control of breathing rather than lung resistance. While there are some studies that claim that Penh is not a reliable measure of respiratory mechanics,^62,64 others have suggested that it correlates with airway resistance and highlights changes in overall lung function in murine models of pulmonary inflammation.^63,65,66

It is thought that the underlying mechanism behind post-burn pulmonary failure involves dysregulated immune responses.⁶⁷ Following burn, a profound amount of pro-inflammatory mediators enter the blood and circulate to the lung’s vast vascular bed, inciting tissue damage and edema formation and ultimately compromising normal function.^3,67 Our lab and others have also shown that burn injury increases intestinal permeability in mice, leading to heightened intestinal bacterial translocation outside of the gut.^6,68 This bacterial translocation induces IL-6 in the liver, which can then contribute to increased pulmonary inflammation.⁶ A majority of burn patients develop pulmonary edema and inflammatory infiltrates, which are histopathological findings of ARDS.³ Previously, our lab has reported an increase in alveolar wall thickness in burned mice in a study that showed that antecedent ethanol exposure in mice worsens post-burn pulmonary histopathological changes.^6,36,37,48 Similar to our previous reports,^6,36,37,48 the present study also showed that at 24 h post-injury, burned mice exhibited an increased alveolar wall thickness and heightened lung tissue cellularity, correlating with greater pulmonary edema and congestion, compared to sham-injured mice. Importantly, these inflammatory histopathological changes in the lung parallel derangements in breathing patterns, suggesting that the burn-induced pulmonary inflammatory response may contribute to respiratory dysfunction.

In addition to examining histopathology, we also evaluated pro-inflammatory mediators in the lung after burn. Recently, we have reported that burn-injured mice have increased pulmonary levels of CXCL1, which is further elevated after either a single or episodic binge of ethanol prior to burn injury.⁶⁹ Consistent with our previous findings,⁶⁹ we also report herein that protein levels of CXCL1 were heightened after burn (Figure 3). CXCL1 is a chemoattractant that recruits several types of inflammatory cells, including neutrophils, to sites of inflammation.⁷⁰ Unlike previous studies, we did not observe a large increase in neutrophils in the lung sections at 24 h after injury (data not shown). A possible explanation for this would be that the neutrophils have not migrated into the lung tissues yet, and therefore, future studies at later time points are warranted.

Elevated levels of circulating IL-6 correlate with heightened mortality risk in trauma and high levels of IL-6 are associated with high mortality in burn patients.⁷¹ Prior studies have shown that burn injury increases lung IL-6,^6,69,72 which was confirmed in the present study (Figure 2). A couple of studies have shown that IL-6 is involved in pathogenesis of pulmonary inflammation, congestion, and tissue injury.^73,74 A part of this pathogenesis may involve CXCL1 expression as one study demonstrated that IL-6 can induce CXLC1 expression in the lung.⁷⁵ It is worth noting that a role of IL-6 in pulmonary inflammation post-trauma appears to dependent on the etiology of the injury. In hypoxia-induced acute lung injury, IL-6 was shown to be protective as mice overexpressing IL-6 had less mortality and tissue injury in the lungs.⁷⁶ Moreover, in IL-6 knockout mice, diminished acute phase response was observed following injury by lipopolysaccharide or infection by Listeria monocytogenes.⁷⁷ However, in immune complex-mediated vascular injury, IL-6 knockout mice had similar pulmonary inflammation, relative to wild-type mice.⁷⁸ In this present study, heightened pulmonary IL-6 coincided with increased lung inflammation and respiratory dysfunction, suggesting that IL-6 plays an essential role in burn-induced inflammatory response.

Our study reports for the first time that burn injury elevated lung levels of Camp mRNA expression and cathelicidin CRAMP protein levels. Interestingly, the increase in lung CRAMP at 24 h post-burn in our murine model parallels pulmonary inflammation. Our data suggest that the inflammatory mileu in the lung may be upregulating pulmonary CRAMP in mice as part of the post-burn inflammatory response. Infection, injury, and inflammation lead to induction of cathelicidin expression in organs such as skin, colon, and lung.^15,79,80 As an AMP, cathelicidins serve as an antibiotic at epithelial surfaces, providing protection against bacteria, fungi, and viruses.^81–85 Much of what we know about cathelicidin is from work that has been done to elucidate their protective role in skin infections (reviewed in^86–88). In the lung, there are studies that suggest that LL-37 or CRAMP augments host defense against bacterial infections such as Pseudomonas aeruginosa and Klebsiella Pneumonia.^89–91 These reports reveal Pseudomonas aeruginosa, a common pathogen seen in burn patients, can induce lung CRAMP.^89,90 In addition, they show that CRAMP knockout mice have reduced survival and impaired pulmonary bacterial clearance when infected with Pseudomonas aeruginosa in the respiratory tract.^89,90 A couple of studies reported that increased sputum levels of LL-37 were negatively correlated with lung function in chronic obstructive pulmonary disease patients.^92,93 In our study, we observed decreased lung function in parallel with heightened CRAMP induction, suggesting that CRAMP level could be useful as a biomarker of lung function in trauma patients. However, one study showed that low plasma cathelicidin expression is associated with reduced lung function in current and former smokers.⁹⁴ Therefore, the etiology of inflammation must be considered in using CRAMP as a biomarker.

In addition to their bactericidal function, evidence from literature suggests that cathelicidins can also act as an inflammatory mediator as they have chemotactic properties.^34,80 LL-37 or CRAMP can attract neutrophils and monocytes both in vitro and in vivo.^{27,89,95–97} Moreover, they can induce chemokine release by immune and epithelial cells,⁹⁸ enhancing leukocyte migration.^99,100 LL-37 can also prevent neutrophil apotosis,¹⁰¹ induce apoptosis of injured airway epithelial cells,^102,103 and show cytotoxicity towards human peripheral blood leukocytes.¹⁰⁴ This evidence demonstrates that cathelicidins not only promote pro-inflammatory activities but also can play a protective role at inflammatory sites by promoting death of damaged cells.

To our knowledge, this study is the first to show that primary lung epithelial cells are the major cellular source of lung Camp post-burn. Reports have shown that Camp can be induced in multiple cell types, including bronchial epithelial cells, alveolar epithelial cells, macrophages, and neutroph ils.^21,90,105 However, in our study, we only found expression in lung epithelial cells, not in AMs. We cannot exclude the possibility that other types of lung macrophages (eg, interstitial macrophage) can express Camp. In addition, this study failed to detect a large influx of neutrophils at 24 h post-burn (data not shown), which further suggests that cells expressing Camp were being activated locally rather than recruited.

S100A8 is a Ca²⁺-binding protein that belongs to the S100 family. It exists in the form of a noncovalent heterodimer with S100A9, called calprotectin (SA100A8/S100A9). These proteins are widely regarded as both AMPs and DAMPs.^17,106 They are upregulated during infections,¹⁰⁷ inflammation¹⁰⁸ and are released as a result of tissue injury.^109,110 Calprotectin has antimicrobial functions, as it can interact directly with Pseudomonas aeruginosa and Staphylococcus aureus¹¹¹ or limit growth of these bacteria by chelating nutrient metals, such as zinc and manganese.¹¹¹ In addition to their antimicrobial activities, S100A8, S100A9, or calprotectin can induce pro-inflammatory cytokines and chemokines from activated immune cells, epithelial, and endothelial cells, forming a positive feedback response.¹¹² Like cathelicidins, they have chemotactic activity and can stimulate macrophage phagocytosis.²⁹ One study showed that blocking S100A8, s100A9, and calprotectin with neutralizing antibodies diminished neutrophil migration into the alveolar space in mice with Streptococcus pneumoniae infection.³⁰ Moreover, levels of S100 molecules have been correlated with severity in inflammatory lung disease¹¹³ and can serve as a candidate biomarker for therapeutic response in inflammatory diseases.¹¹⁴ Future studies exploring plasma level of S100A8, S100A9, or calprotectin to determine mortality and respiratory function would be of interest.

S100A8, S100A9, or calprotectin are primarily produced by neutrophils¹⁷ and can also be made by activated macrophages and lung epithelial cells.¹⁰⁶ While AMs from sham and burn mice expressed low levels of S100a8, there was no significant difference between cells from the 2 groups. However, as with cathelicidin, we cannot rule out whether other types of pulmonary macrophages make additional S100a8 post-burn. It is possible that infiltrating neutrophils could also bring stored calprotectin protein. However, we failed to see a large influx of neutrophils into the lung at 24 h post-burn (data not shown), suggesting that S100a8 is expressed by activated epithelial cells in inflamed lung rather than being recruited.

This study also examined expression of Defa1, Defb1, Defb2, and Defb3 in whole lung tissue after sham and burn injury at 24 h. This set of AMPs was chosen because reports have shown upregulation of their expression in the lung during inflammation.^24–26 Our results show no differences in Defa1 expression between sham and burn-injured mice and no detectable expression in Defb1 through 3, regardless of injury. One possible explanations for these findings could be that neutrophils are a major source of defensins in humans but not in mice.¹¹⁵ Additionally, these AMPs are expressed at a very low level from airway epithelia¹¹⁶ and possibly take more than 24 h post-injury to become detectable. Therefore, future investigations could include whether they are upregulated beyond 24 h in the lung after burn.

In summary, we report that, in a murine model of scald burn injury, burn leads to respiratory dysfunction and pulmonary inflammation, which was observed in parallel with induction of the AMPs Camp (and CRAMP) and S100a8 from lung epithelial cells. Together, these data suggest that pulmonary inflammation post-burn involves an AMP response from lung epithelial cells. Future investigations into the signaling pathway by which these AMPs are upregulated in lungs after burn will pave the way for a therapeutic treatment to prevent bacterial infections of the respiratory tract in patients with burn injury.