key: cord-0261112-h72la3s5
authors: Laggner, M.; Lingitz, M.-T.; Copic, D.; Direder, M.; Klas, K.; Bormann, D.; Gugerell, A.; Moser, B.; Radtke, C.; Hacker, S.; Mildner, M.; Ankersmit, H. J.; Haider, T.
title: Severity of thermal burn injury is associated with systemic neutrophil activation
date: 2021-10-07
journal: nan
DOI: 10.1101/2021.10.07.21264679
sha: a70694714137f0f1b6cae20acda74df798d5f7fc
doc_id: 261112
cord_uid: h72la3s5

Objectives Burn injuries elicit a unique and dynamic stress response which can lead to burn injury progression. Though neutrophils represent crucial players in the burn-induced immunological events, the dynamic secretion pattern and systemic levels of neutrophil-derived factors have not been investigated in detail so far. Methods Serum levels of neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and complement factor C3a were quantified in burn victims over 4 weeks post injury. Furthermore, the potential association with mortality, degree of burn injury, and inhalation trauma was evaluated. In addition, leukocyte, platelet, neutrophil, and lymphocyte counts were assessed. Lastly, we analyzed the association of neutrophil-derived factors with clinical severity scoring systems. Results Serum levels of NE, MPO, CitH3, and C3a were remarkably elevated in burn victims compared to healthy controls. Leukocyte and neutrophil counts were significantly increased on admission day and day 1, while relative lymphocytes were decreased in the first 7 days post burn trauma. Though neutrophil-derived factors did not predict mortality, patients suffering from 3rd degree burn injuries displayed increased CitH3 and NE levels. Accordingly, CitH3 and NE were elevated in cases with higher abbreviated burn severity indices (ABSI). Conclusions Taken together, our data suggest a role for neutrophil activation and NETosis in burn injuries and burn injury progression. Targeting exacerbated neutrophil activation might represent a new therapeutic option for severe cases of burn injury.

burn injuries are still associated with a high mortality rate, most commonly resulting from 71 multiple organ failure, sepsis, and respiratory complications (2). Morbidity and mortality can be 72 increased up to 10 years after the initial insult (3). 73 74 Burn trauma elicits a unique systemic stress response characterized by increased metabolism and 75 inflammation (4) . The early systemic inflammatory response syndrome (SIRS) is characterized 76 by pro-inflammatory cytokines, such as interleukin 6 (IL-6), IL-8, and tumor necrosis factor 77 alpha (TNFα) and usually lasts for several days (5, 6). Later, the milieu is shifted towards an 78 anti-inflammatory response syndrome (AIRS), where the immunosuppressant mediators TGFβ1, 79 and IL-10 prevail (6). Though inflammation is inherent and indispensable for normal wound 80 healing (7), burn injuries can induce a state of distorted inflammatory response which can persist 81 up to several years (8) and which can ultimately lead to host tissue damage and organ 82 dysfunction. Various factors orchestrate early immune reactions, such as the amount of affected 83 body surface area, burn depth and cause, inhalation injury, patient age, and chronic medical 84 conditions (9). Neutrophils (10) and macrophages (11) are early key players infiltrating the burn-85 injured area. Though these immune cells are readily activated by burn injury-induced damage-86 associated molecular patterns (DAMPs), macrophage antigen presentation and neutrophil-87 mediated killing of pathogens have been shown to be diminished following burn injury (12, 13), 88 leading to increased susceptibility to infections. While immunological hyper-activation entails 89 tissue damage, exaggerated immunosuppression predisposes to infections (14). Hence, a delicate 90 balance of immunomodulators determines clinical outcome in burn injuries. 91

A commonly observed phenomenon of burn victims is secondary burn progression (15). While 92 tissue necrosis and ischemia are immediate results of burn injury, tissue damage is often 93 aggravated long after the initial trauma. Formation of red blood cell aggregates and 94 microthrombi and subsequent occlusion of the microvasculature have been identified as the 95 underlying cause of burn progression (16). Though secondary burn progression substantially 96 promotes tissue damage, its role in the post injury tissue response is often underestimated. 97 98 Neutrophils are implicated in the host defense against pathogens, whereby antimicrobial activity 99 is exerted by phagocytosis, release of effector molecules, and formation of neutrophil 100 extracellular traps (NETs) (17). Activated protein-arginine deiminase 4 (PAD4) catalyzes 101 citrullination of histones, which results in chromatin decondensation. Granule proteins, such as 102 myeloperoxidase (MPO) and neutrophil elastase (NE), further promote DNA de-compaction and 103 intracellular DNA together with granule proteins are released following plasma membrane 104 rupture (18, 19) . It was further demonstrated that neutrophils contain intracellular stores of C3a 105 (20), which might be self-synthesized or absorbed from the serum (21). Moreover, C3a was 106 reported to induce neutrophil activation (22). A role for neutrophils in coagulation and 107 microvascular obstruction has been described at several occasions (23-27). The amount of 108 neutrophil-derived circulating, free DNA was proposed as a predictor of mortality in severely 109 burnt patients (28) and elevated human neutrophil elastase DNA and nucleosomes were detected 110 in burn and sepsis patients (29). Though previous studies have reported a role for neutrophils and 111

NETs in burn injury (30, 31), systemic surrogate markers indicating NETosis have not been 112 comprehensively studied so far. Our group was previously able to show activation of the soluble 113 suppressor of tumorigenicity 2 (sST2) / IL-33 axis in sepsis (32) as well as thermal burn injury 114 (33) and found increased soluble ST2 to be a predictor of mortality. In the current study, we 115 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted October 7, 2021. Samples used in the current study have already been analyzed for sST2 and IL-33 (33). 127

Patients > 18 years who were admitted within 24 hours post trauma to burn intensive care unit 128 and presented with a burn injury covering > 10 % of the total body surface area (TBSA) at 129 primary survey were included in this study (Table 1) . Patients with chronic infectious diseases or 130 autoimmune disorders were excluded. Eight healthy volunteers served as controls. 131 Sera were obtained daily within the first week after admission, and weekly up to day 28 after 132 admission. For controls, samples were obtained in the same manner. Whole blood was incubated 133 for thirty minutes at ambient temperature and centrifuged at 2850 relative centrifugal force for 17 134 minutes. Sera were separated and cryopreserved below -70°C. 135

Intensive care management including fluid resuscitation was performed according to institutional 136 guidelines and was not affected by this study. Inhalation trauma was assessed on primary survey 137 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted October 7, 2021. Complete blood counts were determined during routine clinical tests and reference values were 153 adopted from in-house reference ranges. Sequential organ failure assessment (SOFA) score (34), 154 acute physiology and chronic health evaluation II (APACHE II) score (35), and abbreviated burn 155 severity index (ABSI) (36) were assessed on the day of admission. Median values of severity 156 scores were used to assign values to low and high groups. As the SOFA score of our study 157 population displayed a median of 6.5, we chose 

To study the role of burn injury-induced NETosis, we tracked the neutrophil-derived factors 170 MPO, CitH3, NE, and C3a in sera of burn victims up to 4 weeks post trauma. We found that 171

NETosis-associated factors were significantly increased compared to healthy controls ( Figure 1 ). 172 MPO was remarkably increased in the early days post burn injury ( Figure 1A ). While CitH3 did 173 not differ from controls on admission day and day 1, serum CitH3 concentrations showed a 174 delayed increase, peaking on day 4 post admission ( Figure 1B) , indicating a potential 'second 175 burn' hit. Intriguingly, NE levels were elevated on admission day and in the early stress response 176 to burn trauma before approximating levels detected in healthy controls ( Figure 1C ). Serum C3a 177 was strongly elevated in burn victims compared to controls ( Figure 1D ). To test a potential 178 connection between systemic C3a levels and liver parameters in burn victims, we furthermore 179 assessed gamma-glutamyltransferase (γ-GT) levels. γ-GT started increasing 2 weeks post injury, 180 but did not correlate with C3a (Supplemental Figure S1 ). Since lactadherin promoted survival of 181 septic rats (37) and lactadherin prevented coagulopathy following traumatic brain injury (38), we 182 addressed the question whether lactadherin might also be involved in the burn injury-induced 183 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint immune response. When assessing serum lactadherin levels, we observed no difference between 184 burn victims and healthy controls (Supplemental Figure S2 ). We furthermore aimed to 185 investigate the relationship between neutrophil-derived factors and found a positive correlation 186 between MPO and NE, but not between CitH3 and NE or between CitH3 and MPO 187 (Supplemental Figure S3) . We aimed to test whether our set of serum markers might be associated with mortality, inhalation 204 trauma, or 3 rd degree burn injury. Our battery of neutrophil-derived factors did not predict 205 mortality, and C3a was even strongly reduced in deceased burn victims (Supplemental Figure  206 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint S4). We furthermore compared burn victims with inhalation trauma to those without respiratory 207 damage and observed no effect of lung injury on serum levels of neutrophil-derived factors 208 (Supplemental Figure S5) . Similarly, serum MPO levels displayed no difference when 209 comparing 3 rd degree burn victims with patients suffering from 1 st and 2 nd degree burns (Figure  210 3A). By comparison, we observed elevated serum CitH3 and NE levels on days 3 and 4 of 3 rd 211 degree burns compared to first and second degree burns (Figures 3B & C) . Of note, the delayed 212 increase of serum CitH3 3 and 4 days post injury were exclusively detected in 3 rd degree burns. 213

Surprisingly, C3a levels were strongly reduced in higher degree burns on day 3 ( Figure 3D ). 214

These findings suggest that the degree of burn injury determines the extent of neutrophil 215 activation and NETosis. 216 217 CitH3 is elevated in patients with higher severity scores 218

We sought to determine whether neutrophil-derived factors were associated with clinical severity 219 scoring systems and dichotomized SOFA, APACHE II, and ABSI scores into low and high 220 values. We observed increased MPO and CitH3 levels in patients with APACHE II scores above 221 18 (Supplemental Figure S6 ) and NE was significantly higher in SOFA scores ≥ 7 (Supplemental 222 Figure S7 ). Intriguingly, CitH3 and NE levels were elevated in patients with ABSI scores above 223 9, while MPO and C3a showed no difference ( Figure 4) . These data suggest that specific 224

NETosis-associated factors are elevated in more severe burns. 225 226 Burn injury immune cell signature is independent of severity 227 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint Finally, we investigated whether the burn injury-induced alterations in immune cell levels were 228 associated with severity of burn injury. Therefore, cell counts were compared between 3 rd degree 229 and lower degree burn victims and furthermore between patients with higher and lower clinical 230 severity scores. A trend towards increased leukocyte and neutrophil counts in more severe cases 231 was found on admission day, while no difference in platelet and relative neutrophil counts was 232 observed (Supplemental Figures S8 -S11 ). Absolute and relative lymphopenia was more 233 pronounced in the early days following burn injury when comparing low versus high SOFA 234 scores (Supplemental Figure S10 has been identified as a prognostic marker for mortality and an early biomarker of sepsis in burn-248 injured patients (12, 28), though free DNA does not exclusively originate from NETs (39) and is 249 therefore considered a rather indirect evidence of neutrophil involvement. Further studies 250 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted October 7, 2021. We observed remarkably increased absolute leukocyte counts in burn victims compared to 275 healthy controls, while absolute lymphocyte counts remained largely unaltered. In addition, 276 absolute neutrophil levels were strongly increased post burn injury. These data are in line with 277 data reported by Mulder et al. (49) and it is tempting to speculate that the elevated neutrophil 278 amounts detected in our patient cohort might serve as a potential cellular source for MPO, CitH3, 279 NE, and C3a. We furthermore observed that the increase of absolute leukocyte and neutrophil 280 counts was even more pronounced in more severe cases. Intriguingly, relative lymphocyte levels 281

were remarkably decreased in patients with burn injury and lower in patients with higher SOFA 282 score. Lymphopenia has already been reported in septic and burn victims, which might 283 contribute to post-injury immunosuppression, morbidity, and mortality (50-52). Platelet levels 284

were decreased in the early stress response and re-elevated starting 14 days after burn injury. 285

These dynamics are in accordance with previous reports, where a sustained pro-coagulant state 286 of burn patients was described (53). Together, these data connect the systemic factors with 287 clinical blood parameters and contribute to a better understanding of the cellular and molecular 288 processes in the concerted burn injury response. 289 290 Microvascular obstruction was reported as the leading cause of burn injury progression and red 291 blood cell aggregates, microthrombi, and ischemia have been described to promote secondary 292 tissue damage (16). NETs serve as a scaffold for platelets, induce platelet aggregation and 293 coagulation (23, 24) and have furthermore been shown to exert pro-coagulant activities in sepsis 294 (25). Moreover, neutrophil activation and CitH3 were induced by myocardial 295 ischemia/reperfusion injury and showed pro-thrombotic and cytotoxic features, while eliminating 296 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted October 7, 2021. Our data revealed that 3 rd degree burns showed higher CitH3 and NE levels and these results are 307 in line with the clinical severity score ABSI, where cases with higher scoring displayed increased 308

CitH3 and NE. These data suggest that the severity of burn injury dictates the level of neutrophil 309 activation. Intriguingly, patients succumbing to burn trauma did not display elevated neutrophil-310 derived immunomodulators. While we found elevated CitH3 and NE levels in patients with 311 higher degree and severity burns, future studies with larger samples sizes will be required to 312 determine whether elevated NETosis-associated factors might serve as a prognostic factor for 313 mortality following severe burn injury. Moreover, no difference between burn victims suffering 314 from inhalation trauma compared to burn-injured patients without inhalation trauma was 315 observed. Neutrophilia in the bronchoalveolar space upon inhalation trauma has been described 316 and attenuating neutrophil recruitment improved histopathology scores and bacterial clearance 317 (55). Though NETosis markers were not related to inhalation trauma in our patient collective, a 318 role for burn-associated lung injury in neutrophil activation cannot be entirely ruled out (56). 319 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted October 7, 2021. ; https://doi.org/10.1101/2021.10.07.21264679 doi: medRxiv preprint

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