IMMUNE RESPONSE TO BURN INJURY: From Animal and Patient Data Towards In Vitro Modeling Patrick P.G. Mulder
Immune Response to Burn Injury: From Animal and Patient Data Towards In Vitro Modeling Patrick P.G. Mulder
Colophon Immune Response to Burn Injury: From Animal and Patient Data Towards In Vitro Modeling PhD thesis, Radboud University, the Netherlands The research described in this thesis was performed at the Association of Dutch Burn Centres, Beverwijk, the Netherlands and at the Department of Laboratory Medicine, Laboratory of Medical Immunology, Radboud University Medical Center, Nijmegen, the Netherlands. The presented work was supported by a grant from the Dutch Burns Foundation. Distribution of this thesis was financially supported by Stichting Brandwonden Research Instituut, Radboud University, Stichting ETB-BISLIFE, Nederlandse Brandwonden Stichting, Stichting Proefdiervrij and Medskin Solutions dr. Suwelack. Provided by thesis specialist Ridderprint, ridderprint.nl Printing: Ridderprint Layout and design: Katie McGonigal, persoonlijkproefschrift.nl ISBN: 978-94-6483-447-5 © Patrick P.G. Mulder, 2023, Haarlem, The Netherlands All rights reserved. No part of this thesis may be reproduced or transmitted in any form, by any means, electronic or mechanical without prior permission of the author, or, for work that has been published, of the respective journal.
Immune Response to Burn Injury: From Animal and Patient Data Towards In Vitro Modeling Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. J.M. Sanders, volgens besluit van het college voor promoties in het openbaar te verdedigen op maandag 11 december 2023 om 14.30 uur precies door Patrick Petrus Gijsbertus Mulder geboren op 9 mei 1995 te Noordwijkerhout
Promotor: Prof. dr. I. Joosten Copromotoren: Dr. H.J.P.M. Koenen Dr. B.K.H.L. Boekema (Vereniging Samenwerkende Brandwondencentra Nederland) Manuscriptcommissie: Prof. dr. E.M.G.J. de Jong Prof. dr. Y. van Kooyk (Vrije Universiteit Amsterdam) Dr. M. Kox
TABLE OF CONTENT Chapter 1 General Introduction 7 Part 1 Immune Response in Animal Burn Models 23 Chapter 2 Burn-Induced Local and Systemic Immune Response: Systematic Review and Meta-Analysis of Animal Studies 25 Chapter 3 Kinetics of Inflammatory Mediators in the Immune Response to Burns: Systematic Review and Meta-Analysis of Animal Studies 61 Part 2 Immune Response in Burn Patients 115 Chapter 4 Persistent Systemic Inflammation in Patients With Severe Burn Injury Is Accompanied by Influx of Immature Neutrophils and Shifts in T Cell Subsets and Cytokine Profiles 117 Chapter 5 Burn-injured skin is marked by a prolonged local acute inflammatory response of innate immune cells and proinflammatory cytokines 149 Part 3 In Vitro Modeling 177 Chapter 6 Full Skin Equivalent Models for Simulation of Burn Wound Healing, Exploring Skin Regeneration and Cytokine Response 179 Chapter 7 Monocytes and T cells Incorporated in Full Skin Equivalents to Study Innate or Adaptive Immune Reactions after Burn Injury 209 Discussion and Summary 235 Chapter 8 General Discussion 237 Chapter 9 English Summary 257 Chapter 10 Dutch Summary - Nederlandse Samenvatting 265 Appendix 271 Scientific Output 272 Portfolio 276 Data Management Plan 278 Acknowledgements - Dankwoord 280 About the Author 286
CHAPTER 1 General Introduction
8 Chapter 1 Burn injury is a prevalent cause of disability and mortality throughout the world and its consequences affect patients both physically and mentally [1,2]. Depending on the severity of the injury and condition of the victim, healing of burns can be problematic, leading to secondary medical complications [3–5]. Health issues that often occur relatively early after burn injury are systemic inflammatory response syndrome (SIRS), hyper-metabolism, wound deepening, bacterial infection and hypovolemia due to a massive loss of fluids [6–9]. These extreme reactions in the body are likely to hinder and delay the wound healing process and have a major impact on morbidity and mortality of burn survivors [10,11]. Treatment of burn injuries is an intensive and time-consuming process. Complications of burn injury are generally present for the long-term or permanent and even years later new symptoms might occur, especially when patients are growing or when vital organs are irreversibly damaged [8,12]. Among long-term complications of burn injury are (hypertrophic) scar formation, loss of skin elasticity, contractions or diseases related to vital organs. Next to that, problems with mental well-being and reduced quality of life impact patients’ overall health [13,14]. Over the years, it has become more and more evident that the immune system plays an indispensable role in most (patho-) physiological responses to burn injury [15,16]. It remains, however, largely unclear how specific immune reactions lead to burn-related diseases. IMMUNE CELLS AND INFLAMMATORY FACTORS IN WOUND HEALING Next to being a protective, physical barrier to the outside, the skin is an important regulator of homeostasis [17]. Cells in the skin continuously carry out immune surveillance to ensure early and effective defense mechanisms against both internal (e.g. oncogenesis) and external threats (e.g. bacteria or viruses) [18]. The immune system consists of two arms: the innate and the adaptive immune system. The innate immune system reacts in a generic, rapid and nonspecific way, while the adaptive arm is more specialized, organized and takes more time to develop [19]. Granulocytes, mast cells, monocytes, macrophages, dendritic cells and natural killers cells (NK cells) are cells of the innate immune system and react through stimulation of pathogen-recognition receptors (PRRs). Upon interaction with pathogens, they activate cascades and recruit other immune cells via cytokine release and antigen presentation [20]. The adaptive immune system consists of T cells and B cells which are lymphocyte subtypes with a unique repertoire of immune receptors to discriminate auto-antigens from allo-antigens. These cells can react strongly to pathogen antigens by secreting antibodies, toxins and cytokines and are able to build immunological memory that will establish a stronger and more rapid response after subsequent re-encounter [19,21]. Beside fibroblasts and
9 General Introduction keratinocytes, healthy skin is inhabited mainly by lymphocytes and antigen presenting cells (dendritic cells, Langerhans cells and macrophages) that survey the skin and react to foreign structures and danger signals [17,18,22]. The immune response plays a central role during wound healing. It is essential for a proper host defense against invading microbes and coordinates healing processes during the different stages of skin regeneration [23]. The inflammatory response starts immediately after trauma. Injured skin will release damage associated molecular patterns (DAMPs) that emerge from ruptured cells [24,25]. DAMPs such as HMGB1, IL-1α or DNA are structures that act as danger signals and stimulate pattern PRRs on surrounding cells and skin-resident immune cells [26–28]. These cells respond to PRR stimulation by secreting effector molecules such as cytokines and chemokines that attract and navigate immune cells towards the wound site [29]. Immune cells that are active during tissue damage and regeneration include neutrophils, eosinophils, mast cells, monocytes, macrophages, T cells, B cells and NK cells (Figure 1). 1
10 Chapter 1 Figure 1. Immune cells involved in wound healing. Next to the immune cells, there are platelets which are fragments of megakaryocytes that start coagulation to stop the bleeding and produce factors that initiate the inflammatory response [30]. Neutrophils have a short life-span and are primarily needed to phagocytose and destroy cell remnants and invading bacteria [31]. Neutrophils are released from the bone marrow into the blood and undergo different stages of maturity [32]. Neutrophils will accumulate in large numbers at the site of tissue injury and will eventually die via apoptosis [33]. Eosinophils are suggested to play roles during wound healing and might be involved in coagulation, vascular repair and inflammation, however, the exact mechanisms are yet to be discovered [34]. Mast cells proposedly enhance inflammation and vascular permeability through the secretion of histamines early after injury and can stimulate re-epithelization and angiogenesis later on by the release of
11 General Introduction growth factors [35,36]. Monocytes reside in the blood until they migrate into tissues upon inflammatory stimuli [37]. In tissues, monocytes will differentiate into macrophages or dendritic cells to perform immune surveillance and protect against pathogens [37]. The most important macrophage subsets during wound healing are the pro-inflammatory macrophages (M1) and macrophages that support wound healing (M2) [38,39]. NK cells are cytotoxic lymphocytes that are involved in tissue homeostasis and killing of stressed or infected cells [40]. Based on their cytotoxicity and cytokine and marker expression they can be classified as either NKdim cells (less cytokine production, more cytotoxic) and NKbright cells (more cytokine production, less cytotoxic) [41]. T cells and B cells are part of the adaptive immune system and generate tailored responses to pathogens through specific effector T cells and antibody production [42]. After stimulation, naïve T cells can differentiate into specific subtypes with different effector functions: Th1, Th2, T9, Th17, Th22, Tfh cells or regulatory T cells (Tregs) [43]. The Th phenotype will influence other immune cells and the direction and duration of the overall immune response. These immune cells produce and are influenced by inflammatory mediators (Table 1). 1
12 Chapter 1 Table 1. Inflammatory mediators involved in wound healing. Information derived from [44–48]. Category Mediator Source Function Chemokines CCL2 (MCP-1) Monocytes, macrophages, dendritic cells Attracts monocytes, basophils and T cells CCL3 (MIP-1α) Macrophages, eosinophils, Attracts monocytes, macrophages and neutrophils, lymphocytes CCL4 (MIP-1β) Monocytes, macrophages, epithelial cells, fibroblasts Attracts monocytes, macrophages, lymphocytes, dendritic cells CCL11 (eotaxin) Endothelial cells, eosinophils, monocytes, fibroblasts Attracts eosinophils CXCL1 (GROα) Macrophages, neutrophils, epithelial cells Attracts neutrophils CXCL8 (IL-8) Macrophages, epithelial cells, endothelial cells Attracts neutrophils
13 General Introduction Table 1. Continued. Category Mediator Source Function Cytokines IL-1(α/β) Endothelial cells, keratinocytes, neutrophils, others Pro-inflammatory, DAMP, stimulates cell extravasation, T cell activation IL-2 T cells Th1 T cell response, T cell growth factor, activates immune cells IL-4 T cells, eosinophils, basophils, mast cells Anti-inflammatory, Th2 T cell response, regulate allergic responses IL-6 Macrophages, keratinocytes, fibroblasts, lymphocytes, others Pro-/anti-inflammatory, regulates acute phase proteins, stress response IL-10 T cells, B cells, mast cells, macrophages Anti-inflammatory, downregulation of cells IL-12 (p40/p70) Macrophages, dendritic cells, lymphocytes, neutrophils, others Pro-inflammatory, Th1 T cell response, NK cell activation IL-17 (A/F) T cells Pro-inflammatory, Th17 T cell response, activates cells IL-18 Liver, other organs Pro-inflammatory, Th1 T cell response, immune regulation IL-33 Endothelial cells, epithelial cells Pro-inflammatory, induces production of type 2 cytokines, homeostasis IFN-γ T cells, NK cells, macrophages Activation of immune cells, antiviral activity TNF-α Neutrophils, lymphocytes, endothelial cells, mast cells Pro-inflammatory, neutrophil activation 1
14 Chapter 1 Table 1. Continued. Category Mediator Source Function Growth factors EGF Glands, platelets Stimulates growth of epidermal and epithelial cells FGF (1-23) Macrophages Proliferation of fibroblasts, induce production of granulation tissue G-CSF Endothelial cells, macrophages Stimulates bone marrow to release granulocytes into blood GM-CSF T cells, macrophages, endothelial cells, fibroblasts Maturation of granulocytes and monocytes KGF (FGF7) Mesenchymal cells, fibroblasts Induces re-epithelization by keratinocytes PDGF Platelets Stimulates growth of mesenchymal cells, promotes wound healing PGE Macrophages Pro-inflammatory, vasodilator, inhibits aggregation of platelets TGF-β1 Platelets, fibroblasts, monocytes, T cells Anti-inflammatory, regulates cell growth, inhibition of lymphocytes VEGF (A-F) Macrophages, platelets, keratinocytes Stimulates angiogenesis Other mediators CRP Liver Pro-inflammatory, acute phase protein, stress response Histamine Mast cells, basophils Anti-pathogen response, allergic reaction HMGB1 Damaged cells Pro-inflammatory, DAMP, stress response
15 General Introduction DISTORTED WOUND HEALING DURING BURN INJURY During wound healing, the immune homeostasis and tissue repair processes are usually tightly controlled to avoid collateral damage and to ensure a timely recovery [49–51]. Because burn injury often destroys a large portion of skin, it creates a large area of necrotic tissue that can cause an overstimulation of the immune system [27,52]. Fibroblasts, keratinocytes and innate immune cells are highly responsive and release extremely high levels of cytokines that in turn attract massive amounts of inflammatory cells. Extreme influx of pro-inflammatory immune cells can lead to expansion of the wound area, thereby producing additional inflammatory signals [29,53]. Eventually, this can become in a vicious circle of inflammation that will impede tissue repair (Figure 2). Figure 2. Vicious circle of inflammation and tissue damage that can establish after burn injury. During the inflammatory phase after trauma, immune cells will migrate into the wounded skin to remove debris and prevent bacterial colonization [38]. Within days, a portion of these cells disappear through apoptosis while others differentiate into a state that supports wound healing [49]. Generally within one week after injury, lymphocytes will infiltrate the wound site to regulate any ongoing inflammation and, if required, orchestrate a tailored effector response to eliminate infiltrated pathogens [54]. Following the effector phase, reduction of the immune response is needed to establish a proper wound healing process. This will shift the focus from inflammation towards 1
16 Chapter 1 proliferation of keratinocytes and production of collagen, which are required for tissue restoration [55]. After substantial burn injury, it is thought that processes in the immune response are derailed, leading to persistent inflammation. Ongoing inflammation and an aberrant wound healing process can lead to long-term sequalae such as excessive scar formation, hypertrophic scars and contractures [6,38,56]. Such functional and cosmetic impairments will also impact patients’ mental well-being [14,57]. The processes in wound healing are connected to one another and inflammation proposedly plays a central role (Figure 3). Some of these processes are better elucidated than others. For instance, it is still poorly understood which immune reactions are distorted during burn injury or how bone marrow stress response leads to reduced lymphocyte activity and immune deficiency. Also, a great portion of the available evidence comes exclusively from animal studies [58]. It is therefore necessary to shine more light on the reactions in the burn-induced immune response and to bridge the gap between animal data and the human situation [59]. To limit complications and improve wound healing in patients, it is of utmost importance that the involved immune cells and inflammatory mediators are studied in more detail. More information on specific subsets and interplay between cells will help to design more effective ways to improve wound healing after major burn injuries. Figure 3. Scheme of reactions and consequences to burn injury. It highlights a central role of the immune response. SYNTHESIS OF AVAILABLE LITERATURE Researchers have previously investigated burn injury and have sought for ways to improve treatment. Most investigational or interventional studies in the field of burn injury have been performed on experimental animals [58,60,61]. Most of these studies focused on only a few aspects of wound healing and inflammation. Since it is difficult to keep up with all the information and because existing evidence is scattered, there is a strong need for an overview of the available literature [62]. Systematic reviewing
17 General Introduction is a valuable method to synthesize an overview of empirical evidence from separate investigations. These overviews provide insights that will advance experimental design contributing to the reduction and refinement of animal experimentation and will support evidence-based clinical practice [63,64]. BLOOD AND WOUND TISSUE FROM BURN PATIENTS Alternatively, valuable insights into the burn-induced immune response can be generated by investigating patient specimens. Phenotypic characterization and quantification of cells, and analysis of inflammatory mediators in blood and wound tissue inform us of the specific immune cells and factors that are actively involved in inflammation. Patient studies are limited by restrictions in sampling and absence of baseline values. However, valuable information can be generated by using leftover blood and burn tissue specimen originating from routine blood withdrawals and surgeries as part of clinical practice. Laboratory techniques such as flow cytometry, immunoassays and microscopy can uncover cellular activity and processes that are involved in the burn-induced immune response. Moreover, by analyzing patient samples from different time intervals after burn injury, time-dependent effects can be investigated. MODELING THE POST-BURN IMMUNE RESPONSE Growing ethical and scientific concerns drive scientists to search for animal-free approaches to study burn injury. An appealing alternative to animal experimentation is the use of in vitro skin models. Such skin models mimic the tissue architecture of native human skin. The information collected from literature and patient studies can be used to develop and adjust in vitro skin models. Such models can be used to study aspects of wound healing and inflammation after burn injury in a standardized and controlled setting. Since in vitro skin models are not connected to a blood circulation, the influx of immune cells and factors is missing and should be simulated. Therefore, there is a need for more sophisticated in vitro skin models to mimic defined aspects of the burn-induced immune response. Ultimately, the information from animal, patient and skin model studies can spark the design of therapeutic interventions that will improve recovery speed and reduce the side effects of a hyper-inflammatory response such as excessive scarring. Early safety and efficacy tests of promising therapeutic candidates can be performed using the in vitro skin models before progressing to burn patients. 1
18 Chapter 1 AIM AND THESIS OUTLINE To limit secondary complications and thereby improve patients’ overall health and outcome, it is paramount to improve our understanding of the pathophysiological reactions to burn injury. The research aim of this thesis was to improve our understanding of the burn-induced immune response and to develop an in vitro skin model to study cellular reactions without the need for animal experimentation. This thesis is divided into four parts that describe the pursuit of this aim step by step. In Part 1, the empirical evidence regarding burn-induced immune response in animal models is systematically reviewed. Two systematic reviews were performed that synthesize the available literature on the levels of immune cells (Chapter 2) and inflammatory factors (Chapter 3) after burn injury. Meta-analyses and subgroups analyses were performed to reveal time-depend effects and to identify factors of influence. Part 2 of this thesis is focused on the immune response in burn patients. These data were generated using blood and post-operative burn tissue samples from patients. The systemic and local immune profile after burn injury in time was studied by analyzing immune cells and cytokines using flow cytometry and immunohistochemistry. For comparison, blood and skin from healthy subjects were used. Chapter 4 describes the dynamics and phenotypic changes of immune cells and response levels of effector molecules in patient blood. In Chapter 5 the effect of burn injury on immune cells and inflammatory mediators in burn wound tissue is displayed. Part 3 of this thesis contains the experimental work with full skin equivalent models to simulate aspects of burn injury in vitro. Chapter 6 shows the optimization and validation of our full skin equivalent model that can be used to study burn wound healing and the concomitant cytokine response. In Chapter 7 the full skin equivalent model was supplemented with T cells or monocyte-derived macrophages to study their phenotype and reactions within this model of burn injury. The findings in this thesis are put into a broader perspective in Chapter 8, Chapter 9 and Chapter 10, which contain the General Discussion, English Summary and Dutch Summary (Nederlandse Samenvatting).
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21 General Introduction 1
PART 1 Immune Response in Animal Burn Models
CHAPTER 2 Burn-Induced Local and Systemic Immune Response: Systematic Review and MetaAnalysis of Animal Studies Published in Journal of Investigative Dermatology, 2022, 142, 3093-3109 DOI: 10.1016/j.jid.2022.05.004 By Patrick P.G. Mulder1,2, Hans J.P.M. Koenen2, Marcel Vlig1, Irma Joosten2, Rob B.M. de Vries3, and Bouke K.H.L. Boekema1,4 1Preclinical Research, Association of Dutch Burn Centres (ADBC), Beverwijk, The Netherlands. 2Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, The Netherlands. 3SYstematic Review Centre for Laboratory animal Experimentation (SYRCLE), Department for Health Evidence, Radboud University Medical Center, Nijmegen, The Netherlands. 4Department of Plastic, Reconstructive and Hand Surgery, Amsterdam UMC, VU University, Amsterdam, The Netherlands.
26 Chapter 2 ABSTRACT Because burn injuries are often followed by a derailed immune response and excessive inflammation, a thorough understanding of the occurring reactions is key to preventing secondary complications. This systematic review, which includes 247 animal studies, shows the post burn response of 14 different immune cell types involved in immediate and long-term effects in both wound tissue and circulation. Peripheral blood neutrophil and monocyte numbers increased directly after burns, whereas thrombocyte numbers increased near the end of the first week. However, lymphocyte numbers were decreased for at least 2 weeks. In burn wound tissue, neutrophil and macrophage numbers accumulated during the first 3 weeks. Burns also altered cellular functions because we found an increased migratory potential of leukocytes, impaired antibacterial activity of neutrophils, and enhanced inflammatory mediator production by macrophages. Neutrophil surges were positively associated with burn size and were highest in rats. Altogether, this comprehensive overview of the temporal immune cell dynamics shows that unlike normal wound healing, burn injury induces a long-lasting inflammatory response. It provides a fundamental research basis to improve experimental set-ups, burn care, and outcomes.
27 Review Immune Cells in Animal Burn Models INTRODUCTION Burn trauma often induces an overreaction of the immune system, known as systemic inflammatory response syndrome, which can cause damage to surrounding tissues and even distant organs [1,2]. Hyperactive inflammation and obstruction of wound healing can lead to excessive scarring [3] and psychological distress [4]. Information on the specific immune cells and inflammatory factors involved in the different phases of burn wound healing in humans is however scattered and incomplete. Human studies are limited by the absence of baseline values, heterogeneity among cases, and restrictions in (the timing of) blood and wound sampling. Animal experiments, executed in controlled and standardized settings [5], could improve our understanding of the mechanisms underlying the burn-induced immune response in humans. Undoubtedly, various genomic and physiological processes of the human response to trauma differ from that of animals, such as signaling pathways, wound contraction, and scar formation [6–8]. Nevertheless, animal studies contain valuable information that will improve our understanding of the cellular immune response to burn trauma. In this study, we aimed to identify the immune cells involved in the local and systemic inflammatory response to burn injury in animal models. Ultimately, we anticipate that this review leads to new perspectives in burn care and will support the improvement of treatment for patients. RESULTS Study selection, characteristics, and quality Our search generated 10,733 citations, of which 1,224 were considered relevant during title and abstract screening. From this selection, 111 studies were inaccessible, 247 were included in the systematic review (Figure 1), and 182 were used in meta-analyses (Supplementary File 1, Supplementary File 2). An overview of the study characteristics (Figure 2A-G) showed that most experiments were performed on young mice or rats. Full-thickness dorsal injury using hot water was the most common burn technique. It is worth noting that underreporting complicated the assessment of the overall study quality. Risk of bias (RoB) analysis showed that 33.5% of the included studies reported the use of randomization of animals before experimentation (Figure 2H). The majority of studies (94.0%) did not report the use of blinding, and a conflict-of-interest statement was present in 33.9% of the studies, in which four studies reported an actual conflict (Figure 2I,J). Overall, there was no significant indication of publication bias for the overall outcomes, but we did find a substantial risk of selection and performance bias. 2
28 Chapter 2 Figure 1. PRISMA flowchart of study identification, screening, and inclusion. Representation of the steps taken to select the relevant studies for the systematic review and meta-analyses [9]. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.
29 Review Immune Cells in Animal Burn Models Figure 2. Characteristics of studies in systematic review and risk of bias assessment. Numbers indicate the number of studies. (A) Types of animal species and strains. (B) Age of study animals. (C) Sex of study animals. (D) Location of burn injury. (E) Depth of burn injury. (F) Type of burn agent. (G) TBSA that was burned as a percentage. (H) Quality of reporting of all included studies. (I) Risk of bias assessment of all baseline-controlled studies. (J) Complete risk of bias assessment of a random sample consisting of 25 of the included studies. D, dermis; E. Epidermis; H, hypodermis; NR, not reported; TBSA, total body surface area. 2
30 Chapter 2 Burn-induced immune response is dominated by innate immune cells Meta-analyses were performed on outcome measures for which at least five articles were available (Supplementary Table 1). Immune cell counts in blood or wound tissue from burn-injured animals were compared with immune cell counts in blood or skin from uninjured animals (baseline or control group). Overall, there was a significant increase in leukocytes in both peripheral blood and wound tissue (Figure 3). Systemically, the numbers of neutrophils and monocytes were significantly elevated, whereas lymphocyte numbers decreased. Total leukocyte counts were higher in baseline-controlled studies than in studies with separate uninjured controls. There was no significant change in overall eosinophil or thrombocyte counts. The higher standardized mean difference of neutrophils than of total leukocytes might be caused by the decrease in lymphocyte counts. Within the lymphocyte population, only B-cell counts were significantly decreased (Figure 3B). In burn wound tissue, the numbers of neutrophils, macrophages, and mast cells were increased (Figure 3C). Cell migratory activity, mainly tested by adherence to endothelium or in vitro migration assays, was increased in total leukocytes but not in neutrophils (Figure 3D). Migratory activity of leukocytes was lower in baseline-controlled studies than in studies with separate uninjured controls. Antibacterial function of neutrophils was decreased after burn injury, whereas there was no significant effect on ROS production or inflammatory mediator secretion by neutrophils. The secretion of inflammatory mediators by macrophages was increased. There were not enough studies reporting total lymphocyte counts in wound tissue to be included in the meta-analysis.
31 Review Immune Cells in Animal Burn Models Figure 3. Overall outcome of immune cell counts and function after burn injury. Overall metaanalysis of(A) blood immune cell counts, (B) blood lymphocyte counts, (C) wound immune cell counts, and (D) immune cell functions. Results are shown as SMD of immune cell counts in the blood or wound tissue from burn-injured animals compared with immune cell counts in blood or skin from uninjured animals (baseline or control group) ± CI95%. The I 2 statistic, number of studies, and the total number of animals used in the burn group for each meta-analysis are shown below the graphs. CI95%, 95% confidence interval; SMD, standardized mean difference. Blood innate response intensifies and is persistent We performed longitudinal analysis on selected time intervals encompassing the four different biological phases of wound healing: hemostasis, inflammation, proliferation, and remodeling (Figure 4A-G). Meta-regression analyses were performed from post burn day (PBD) 0 until PBD 21 (Figure 4H). Blood leukocytes displayed a steady increase, with the highest counts from PBD 5 until PBD 28 (Figure 4A). Neutrophil counts were immediately increased during injury and remained elevated up to PBDs 15‒21 (Figure 4B). Monocyte counts were increased from PBD 5 until PBD 14 (Figure 4C). Thrombocyte counts were decreased on PBDs 0‒1 and later increased on PBDs 5‒9 (Figure 4D). The decline of lymphocytes was most predominant directly after burn injury, whereas on PBDs 10‒14, counts returned to control levels (Figure 4E). We detected a decrease in B-cell counts on PBDs 5‒9 but found no significant differences in T-cell counts (Figure 4F,G). To further investigate the opposed dynamics of neutrophils and lymphocytes during burn injury, we calculated the neutrophil/lymphocyte ratio (NLR) for studies that reported both neutrophil and lymphocyte counts (Supplementary Figure 1). During the first 9 days, significantly higher NLRs were observed in burn-injured animals, which is an indication of systemic inflammatory response syndrome [10]. Overall, the temporal analysis revealed that whereas the increase in neutrophil counts was immediate, total 2
32 Chapter 2 leukocyte, monocyte, and thrombocyte counts increased during the first week, whereas lymphocyte numbers decreased. Figure 4. Longitudinal analyses of blood immune cell counts after burn injury. Longitudinal meta-analysis of blood cell counts: (A) leukocytes, (B) neutrophils, (C) monocytes, (D) thrombocytes, (E) lymphocytes, (F) B cells, and (G) T cells. (H) Meta-regression with immediate effect (intercept) and linear coefficient of time after burn (PBD 0 until PBD 21). Results are shown as SMD of immune cell counts in blood from burn-injured animals compared with immune cell counts in blood from uninjured animals (baseline or control group) ± CI95%. The I 2 statistic, number of studies, and the total number of animals used in the burn group for each interval are shown below the graphs. Bonferroni-corrected P-values of significant differences between time intervals are given in the graphs. CI95%, 95% confidence interval; PBD, post burn day; SMD, standardized mean difference. Direct innate response in wound is accompanied by altered functions Longitudinal analyses were performed on cell counts in wound tissue as well as on cell function (Figure 5) and revealed an instant increase in leukocyte migratory activity on PBDs 0‒4 and an increase in wound leukocyte numbers on PBDs 0‒1 and 5‒9 (Figure 5A,B). Mast cell numbers showed a decrease around PBDs 2‒4 and a subsequent increase from PBD 10 until PBD 21 (Figure 5C). On the other hand, neutrophil numbers increased instantly and remained elevated until at least PBD 14 (Figure 5D). Although the production of ROS by neutrophils was not significantly altered by burn injury, we did detect an increase in inflammatory mediator secretion by neutrophils on PBDs 0‒1
33 Review Immune Cells in Animal Burn Models and decreased neutrophil antibacterial activity on PBDs 5‒9 (Figure 5E-G). Macrophage numbers increased immediately and remained elevated until PBD 14 (Figure 5H). Release of inflammatory mediators by macrophages was increased on PBDs 0‒4 (Figure 5I). Altogether, the instant increase of innate immune cells in wound tissue persisted for at least 2 weeks, whereas certain functions were affected. Figure 5. Longitudinal analyses of wound immune cell counts and cell function after burn injury. Longitudinal meta-analysis of (A) burn wound leukocyte counts, (B) leukocyte migration, (C) burn wound mast cell counts, (D) burn wound neutrophil counts, (E) neutrophil antibacterial activity, (F) neutrophil ROS production, (G) neutrophil inflammatory mediator production, (H)burn wound macrophage counts, and (I) macrophage inflammatory mediator production. (J) Metaregression with the immediate effect (intercept) and linear coefficient of time after burn (PBD 0 until PBD 21). Results are shown as SMD of immune cell counts in wound tissue from burn-injured animals compared with immune cell counts in the skin from uninjured animals (baseline or control group) ± CI95%. The I 2 statistic, number of studies, and the total number of animals in the burn group for each interval are shown below the graphs. Bonferroni-corrected P-values of significant differences between intervals are given in the graphs. CI95%, 95% confidence interval; inflamm., inflammatory; med., mediator; NS, not significant; PBD, post burn day; prod., production; SMD, standardized mean difference. Immune response depends on animal characteristics and burn technique To investigate the differences between experimental models, subgroup analyses were performed (Figure 6). The highest blood leukocyte counts were found in rats or in adult animals. Sensitivity analyses confirmed that the interspecies effect was still present when only young animals were compared and that the difference from aging remained when only rats were analyzed. Neutrophil counts were higher in studies using >25% 2
34 Chapter 2 total body surface area (TBSA) than in those using 5‒25% TBSA and were highest in rats. Sensitivity analysis showed that the effect of TBSA was present in mice but not in rats. Surprisingly, neutrophil wound counts in studies using 5‒25% TBSA were lower than in those using ≤5% TBSA, in both mice and rats. Blood neutrophil counts were higher in males than in females. Interestingly, both wound leukocyte and neutrophil counts were lower in scalds than in metal burns. Within TBSA groups, the difference in neutrophil counts between species was still present in wound tissue but not in blood, indicating that collinearity could play a role. The difference between sexes for blood counts and the effect of metal burns on wound neutrophil counts were not influenced by TBSA or species. Because the majority of the studies used full-thickness burns, subgroup analysis on wound depth could only be performed for wound neutrophil counts. Overall, the leukocyte response was affected by type of species, animal age, and burn agent, whereas the neutrophil counts depended on species, sex, wound size, and burn agent.
35 Review Immune Cells in Animal Burn Models Figure 6. Subgroup analysis of immune cell counts after burn injury. Subgroup analysis of (A) burned TBSA, (B) species, (C) burn agent, (D) age, (E) sex, and (F) wound depth. Only subgroups for which at least five articles were available were used in the analysis. Results are shown as SMD of immune cell counts in blood or wound tissue from burn-injured animals compared with immune cell counts in blood or skin from uninjured animals (baseline or control group) ± CI95%. The I 2 statistic, number of studies, and the total number of animals in the burn group for each subgroup are shown below the graphs. Bonferroni-corrected P-values of significant differences between subgroups are given in the graphs. CI95%, 95% confidence interval; FT, full-thickness; PT, partial-thickness; SMD, standardized mean difference; TBSA, total body surface area. 2
36 Chapter 2 DISCUSSION An improved understanding of the burn-induced immune response is necessary to prevent secondary pathologies in patients with burns as much as possible. In this study, we synthesized available literature on the post burn immune response in animals into a comprehensive systematic overview. Even though there was great heterogeneity and variation among the studies, the meta-analyses clearly displayed the dynamics of innate and adaptive immune cells after burn injury. In peripheral blood, the numbers of neutrophils, monocytes, and thrombocytes increased shortly or within 1 week after burn injury and remained increased over the first month. In contrast, lymphocyte numbers were reduced during the first 2 weeks, indicating that the response is driven by the innate arm of the immune system and that resolution of inflammation is delayed. In wound tissue, we observed an immediate surge of neutrophils and macrophages during the first 2 weeks, whereas for mast cells, a time-dependent response was observed because numbers decreased near the end of the first week and steadily increased from PBD 10 onward. Although several studies investigated the specific subsets of lymphocytes in wound tissue, there were not enough data available on total lymphocyte counts. Furthermore, burn injury affected cell function because we showed that migration of leukocytes and inflammatory mediator production by neutrophils and macrophages were increased earlier on and that antibacterial activity of neutrophils was reduced on PBDs 5‒9. In general, wound healing entails four biological phases, namely hemostasis, inflammation, proliferation, and remodeling. The immediate increase in thrombocyte and neutrophil numbers during the inflammation phase is attenuated within the first week [8,11,12]. Macrophage numbers, which are important for the transition from inflammation to proliferation [13], normalize later on, whereas lymphocyte numbers increase from the second week onward [14]. In this study, we show that at least in animals, these processes are derailed and that high numbers of circulatory thrombocytes, neutrophils, and monocytes are persistent, whereas lymphocyte numbers are actually reduced. This suggests that the timing in typical schematic depictions of the cellular immune response during wound healing does not hold true for burn injury. Unlike in humans, B-cell counts in uninjured rodents are higher than their T-cell counts[15], which could explain the larger effect of burn injury on B cells than on T cells that we found in animals. A relative increase in innate immune cells and a decrease in lymphocytes have also been detected in patients with burns [16,17]. Danger-associated molecular patterns that are released by wounded tissues are suggested to cause a continuous activation of the immune system [18,19]. In turn, a hyperactive immune system can cause damage to
37 Review Immune Cells in Animal Burn Models surrounding tissues, thereby producing additional danger-associated molecular patterns and cytokines that uphold the inflammation. The time-dependent response of thrombocytes is similar to the early thrombocyte response in burn patients [20]. The typical early trauma-induced leukopenia in patients with burn wounds that is caused by exsanguination, resuscitation, and emigration of immune cells from the blood circulation was in our meta-analysis only visible when the early time points were analyzed per day. Leukopenia is naturally restored by the bone marrow [21,22]. During acute inflammation, predominantly, neutrophils and monocytes are replenished by the bone marrow, which can lead to reduced lymphopoiesis and overrepresentation of innate immune cells in the circulation [23]. Moreover, the NLR, a marker for systemic inflammatory response syndrome in humans, was in animals also highly increased during the first 9 days after burns. In patients with burns, persistent leukocytosis in combination with lymphopenia is associated with persistent inflammation, arrested wound healing, increased susceptibility to opportunistic infection, and increased mortality [2,24,25]. Because the thrombocyte count and NLR correspond with systemic inflammatory response syndrome and septic events, they are of prognostic and diagnostic value [10,26]. In wound tissue of animals, increased levels of neutrophils, macrophages, and mast cells were detected until at least PBD 14. The transition of macrophages from an M1 phenotype toward an M2 phenotype is essential to facilitate proper wound healing [27,28]. Although monocyte or macrophage subtypes could not be investigated, we found that total wound macrophage numbers were increased and that the production of inflammatory mediators by macrophages was enhanced. The activity of neutrophils is altered after severe trauma in animals [29–32], but it remains unclear whether trauma, in general, enhances or weakens neutrophil activity (Figure 5). Presumably, the emergency release of neutrophils into the circulation is responsible for reduced chemotactic activity owing to the inflexibility of the banded nucleus of immature neutrophils [33], whereas rapid activation can lead to impaired antibacterial activity [31]. On the other hand, the immaturity of neutrophils could amplify the granule content and increase the release of inflammatory factors [34,35]. Mast cells have also been proposed to play an active role during wound healing in both animals and humans. They might enhance inflammation and vascular permeability through the secretion of histamines early after injury and stimulate re-epithelization and angiogenesis later on by the release of GFs [36,37]. This coincides with increased numbers of mast cells on PBDs 0‒1 and on PBDs 15‒21. Only a minority of studies used porcine or canine models, and therefore it was unfeasible to study the differences between species other than mice and rats. Although pigs come 2
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