Effect of severity of injury on immune system and its role in patient outcome in patients with Blunt Trauma to the chest

Effect of severity of injury on immune system and its role in patient outcome in patients with Blunt Trauma to the chest | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of severity of injury on immune system and its role in patient outcome in patients with Blunt Trauma to the chest Dr.Pranay Kabiraj, Dr. Purva Mathur, Nigam Rajput, Dr.Pratyusha Priyadarshini, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4253254/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND Chest injuries are one of the most common injuries which are admitted in the trauma and emergency departments. It is known that the immune system affects the outcome of patients with significant trauma though the specific role of the components of immune system still remains to be explored. This study aimed to explore the temporal association of immune system with severity and development of complications in patients with blunt chest trauma (BCT). METHODS 75 patients (50 non-intubated and 25 intubated) satisfying the inclusion and exclusion criteria were enrolled in the study and blood samples were collected on Day 0, Day 2, Day 5 and On the day of discharge. Broncho- alveolar lavage fluid was collected once in the intubated patients. The samples were processed to estimate the levels of intracellular cytokines of Neutrophils, T-regulatory cells and T-helper cells. Extracellular levels of IL-9, IFN-γ, TNF-α, IL-4, IL-22, IL-1β, IL-17A, IL-6, IL-10, TGF-β were estimated in the serum as well as in the BAL fluid. 30 healthy volunteers and 10 patients with esophageal cancer undergoing routine bronchoscopy without tracheobronchial involvement were used as controls. The levels of cytokines were compared between 1) the injured and healthy patients 2) patients with Injury severity Score 15 3) Patients with thoracic trauma severity score 5 , 4) patients who had uneventful recovery and patients who had uneventful recovery RESULTS Intraneutrophilic levels of IL-6((p<0.001**) ,IL-1β((p<0.001**) , Th22( p =0.0001), Th1(p=0.0012), Intracellular T-reg cytokines levels of IL-10(p=0.0001) and FOX-P3(p<0.001) levels were higher in the patients group as compared to the control group on day-0 of injury. Extracellular cytokines levels of TNF-α (p=0.0433), TGF-β (p=0.0069) were significantly raised in the patient group. In the BAL fluid sample intraneutrophilic levels of IL-6(p<0.001), IL-1β(p<0.0002), Th22(p=0.016) and Th1(0.0049), Intracellular T-reg cytokines levels of TGF-β(p=0.0004) and FOX-P3(p=0.0003) were significantly increased in the patient group as compared to the control group. Extracellular levels of IL-4 (p=0.0271) and IL-1β (p=0.0041) were significantly elevated in the BAL fluid of patients. Patients with overall higher injury severity score (ISS>15) had significantly increased intraneutrophilic cytokines as compared to those with ISS<15. The intracellular T-regulatory cytokines and intracellular T helper cell cytokines did not show such consistent significant results. The extracellular cytokines levels also did not show any statistically significant results in severely injured patients as compared to lesser injured group(ISS>15 vs ISS5 vs TTSS5 group on the day of discharge. The extracellular levels of IL-9, IL-1β, IL-17A, IL-6, IL-10, and TGF-β were significantly elevated in TTSS >5 group even on the day of discharge. Out of 75 patients 22 (29.33%) developed complications. In the complications group the intraneutrophilic cytokines levels of IL-6(p=0.0251) and IL-1β(p=0.0143) were significantly higher on the day of admission as compared to patients who did not develop any complications. The extracellular cytokines levels showed higher values on day 2 of injury of which IFN-γ(p=0.0022), TNF-α(0.0062), IL-4(p=0.0128), IL-1β(p=0.0241), IL-17A(p=0.0035), IL-6(p=0.0279) and IL-10(p=0.023) were raised significantly on day 2 in complications group as compared to patients without complications. CONCLUSIONS The immune system plays a very significant role in final outcome of patients with chest injury in addition to the anatomical injury. The severity of injury activates the immune system in a proportional manner and further exploration of this role can help in development of prognostic indicators and therapeutic immunomodulation. Blunt Chest Trauma Intracellular cytokines T- regulatory cytokines T-helper cells BAL Fluid ISS TTSS Complications Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Trauma is a major public health problem in India and across the globe. Over One-fifth of the global trauma deaths occur in India of which almost 50% are deemed preventable( 1 , 2 ). Isolated chest injuries account for almost 20–25% of the trauma burden and is only next to head and abdominal injuries in causing the most deaths in a multiply injured person( 3 , 4 ). Even in patients with other systems trauma, chest injuries play a major role in the clinical course, management and outcome of the patient. In addition to the anatomical severity the management of chest injury patients is complicated by development of clinical conditions such as Multiple Organ Dysfunction Syndrome (MODS), Acute Respiratory Distress Syndrome (ARDS), pneumonia, Systemic Inflammatory and Anti-Inflammatory Response Syndrome. Trauma activates the immune system of the body at both the local tissue level as well as the systemic level. The role of immune system and the disorders associated with it significantly effects the outcome of the patients. Hence it is important that these mechanisms involving the hyperimmune response driven by Th1 cells and the immunosuppressive response driven by Th2 response be understood and correlated with the anatomical severity of injury and outcomes in patients with chest trauma. Conventional teaching and early research states that significant trauma leads to a hyperimmune response and any derangement in this response can cause a systemic inflammatory response syndrome. This was believed to be the reason for early peak in mortality following trauma. The systemic anti-inflammatory mechanisms then comes into play to counter and limit the acute hyperimmune response. It was believed that uncontrolled systemic anti-inflammatory response increases the susceptibility to nosocomial infections in the later weeks, with various clinical manifestations such as ARDS, MODS, Ventilator Associated Pneumonia (VAP) and Hospital Associated Pneumonia (HAP). Contrary to the earlier belief new research using genome wide expression analysis shows activation of the pro-inflammatory and anti-inflammatory genes simultaneously, rather than one following the other as believed earlier( 5 ). There is now enough evidence to show that a multi system trauma might lead to an uncontrolled cytokine storm. This cytokine storm is dynamic in nature and the early cytokine response differs from the late cytokine responses. While some patients have an uneventful recovery, others with same kind of injury might have a complicated hospital course. The innate as well as the adaptive immune system play an integral role in the pathophysiology of these patients but unfortunately is poorly understood. This study was designed to understand the effect of trauma and its severity on the innate and adaptive immune effector cell response both at the systemic and local level and investigate the role of these immune responses in development of complications. METHODOLOGY Study Population The study was done with a sample size of 75 blunt chest trauma (BCT) patients which were divided into two arms 50 patients in the non-intubated and 25 patients in the intubated arm (i.e., those patients who required intubation on admission). To measure the systemic response and to investigate the temporal response multiple blood samples were collected on day 0, day 2, day 5 and on the day of discharge. Single sample of bronchoalveolar lavage fluid (BAL) was collected from the intubated patients whenever they underwent fiberoptic bronchoscopy (FOB) during their routine course of the treatment, to study the local immune response. In the control arm blood samples from 30 healthy volunteers were collected and BAL control samples were collected from 10 diseased controls, who were patients of carcinoma of oesophagus who underwent bronchoscopy for evaluation of tracheobronchial involvement of oesophageal malignancy during evaluation of their primary illness in Department of Pulmonology. Only those samples were processed which had no tracheobronchial involvement. Inclusion and Exclusion Criteria Patients admitted to the hospital within 24 hours of the injury with BCT between the age group of 18yrs to 65yrs were included in the study. Those who arrived later than 24hrs, pregnant patients, patients receiving chemotherapy and immunomodulators, patients with hollow viscus injury, and patients with splenectomy and those suffering from pre-existing co-morbidities e.g. , Diabetes, Hypertension, Chronic Obstructive Pulmonary Diseases, Chronic Kidney Disease, Chronic Liver Disease were excluded. Sample Collection and Study Blood and Serum collection and Study Peripheral blood samples (5 ml) were collected from BCT patients on days 0,2, 5 and day of discharge. 5ml of blood sample was collected once from hospital staffs for control. Broncho-Alveolar Lavage (BAL) Fluid Study and collection of BAL fluid Bronchoalveolar lavage (BAL) fluid 5ml was collected from patients who required intubation and FOB during their routine clinical course. 5ml of BAL sample was collected from the diseased controls. BAL sample was analyzed for intracellular, extracellular cytokine study. Parameters studied Injury severity score(ISS)( 6 ) and thoracic trauma severity score(TTSS)( 7 ) was calculated for each patient. Intracellular as well as extracellular levels of Cytokines were measured in both the blood as well as in the BAL fluid as mentioned below. Intracellular cytokines levels of Neutrophils (CD66b+), T-regulatory(T-reg) cells (CD25+), and T helper cells (CD4+) (Th22, Th1, Th2, Th9 and Th17) levels were measured. Intraneutrophilic cytokine levels of IL6 and IL1β were measured along with intra T regulatory cell cytokines IL-10, TGFβ and FOX-P3 levels. Extracellular levels of IL-9, IFN-γ, TNF-α, IL-4, IL-22, IL-1β, IL-17A, IL-6, IL-10, TGF-β were measured. The clinical course was thoroughly followed till final disposition and the daily developments such as vitals chartings, daily progress, development of complications such as Sepsis( 8 ), HAP, VAP( 9 , 10 ), ARDS( 11 ) including death of the patient was studied and noted. Standard definition of sepsis, HAP, VAP and ARDS were used. Statistical Analysis Statistical analysis was done using IBM SPSS Statistics version 29 software. Data was presented as mean ± SD. Unpaired student's t-test was used to determine the significance of difference between the two independent groups among continuous variables. For skewed data a corresponding non-parametric test, that is Mann-Whitney test was applied. Categorical variables were compared by chi- squared test and Fischer's test. Continuous variables were compared using independent t test (for normal distribution) and Wilcoxon rank-sum (for non-normal distribution). Significance was set at 5% level and a p-value less than 0.05 was taken as significant. Prior approval for the study was taken by the institutional ethical committee IECPG-88/27.02.2020 RESULTS AND ANALYSIS Demography and clinical characteristics A total of 75 patients with BTC who fulfilled the inclusion and exclusion criteria were included in the study, of which 50 patients did not require ventilatory support and 25 patients required ventilation. Blood and serum samples of these 75 patients were analyzed and compared with 30 healthy volunteers. BAL fluid from 25 intubated patients were compared with 10 diseased controls. Out of these 66 (88%) patients were males and 9 (12%) were females. Mean age of the patients was 43.7 ± 14.1 years, the most common age group was between 31–40 years 26 (34.6%). Most common mode of injury was road traffic injury 44 (58.6%) patients, followed by fall from height 19(25.3%) patients. Of these 26 (34.6%) patients had an ISS score of \(\le\) 15 and 49(65.3%) had and ISS score >15. 27 (36%) patients had a TTSS score of \(\le\) 5 and 48(64%) patients had a TTSS >5. 53 (70.66%) patients had uncomplicated clinical course whereas 22 (29.33%) patients had complicated clinical course. Part 1: Analysis of effect of trauma on immune response; Cases Vs Controls Comparison of intracellular cytokines in cases versus heathy controls (day 0) To study the change in the baseline cytokines profile of BCT Patients, the intracellular level of neutrophilic and lymphocytic cytokines on the day of admission (day 0) of the BCT patients were compared with healthy controls. Neutrophils Comparative analysis showed that the CD66b + , IL-1β, IL-6 response was significant higher in the patients’ group than in the control group (p < 0.001) on day 0.(Fig. 1) T helper cells Comparison of the T-helper intracellular cytokines level between the patients and controls (day 0) shows higher levels of CD4( p < 0.001) , Th22( p = 0.0001) , Th2(0.2158), Th1( p = 0.0012) and lower levels of Th9(0.0592), Th17(0.1317). (Fig. 2). T regulatory cells Comparison of T regulatory cells intracellular cytokines showed significantly increased expression of IL-10( p = 0.0001 ) and FOX-P3 (p < 0.001) in cases immediately after trauma (day0). (Fig. 3) Comparison of extracellular cytokines in cases versus heathy controls (day 0) Comparison of the extracellular cytokines levels between the patients and controls shows higher levels of IL-9, IFN-γ, TNF-α, IL-4, IL-1β, IL-22, IL-10, TGF-β and lower levels of IL-6, IL-17A in cases, TNF-α ( p = 0.0433 ), TGF-β ( p = 0.0069) cytokines attained statistically significant p values.. Mixed patterns of increase in proinflammatory and anti-inflammatory cytokines were noted. Bronchoalveolar Lavage (BAL) fluid analysis for Intracellular cytokines in BCT patients vs Diseased Controls. Neutrophils Significantly raised levels of intracellular neutrophilic cytokines [CD66b (p < 0.001) , IL-6 (p < 0.001) , IL-1β( p < 0.0002)] in BAL fluid of BCT patients as compared to the diseased controls. (Fig. 4) T regulatory cells Increased expression of T regulatory cell cytokines in BAL fluids of BCT patients as compared to the diseased controls. Statistically significant levels were seen in expression of CD25 (p < 0.001) , TGF-β (p = 0.0004) and FOX-P3 (p = 0.0003). (Fig. 5) T helper cells Increased levels of T helper cell cytokines were noted in BAL fluids of BCT patients when compared to the diseased controls. Significantly increased levels of expression were noted in CD4 (p < 0.001) , Th22 (p = 0.016) and Th1 (0.0049) cytokines. Similar response was noted in the BAL fluid when compared with the systemic response with increased CD4 expression and increased levels of Th1 and Th2 expression. Also, the Th22 levels showed increased expression in the BAL fluid as was seen in the systemic response between cases and controls. (Fig. 6) Bronchoalveolar Lavage (BAL) fluid analysis for extracellular cytokines in BCT patients vs Diseased Controls. Comparison of extracellular cytokines in BAL fluid of patients in BAL fluid showed increased expression of IL-9, IL-4, IL-22, IL-1β, IL-17A, IL-6 and TGF-β. Statistically significant levels were noted in IL-4 (p = 0.0271) and IL-1β (p = 0.0041). Mixed pattern of pro and anti-inflammatory cytokines was noted, similar to what was seen in the serum analysis. Part 2: Analysis of overall injury severity and thoracic injury severity on immune response The study effect of severity of trauma on the immune response the ISS was calculated and the patients were divided into two groups of ISS ≤ 15 and ISS > 15( 12 ). Similarly, to study the effect of severity of thoracic injuries on the immune response, specific scoring system for thoracic injury i.e., TTSS was used and the patients were divided into two groups of TTSS ≤ 5 and TTSS > 5( 7 , 13 ). Comparison of intracellular/ extracellular cytokines based on Injury severity Score (ISS ≤ 15 Vs ISS > 15) Neutrophils The levels of intraneutrophilic cytokines were increased on all days (Day 0, day 2, day 5 and day of discharge) in patients with ISS > 15 as compared to ISS 15. IL-1β levels were significantly increased on Day 0, Day 2 and Day 5. T regulatory Cell The CD25 levels consistently decreased over time in ISS > 15 as compared to ISS ≤ 15 group but were insignificant. FOX-P3 levels were increased in ISS > 15 group but were of significant only on day 0. T helper Cells All T helper cells intracellular cytokines (CD4, Th22, Th1, Th2, Th17 and Th9) were increased on the day of injury in patients with severe trauma ISS > 15. Only on day 0 levels of CD4+, Th22, Th1, and Th9 were significantly higher in ISS > 15 group. The levels of CD4+, Th22, Th1 and Th2 were increased on all days but were insignificant. Extracellular cytokines The extracellular cytokines levels of IL-9, IFN-γ, TNF-α, IL-1β, IL-6, and IL-10 were elevated in ISS > 15 group as compared to the ISS ≤ 15 group. Though most of the cytokines reached similar levels on the day of discharge. The IL-4 and IL-22 levels were lower in ISS > 15 group when compared with ISS ≤ 15 group. None of these achieved levels of statistical significance. Effect of severity of thoracic trauma severity on immune response Neutrophils The initial levels (Day 0) of intraneutrophilic cytokines CD66b and IL-1β were increased in TTSS > 5 group, but the cytokines levels gradually decreased or became comparable to TTSS 5 group as compared to TTSS ≤ 5. IL-10(p = 0.0154), TGF-β(p = o.0006), FOX-P3(0.0001) levels were significantly raised on the day of discharge. (Fig. 7) T helper cells Except for the levels of Th22 all the T helper intracellular cytokines were supressed in TTSS > 5 group as compared to TTSS ≤ 5 group, but did not reach any statistical significance. Extracellular cytokines The levels of TNF-α, IL-4, IL-1β, IL-17A, IL-6, IL-10 and TGF-β were high in TTSS > 5 group as compared to TTSS ≤ 5 group. The levels of IL-9(p = 0.0064), IL-1β(p = 0.0038), IL-17A(p = 0.0019), IL-6(p = 0.0017), IL-10(p = 0.0004), and TGF-β(p = 0.0001) were significantly raised in TTSS > 5 group even on the day of discharge. (Fig. 8) Part 3. Analysis of immune response in patients of chest trauma developing complications Out of the 75 patients included in the study 22 patients (29.33%) developed complications. The immune response of patients developing complications were compared to those who did not develop any complication. Neutrophils The intraneutrophilic cytokines were persistently increased in the complications group with the IL-6(p = 0.0251) and IL-1β(0.0143) levels being significantly higher on the day of admission, but decreased to a comparable level till the day of discharge. The CD66 + levels were significantly lower in the complication group as compared to the counterpart. (Fig. 9) T regulatory cells All the intra T regulatory cell cytokine levels were suppressed in the complications group as compared to the non- complications group with CD25 levels being significantly decreased on Day 5 and on the Day of discharge T helper cells All the intra T helper cell cytokines levels were higher on day of admission in the complications group as compared to those who did not develop complication. But, all the intra T-helper Cell Cytokines gradually decreased in the complications group to levels below those of the non-complications group. None of the results were statistically significant Extracellular Cytokines All the extracellular cytokines with elevated in the complications group as compared to the non-complication group. IFN-γ(p = 0.0022), TNF-α(0.0062), IL-4(p = 0.0128), IL-1β(p = 0.0241), IL-17A(p = 0.0035), IL-6(p = 0.0279) and IL-10(p = 0.023) were raised significantly on day 2 of injury. TNF-α, IL-6 and IL-10 were significantly increased even on day of the discharge. The peak level of all these cytokines were increased on day 2 of the injury. DISCUSSION The last two decades have improved our understanding of the post traumatic immune activation and its role in the prognosis and final clinical outcomes. Emerging evidences point towards the fact that both hyperimmune response by the innate immune system and the counterbalancing suppressive response by the adaptive immune system occur simultaneously and are equally responsible for the final outcome and prognosis ( 5 ). Exploring the role of immune system to manipulate outcomes is the new logical frontier, as is already being done in the fields of oncology and rheumatology and hence further research is warranted in this direction. This study is an attempt to understand the relationship of the severity of trauma and its effect on the local as well as the systemic immune response in a homogenous group of patients with BTC. We also tried to understand the immune response associated with patients who developed complications during their in-hospital stay. Most common mode on injury in our study population was Road traffic injuries 58.6%, which were consistent with previous studies by Kulshrestha et al and Demirhan et al ( 4 , 14 ). The most commonly affected were young males (88%) ,31–40 years of age was the most common age group (26%) affected. This was in agreement with previous studies( 15 ). Neutrophils Visser et al reported transient activation of the neutrophils following BTC( 16 ). We in our study also noted similar findings. The CD66 and the intraneutrophilic cytokines IL6 and IL-1β were all increased in serum as well as in the BAL fluid when compared between the cases and controls. When examined over a period of time the neutrophilic cytokines showed increase in patient with ISS > 15 and TTSS > 5 as compared to the ISS ≤ 15 and TTSS ≤ 5 initially but were comparable more or less on the day of discharge. But the pattern of expression showed a peculiarity where though the CD66 expression followed the above pattern the expression of Intraneutrophilic cytokines did not follow the same pattern. The same pattern could be seen in patients developing complications, the level of neutrophils decreased quickly in patients with complications as compared to those who had an uneventful recovery, but the levels of IL-1β and IL-6 levels did not show decline. This might point towards a differential expression and regulation at the genetic level. Systematic review by Finlay et al reported an initial peak of neutrophils followed by decline in the levels of circulating neutrophils following major trauma between day3 and day5, followed by increase to normal levels and was consistent with this study( 17 ). Following major trauma immature neutrophils replace more mature neutrophils in circulation and the mature neutrophils get sequestrated in the tissues( 18 – 20 ). Dysregulated apoptosis of immune cells following major trauma has also been reported in literature ( 21 ) and all these factors need to be adequately factored in and studied to understand the neutrophil kinetics following major trauma and its effect on the final outcome of the patients. T regulatory cells The T regulatory cells have an important role in modulation of Th1(cell mediated immune response)/Th2 response (humoral immune response). It has been postulated that trauma tilts the immune response to Th 2 response( 22 – 24 ) and maintains peripheral immune tolerance( 25 ). Foxp3 is the signature transcription factor which is important for the lineage commitment and regulatory functions of these T regulatory cells( 26 , 27 ).Cytokines mostly associated with the Treg cells are IL-10 and TGF-β( 27 ). There exists a paracrine positive feedback loop mechanism between FoxP3 and TGF-β helps maintain peripheral immune tolerance and is the principal factor in maintenance of the Treg cells( 28 , 29 ).In our study FOXP3 levels were elevated in trauma patients as compared to the control group both in the serum as well as in the BAL fluid. The FOXP3 levels were elevated in ISS > 15 and TTSS > 5 group on all days as compared to their counterparts. But interestingly the levels of FOP3 were not raised in patients who developed complications. This might point towards a protective role of these T regulatory cells. Intracellular TGF-β and IL-10 levels also showed decline in patients who developed complications. Yamakawa et al demonstrated in a mice model that trauma activates a sub-population of memory like Treg cells by a MHC class II dependent TCR signaling( 30 ). The protective role of Treg cells have been verified by multiple animal-based studies( 31 , 32 ). T helper cells (Th1, Th2, Th9, Th17, Th22) The earlier notion of two terminally differentiated Th1 and Th2 subsets of the T helper cells playing a role in the immune cacophony has been replaced by discovery of newer subsets of the T helper cells (Th9, Th17, Th22). But the exact mechanism and the effect of the microenvironment on the plasticity and the secondary immune response of these cells are poorly understood. Zhang et al studied the dynamic response of the T helper subsets in 7 ICU patients with BTC with 21 healthy volunteers( 33 ). They reported an initial increase in Th1 response which decreased by 2nd week, they reported a statistically significant increase in Th2 response but after 2 weeks. We found a significantly increased levels of Th1 in serum as well as in BAL fluid of cases as compared to controls, which was in agreement with study by Zhang et al ( 33 ). This points towards an early and definite role of cell mediated immunity in the initial response to trauma. The Th2 levels were also elevated in both but without any statistical significance. When compared on the basis of severity of trauma (based on injury scores) the Th1 and Th2 levels were increased on all days in patients with ISS > 15 as compared to patients with ISS ≤ 15, but without any statistical significance. This was in contrary to the results obtained based on the thoracic trauma severity score, the levels of Th1 and Th2 were higher in patients with lower thoracic trauma score (TTSS ≤ 5) as compared to those with higher scores (TTSS > 5). Those patients who developed complications had an initial higher response of Th1 and Th2 levels which declined later, though these levels were without any statistical significance. Khurana et al in a study with 49 polytrauma patients studied the dynamics of Th1 and Th2 in survivors and non survivors reported an higher levels of Th1 and Th2 in survivors, which is in agreement with our results( 34 ). The knowledge about the physiological and pathological roles played by the T regulator cells and Th17 cells in trauma setting is very limited. The stability and plasticity of the Th17 cells is highly debated. Th17 is highly heterogenous with multiple subsets and the functions of these cells remains to be explored( 35 , 36 ). In our study we found the levels of Th17 to be elevated only in the BAL fluid of cases as compared to the controls but without any statistical significance. When studied based on the severity of trauma the Th17 levels were raised only on day 0 in ISS > 15 as well as in TTSS > 5 group as compared to their respective counterparts and the levels gradually decreased, but the results were without any statistical significance. Similarly, the Th17 levels were raised only on day 0 in patients who developed complications and gradually decreased over time as compared to those who did not have any complications. None of the results were statistically significant. The initial increase in Th17 followed by decrease in trauma group was consistent with studies by Khurana et al and Zhang et al ( 33 , 34 ). Additionally, Khurana et al also reported increased Th17 levels in patients who survived as compared to those who succumbed to chest injuries. In our study we found that the levels of Th17 was higher in patients who did not develop any complication as compared to those who developed complications and this was in coherence with our results. Th22 is unique subset of T helper cells as it does not work on other immune effector cells but on the mucosal and epithelial cells and thereby exerting its effect through the innate immune cells( 37 , 38 ). Though they have been largely attributed to be pathological( 39 ). In our study, Th22 levels were significantly increased in both serum as well as in BAL fluid of trauma patients. Th22 levels were elevated in ISS > 15 group as well as in TTSS > 5 group, with statistical significance on day 0. In patients who developed complications the Th22 levels were higher compared to the non-complications group, on all days except for on the day of discharge, results were though not statistically significant. Th22 remains one of the least studied subsets of the T helper cells in a trauma setting. Khurana et al in their study reported increased levels of Th22 in survivors as compared to non-survivors( 34 ). The role Th9 has primarily been evaluated in context of allergic inflammation and autoimmunity and is thought to be associated with Th2 type immune response with immunesuppressive role( 40 ).Our study showed decreased levels of Th9 in chest trauma patients as compared to controls. The levels of Th9 were decreased in ISS > 15 group and TTSS > 5 and complications group except for day 0. The level of Th9 was slightly elevated in the BAL fluids of the cases as compared to controls. Though none of the results were statistically significant. Khurana et al in their study also reported decreasing levels of Th9 cells in chest injury patients and suppressed levels in those who succumbed to their injuries( 34 ). Extracellular cytokines We found an elevated levels of both proinflammatory and anti-inflammatory cytokines in BTC patients. All extracellular cytokine levels except IL-6 were raised on day 0 in chest injury patients, but only TNF-α and TGF-β were significantly raised. The BAL fluid analysis of cases as compared to diseased control showed elevated levels of IL-9, IL-22, IL-17A, IL-6, TGF-β along with IL-4 and Il-1β which were significantly elevated in BAL fluids of the BTC patients. Cytokines IFN-γ, TNF-α, IL-1β and IL-10 raised in the ISS > 15 group and TNF-α, IL-4, IL-17A, IL-06 and IL-10 were raised in the TTSS > 5 group but without a significant p-value. All the cytokines were raised in the complications group as compared to the non-complications group without any statistical significance. An interesting observation was that the cytokines reached its peak on day 2. Bagaria et al in a similar study found elevated levels of IL-1β and IL-10 in both serum and BAL fluid of chest injury patients as compared to controls and elevated levels IL-6 in BAL fluid( 41 ). Khurana et al in their study comparing survivors of chest injury with non survivors reported increased levels of TNF-α, IFN-γ, IL-2, IL-4, IL-9, IL-17A and IL-22 in the survivor group( 34 ). Kumari et al too reported elevated levels of both pro and anti-inflammatory cytokines post chest injury (IL-2, IL-6, IL-9, IL-1β, IFN-γ, TNF-α, IL-17A and IL-22). Elevated levels of IL-1β and IL-22 in the BAL fluid were reported by the same group, both these results were in agreement with our results too( 42 ). This study attempts to understand the complexity of the post trauma immune response and its role in the clinical outcome of patients with chest injuries, with an effort to identify potential target points for further research and immunomodulation. The lack of evidence for efficacy of immunotherapy in trauma may reflect the heterogeneity of immune system and our inability to identify patients who would benefit from such intervention along with paucity of research in this direction. Therefore, there is an urgent need to identify biomarkers which will help in day-to-day clinical practice to accurately predict and treat immunosuppression. Artificial Intelligence (AI) and Machine Learning (ML) Algorithms can be used to identify early intervention markers and develop predictive and prognostication models for complex trauma patients. Similar studies using AI and ML algorithms for predicting risk and identifying immunophenotypes predicting disease severity( 43 – 45 ). Strengths and limitations of the study This study was based on a homogenous group of patients (chest injury) and estimated both intracellular and extra cellular cytokine response along with T helper response and T regulatory response at both local (BAL) and global level(serum). We tried to correlate these levels to the injury severity and development of complications. There were certain limitations to the study like it was a single centre study with only 75 patients restricting generalization. Also, there was no follow up of the patients. Declarations Author Contribution P.K. and P.M. conceptualized and wrote the main manuscript, reviewed and edited both share first authorship, N.R. was involved in methodology, Laboratory processing of samples , analysis and review of resultsAll Authors were involved in conceptualization, analysis of results and review of results References Vos T, Lim SS, Abbafati C, Abbas KM, Abbasi M, Abbasifard M, et al. Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. The Lancet. 2020 Oct;396(10258):1204–22. Roy N, Kizhakke Veetil D, Khajanchi MU, Kumar V, Solomon H, Kamble J, et al. Learning from 2523 trauma deaths in India- opportunities to prevent in-hospital deaths. BMC Health Serv Res. 2017 Dec;17(1):142. Lecky FE, Bouamra O, Woodford M, Alexandrescu R, O’Brien SJ. Epidemiology of Polytrauma. In: Pape HC, Peitzman A, Schwab CW, Giannoudis PV, editors. Damage Control Management in the Polytrauma Patient [Internet]. New York, NY: Springer New York; 2010 [cited 2023 Jul 26]. p. 13–24. Available from: http://link.springer.com/10.1007/978-0-387-89508-6_2 Demirhan R, Onan B, Oz K, Halezeroglu S. Comprehensive analysis of 4205 patients with chest trauma: a 10-year experience☆. Interact Cardiovasc Thorac Surg. 2009 Sep;9(3):450–3. Xiao W, Mindrinos MN, Seok J, Cuschieri J, Cuenca AG, Gao H, et al. A genomic storm in critically injured humans. J Exp Med. 2011 Dec 19;208(13):2581–90. Baker SP, O’Neill B, Haddon W, Long WB. The injury severity score: a method for describing patients with multiple injuries and evaluating emergency care. J Trauma. 1974 Mar;14(3):187–96. Pape HC, Remmers D, Rice J, Ebisch M, Krettek C, Tscherne H. Appraisal of Early Evaluation of Blunt Chest Trauma: Development of a Standardized Scoring System for Initial Clinical Decision Making: J Trauma Inj Infect Crit Care. 2000 Sep;49(3):496–504. 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De AK, Kodys KM, Pellegrini J, Yeh B, Furse RK, Bankey P, et al. Induction of Global Anergy Rather Than Inhibitory Th2 Lymphokines Mediates Posttrauma T Cell Immunodepression. Clin Immunol. 2000 Jul;96(1):52–66. Miller AC, Rashid RM, Elamin EM. The “T” in Trauma: the Helper T-cell Response and the Role of Immunomodulation in Trauma and Burn Patients. J Trauma Inj Infect Crit Care. 2007 Dec;63(6):1407–17. Rocamora-Reverte L, Melzer FL, Würzner R, Weinberger B. The Complex Role of Regulatory T Cells in Immunity and Aging. Front Immunol. 2021 Jan 27;11:616949. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003 Apr;4(4):330–6. Workman CJ, Szymczak-Workman AL, Collison LW, Pillai MR, Vignali DAA. The development and function of regulatory T cells. Cell Mol Life Sci. 2009 Aug;66(16):2603–22. Pyzik M, Piccirillo CA. TGF-β1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets. 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Crosstalk between T Helper Cell Subsets and Their Roles in Immunopathogenesis and Outcome of Polytrauma Patients. Indian J Crit Care Med. 2020 Dec 16;24(11):1037–44. Chen Z, O’Shea JJ. Th17 cells: a new fate for differentiating helper T cells. Immunol Res. 2008 Jun;41(2):87–102. Serve R, Sturm R, Schimunek L, Störmann P, Heftrig D, Teuben MPJ, et al. Comparative Analysis of the Regulatory T Cells Dynamics in Peripheral Blood in Human and Porcine Polytrauma. Front Immunol. 2018 Mar 13;9:435. Sonnenberg GF, Monticelli LA, Alenghat T, Fung TC, Hutnick NA, Kunisawa J, et al. Innate Lymphoid Cells Promote Anatomical Containment of Lymphoid-Resident Commensal Bacteria. Science. 2012 Jun 8;336(6086):1321–5. Stange J, Hepworth MR, Rausch S, Zajic L, Kühl AA, Uyttenhove C, et al. IL-22 Mediates Host Defense against an Intestinal Intracellular Parasite in the Absence of IFN-γ at the Cost of Th17-Driven Immunopathology. J Immunol. 2012 Mar 1;188(5):2410–8. Raphael I, Nalawade S, Eagar TN, Forsthuber TG. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 2015 Jul;74(1):5–17. Tan C, Gery I. The Unique Features of Th9 Cells and their Products. Crit Rev Immunol. 2012;32(1):1–10. Bagaria V, Mathur P, Madan K, Kumari M, Sagar S, Gupta A, et al. Predicting Outcomes After Blunt Chest Trauma—Utility of Thoracic Trauma Severity Score, Cytokines (IL-1β, IL-6, IL-8, IL-10, and TNF-α), and Biomarkers (vWF and CC-16). Indian J Surg. 2021 Apr;83(S1):113–9. Kumari M, Mathur P, Aggarwal R, Madan K, Sagar S, Gupta A, et al. Changes in extracellular cytokines in predicting disease severity and final clinical outcome of patients with blunt chest trauma. Immunobiology. 2021 May;226(3):152087. Saharan SS, Nagar P, Creasy KT, Stock EO, Feng J, Malloy MJ, et al. Machine learning and statistical approaches for classification of risk of coronary artery disease using plasma cytokines. BioData Min. 2021 Dec;14(1):26. Goyal M, Khanna D, Rana PS, Khaibullin T, Martynova E, Rizvanov AA, et al. Computational Intelligence Technique for Prediction of Multiple Sclerosis Based on Serum Cytokines. Front Neurol. 2019 Jul 18;10:781. Cao B, Zhang N, Zhang Y, Fu Y, Zhao D. Plasma cytokines for predicting diabetic retinopathy among type 2 diabetic patients via machine learning algorithms. Aging. 2021 Jan 31;13(2):1972–88. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4253254","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290536110,"identity":"20930e1f-40a7-41eb-a525-05ea4e6ab1db","order_by":0,"name":"Dr.Pranay 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7","display":"","copyAsset":false,"role":"figure","size":56784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of intracellular T regulatory cell cytokine levels based on the severity of thoracic trauma (TTSS).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4253254/v1/fd70a272011a9f3fd81a5cb0.png"},{"id":55007797,"identity":"84b39a68-b101-4340-a6aa-c784a2dbed9e","added_by":"auto","created_at":"2024-04-19 19:01:10","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":559041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of extracellular cytokine levels based on the severity of thoracic trauma (TTSS).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4253254/v1/f3f3a0407b73ecd9fb516957.jpeg"},{"id":55007796,"identity":"05d765cd-af3c-4f47-90e8-dc0ec1aac93d","added_by":"auto","created_at":"2024-04-19 19:01:10","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":39957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of intraneutrophilic cytokines levels of patients who developed complication with those who had an uneventful recovery. Complication group denoted by\u003c/strong\u003e \u003cstrong\u003e(YES).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4253254/v1/fdbaa3db78722846cc6fd297.png"},{"id":57229625,"identity":"c91646c0-1ca8-4f22-8647-b428b366d5d3","added_by":"auto","created_at":"2024-05-27 20:47:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1779629,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4253254/v1/9a452967-6066-4b9e-935b-b82027df71c7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of severity of injury on immune system and its role in patient outcome in patients with Blunt Trauma to the chest","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTrauma is a major public health problem in India and across the globe. Over One-fifth of the global trauma deaths occur in India of which almost 50% are deemed preventable(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Isolated chest injuries account for almost 20\u0026ndash;25% of the trauma burden and is only next to head and abdominal injuries in causing the most deaths in a multiply injured person(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Even in patients with other systems trauma, chest injuries play a major role in the clinical course, management and outcome of the patient. In addition to the anatomical severity the management of chest injury patients is complicated by development of clinical conditions such as Multiple Organ Dysfunction Syndrome (MODS), Acute Respiratory Distress Syndrome (ARDS), pneumonia, Systemic Inflammatory and Anti-Inflammatory Response Syndrome.\u003c/p\u003e \u003cp\u003eTrauma activates the immune system of the body at both the local tissue level as well as the systemic level. The role of immune system and the disorders associated with it significantly effects the outcome of the patients. Hence it is important that these mechanisms involving the hyperimmune response driven by Th1 cells and the immunosuppressive response driven by Th2 response be understood and correlated with the anatomical severity of injury and outcomes in patients with chest trauma. Conventional teaching and early research states that significant trauma leads to a hyperimmune response and any derangement in this response can cause a systemic inflammatory response syndrome. This was believed to be the reason for early peak in mortality following trauma. The systemic anti-inflammatory mechanisms then comes into play to counter and limit the acute hyperimmune response. It was believed that uncontrolled systemic anti-inflammatory response increases the susceptibility to nosocomial infections in the later weeks, with various clinical manifestations such as ARDS, MODS, Ventilator Associated Pneumonia (VAP) and Hospital Associated Pneumonia (HAP). Contrary to the earlier belief new research using genome wide expression analysis shows activation of the pro-inflammatory and anti-inflammatory genes simultaneously, rather than one following the other as believed earlier(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). There is now enough evidence to show that a multi system trauma might lead to an uncontrolled cytokine storm. This cytokine storm is dynamic in nature and the early cytokine response differs from the late cytokine responses.\u003c/p\u003e \u003cp\u003eWhile some patients have an uneventful recovery, others with same kind of injury might have a complicated hospital course. The innate as well as the adaptive immune system play an integral role in the pathophysiology of these patients but unfortunately is poorly understood. This study was designed to understand the effect of trauma and its severity on the innate and adaptive immune effector cell response both at the systemic and local level and investigate the role of these immune responses in development of complications.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Population\u003c/h2\u003e \u003cp\u003eThe study was done with a sample size of 75 blunt chest trauma (BCT) patients which were divided into two arms 50 patients in the non-intubated and 25 patients in the intubated arm (i.e., those patients who required intubation on admission). To measure the systemic response and to investigate the temporal response multiple blood samples were collected on day 0, day 2, day 5 and on the day of discharge. Single sample of bronchoalveolar lavage fluid (BAL) was collected from the intubated patients whenever they underwent fiberoptic bronchoscopy (FOB) during their routine course of the treatment, to study the local immune response. In the control arm blood samples from 30 healthy volunteers were collected and BAL control samples were collected from 10 diseased controls, who were patients of carcinoma of oesophagus who underwent bronchoscopy for evaluation of tracheobronchial involvement of oesophageal malignancy during evaluation of their primary illness in Department of Pulmonology. Only those samples were processed which had no tracheobronchial involvement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInclusion and Exclusion Criteria\u003c/h2\u003e \u003cp\u003ePatients admitted to the hospital within 24 hours of the injury with BCT between the age group of 18yrs to 65yrs were included in the study. Those who arrived later than 24hrs, pregnant patients, patients receiving chemotherapy and immunomodulators, patients with hollow viscus injury, and patients with splenectomy and those suffering from pre-existing co-morbidities \u003cem\u003ee.g.\u003c/em\u003e, Diabetes, Hypertension, Chronic Obstructive Pulmonary Diseases, Chronic Kidney Disease, Chronic Liver Disease were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection and Study\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eBlood and Serum collection and Study\u003c/h2\u003e \u003cp\u003ePeripheral blood samples (5 ml) were collected from BCT patients on days 0,2, 5 and day of discharge. 5ml of blood sample was collected once from hospital staffs for control.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBroncho-Alveolar Lavage (BAL) Fluid Study and collection of BAL fluid\u003c/h2\u003e \u003cp\u003eBronchoalveolar lavage (BAL) fluid 5ml was collected from patients who required intubation and FOB during their routine clinical course. 5ml of BAL sample was collected from the diseased controls. BAL sample was analyzed for intracellular, extracellular cytokine study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eParameters studied\u003c/h2\u003e \u003cp\u003eInjury severity score(ISS)(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) and thoracic trauma severity score(TTSS)(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) was calculated for each patient. Intracellular as well as extracellular levels of Cytokines were measured in both the blood as well as in the BAL fluid as mentioned below.\u003c/p\u003e \u003cp\u003eIntracellular cytokines levels of Neutrophils (CD66b+), T-regulatory(T-reg) cells (CD25+), and T helper cells (CD4+) (Th22, Th1, Th2, Th9 and Th17) levels were measured. Intraneutrophilic cytokine levels of IL6 and IL1β were measured along with intra T regulatory cell cytokines IL-10, TGFβ and FOX-P3 levels.\u003c/p\u003e \u003cp\u003eExtracellular levels of IL-9, IFN-γ, TNF-α, IL-4, IL-22, IL-1β, IL-17A, IL-6, IL-10, TGF-β were measured.\u003c/p\u003e \u003cp\u003eThe clinical course was thoroughly followed till final disposition and the daily developments such as vitals chartings, daily progress, development of complications such as Sepsis(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), HAP, VAP(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), ARDS(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) including death of the patient was studied and noted. Standard definition of sepsis, HAP, VAP and ARDS were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was done using IBM SPSS Statistics version 29 software. Data was presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Unpaired student's t-test was used to determine the significance of difference between the two independent groups among continuous variables. For skewed data a corresponding non-parametric test, that is Mann-Whitney test was applied. Categorical variables were compared by chi- squared test and Fischer's test. Continuous variables were compared using independent t test (for normal distribution) and Wilcoxon rank-sum (for non-normal distribution). Significance was set at 5% level and a p-value less than 0.05 was taken as significant.\u003c/p\u003e \u003cp\u003e Prior approval for the study was taken by the institutional ethical committee IECPG-88/27.02.2020\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND ANALYSIS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eDemography and clinical characteristics\u003c/h2\u003e\n\u003cp\u003eA total of 75 patients with BTC who fulfilled the inclusion and exclusion criteria were included in the study, of which 50 patients did not require ventilatory support and 25 patients required ventilation. Blood and serum samples of these 75 patients were analyzed and compared with 30 healthy volunteers. BAL fluid from 25 intubated patients were compared with 10 diseased controls. Out of these 66 (88%) patients were males and 9 (12%) were females. Mean age of the patients was 43.7\u0026thinsp;\u0026plusmn;\u0026thinsp;14.1 years, the most common age group was between 31\u0026ndash;40 years 26 (34.6%). Most common mode of injury was road traffic injury 44 (58.6%) patients, followed by fall from height 19(25.3%) patients. Of these 26 (34.6%) patients had an ISS score of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e15 and 49(65.3%) had and ISS score \u0026gt;15. 27 (36%) patients had a TTSS score of\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\le\\)\u003c/span\u003e\u003c/span\u003e5 and 48(64%) patients had a TTSS \u0026gt;5. 53 (70.66%) patients had uncomplicated clinical course whereas 22 (29.33%) patients had complicated clinical course.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003ePart 1: Analysis of effect of trauma on immune response; Cases Vs Controls\u003c/h2\u003e\n\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\n\u003ch2\u003eComparison of intracellular cytokines in cases versus heathy controls (day 0)\u003c/h2\u003e\n\u003cp\u003eTo study the change in the baseline cytokines profile of BCT Patients, the intracellular level of neutrophilic and lymphocytic cytokines on the day of admission (day 0) of the BCT patients were compared with healthy controls.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eNeutrophils\u003c/h2\u003e\n\u003cp\u003eComparative analysis showed that the CD66b\u003csup\u003e+\u003c/sup\u003e, IL-1\u0026beta;, IL-6 response was significant higher in the patients\u0026rsquo; group than in the control group \u003cstrong\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e on day 0.(Fig.\u0026nbsp;1)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eT helper cells\u003c/h2\u003e\n\u003cp\u003eComparison of the T-helper intracellular cytokines level between the patients and controls (day 0) shows higher levels of CD4(\u003cstrong\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e, Th22(\u003cspan class=\"BoldUnderline\"\u003ep\u003c/span\u003e\u0026thinsp;\u003cstrong\u003e=\u0026thinsp;0.0001)\u003c/strong\u003e, Th2(0.2158), Th1(\u003cstrong\u003ep\u0026thinsp;=\u0026thinsp;0.0012)\u003c/strong\u003e and lower levels of Th9(0.0592), Th17(0.1317). (Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eT regulatory cells\u003c/h2\u003e\n\u003cp\u003eComparison of T regulatory cells intracellular cytokines showed significantly increased expression of IL-10(\u003cstrong\u003ep\u0026thinsp;=\u0026thinsp;0.0001\u003c/strong\u003e) and FOX-P3 \u003cstrong\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e in cases immediately after trauma (day0). (Fig.\u0026nbsp;3)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eComparison of extracellular cytokines in cases versus heathy controls (day 0)\u003c/h2\u003e\n\u003cp\u003eComparison of the extracellular cytokines levels between the patients and controls shows higher levels of IL-9, IFN-\u0026gamma;, TNF-\u0026alpha;, IL-4, IL-1\u0026beta;, IL-22, IL-10, TGF-\u0026beta; and lower levels of IL-6, IL-17A in cases, TNF-\u0026alpha; (\u003cstrong\u003ep\u0026thinsp;=\u0026thinsp;0.0433\u003c/strong\u003e), TGF-\u0026beta; (\u003cstrong\u003ep\u0026thinsp;=\u0026thinsp;0.0069)\u003c/strong\u003e cytokines attained statistically significant p values.. Mixed patterns of increase in proinflammatory and anti-inflammatory cytokines were noted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBronchoalveolar Lavage (BAL) fluid analysis for Intracellular cytokines in BCT patients vs Diseased Controls.\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003eNeutrophils\u003c/h2\u003e\n\u003cp\u003eSignificantly raised levels of intracellular neutrophilic cytokines [CD66b\u003cstrong\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e, IL-6\u003cstrong\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e, IL-1\u0026beta;(\u003cstrong\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0002)]\u003c/strong\u003e in BAL fluid of BCT patients as compared to the diseased controls. (Fig.\u0026nbsp;4)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003eT regulatory cells\u003c/h2\u003e\n\u003cp\u003eIncreased expression of T regulatory cell cytokines in BAL fluids of BCT patients as compared to the diseased controls. Statistically significant levels were seen in expression of CD25\u003cstrong\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e, TGF-\u0026beta;\u003cstrong\u003e(p\u0026thinsp;=\u0026thinsp;0.0004)\u003c/strong\u003e and FOX-P3\u003cstrong\u003e(p\u0026thinsp;=\u0026thinsp;0.0003).\u003c/strong\u003e (Fig.\u0026nbsp;5)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003eT helper cells\u003c/h2\u003e\n\u003cp\u003eIncreased levels of T helper cell cytokines were noted in BAL fluids of BCT patients when compared to the diseased controls. Significantly increased levels of expression were noted in CD4\u003cstrong\u003e(p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/strong\u003e, Th22\u003cstrong\u003e(p\u0026thinsp;=\u0026thinsp;0.016)\u003c/strong\u003e and Th1\u003cstrong\u003e(0.0049)\u003c/strong\u003e cytokines. Similar response was noted in the BAL fluid when compared with the systemic response with increased CD4 expression and increased levels of Th1 and Th2 expression. Also, the Th22 levels showed increased expression in the BAL fluid as was seen in the systemic response between cases and controls. (Fig.\u0026nbsp;6)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBronchoalveolar Lavage (BAL) fluid analysis for extracellular cytokines in BCT patients vs Diseased Controls.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparison of extracellular cytokines in BAL fluid of patients in BAL fluid showed increased expression of IL-9, IL-4, IL-22, IL-1\u0026beta;, IL-17A, IL-6 and TGF-\u0026beta;. Statistically significant levels were noted in IL-4 \u003cstrong\u003e(p\u0026thinsp;=\u0026thinsp;0.0271)\u003c/strong\u003e and IL-1\u0026beta; \u003cstrong\u003e(p\u0026thinsp;=\u0026thinsp;0.0041).\u003c/strong\u003e Mixed pattern of pro and anti-inflammatory cytokines was noted, similar to what was seen in the serum analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003ePart 2: Analysis of overall injury severity and thoracic injury severity on immune response\u003c/h2\u003e\n\u003cp\u003eThe study effect of severity of trauma on the immune response the ISS was calculated and the patients were divided into two groups of ISS\u0026thinsp;\u0026le;\u0026thinsp;15 and ISS\u0026thinsp;\u0026gt;\u0026thinsp;15(\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e). Similarly, to study the effect of severity of thoracic injuries on the immune response, specific scoring system for thoracic injury i.e., TTSS was used and the patients were divided into two groups of TTSS\u0026thinsp;\u0026le;\u0026thinsp;5 and TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5(\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003eComparison of intracellular/ extracellular cytokines based on Injury severity Score (ISS\u0026thinsp;\u0026le;\u0026thinsp;15 Vs ISS\u0026thinsp;\u0026gt;\u0026thinsp;15)\u003c/h2\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003eNeutrophils\u003c/h2\u003e\n\u003cp\u003eThe levels of intraneutrophilic cytokines were increased on all days (Day 0, day 2, day 5 and day of discharge) in patients with ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 as compared to ISS\u0026thinsp;\u0026lt;\u0026thinsp;15. The level of IL-6 was significantly increased on Day 0 in patients with ISS\u0026thinsp;\u0026gt;\u0026thinsp;15. IL-1\u0026beta; levels were significantly increased on Day 0, Day 2 and Day 5.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003eT regulatory Cell\u003c/h2\u003e\n\u003cp\u003eThe CD25 levels consistently decreased over time in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 as compared to ISS\u0026thinsp;\u0026le;\u0026thinsp;15 group but were insignificant. FOX-P3 levels were increased in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group but were of significant only on day 0.\u003c/p\u003e\n\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n\u003ch2\u003eT helper Cells\u003c/h2\u003e\n\u003cp\u003eAll T helper cells intracellular cytokines (CD4, Th22, Th1, Th2, Th17 and Th9) were increased on the day of injury in patients with severe trauma ISS\u0026thinsp;\u0026gt;\u0026thinsp;15. Only on day 0 levels of CD4+, Th22, Th1, and Th9 were significantly higher in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group. The levels of CD4+, Th22, Th1 and Th2 were increased on all days but were insignificant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n\u003ch2\u003eExtracellular cytokines\u003c/h2\u003e\n\u003cp\u003eThe extracellular cytokines levels of IL-9, IFN-\u0026gamma;, TNF-\u0026alpha;, IL-1\u0026beta;, IL-6, and IL-10 were elevated in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group as compared to the ISS\u0026thinsp;\u0026le;\u0026thinsp;15 group. Though most of the cytokines reached similar levels on the day of discharge. The IL-4 and IL-22 levels were lower in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group when compared with ISS\u0026thinsp;\u0026le;\u0026thinsp;15 group. None of these achieved levels of statistical significance.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n\u003ch2\u003eEffect of severity of thoracic trauma severity on immune response\u003c/h2\u003e\n\u003cdiv id=\"Sec28\" class=\"Section4\"\u003e\n\u003ch2\u003eNeutrophils\u003c/h2\u003e\n\u003cp\u003eThe initial levels (Day 0) of intraneutrophilic cytokines CD66b and IL-1\u0026beta; were increased in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group, but the cytokines levels gradually decreased or became comparable to TTSS\u0026thinsp;\u0026lt;\u0026thinsp;5 group. Though none of the results were statistically relevant.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n\u003ch2\u003eT regulatory cells\u003c/h2\u003e\n\u003cp\u003eCD25 levels were supressed on all time periods on day 0, day 2, day 5 and on the day of discharge in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group as compared to TTSS\u0026thinsp;\u0026le;\u0026thinsp;5. IL-10(p\u0026thinsp;=\u0026thinsp;0.0154), TGF-\u0026beta;(p\u0026thinsp;=\u0026thinsp;o.0006), FOX-P3(0.0001) levels were significantly raised on the day of discharge. (Fig.\u0026nbsp;7)\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eT helper cells\u003c/h3\u003e\n\u003cp\u003eExcept for the levels of Th22 all the T helper intracellular cytokines were supressed in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group as compared to TTSS\u0026thinsp;\u0026le;\u0026thinsp;5 group, but did not reach any statistical significance.\u003c/p\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n\u003ch2\u003eExtracellular cytokines\u003c/h2\u003e\n\u003cp\u003eThe levels of TNF-\u0026alpha;, IL-4, IL-1\u0026beta;, IL-17A, IL-6, IL-10 and TGF-\u0026beta; were high in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group as compared to TTSS\u0026thinsp;\u0026le;\u0026thinsp;5 group. The levels of IL-9(p\u0026thinsp;=\u0026thinsp;0.0064), IL-1\u0026beta;(p\u0026thinsp;=\u0026thinsp;0.0038), IL-17A(p\u0026thinsp;=\u0026thinsp;0.0019), IL-6(p\u0026thinsp;=\u0026thinsp;0.0017), IL-10(p\u0026thinsp;=\u0026thinsp;0.0004), and TGF-\u0026beta;(p\u0026thinsp;=\u0026thinsp;0.0001) were significantly raised in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group even on the day of discharge. (Fig.\u0026nbsp;8)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\n\u003ch2\u003ePart 3. Analysis of immune response in patients of chest trauma developing complications\u003c/h2\u003e\n\u003cp\u003eOut of the 75 patients included in the study 22 patients (29.33%) developed complications. The immune response of patients developing complications were compared to those who did not develop any complication.\u003c/p\u003e\n\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\n\u003ch2\u003eNeutrophils\u003c/h2\u003e\n\u003cp\u003eThe intraneutrophilic cytokines were persistently increased in the complications group with the IL-6(p\u0026thinsp;=\u0026thinsp;0.0251) and IL-1\u0026beta;(0.0143) levels being significantly higher on the day of admission, but decreased to a comparable level till the day of discharge. The CD66\u0026thinsp;+\u0026thinsp;levels were significantly lower in the complication group as compared to the counterpart. (Fig.\u0026nbsp;9)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\n\u003ch2\u003eT regulatory cells\u003c/h2\u003e\n\u003cp\u003eAll the intra T regulatory cell cytokine levels were suppressed in the complications group as compared to the non- complications group with CD25 levels being significantly decreased on Day 5 and on the Day of discharge\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eT helper cells\u003c/h3\u003e\n\u003cp\u003eAll the intra T helper cell cytokines levels were higher on day of admission in the complications group as compared to those who did not develop complication. But, all the intra T-helper Cell Cytokines gradually decreased in the complications group to levels below those of the non-complications group. None of the results were statistically significant\u003c/p\u003e\n\u003ch3\u003eExtracellular Cytokines\u003c/h3\u003e\n\u003cp\u003eAll the extracellular cytokines with elevated in the complications group as compared to the non-complication group. IFN-\u0026gamma;(p\u0026thinsp;=\u0026thinsp;0.0022), TNF-\u0026alpha;(0.0062), IL-4(p\u0026thinsp;=\u0026thinsp;0.0128), IL-1\u0026beta;(p\u0026thinsp;=\u0026thinsp;0.0241), IL-17A(p\u0026thinsp;=\u0026thinsp;0.0035), IL-6(p\u0026thinsp;=\u0026thinsp;0.0279) and IL-10(p\u0026thinsp;=\u0026thinsp;0.023) were raised significantly on day 2 of injury. TNF-\u0026alpha;, IL-6 and IL-10 were significantly increased even on day of the discharge. The peak level of all these cytokines were increased on day 2 of the injury.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe last two decades have improved our understanding of the post traumatic immune activation and its role in the prognosis and final clinical outcomes. Emerging evidences point towards the fact that both hyperimmune response by the innate immune system and the counterbalancing suppressive response by the adaptive immune system occur simultaneously and are equally responsible for the final outcome and prognosis (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Exploring the role of immune system to manipulate outcomes is the new logical frontier, as is already being done in the fields of oncology and rheumatology and hence further research is warranted in this direction. This study is an attempt to understand the relationship of the severity of trauma and its effect on the local as well as the systemic immune response in a homogenous group of patients with BTC. We also tried to understand the immune response associated with patients who developed complications during their in-hospital stay.\u003c/p\u003e \u003cp\u003eMost common mode on injury in our study population was Road traffic injuries 58.6%, which were consistent with previous studies by \u003cem\u003eKulshrestha et al\u003c/em\u003e and \u003cem\u003eDemirhan et al\u003c/em\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The most commonly affected were young males (88%) ,31\u0026ndash;40 years of age was the most common age group (26%) affected. This was in agreement with previous studies(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section2\"\u003e \u003ch2\u003eNeutrophils\u003c/h2\u003e \u003cp\u003e \u003cem\u003eVisser et al\u003c/em\u003e reported transient activation of the neutrophils following BTC(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). We in our study also noted similar findings. The CD66 and the intraneutrophilic cytokines IL6 and IL-1β were all increased in serum as well as in the BAL fluid when compared between the cases and controls. When examined over a period of time the neutrophilic cytokines showed increase in patient with ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 and TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 as compared to the ISS\u0026thinsp;\u0026le;\u0026thinsp;15 and TTSS\u0026thinsp;\u0026le;\u0026thinsp;5 initially but were comparable more or less on the day of discharge. But the pattern of expression showed a peculiarity where though the CD66 expression followed the above pattern the expression of Intraneutrophilic cytokines did not follow the same pattern. The same pattern could be seen in patients developing complications, the level of neutrophils decreased quickly in patients with complications as compared to those who had an uneventful recovery, but the levels of IL-1β and IL-6 levels did not show decline. This might point towards a differential expression and regulation at the genetic level. Systematic review by \u003cem\u003eFinlay et al\u003c/em\u003e reported an initial peak of neutrophils followed by decline in the levels of circulating neutrophils following major trauma between day3 and day5, followed by increase to normal levels and was consistent with this study(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Following major trauma immature neutrophils replace more mature neutrophils in circulation and the mature neutrophils get sequestrated in the tissues(\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Dysregulated apoptosis of immune cells following major trauma has also been reported in literature (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and all these factors need to be adequately factored in and studied to understand the neutrophil kinetics following major trauma and its effect on the final outcome of the patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eT regulatory cells\u003c/h2\u003e \u003cp\u003eThe T regulatory cells have an important role in modulation of Th1(cell mediated immune response)/Th2 response (humoral immune response). It has been postulated that trauma tilts the immune response to Th 2 response(\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and maintains peripheral immune tolerance(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Foxp3 is the signature transcription factor which is important for the lineage commitment and regulatory functions of these T regulatory cells(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).Cytokines mostly associated with the Treg cells are IL-10 and TGF-β(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). There exists a paracrine positive feedback loop mechanism between FoxP3 and TGF-β helps maintain peripheral immune tolerance and is the principal factor in maintenance of the Treg cells(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).In our study FOXP3 levels were elevated in trauma patients as compared to the control group both in the serum as well as in the BAL fluid. The FOXP3 levels were elevated in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 and TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group on all days as compared to their counterparts. But interestingly the levels of FOP3 were not raised in patients who developed complications. This might point towards a protective role of these T regulatory cells. Intracellular TGF-β and IL-10 levels also showed decline in patients who developed complications. \u003cem\u003eYamakawa et al\u003c/em\u003e demonstrated in a mice model that trauma activates a sub-population of memory like Treg cells by a MHC class II dependent TCR signaling(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The protective role of Treg cells have been verified by multiple animal-based studies(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003ch2\u003eT helper cells (Th1, Th2, Th9, Th17, Th22)\u003c/h2\u003e \u003cp\u003eThe earlier notion of two terminally differentiated Th1 and Th2 subsets of the T helper cells playing a role in the immune cacophony has been replaced by discovery of newer subsets of the T helper cells (Th9, Th17, Th22). But the exact mechanism and the effect of the microenvironment on the plasticity and the secondary immune response of these cells are poorly understood. \u003cem\u003eZhang et al\u003c/em\u003e studied the dynamic response of the T helper subsets in 7 ICU patients with BTC with 21 healthy volunteers(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). They reported an initial increase in Th1 response which decreased by 2nd week, they reported a statistically significant increase in Th2 response but after 2 weeks. We found a significantly increased levels of Th1 in serum as well as in BAL fluid of cases as compared to controls, which was in agreement with study by \u003cem\u003eZhang et al\u003c/em\u003e(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This points towards an early and definite role of cell mediated immunity in the initial response to trauma. The Th2 levels were also elevated in both but without any statistical significance. When compared on the basis of severity of trauma (based on injury scores) the Th1 and Th2 levels were increased on all days in patients with ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 as compared to patients with ISS\u0026thinsp;\u0026le;\u0026thinsp;15, but without any statistical significance. This was in contrary to the results obtained based on the thoracic trauma severity score, the levels of Th1 and Th2 were higher in patients with lower thoracic trauma score (TTSS\u0026thinsp;\u0026le;\u0026thinsp;5) as compared to those with higher scores (TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5). Those patients who developed complications had an initial higher response of Th1 and Th2 levels which declined later, though these levels were without any statistical significance. \u003cem\u003eKhurana et al\u003c/em\u003e in a study with 49 polytrauma patients studied the dynamics of Th1 and Th2 in survivors and non survivors reported an higher levels of Th1 and Th2 in survivors, which is in agreement with our results(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe knowledge about the physiological and pathological roles played by the T regulator cells and Th17 cells in trauma setting is very limited. The stability and plasticity of the Th17 cells is highly debated. Th17 is highly heterogenous with multiple subsets and the functions of these cells remains to be explored(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). In our study we found the levels of Th17 to be elevated only in the BAL fluid of cases as compared to the controls but without any statistical significance. When studied based on the severity of trauma the Th17 levels were raised only on day 0 in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 as well as in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group as compared to their respective counterparts and the levels gradually decreased, but the results were without any statistical significance. Similarly, the Th17 levels were raised only on day 0 in patients who developed complications and gradually decreased over time as compared to those who did not have any complications. None of the results were statistically significant. The initial increase in Th17 followed by decrease in trauma group was consistent with studies by \u003cem\u003eKhurana et al\u003c/em\u003e and \u003cem\u003eZhang et al\u003c/em\u003e(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Additionally, \u003cem\u003eKhurana et al\u003c/em\u003e also reported increased Th17 levels in patients who survived as compared to those who succumbed to chest injuries. In our study we found that the levels of Th17 was higher in patients who did not develop any complication as compared to those who developed complications and this was in coherence with our results.\u003c/p\u003e \u003cp\u003eTh22 is unique subset of T helper cells as it does not work on other immune effector cells but on the mucosal and epithelial cells and thereby exerting its effect through the innate immune cells(\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Though they have been largely attributed to be pathological(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In our study, Th22 levels were significantly increased in both serum as well as in BAL fluid of trauma patients. Th22 levels were elevated in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group as well as in TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group, with statistical significance on day 0. In patients who developed complications the Th22 levels were higher compared to the non-complications group, on all days except for on the day of discharge, results were though not statistically significant. Th22 remains one of the least studied subsets of the T helper cells in a trauma setting. \u003cem\u003eKhurana et al\u003c/em\u003e in their study reported increased levels of Th22 in survivors as compared to non-survivors(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe role Th9 has primarily been evaluated in context of allergic inflammation and autoimmunity and is thought to be associated with Th2 type immune response with immunesuppressive role(\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).Our study showed decreased levels of Th9 in chest trauma patients as compared to controls. The levels of Th9 were decreased in ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group and TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 and complications group except for day 0. The level of Th9 was slightly elevated in the BAL fluids of the cases as compared to controls. Though none of the results were statistically significant. \u003cem\u003eKhurana et al\u003c/em\u003e in their study also reported decreasing levels of Th9 cells in chest injury patients and suppressed levels in those who succumbed to their injuries(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eExtracellular cytokines\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe found an elevated levels of both proinflammatory and anti-inflammatory cytokines in BTC patients. All extracellular cytokine levels except IL-6 were raised on day 0 in chest injury patients, but only TNF-α and TGF-β were significantly raised. The BAL fluid analysis of cases as compared to diseased control showed elevated levels of IL-9, IL-22, IL-17A, IL-6, TGF-β along with IL-4 and Il-1β which were significantly elevated in BAL fluids of the BTC patients. Cytokines IFN-γ, TNF-α, IL-1β and IL-10 raised in the ISS\u0026thinsp;\u0026gt;\u0026thinsp;15 group and TNF-α, IL-4, IL-17A, IL-06 and IL-10 were raised in the TTSS\u0026thinsp;\u0026gt;\u0026thinsp;5 group but without a significant p-value. All the cytokines were raised in the complications group as compared to the non-complications group without any statistical significance. An interesting observation was that the cytokines reached its peak on day 2. \u003cem\u003eBagaria et al\u003c/em\u003e in a similar study found elevated levels of IL-1β and IL-10 in both serum and BAL fluid of chest injury patients as compared to controls and elevated levels IL-6 in BAL fluid(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). \u003cem\u003eKhurana et al\u003c/em\u003e in their study comparing survivors of chest injury with non survivors reported increased levels of TNF-α, IFN-γ, IL-2, IL-4, IL-9, IL-17A and IL-22 in the survivor group(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). \u003cem\u003eKumari et al\u003c/em\u003e too reported elevated levels of both pro and anti-inflammatory cytokines post chest injury (IL-2, IL-6, IL-9, IL-1β, IFN-γ, TNF-α, IL-17A and IL-22). Elevated levels of IL-1β and IL-22 in the BAL fluid were reported by the same group, both these results were in agreement with our results too(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study attempts to understand the complexity of the post trauma immune response and its role in the clinical outcome of patients with chest injuries, with an effort to identify potential target points for further research and immunomodulation. The lack of evidence for efficacy of immunotherapy in trauma may reflect the heterogeneity of immune system and our inability to identify patients who would benefit from such intervention along with paucity of research in this direction. Therefore, there is an urgent need to identify biomarkers which will help in day-to-day clinical practice to accurately predict and treat immunosuppression. Artificial Intelligence (AI) and Machine Learning (ML) Algorithms can be used to identify early intervention markers and develop predictive and prognostication models for complex trauma patients. Similar studies using AI and ML algorithms for predicting risk and identifying immunophenotypes predicting disease severity(\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eStrengths and limitations of the study\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThis study was based on a homogenous group of patients (chest injury) and estimated both intracellular and extra cellular cytokine response along with T helper response and T regulatory response at both local (BAL) and global level(serum). We tried to correlate these levels to the injury severity and development of complications.\u003c/p\u003e \u003cp\u003eThere were certain limitations to the study like it was a single centre study with only 75 patients restricting generalization. Also, there was no follow up of the patients.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eP.K. and P.M. conceptualized and wrote the main manuscript, reviewed and edited both share first authorship, N.R. was involved in methodology, Laboratory processing of samples , analysis and review of resultsAll Authors were involved in conceptualization, analysis of results and review of results\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVos T, Lim SS, Abbafati C, Abbas KM, Abbasi M, Abbasifard M, et al. 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J Trauma Inj Infect Crit Care. 1995 Sep;411\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eMortaz E, Zadian SS, Shahir M, Folkerts G, Garssen J, Mumby S, et al. Does Neutrophil Phenotype Predict the Survival of Trauma Patients? Front Immunol. 2019 Sep 6;10:2122. \u003c/li\u003e\n\u003cli\u003eOʼSullivan ST, Lederer JA, Horgan AF, Chin DHL, Mannick JA, Rodrick ML. Major Injury Leads to Predominance of the T Helper-2 Lymphocyte Phenotype and Diminished Interleukin-12 Production Associated with Decreased Resistance to Infection: Ann Surg. 1995 Oct;222(4):482\u0026ndash;92. \u003c/li\u003e\n\u003cli\u003eDe AK, Kodys KM, Pellegrini J, Yeh B, Furse RK, Bankey P, et al. Induction of Global Anergy Rather Than Inhibitory Th2 Lymphokines Mediates Posttrauma T Cell Immunodepression. Clin Immunol. 2000 Jul;96(1):52\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eMiller AC, Rashid RM, Elamin EM. The \u0026ldquo;T\u0026rdquo; in Trauma: the Helper T-cell Response and the Role of Immunomodulation in Trauma and Burn Patients. J Trauma Inj Infect Crit Care. 2007 Dec;63(6):1407\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eRocamora-Reverte L, Melzer FL, W\u0026uuml;rzner R, Weinberger B. The Complex Role of Regulatory T Cells in Immunity and Aging. Front Immunol. 2021 Jan 27;11:616949. \u003c/li\u003e\n\u003cli\u003eFontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003 Apr;4(4):330\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eWorkman CJ, Szymczak-Workman AL, Collison LW, Pillai MR, Vignali DAA. The development and function of regulatory T cells. Cell Mol Life Sci. 2009 Aug;66(16):2603\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003ePyzik M, Piccirillo CA. TGF-\u0026beta;1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets. J Leukoc Biol. 2007 Aug 1;82(2):335\u0026ndash;46. \u003c/li\u003e\n\u003cli\u003eAwasthi A, Carrier Y, Peron JPS, Bettelli E, Kamanaka M, Flavell RA, et al. A dominant function for interleukin 27 in generating interleukin 10\u0026ndash;producing anti-inflammatory T cells. Nat Immunol. 2007 Dec;8(12):1380\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eYamakawa K, Tajima G, Keegan JW, Nakahori Y, Guo F, Seshadri AJ, et al. Trauma induces expansion and activation of a memory-like Treg population. J Leukoc Biol. 2021 Mar 1;109(3):645\u0026ndash;56. \u003c/li\u003e\n\u003cli\u003eJia W, Cao L, Yang S, Dong H, Zhang Y, Wei H, et al. Regulatory T Cells Are Protective in Systemic Inflammation Response Syndrome Induced by Zymosan in Mice. Mosley RL, editor. PLoS ONE. 2013 May 10;8(5):e64397. \u003c/li\u003e\n\u003cli\u003eLi M, Lin YP, Chen JL, Li H, Jiang RC, Zhang JN. Role of Regulatory T cell in Clinical Outcome of Traumatic Brain Injury. Chin Med J (Engl). 2015 Apr 20;128(8):1072\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eZhang Y, Li XF, Wu W, Chen Y. Dynamic changes of circulating T-helper cell subsets following severe thoracic trauma. \u003c/li\u003e\n\u003cli\u003eSagar S, Bhardwaj N, Khurana S, Mathur P, Pal R. Crosstalk between T Helper Cell Subsets and Their Roles in Immunopathogenesis and Outcome of Polytrauma Patients. Indian J Crit Care Med. 2020 Dec 16;24(11):1037\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eChen Z, O\u0026rsquo;Shea JJ. Th17 cells: a new fate for differentiating helper T cells. Immunol Res. 2008 Jun;41(2):87\u0026ndash;102. \u003c/li\u003e\n\u003cli\u003eServe R, Sturm R, Schimunek L, St\u0026ouml;rmann P, Heftrig D, Teuben MPJ, et al. Comparative Analysis of the Regulatory T Cells Dynamics in Peripheral Blood in Human and Porcine Polytrauma. Front Immunol. 2018 Mar 13;9:435. \u003c/li\u003e\n\u003cli\u003eSonnenberg GF, Monticelli LA, Alenghat T, Fung TC, Hutnick NA, Kunisawa J, et al. Innate Lymphoid Cells Promote Anatomical Containment of Lymphoid-Resident Commensal Bacteria. Science. 2012 Jun 8;336(6086):1321\u0026ndash;5. \u003c/li\u003e\n\u003cli\u003eStange J, Hepworth MR, Rausch S, Zajic L, K\u0026uuml;hl AA, Uyttenhove C, et al. IL-22 Mediates Host Defense against an Intestinal Intracellular Parasite in the Absence of IFN-\u0026gamma; at the Cost of Th17-Driven Immunopathology. J Immunol. 2012 Mar 1;188(5):2410\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eRaphael I, Nalawade S, Eagar TN, Forsthuber TG. T cell subsets and their signature cytokines in autoimmune and inflammatory diseases. Cytokine. 2015 Jul;74(1):5\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eTan C, Gery I. The Unique Features of Th9 Cells and their Products. Crit Rev Immunol. 2012;32(1):1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eBagaria V, Mathur P, Madan K, Kumari M, Sagar S, Gupta A, et al. Predicting Outcomes After Blunt Chest Trauma\u0026mdash;Utility of Thoracic Trauma Severity Score, Cytokines (IL-1\u0026beta;, IL-6, IL-8, IL-10, and TNF-\u0026alpha;), and Biomarkers (vWF and CC-16). Indian J Surg. 2021 Apr;83(S1):113\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eKumari M, Mathur P, Aggarwal R, Madan K, Sagar S, Gupta A, et al. Changes in extracellular cytokines in predicting disease severity and final clinical outcome of patients with blunt chest trauma. Immunobiology. 2021 May;226(3):152087. \u003c/li\u003e\n\u003cli\u003eSaharan SS, Nagar P, Creasy KT, Stock EO, Feng J, Malloy MJ, et al. Machine learning and statistical approaches for classification of risk of coronary artery disease using plasma cytokines. BioData Min. 2021 Dec;14(1):26. \u003c/li\u003e\n\u003cli\u003eGoyal M, Khanna D, Rana PS, Khaibullin T, Martynova E, Rizvanov AA, et al. Computational Intelligence Technique for Prediction of Multiple Sclerosis Based on Serum Cytokines. Front Neurol. 2019 Jul 18;10:781. \u003c/li\u003e\n\u003cli\u003eCao B, Zhang N, Zhang Y, Fu Y, Zhao D. Plasma cytokines for predicting diabetic retinopathy among type 2 diabetic patients via machine learning algorithms. Aging. 2021 Jan 31;13(2):1972\u0026ndash;88. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Blunt Chest Trauma, Intracellular cytokines, T- regulatory cytokines, T-helper cells, BAL Fluid, ISS, TTSS, Complications","lastPublishedDoi":"10.21203/rs.3.rs-4253254/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4253254/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChest injuries are one of the most common injuries which are admitted in the trauma and emergency departments. It is known that the immune system affects the outcome of patients with significant trauma though the specific role of the components of immune system still remains to be explored. This study aimed to explore the temporal association of immune system with severity and development of complications in patients with blunt chest trauma (BCT).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e75 patients (50 non-intubated and 25 intubated) satisfying the inclusion and exclusion criteria were enrolled in the study and blood samples were collected on Day 0, Day 2, Day 5 and On the day of discharge. Broncho- alveolar lavage fluid was collected once in the intubated patients. The samples were processed to estimate the levels of intracellular cytokines of Neutrophils, T-regulatory cells and T-helper cells. Extracellular levels of IL-9, IFN-γ, TNF-α, IL-4, IL-22, IL-1β, IL-17A, IL-6, IL-10, TGF-β were estimated in the serum as well as in the BAL fluid. 30 healthy volunteers and 10 patients with esophageal cancer undergoing routine bronchoscopy without tracheobronchial involvement were used as controls. The levels of cytokines were compared between 1) the injured and healthy patients 2) patients with Injury severity Score \u0026lt;15 and \u0026gt;15 3) Patients with thoracic trauma severity score \u0026lt;5 and \u0026gt;5 , 4) patients who had uneventful recovery and patients who had uneventful recovery\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntraneutrophilic levels of IL-6((p\u0026lt;0.001**) ,IL-1β((p\u0026lt;0.001**) , Th22(\u003cu\u003ep\u003c/u\u003e=0.0001), Th1(p=0.0012), Intracellular T-reg cytokines levels of IL-10(p=0.0001) and FOX-P3(p\u0026lt;0.001) levels were higher in the patients group as compared to the control group on day-0 of injury. Extracellular cytokines levels of TNF-α (p=0.0433), TGF-β (p=0.0069) were significantly raised in the patient group.\u003c/p\u003e\n\u003cp\u003eIn the BAL fluid sample intraneutrophilic levels of IL-6(p\u0026lt;0.001), IL-1β(p\u0026lt;0.0002), Th22(p=0.016) and Th1(0.0049), Intracellular T-reg cytokines levels of TGF-β(p=0.0004) and FOX-P3(p=0.0003) were significantly increased in the patient group as compared to the control group. Extracellular levels of IL-4 (p=0.0271) and IL-1β (p=0.0041) were significantly elevated in the BAL fluid of patients.\u003c/p\u003e\n\u003cp\u003ePatients with overall higher injury severity score (ISS\u0026gt;15) had significantly increased intraneutrophilic cytokines as compared to those with ISS\u0026lt;15. The intracellular T-regulatory cytokines and intracellular T helper cell cytokines did not show such consistent significant results. The extracellular cytokines levels also did not show any statistically significant results in severely injured patients as compared to lesser injured group(ISS\u0026gt;15 vs ISS\u0026lt;15)\u003c/p\u003e\n\u003cp\u003eWhen compared on the basis of thoracic trauma severity intraneutrophilic cytokines levels did not show any significant results in patients with TTSS\u0026gt;5 vs TTSS\u0026lt;5. Intracellular T-regulatory cytokine levels of IL-10(p=0.0154), TGF-β(p=o.0006), FOX-P3(0.0001) were significantly increased in TTSS\u0026gt;5 group on the day of discharge. The extracellular levels of IL-9, IL-1β, IL-17A, IL-6, IL-10, and TGF-β were significantly elevated in TTSS \u0026gt;5 group even on the day of discharge.\u003c/p\u003e\n\u003cp\u003eOut of 75 patients 22 (29.33%) developed complications. In the complications group the intraneutrophilic cytokines levels of IL-6(p=0.0251) and IL-1β(p=0.0143) were significantly higher on the day of admission as compared to patients who did not develop any complications. The extracellular cytokines levels showed higher values on day 2 of injury of which IFN-γ(p=0.0022), TNF-α(0.0062), IL-4(p=0.0128), IL-1β(p=0.0241), IL-17A(p=0.0035), IL-6(p=0.0279) and IL-10(p=0.023) were raised significantly on day 2 in complications group as compared to patients without complications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe immune system plays a very significant role in final outcome of patients with chest injury in addition to the anatomical injury. The severity of injury activates the immune system in a proportional manner and further exploration of this role can help in development of prognostic indicators and therapeutic immunomodulation.\u003c/p\u003e","manuscriptTitle":"Effect of severity of injury on immune system and its role in patient outcome in patients with Blunt Trauma to the chest","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-19 19:01:05","doi":"10.21203/rs.3.rs-4253254/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d17fdc5c-c0ec-4aec-8d54-b03e697aaabf","owner":[],"postedDate":"April 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-27T20:39:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-19 19:01:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4253254","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4253254","identity":"rs-4253254","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}