AAST 2013 PLENARY PAPER
Vagal nerve stimulation modulates the dendritic cell profile in posthemorrhagic shock mesenteric lymph Koji Morishita, MD, PhD, Todd W. Costantini, MD, Brian Eliceiri, PhD, Vishal Bansal, MD, and Raul Coimbra, MD, PhD, San Diego, California
BACKGROUND: Previous studies have established that posthemorrhagic shock mesenteric lymph (PHSML) contains proinflammatory mediators, while the cellular basis of PHSML is less well characterized in acute models of injury. CD103+ dendritic cells (DCs) have been identified in the mesenteric lymph (ML) in models of chronic intestinal inflammation, suggesting an important role in the gut response to injury. We have previously demonstrated the ability of vagal nerve stimulation (VNS) to prevent gut barrier failure after trauma/hemorrhagic shock (T/HS); however, the ability of VNS to alter ML DCs is unknown. We hypothesized that the CD103+ MHC-II+ DC population would change in PHSML and that VNS would prevent injury-induced changes in this population in PHSML. METHODS: Male Sprague-Dawley rats were randomly assigned to trauma/sham shock or T/HS. T/HS was induced by midline laparotomy and 60 minutes of HS (blood pressure, 35 mm Hg), followed by fluid resuscitation. A separate cohort of animals underwent cervical VNS after the HS phase. Gut tissue was harvested at 2 hours after injury for histologic analysis. ML was collected during the pre-HS, HS, and post-HS phase. For flow cytometric analysis, ML cells were subjected to staining with CD103 and MHC-II antibodies, and this cell population was compared in the pre-HS and post-HS phase from the same animal. The CD4+Foxp3+ cell (T reg) population in the ML node (MLN) was also tested to determine effects of CD103+ DC modulation in the ML. RESULTS: VNS reduced histologic gut injury and ML flow seen after injury. The CD103+ MHC-II+ DC population in the PHSML was significantly decreased compared with pre-HS and was associated with decreased T reg expression in the MLN. VNS prevented the injury-induced decrease in the CD103+ MHC-II+ DC population in the ML and restored the T reg population in the MLN. CONCLUSION: These findings suggest that VNS mediates the inflammatory responses in ML DCs and MLN T reg cells by affecting the set point of T/HS responsiveness. (J Trauma Acute Care Surg. 2014;76: 610Y618. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Hemorrhagic shock; mesenteric lymph; intestinal inflammation; inflammatory cells; rats.
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rauma/hemorrhagic shock (T/HS)Yinduced gut barrier failure is known to initiate a systemic inflammatory response.1 Following T/HS, the injured gut releases inflammatory mediators into the mesenteric lymph (ML)2 that cause endothelial and red blood cell dysfunction, activate neutrophils, and cause distant organ injury.2 Identification of the specific proinflammatory mediators that travel through the ML and drive the systemic inflammatory response syndrome response has been the subject of investigation and has previously focused on the activity of biologically active lipids in the ML.3,4 Changes in activation and trafficking of inflammatory cells from the injured intestine through the ML may also be responsible for driving the systemic inflammatory response syndrome response to injury.
Submitted: July 31, 2013, Revised: November 20, 2013, Accepted: November 21, 2013. From the Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California, San Diego Health Sciences, San Diego, California. This study was presented at the 72nd annual meeting of the American Association for the Surgery of Trauma, September 18Y21, 2013, in San Francisco, California. Address for reprints: Todd W. Costantini, MD, Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California, San Diego Health Sciences, 200 W Arbor Dr, #8896, San Diego, CA 92103-8896; email: [email protected]. DOI: 10.1097/TA.0000000000000137
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The cellular component of ML consists of lymphocytes and nonlymphoid cells such as dendritic cells (DCs).5 DCs are located in the lamina propria of the gut and sample luminal antigens that may be present after gut barrier failure. The DCs continuously migrate from the intestine to the ML nodes (MLNs) via ML and are crucial in balancing immunity and tolerance in the intestine.6 While intestinal DCs have been shown to be altered in gut inflammation,7 ML DCs are less well characterized in acute models of injury. Rat ML DCs are identified by their high cell surface expression of the integrin >E (CD103) and MHC-II.6 Previous study demonstrated that CD103+ DCs were dramatically reduced in the gut of colitic mice,7 which suggests an important role for this cell population in the gut immune responses. Furthermore, CD103+ DCs are thought to play an important role in the generation of Foxp3+ T reg cells in the gut draining lymphoid tissues, where Foxp3+ T regs are critical in promoting tolerance to inflammation.8 Vagal nerve stimulation (VNS) attenuates the systemic inflammatory response to infection, reduces systemic cytokine release, and prevents the development of shock in animals with lethal endotoxemia.9 Our laboratory has demonstrated that VNS has a marked effect on intestinal barrier function and gut inflammation after acute injury10,11 and attenuates acute lung injury.12 Other groups have shown that VNS prevents T/HS-induced gut injury, decreases toxic ML, and attenuates acute lung injury; however, the mechanism by which VNS alters the inflammatory state of the ML is unknown.13 J Trauma Acute Care Surg Volume 76, Number 3
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Moreover, VNS has recently been considered to control intestinal immune homeostasis by altering the balance between tolerance and inflammation in the gut.14 The aim of our study was to measure changes in the DC population in the ML after T/HS. We hypothesized that (1) the CD103+ MHC-II+ ML DC and CD4+Foxp3+ MLN T reg cell populations would change after T/HS, indicating a shift toward an inflammatory phenotype, and that (2) VNS would prevent T/HS-induced change in these populations of DCs and T reg cells, thus altering the inflammatory set point in the gut.
MATERIALS AND METHODS
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identical anesthesia and surgical procedure without hemorrhage. Each animal’s body temperature was maintained at 37-C.
VNS A cohort of animals underwent right cervical neck incision followed by right cervical VNS immediately after T/HS insult. Stimulation of vagus nerve was performed using a VariStim II probe (Medtronic Xomed, Jacksonville, FL) at 2 mA, intermittently for 10 minutes. Following nerve stimulation, the incision was closed with 4-0 silk suture. Sham animals underwent right cervical incision and exposure of the vagus nerve but did not receive electrical stimulation.
T/HS Model Male Sprague-Dawley rats weighing 280 g to 300 g were obtained from Harlan Laboratories (Placentia, CA). All animal experiments were approved by the University of California San Diego Institutional Animal Care and Use Committee. Animals were anesthetized with ketamine (50 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg; Sigma Chemical, St. Louis, MO), and the left femoral artery and vein were cannulated with a polyethylene tube (PE-50). Nonlethal hemorrhagic shock was induced via femoral vein cannulation until the mean arterial pressure (MAP) was reduced to 35 mm Hg and maintained for 60 minutes. At the end of shock period, animals in the T/HS group were resuscitated with shed blood plus two times shed blood volume in normal saline (Baxter, Deerfield, IL). The MAP was continuously monitored using the femoral arterial catheter (Philips V24/26, Andover, MA). The trauma/sham shock (T/SS) animals underwent the
Collection of ML and MLNs The ML duct was exposed, and the efferent mesenteric lymphatic was cannulated (PE-50). ML was collected on ice during the pre-HS phase (30 minutes), HS phase (60 minutes), and post-HS phase (120 minutes) by definition (Fig. 1A). The cell pellet was prepared for flow cytometric analysis as previously described.15 MLNs were harvested at 24 hours following resuscitation and cut into pieces and incubated for 20 minutes under agitation at 37-C in the presence of Collagenase/Dispase (Roche Diagnostic, Indianapolis, IN) and DNase I (NEW ENGLAND BioLabs, Ipswich, MA). The tissue was then passed through a 70-Km membrane to generate single-cell suspensions and prepared for flow cytometric analysis. Cell count and viability were obtained using a hemocytometer (Countess automatched cell counter, Invitrogen, Grand Island, NY).
Figure 1. Effects of VNS on ML after T/HS. A, ML was collected during the pre-HS phase (30 minutes), HS phase (60 minutes), and post-HS phase (60 minutes 2). ML flow (KL/h) (B), ML cell viability (%) (C), and total ML cell (106 cells/mL) (D) were evaluated at each phase. n = 5 in all groups, Data shown as mean (SD). *p G 0.05 pre-HS vs. post-HS. †p G 0.05 T/HS vs. T/HS + VNS. * 2014 Lippincott Williams & Wilkins
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Histologic Evaluation At 2 hours after injury, segments of distal ileum were removed and fixed in 10% buffered formalin, embedded in paraffin, and sectioned. Hematoxylin and eosin staining of the intestine was performed by the University California, San Diego Histology Core Services. An investigator blinded to experimental groups analyzed multiple fields from section of ileum imaged at 20 and 60 with a light microscope. The ileum sections were classified according to the degree of tissue lesion according Chiu et al.16
Flow Cytometric Analysis The expression of cell surface molecules on DCs and regulatory T cell (T reg) cells were determined by flow cytometry. ML and MLNs cells (1.0 106 cells/mL) were incubated with anti-rat RTB1 (MHC-II)-PerCP (clone OX-6), anti-rat CD4-APC (OX-35), PerCP mouse IgG1 J isotype control antibody (MOCP-31C) (BD Bioscience, San Diego, CA), anti-rat CD103-FITC (OX-62), anti-rat CD11bc-APC (OX-42), anti-rat CD80-PE (3H5), anti-rat CD86-PE (24F), FITC-mouse IgG1 J isotype control antibody (MOCP-21), PEmouse IgG1 J isotype control (FC) antibody (MOCP-21), and APC-mouse IgG2a J isotype control antibody (MOPC-173) (BioLegend, San Diego, CA) and for 30 minutes at 4-C. Intracellular staining for Fox3 (FoxP3-Alexa Fluor 488 [150 D], BioLegend) was performed using Cytofix/Cytoperm kit (BD Biosciences). Flow cytometry was performed with a BD Accuri C6 (BD Bioscience), and 10,000 events were collected for analysis. Data were analyzed with FlowJo (Three Star, Ashland, OR) software.
Statistical Analysis Data were expressed as mean (SD). Analysis of variance with Student-Newman-Keuls post hoc analysis or Student’s t test was performed with appropriate. Analysis of gut injury grading was performed using Kruskal-Wallis nonparametric analysis of variance test. Statistical significance was determined based on p G 0.05.
RESULTS Effects of VNS on ML After T/HS There was no difference in MAP between T/HS and T/ HS + VNS group before, during, or after HS. The total shed blood volume was similar between T/HS and T/HS + VNS groups (Table 1). The ML flow volume at 60 minutes post-HS increased significantly when compared with the pre-HS phase (2,145.0 [500.5] KL/h vs. 792.0 [317.7] KL/h, p G 0.05). VNS
limited the T/HS-induced increase in ML flow at 60 minutes (1,370.0 [432.4] KL/h vs. 2,145.0 [500.5] KL/h, p G 0.05) and 120 minutes post-HS (238.0 [82.2] KL/h vs. 474.0 [150.1] KL/h, p G 0.05) (Fig. 1B). The cell viability of ML was greater than 85% in all groups (Fig. 1C). ML cell counts at 60 minutes postHS and 120 minutes post-HS were decreased compared with that of pre-HS (p G 0.05). However, there was no difference in the cell count between T/HS and T/HS + VNS at each phase (Fig. 1D).
Effects of VNS on Gut After T/HS Based on the previously established effect of T/HS on the integrity of the gut barrier,1 histologic analysis was performed on representative tissue samples. T/HS caused histologic gut injury characterized by villous blunting and necrosis of the villous tips, consistent with previous report.1,16 At 2 hours after injury, the histologic appearance of the distal ileum of VNStreated animals was similar to that of the T/SS animals, demonstrating the protective effects of VNS on the development of intestinal injury after T/HS (Fig. 2A). In addition, the histologic injury score of the T/HS group was significantly higher than those obtained for the T/SS, T/SS+VNS, and T/HS + VNS groups (p G 0.05) (Fig. 2B).
Surface Phenotype of ML Cells Following T/HS Classically, DCs express MHC-II, CD103, CD11c, CD80, and CD8617 on their surface. To determine the surface expression of ML cells from sham animals, cells were stained with MHC-II, CD103, CD11bc, CD80, CD86, CD4 antibodies, and isotype-matched control antibodies for each of the surface marker antibodies and assessed using flow cytometry. As shown in Figure 3A, CD103, MHC-II, and CD4 expressions were detected on ML cell surface from sham animals. Next, we examined whether the expressions of CD103, MHC-II, and CD4 on ML cells changed after T/HS. The serial analysis of ML established consistent baseline measurements in each animal and allowed us to quantify the relative change in various cell populations after injury. ML cells were compared in the pre-HS (baseline) and post-HS (120 minutes) phases from the same animals. MHC-II expression on ML cells was significantly decreased following T/HS (n = 5, p G 0.05), while CD103 and CD4 expression on ML cells were unchanged (Fig. 3B and C).
VNS Prevents the Shock-Induced Decrease of CD103+ MHC-II+ DCs in the ML Based on our previous studies demonstrating VNSmediated protection of the gut in acute injury models,10,11
TABLE 1. T/HS Model MAP (Pre-HS), mm Hg MAP (HS), mm Hg MAP (Post-HS), mm Hg Shed blood volume, mL
T/SS
T/SS + VNS
T/HS
T/HS + VNS
p
97.0 (19.8) V 116.3 (26.4) V
84.2 (9.7) V 114.0 (23.3) V
85.6 (4.5) 35.1 (1.1) 100.3 (12.7) 7.5 (0.7)
94.2 (12.0) 35.5 (2.0) 121 (5.1) 7.8 (1.4)
0.33 0.19 0.21 0.72
Male Sprague-Dawley rats were bled to a MAP of 35 mm Hg for 1 hour and then reperfused with their own shed blood and normal saline. A separate cohort of animals underwent cervical VNS after the HS phase. The MAP was continuously monitored during the experiments. n = 5 in all groups, Data shown as mean (SD).
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Figure 2. Effects of VNS on gut after T/HS. A, Section of distal intestine harvested 2 hours after T/HS and hematoxylin and eosin staining of small intestine was performed. Top row, 20 magnification; bottom row, 60 magnification. B, Graph displays gut injury scores (Chiu score) n = 4Y6, *p G 0.05 compared with T/HS group.
we considered that VNS treatment after T/HS might limit the injury-induced decrease in CD103+ MHC-II+ ML DC population. As shown in Figure 4A and B, the CD103+ MHCII+ ML DC population at 120 minutes post-HS was significantly decreased compared with that of pre-HS (pre-HS, 8.5% [0.7%] vs. 5.2% [0.7%], p G 0.05). Performing VNS prevented the decrease in this population caused by acute injury (pre-HS, 8.0% [0.6] vs. 8.2% [2.9%], p = 0.90). In addition, the serial analysis of CD103+ MHC-II+ ML DC population revealed that this population was significantly decreased at 60 minutes (65.9 [5.7] vs. 100%, p G 0.05) and 120 minutes (60.4 [18.9] vs. 100%, p G 0.05) post-HS when compared with that of pre-HS and performing VNS prevented the decrease in this population at 120 minutes post-HS caused by acute injury (102.4% [35.3%] vs. 60.4% [18.9%], p G 0.05) (Fig. 4C).
VNS Prevents the T/HS-Induced Decrease in Foxp3+ T reg Cells in the MLNs CD103+ DCs are thought to play an important role in the generation of Foxp3+ T reg in MLNs.8 We therefore examined the Foxp3+ T reg cell population in MLNs after T/HS
to determine the downstream effects of changes in the CD103+ DC population. The CD4+Foxp3+ MLN cell population was significantly decreased after T/HS. VNS prevented the injury-induced decrease in the population of CD4+Foxp3+ cells in the MLN (1.9% [0.8%] vs. 4.7% [0.8%], p G 0.05) (Fig. 5A and B).
DISCUSSION The development of trauma systems, resuscitation protocols, and advances in critical care has improved survival in patients with severe injury.18 Seriously injured patients frequently develop late complications including nosocomial infections and organ failure, which continue to be the leading cause of death after acute injury.18,19 Previous studies have demonstrated that T/HS causes a marked alternation in many immune functions, including T-cell activation and proliferation, cytokine release, and the antigen presentation functions of DCs.20Y22 Murine and human studies have shown that there is a significant loss of DCs after T/HS.20,23 T/HS induces depressed splenic DC maturation and suppressed DC antigen presentation
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Figure 3. Surface phenotype of ML cells following T/HS. A, ML cells from sham animals were stained with antibodies and analyzed by flow cytometry. Filled histograms are isotype controls; unfilled histograms show staining for the named (CD103, MHC II, CD11bc, CD80, CD86, and CD4) antigens. B, ML was collected during the pre-HS and post-HS phases. Cells were stained with MHC-II and CD103 antibodies and analyzed by flow cytometry. Histograms represent surface expression of CD103 and MHC-II on ML cells. Overlays indicate pre-HS versus post-HS (120 minutes) data. Unfilled histogram: pre-HS; filled histogram: post-HS (120 minutes). C, Bar graphs show the kinetics of CD103+, MHC-II+, and CD4+ ML cells after T/HS. n = 5 in all groups. Data shown as mean (SD). *pG 0.01, pre-HS versus post-HS (120 minutes)
function in mice.20 It has been reported that in trauma patients, monocyte conversion to immature DCs is impaired after T/HS as well.23 The intestinal mucosa contains a complex population of inflammatory cells, including macrophages and DCs, which together are thought to play a central role in regulating mucosal innate and adaptive immune responses in both the steady-state and inflammatory setting.24 DCs are potent antigen-presenting cells and are key modulators of the immune response.12 Phenotypically different populations of DCs have been identified in the intestine lamina propria, Peyer’s patches, intestinal lymphoid follicles, MLNs, and ML.6,25 DCs migrate from the intestine to the MLNs through the intestinal lymphatics. Murine intestinal DC populations are defined by expression of the integrins CD103 and CD11c and have been further subdivided according to CD11b expression.26 Moreover, thoracic and ML DCs can be identified by their expression of both CD103 and MHC-II.15,26 CD103+ DCs have been shown in the gut and MLNs in models of chronic gut inflammation.7,27 614
CD103+ DCs are reduced in the gut and MLNs of colitic mice.7 In addition, CD103+ DCs are absent from inflamed area in ileal sections in a rat model of indomethacin-induced enteritis during acute stage of inflammation.28 Of interest, CD103+ DC deficiency contributes to the perpetuation of ileitis, supporting a protective function for this population.29 Therefore, CD103+ DCs could play important roles in the regulation of homeostatic balance between mucosal immunity and tolerance in the gastrointestinal tract. Recent work has demonstrated that migrating gut CD103+ DCs prominently induce the development of Foxp3+ T reg cells in the MLNs.8 Foxp3 is a transcription factor required for the development of T reg cells. Foxp3+ T reg cells function to maintain immune tolerance and prevent inflammatory disease.30 Among DCs, the enzymes that convert vitamin A into retinoic acid are most prominently expressed in a population of CD103+ DCs. Retinoic acid promotes the peripheral differentiation of induced Foxp3+ T reg cell numbers and function during inflammatory responses. Thus, CD103+ DCs are thought to be the key factors involved in the * 2014 Lippincott Williams & Wilkins
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Figure 4. VNS prevents the shock-induced decrease of CD103+ MHC-II+ ML DCs. A, Numbers in the FACS plot show the percent frequency of the CD103+ MHC-II+ DC population. The percent frequency of this cell population was compared in the pre-HS and post-HS phases (120 minutes) from the same animals. B, Bar graphs show the percent frequency of CD103+ MHC-II+ ML DC populations. This cell population was compared between pre-HS and post-HS phases (120 minutes). Values (%) are expressed as mean (SD), n = 4Y5. *p G 0.05, pre-HS versus post-HS (120 minutes). C, The serial analysis of ML established consistent baseline measurements in each animal and allowed us to quantify the relative change in various cell populations after injury. Percent changes in CD103+ MHC-II+ DCs were determined to be compared in the pre-HS phase (baseline) and post-HS phase from the same animals. Data (%) shown as mean (SD). *p G 0.05, pre-HS versus post-HS, †p G 0.05, T/HS versus T/HS + VNS.
induction of T reg cells and promote tolerance to inflammation in the gut.8 In the current study, CD103+ ML DCs and Foxp3+ MLNs T reg cells were significantly decreased after T/HS, suggesting a shift to an inflammatory phenotype in the gut. Previous investigators demonstrated the ability of VNS to prevent intestinal barrier failure, intestinal inflammation, and the production of biologically active ML in models of acute injury.1,10,11,13,31,32 In the present study, we found that performing VNS after acute injury alters T/HS-induced gut injury. This protective effect correlates with our previous published study, showing the effect of VNS after burn injury.11 In addition, we demonstrated that VNS significantly limited injury-induced increased in ML flow. ML flow is known to be modulated by multiple factors, such as gastrointestinal peristalsis, autonomic nerves, and hemorrhage.5 Recent studies have shown that the vagal innervations of the gastrointestinal
tract play a major role in controlling intestinal immune activation;14 however, the mechanism by which VNS exerts its anti-inflammatory effects has not been defined. Work in our laboratory first demonstrated that VNS-induced gut protection is independent of the spleen,33 a departure from previous studies by Tracey et al.34 looking at systemic inflammation where VNS protection is spleen dependent. This finding was recently advanced in a study by Matteoli et al.,35 which found that VNS protection in the gut is independent of T cells and signals through myeloid cells in the gut. Our present results demonstrated that VNS prevents the T/HS-induced decrease in CD103+ MHC-II+ ML DC and Foxp3+ MLNs T reg cell populations in the early phases of injury. To our knowledge, this is the first study directly examining the effects of VNS on the ML DCs and MLNs T reg cells after acute injury. Several immune cells express
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Figure 5. VNS prevents the shock-induced decrease of CD4+ Foxp3+ T reg cells in the MLNs. At 24 hours following resuscitation, MLN cells were isolated and stained with CD4 and Foxp3 antibodies and analyzed by flow cytometry. A, Representative flow cytometry data demonstrate a decrease in CD4+Foxp3+ T regs after T/HS. VNS normalizes the expression of CD4+Foxp3+ T reg in the MLN. B, Graph demonstrating the percentage of CD4+Foxp3+ T regs present in the MLN. Values are expressed as mean (SD), n = 4Y5. *p G 0.05 compared with T/HS group.
various nicotinic acetylcholine receptor (nAChR) subtypes. Mouse DCs are known to express nAChR >2, >5, >6, >7, A2, A4 subunits.36 In vitro studies have shown that nicotine activates DCs and augments their capacity to stimulate T-cell proliferation and cytokines secretion.37 >7 nAChR is also thought to be expressed on CD4+CD25+ T reg cells, and the upregulation of Foxp3 expression on CD4+CD25+ T reg cells occurs in the presence of nicotine stimulation in vitro study.38 These effects of nicotine on the functional properties of DCs and T reg cells might correlate with the results described in our current study regarding the immunomodulatory effect of VNS after T/HS. Finally, CD103j and CD103+ lymph DCs have distinct effects on the differentiation of primed T lymphocytes.39 The balance between CD103j DCs, CD103+ DCs, and macrophages is thought to be critical for maintaining intestinal immune homeostasis.39,40 Therefore, investigating how the functions of CD103j and CD103+ lymph DCs change in response to acute injury of intestinal immune responses will provide essential insights into the development of intestinal immunopathology. 616
In summary, T/HS induced gut injury and depressed ML flow, CD103+ MHC-II+ ML DC, and Foxp3+ MLN T reg cell populations. VNS modulates ML CD103+ DCs trafficking to the MLNs and T reg cell generation in the MLNs, thus altering the gut inflammatory response to acute injury. Treatments that cause signaling via the vagus nerve may represent an ideal strategy to limit the systemic inflammatory response to severe trauma. AUTHORSHIP K.M. performed the surgery and sample collection in the animal experiment. K.M. and B.E. performed the data collection and analysis of flow cytometry. K.M., T.W.C., B.E., V.B., and R.C. conceived the study and participated in its design and coordination. K.M., T.W.C., B.E., and R.C. drafted the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGMENT We thank Ann-Marie Hageny for her technical assistance with the flow cytometry.
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DISCLOSURE This study was funded in part by the American Association for the Surgery of Trauma Research and Education Foundation Scholarship Award (to T.W.C.).
REFERENCES 1. Reys LG, Ortiz-Pomales YT, Lopez N, Cheadle G, de Oliveira PG, Eliceiri B, Bansal V, Costantini TW, Coimbra R. Uncovering the neuroentericpulmonary axis: vagal nerve stimulation prevents acute lung injury following hemorrhagic shock. Life Sci. 2013;92:783Y792. 2. Deitch EA. Gut lymph and lymphatics: a source of factors leading to organ injury and dysfunction. Ann N Y Acad Sci. 2010;1207(Suppl 1): E103YE111. 3. Morishita K, Aiboshi J, Kobayashi T, Mikami S, Yokoyama Y, Ogawa K, Yokota H, Otomo Y. Lipidomics analysis of mesenteric lymph after trauma and hemorrhagic shock. J TraumaAcute Care Surg. 2012;72:1541Y1547. 4. Moore EE, Organ CH Jr. Memorial lecture: splanchnic hypoperfusion provokes acute lung injury via a 5-lipoxygenase-dependent mechanism. Am J Surg. 2010;200:681Y689. 5. Fanous MY, Phillips AJ, Windsor JA. Mesenteric lymph: the bridge to future management of critical illness. JOP. 2007;8:374Y399. 6. Milling S, Yrlid U, Cerovic V, MacPherson G. Subsets of migrating intestinal dendritic cells. Immunol Rev. 2010;234:259Y267. 7. Strauch UG, Grunwald N, Obermeier F, Gurster S, Rath HC. Loss of CD103+ intestinal dendritic cells during colonic inflammation. World J Gastroenterol. 2010;16:21Y29. 8. Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, Belkaid Y, Powrie F. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acidYdependent mechanism. J Exp Med. 2007;204:1757Y1764. 9. Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI, Watkins LR, Abumrad N, Eaton JW, Tracey KJ. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature. 2000;405:458Y462. 10. Costantini TW, Bansal V, Peterson CY, Loomis WH, Putnam JG, Rankin F, Wolf P, Eliceiri BP, Baird A, Coimbra R. Efferent vagal nerve stimulation attenuates gut barrier injury after burn: modulation of intestinal occludin expression. J Trauma. 2010;68:1349Y1354. 11. Krzyzaniak M, Peterson C, Loomis W, Hageny AM, Wolf P, Reys L, Putnam J, Eliceiri B, Baird A, Bansal V, et al. Postinjury vagal nerve stimulation protects against intestinal epithelial barrier breakdown. J Trauma. 2011;70:1168Y1175. 12. Ruane DT, Lavelle EC. The role of CD103(+) dendritic cells in the intestinal mucosal immune system. Front Immunol. 2011;2:25. 13. Levy G, Fishman JE, Xu D, Chandler BT, Feketova E, Dong W, Qui Y, Alli V, Ulloa L, Deitch EA. Parasympathetic stimulation via the vagus nerve prevents systemic organ dysfunction by abrogating gut injury and lymph toxicity in trauma and hemorrhagic shock. Shock. 2013;39:39Y44. 14. Matteoli G, Boeckxstaens GE. The vagal innervation of the gut and immune homeostasis. Gut. 2012;62:1214Y1222. 15. Milling SW, Jenkins C, MacPherson G. Collection of lymph-borne dendritic cells in the rat. Nat Protoc. 2006;1:2263Y2270. 16. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg. 1970;101:478Y483. 17. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245Y252. 18. Diebel LN, Liberati DM. Estrogen impairs pulmonary microvascular response to gut-derived mediators after shock conditions. J Trauma. 2011;71:656Y662. 19. Baue AE. Multiple organ failure, multiple organ dysfunction syndrome, and systemic inflammatory response syndrome. Why no magic bullets? Arch Surg. 1997;132:703Y707. 20. Kawasaki T, Fujimi S, Lederer JA, Hubbard WJ, Choudhry MA, Schwacha MG, Bland KI, Chaudry IH. Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice. J Immunol. 2006;177:4514Y4520. 21. Xu YX, Ayala A, Chaudry IH. Prolonged immunodepression after trauma and hemorrhagic shock. J Trauma. 1998;44:335Y341.
22. Zellweger R, Ayala A, DeMaso CM, Chaudry IH. Trauma-hemorrhage causes prolonged depression in cellular immunity. Shock. 1995;4:149Y153. 23. De AK, Laudanski K, Miller-Graziano CL. Failure of monocytes of trauma patients to convert to immature dendritic cells is related to preferential macrophage-colony-stimulating factor-driven macrophage differentiation. J Immunol. 2003;170:6355Y6362. 24. Kelsall B. Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages. Mucosal Immunol. 2008;1: 460Y469. 25. Iwasaki A. Mucosal dendritic cells. Annu Rev Immunol. 2007;25:381Y418. 26. Farache J, Zigmond E, Shakhar G, Jung S. Contributions of dendritic cells and macrophages to intestinal homeostasis and immune defense. Immunol Cell Biol. 2013;91:232Y239. 27. Rutella S, Locatelli F. Intestinal dendritic cells in the pathogenesis of inflammatory bowel disease. World J Gastroenterol. 2011;17:3761Y3775. 28. Silva MA, Porras M, Jury J, Vergara P, Perdue MH. Characterization of ileal dendritic cell distribution in a rat model of acute and chronic inflammation. Inflamm Bowel Dis. 2006;12:457Y470. 29. Annacker O, Coombes JL, Malmstrom V, Uhlig HH, Bourne T, JohanssonLindbom B, Agace WW, Parker CM, Powrie F. Essential role for CD103 in the T cell-mediated regulation of experimental colitis. J Exp Med. 2005;202:1051Y1061. 30. Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol. 2011;11:119Y130. 31. Krzyzaniak MJ, Peterson CY, Cheadle G, Loomis W, Wolf P, Kennedy V, Putnam JG, Bansal V, Eliceiri B, Baird A, et al. Efferent vagal nerve stimulation attenuates acute lung injury following burn: the importance of the gut-lung axis. Surgery. 2011;150:379Y389. 32. Costantini TW, Krzyzaniak M, Cheadle GA, Putnam JG, Hageny AM, Lopez N, Eliceiri BP, Bansal V, Coimbra R. Targeting alpha-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury. Am J Pathol. 2012;181:478Y486. 33. Costantini TW, Bansal V, Krzyzaniak M, Putnam JG, Peterson CY, Loomis WH, Wolf P, Baird A, Eliceiri BP, Coimbra R. Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. Am J Physiol Gastrointest Liver Physiol. 2010;299: G1308YG1318. 34. Huston JM, Gallowitsch-Puerta M, Ochani M, Ochani K, Yuan R, RosasBallina M, Ashok M, Goldstein RS, Chavan S, Pavlov VA, et al. Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis. Crit Care Med. 2007;35: 2762Y2768. 35. Matteoli G, Gomez-Pinilla PJ, Nemethova A, et al. A distinct vagal antiinflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen. Gut. 2013, Aug 23 [epub ahead of print]. 36. Kawashima K, Yoshikawa K, Fujii YX, Moriwaki Y, Misawa H. Expression and function of genes encoding cholinergic components in murine immune cells. Life Sci. 2007;80:2314Y2319. 37. Aicher A, Heeschen C, Mohaupt M, Cooke JP, Zeiher AM, Dimmeler S. Nicotine strongly activates dendritic cell-mediated adaptive immunity: potential role for progression of atherosclerotic lesions. Circulation. 2003;107:604Y611. 38. Wang DW, Zhou RB, Yao YM, Zhu XM, Yin YM, Zhao GJ, Dong N, Sheng ZY. Stimulation of alpha7 nicotinic acetylcholine receptor by nicotine increases suppressive capacity of naturally occurring CD4+CD25+ regulatory T cells in mice in vitro. J Pharmacol Exp Ther. 2010;335:553Y561. 39. Cerovic V, Houston SA, Scott CL, Aumeunier A, Yrlid U, Mowat AM, Milling SW. Intestinal CD103(j) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol. 2013;6:104Y113. 40. Schulz O, Jaensson E, Persson EK, Liu X, Worbs T, Agace WW, Pabst O. Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med. 2009;206:3101Y3114.
DISCUSSION Dr. William G. Cioffi (Providence, Rhode Island): Dr. Constantini has presented a nice extension of prior work
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from this group advancing our understanding of how vagal nerve stimulation may protect animals from gut barrier failure and remote organ injury following trauma and hemorrhagic shock. They have hypothesized that mesenteric lymph dendritic cells may play a role in the prevention of lung injury and their new finding is that vagal nerve stimulation blunts the loss of dendritic cells in mesenteric lymph. In non-stimulated animals it was a 50% reduction in CD103 MHC2 positive cells, presumably mature dendritic cells. These same animals had a marked increase in lymph flow. So at present they have two observations: one that vagal nerve stimulation protects against gut barrier function and remote organ injury, in this case the lung; and two, that there is a change in dendritic cell populations but these may or may not be related. I have several questions. What was the absolute number of dendritic cells, especially given the marked differences in lymph flow? Since a single dendritic cell may affect hundreds if not thousands of T-cells, are the changes that you observed clinically relevant? What’s the biological activity of these cells in the lymph? Are they mature and functional? Dendritic cells interact when presented with antigen interact with T-cells and help regulate the T-cell cytokine phenotypic response. So do you have any data either distally in the lung or proximally in the gut that indeed with these changes in the dendritic subpopulation you are seeing a different T-cell phenotypic response? And I guess another way to ask the question is, what is your proposed mechanism for dendritic cells protection of lung injury? Finally, can you give us an update on your progress on a clinically-relevant mechanism to provide vagal nerve stimulation at the gut level? This is really a nice paper, a great extension, and bringing a new hypothesis to your findings but at present I’m just not sure that the two are related. Thank you. Dr. Todd W. Constantini (San Diego, California): Dr. Cioffi, thank you very much for your comments. To address the first question, you asked if there was a change in the absolute number of dendritic cells given the differences in mesenteric lymph flow. While there is a an increase in mesenteric lymph flow after hemorrhagic shock, there was no difference in the total number of cells in that lymph fluid. We measured changes in the dendritic cell population by comparing pre-shock and post-shock values for each animal. Using that technique, each animal served as its own control. So, yes we were able to measure a change in the absolute number of dendritic cells. Next you asked whether or not the dendritic cells are mature and functional. Based on two factors it seems at least clear to us that they are mature. Once dendritic cells become activated they won’t leave the gut unless they are mature and functional cells so there is no reason for them to be in the mesenteric lymph unless they are functional. Also, the fact that the dendritic cells display the MHC2 marker suggests also that this is a mature or activated cell.
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Next you asked about the relevance of migration of this dendritic cell population through the mesenteric lymph into the mesenteric lymph node. You also asked whether or not this change in dendritic cells will alter T-cell activation and how it affects the lung. I will answer those two questions together. Based on our knowledge of CD103 positive cells and their role in mediating tolerance to injury, it is believed that CD103 positive cells go on to increase activation of regulatory T-cells or T-regs which has a general anti-inflammatory effect, basically keeping things at homeostasis by restraining effector T cell responses. After injury we see a decrease in the CD103 population which suggests that this tolerance signal is going away and potentially shifting to a more inflammatory phenotype. Part of our current studies are trying to understand what is happening in regards to the balance between tolerance and inflammation after injury. The current thinking is that there is a population of dendritic cells, CD103 negative dendritic cells, which tip the balance to an effector T-cell or a TH-17 response which then sets up for an IL-17 cytokine response and increased systemic inflammation. Interestingly, this IL-17 response is seen at very early time points after injury, generally targets mucosal surfaces, i.e., the gut and the lung, and also can cause neutrophil recruitment. So I think that the migration of cells through the mesenteric lymph may alter the balance between tolerance and immunity, where a decrease in the CD103 population demonstrated here may decrease Treg activation and tip the balance to a TH-17 response and ultimately result in tissue injury. This is the focus of the current studies in our lab. And then, finally, you asked us to update you on the clinical relevance which is a very important question. Obviously, we don’t support the idea that we’re going to directly electrically stimulate the vagus nerve in the neck of patients after injury. We’re really addressing the translational potential of vagal nerve signaling on one main front. We have a relationship with a pharmaceutical group who has a pharmacologic vagal agonist, a vagal-mimetic, for which we are going to use in some preclinical studies in our animal models to see if we can reproduce our findings of direct electrical vagal nerve stimulation. Our initial studies have focused on characterizing the mechanism of action of this drug as it is unclear the exact mechanism through which this vagal agonists exerts its anti-inflammatory effects. I’d say the other interesting possibility on the horizon is the idea of something called an auricular vagal nerve stimulator, something that is being used a little bit in Europe in patients with epilepsy. You can imagine having a device the size of your iPhone with an ear bud on it that goes into your ear and actually stimulates the auricular branch of the vagas nerve which can cause vagal nerve outflow. That’s clearly a more innovative technology that has only recently been implemented in patients but interesting, nonetheless. So stay tuned as we continue this work and hopefully bring this to clinical relevance. Thank you.
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Vagal nerve stimulation modulates the dendritic cell profile in posthemorrhagic shock mesenteric lymph Koji Morishita, MD, PhD, Todd W. Costantini, MD, Brian Eliceiri, PhD, Vishal Bansal, MD, and Raul Coimbra, MD, PhD, San Diego, California
BACKGROUND: Previous studies have established that posthemorrhagic shock mesenteric lymph (PHSML) contains proinflammatory mediators, while the cellular basis of PHSML is less well characterized in acute models of injury. CD103+ dendritic cells (DCs) have been identified in the mesenteric lymph (ML) in models of chronic intestinal inflammation, suggesting an important role in the gut response to injury. We have previously demonstrated the ability of vagal nerve stimulation (VNS) to prevent gut barrier failure after trauma/hemorrhagic shock (T/HS); however, the ability of VNS to alter ML DCs is unknown. We hypothesized that the CD103+ MHC-II+ DC population would change in PHSML and that VNS would prevent injury-induced changes in this population in PHSML. METHODS: Male Sprague-Dawley rats were randomly assigned to trauma/sham shock or T/HS. T/HS was induced by midline laparotomy and 60 minutes of HS (blood pressure, 35 mm Hg), followed by fluid resuscitation. A separate cohort of animals underwent cervical VNS after the HS phase. Gut tissue was harvested at 2 hours after injury for histologic analysis. ML was collected during the pre-HS, HS, and post-HS phase. For flow cytometric analysis, ML cells were subjected to staining with CD103 and MHC-II antibodies, and this cell population was compared in the pre-HS and post-HS phase from the same animal. The CD4+Foxp3+ cell (T reg) population in the ML node (MLN) was also tested to determine effects of CD103+ DC modulation in the ML. RESULTS: VNS reduced histologic gut injury and ML flow seen after injury. The CD103+ MHC-II+ DC population in the PHSML was significantly decreased compared with pre-HS and was associated with decreased T reg expression in the MLN. VNS prevented the injury-induced decrease in the CD103+ MHC-II+ DC population in the ML and restored the T reg population in the MLN. CONCLUSION: These findings suggest that VNS mediates the inflammatory responses in ML DCs and MLN T reg cells by affecting the set point of T/HS responsiveness. (J Trauma Acute Care Surg. 2014;76: 610Y618. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: Hemorrhagic shock; mesenteric lymph; intestinal inflammation; inflammatory cells; rats.
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rauma/hemorrhagic shock (T/HS)Yinduced gut barrier failure is known to initiate a systemic inflammatory response.1 Following T/HS, the injured gut releases inflammatory mediators into the mesenteric lymph (ML)2 that cause endothelial and red blood cell dysfunction, activate neutrophils, and cause distant organ injury.2 Identification of the specific proinflammatory mediators that travel through the ML and drive the systemic inflammatory response syndrome response has been the subject of investigation and has previously focused on the activity of biologically active lipids in the ML.3,4 Changes in activation and trafficking of inflammatory cells from the injured intestine through the ML may also be responsible for driving the systemic inflammatory response syndrome response to injury.
Submitted: July 31, 2013, Revised: November 20, 2013, Accepted: November 21, 2013. From the Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California, San Diego Health Sciences, San Diego, California. This study was presented at the 72nd annual meeting of the American Association for the Surgery of Trauma, September 18Y21, 2013, in San Francisco, California. Address for reprints: Todd W. Costantini, MD, Division of Trauma, Surgical Critical Care, and Burns, Department of Surgery, University of California, San Diego Health Sciences, 200 W Arbor Dr, #8896, San Diego, CA 92103-8896; email: [email protected]. DOI: 10.1097/TA.0000000000000137
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The cellular component of ML consists of lymphocytes and nonlymphoid cells such as dendritic cells (DCs).5 DCs are located in the lamina propria of the gut and sample luminal antigens that may be present after gut barrier failure. The DCs continuously migrate from the intestine to the ML nodes (MLNs) via ML and are crucial in balancing immunity and tolerance in the intestine.6 While intestinal DCs have been shown to be altered in gut inflammation,7 ML DCs are less well characterized in acute models of injury. Rat ML DCs are identified by their high cell surface expression of the integrin >E (CD103) and MHC-II.6 Previous study demonstrated that CD103+ DCs were dramatically reduced in the gut of colitic mice,7 which suggests an important role for this cell population in the gut immune responses. Furthermore, CD103+ DCs are thought to play an important role in the generation of Foxp3+ T reg cells in the gut draining lymphoid tissues, where Foxp3+ T regs are critical in promoting tolerance to inflammation.8 Vagal nerve stimulation (VNS) attenuates the systemic inflammatory response to infection, reduces systemic cytokine release, and prevents the development of shock in animals with lethal endotoxemia.9 Our laboratory has demonstrated that VNS has a marked effect on intestinal barrier function and gut inflammation after acute injury10,11 and attenuates acute lung injury.12 Other groups have shown that VNS prevents T/HS-induced gut injury, decreases toxic ML, and attenuates acute lung injury; however, the mechanism by which VNS alters the inflammatory state of the ML is unknown.13 J Trauma Acute Care Surg Volume 76, Number 3
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Moreover, VNS has recently been considered to control intestinal immune homeostasis by altering the balance between tolerance and inflammation in the gut.14 The aim of our study was to measure changes in the DC population in the ML after T/HS. We hypothesized that (1) the CD103+ MHC-II+ ML DC and CD4+Foxp3+ MLN T reg cell populations would change after T/HS, indicating a shift toward an inflammatory phenotype, and that (2) VNS would prevent T/HS-induced change in these populations of DCs and T reg cells, thus altering the inflammatory set point in the gut.
MATERIALS AND METHODS
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identical anesthesia and surgical procedure without hemorrhage. Each animal’s body temperature was maintained at 37-C.
VNS A cohort of animals underwent right cervical neck incision followed by right cervical VNS immediately after T/HS insult. Stimulation of vagus nerve was performed using a VariStim II probe (Medtronic Xomed, Jacksonville, FL) at 2 mA, intermittently for 10 minutes. Following nerve stimulation, the incision was closed with 4-0 silk suture. Sham animals underwent right cervical incision and exposure of the vagus nerve but did not receive electrical stimulation.
T/HS Model Male Sprague-Dawley rats weighing 280 g to 300 g were obtained from Harlan Laboratories (Placentia, CA). All animal experiments were approved by the University of California San Diego Institutional Animal Care and Use Committee. Animals were anesthetized with ketamine (50 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg; Sigma Chemical, St. Louis, MO), and the left femoral artery and vein were cannulated with a polyethylene tube (PE-50). Nonlethal hemorrhagic shock was induced via femoral vein cannulation until the mean arterial pressure (MAP) was reduced to 35 mm Hg and maintained for 60 minutes. At the end of shock period, animals in the T/HS group were resuscitated with shed blood plus two times shed blood volume in normal saline (Baxter, Deerfield, IL). The MAP was continuously monitored using the femoral arterial catheter (Philips V24/26, Andover, MA). The trauma/sham shock (T/SS) animals underwent the
Collection of ML and MLNs The ML duct was exposed, and the efferent mesenteric lymphatic was cannulated (PE-50). ML was collected on ice during the pre-HS phase (30 minutes), HS phase (60 minutes), and post-HS phase (120 minutes) by definition (Fig. 1A). The cell pellet was prepared for flow cytometric analysis as previously described.15 MLNs were harvested at 24 hours following resuscitation and cut into pieces and incubated for 20 minutes under agitation at 37-C in the presence of Collagenase/Dispase (Roche Diagnostic, Indianapolis, IN) and DNase I (NEW ENGLAND BioLabs, Ipswich, MA). The tissue was then passed through a 70-Km membrane to generate single-cell suspensions and prepared for flow cytometric analysis. Cell count and viability were obtained using a hemocytometer (Countess automatched cell counter, Invitrogen, Grand Island, NY).
Figure 1. Effects of VNS on ML after T/HS. A, ML was collected during the pre-HS phase (30 minutes), HS phase (60 minutes), and post-HS phase (60 minutes 2). ML flow (KL/h) (B), ML cell viability (%) (C), and total ML cell (106 cells/mL) (D) were evaluated at each phase. n = 5 in all groups, Data shown as mean (SD). *p G 0.05 pre-HS vs. post-HS. †p G 0.05 T/HS vs. T/HS + VNS. * 2014 Lippincott Williams & Wilkins
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Histologic Evaluation At 2 hours after injury, segments of distal ileum were removed and fixed in 10% buffered formalin, embedded in paraffin, and sectioned. Hematoxylin and eosin staining of the intestine was performed by the University California, San Diego Histology Core Services. An investigator blinded to experimental groups analyzed multiple fields from section of ileum imaged at 20 and 60 with a light microscope. The ileum sections were classified according to the degree of tissue lesion according Chiu et al.16
Flow Cytometric Analysis The expression of cell surface molecules on DCs and regulatory T cell (T reg) cells were determined by flow cytometry. ML and MLNs cells (1.0 106 cells/mL) were incubated with anti-rat RTB1 (MHC-II)-PerCP (clone OX-6), anti-rat CD4-APC (OX-35), PerCP mouse IgG1 J isotype control antibody (MOCP-31C) (BD Bioscience, San Diego, CA), anti-rat CD103-FITC (OX-62), anti-rat CD11bc-APC (OX-42), anti-rat CD80-PE (3H5), anti-rat CD86-PE (24F), FITC-mouse IgG1 J isotype control antibody (MOCP-21), PEmouse IgG1 J isotype control (FC) antibody (MOCP-21), and APC-mouse IgG2a J isotype control antibody (MOPC-173) (BioLegend, San Diego, CA) and for 30 minutes at 4-C. Intracellular staining for Fox3 (FoxP3-Alexa Fluor 488 [150 D], BioLegend) was performed using Cytofix/Cytoperm kit (BD Biosciences). Flow cytometry was performed with a BD Accuri C6 (BD Bioscience), and 10,000 events were collected for analysis. Data were analyzed with FlowJo (Three Star, Ashland, OR) software.
Statistical Analysis Data were expressed as mean (SD). Analysis of variance with Student-Newman-Keuls post hoc analysis or Student’s t test was performed with appropriate. Analysis of gut injury grading was performed using Kruskal-Wallis nonparametric analysis of variance test. Statistical significance was determined based on p G 0.05.
RESULTS Effects of VNS on ML After T/HS There was no difference in MAP between T/HS and T/ HS + VNS group before, during, or after HS. The total shed blood volume was similar between T/HS and T/HS + VNS groups (Table 1). The ML flow volume at 60 minutes post-HS increased significantly when compared with the pre-HS phase (2,145.0 [500.5] KL/h vs. 792.0 [317.7] KL/h, p G 0.05). VNS
limited the T/HS-induced increase in ML flow at 60 minutes (1,370.0 [432.4] KL/h vs. 2,145.0 [500.5] KL/h, p G 0.05) and 120 minutes post-HS (238.0 [82.2] KL/h vs. 474.0 [150.1] KL/h, p G 0.05) (Fig. 1B). The cell viability of ML was greater than 85% in all groups (Fig. 1C). ML cell counts at 60 minutes postHS and 120 minutes post-HS were decreased compared with that of pre-HS (p G 0.05). However, there was no difference in the cell count between T/HS and T/HS + VNS at each phase (Fig. 1D).
Effects of VNS on Gut After T/HS Based on the previously established effect of T/HS on the integrity of the gut barrier,1 histologic analysis was performed on representative tissue samples. T/HS caused histologic gut injury characterized by villous blunting and necrosis of the villous tips, consistent with previous report.1,16 At 2 hours after injury, the histologic appearance of the distal ileum of VNStreated animals was similar to that of the T/SS animals, demonstrating the protective effects of VNS on the development of intestinal injury after T/HS (Fig. 2A). In addition, the histologic injury score of the T/HS group was significantly higher than those obtained for the T/SS, T/SS+VNS, and T/HS + VNS groups (p G 0.05) (Fig. 2B).
Surface Phenotype of ML Cells Following T/HS Classically, DCs express MHC-II, CD103, CD11c, CD80, and CD8617 on their surface. To determine the surface expression of ML cells from sham animals, cells were stained with MHC-II, CD103, CD11bc, CD80, CD86, CD4 antibodies, and isotype-matched control antibodies for each of the surface marker antibodies and assessed using flow cytometry. As shown in Figure 3A, CD103, MHC-II, and CD4 expressions were detected on ML cell surface from sham animals. Next, we examined whether the expressions of CD103, MHC-II, and CD4 on ML cells changed after T/HS. The serial analysis of ML established consistent baseline measurements in each animal and allowed us to quantify the relative change in various cell populations after injury. ML cells were compared in the pre-HS (baseline) and post-HS (120 minutes) phases from the same animals. MHC-II expression on ML cells was significantly decreased following T/HS (n = 5, p G 0.05), while CD103 and CD4 expression on ML cells were unchanged (Fig. 3B and C).
VNS Prevents the Shock-Induced Decrease of CD103+ MHC-II+ DCs in the ML Based on our previous studies demonstrating VNSmediated protection of the gut in acute injury models,10,11
TABLE 1. T/HS Model MAP (Pre-HS), mm Hg MAP (HS), mm Hg MAP (Post-HS), mm Hg Shed blood volume, mL
T/SS
T/SS + VNS
T/HS
T/HS + VNS
p
97.0 (19.8) V 116.3 (26.4) V
84.2 (9.7) V 114.0 (23.3) V
85.6 (4.5) 35.1 (1.1) 100.3 (12.7) 7.5 (0.7)
94.2 (12.0) 35.5 (2.0) 121 (5.1) 7.8 (1.4)
0.33 0.19 0.21 0.72
Male Sprague-Dawley rats were bled to a MAP of 35 mm Hg for 1 hour and then reperfused with their own shed blood and normal saline. A separate cohort of animals underwent cervical VNS after the HS phase. The MAP was continuously monitored during the experiments. n = 5 in all groups, Data shown as mean (SD).
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Figure 2. Effects of VNS on gut after T/HS. A, Section of distal intestine harvested 2 hours after T/HS and hematoxylin and eosin staining of small intestine was performed. Top row, 20 magnification; bottom row, 60 magnification. B, Graph displays gut injury scores (Chiu score) n = 4Y6, *p G 0.05 compared with T/HS group.
we considered that VNS treatment after T/HS might limit the injury-induced decrease in CD103+ MHC-II+ ML DC population. As shown in Figure 4A and B, the CD103+ MHCII+ ML DC population at 120 minutes post-HS was significantly decreased compared with that of pre-HS (pre-HS, 8.5% [0.7%] vs. 5.2% [0.7%], p G 0.05). Performing VNS prevented the decrease in this population caused by acute injury (pre-HS, 8.0% [0.6] vs. 8.2% [2.9%], p = 0.90). In addition, the serial analysis of CD103+ MHC-II+ ML DC population revealed that this population was significantly decreased at 60 minutes (65.9 [5.7] vs. 100%, p G 0.05) and 120 minutes (60.4 [18.9] vs. 100%, p G 0.05) post-HS when compared with that of pre-HS and performing VNS prevented the decrease in this population at 120 minutes post-HS caused by acute injury (102.4% [35.3%] vs. 60.4% [18.9%], p G 0.05) (Fig. 4C).
VNS Prevents the T/HS-Induced Decrease in Foxp3+ T reg Cells in the MLNs CD103+ DCs are thought to play an important role in the generation of Foxp3+ T reg in MLNs.8 We therefore examined the Foxp3+ T reg cell population in MLNs after T/HS
to determine the downstream effects of changes in the CD103+ DC population. The CD4+Foxp3+ MLN cell population was significantly decreased after T/HS. VNS prevented the injury-induced decrease in the population of CD4+Foxp3+ cells in the MLN (1.9% [0.8%] vs. 4.7% [0.8%], p G 0.05) (Fig. 5A and B).
DISCUSSION The development of trauma systems, resuscitation protocols, and advances in critical care has improved survival in patients with severe injury.18 Seriously injured patients frequently develop late complications including nosocomial infections and organ failure, which continue to be the leading cause of death after acute injury.18,19 Previous studies have demonstrated that T/HS causes a marked alternation in many immune functions, including T-cell activation and proliferation, cytokine release, and the antigen presentation functions of DCs.20Y22 Murine and human studies have shown that there is a significant loss of DCs after T/HS.20,23 T/HS induces depressed splenic DC maturation and suppressed DC antigen presentation
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Figure 3. Surface phenotype of ML cells following T/HS. A, ML cells from sham animals were stained with antibodies and analyzed by flow cytometry. Filled histograms are isotype controls; unfilled histograms show staining for the named (CD103, MHC II, CD11bc, CD80, CD86, and CD4) antigens. B, ML was collected during the pre-HS and post-HS phases. Cells were stained with MHC-II and CD103 antibodies and analyzed by flow cytometry. Histograms represent surface expression of CD103 and MHC-II on ML cells. Overlays indicate pre-HS versus post-HS (120 minutes) data. Unfilled histogram: pre-HS; filled histogram: post-HS (120 minutes). C, Bar graphs show the kinetics of CD103+, MHC-II+, and CD4+ ML cells after T/HS. n = 5 in all groups. Data shown as mean (SD). *pG 0.01, pre-HS versus post-HS (120 minutes)
function in mice.20 It has been reported that in trauma patients, monocyte conversion to immature DCs is impaired after T/HS as well.23 The intestinal mucosa contains a complex population of inflammatory cells, including macrophages and DCs, which together are thought to play a central role in regulating mucosal innate and adaptive immune responses in both the steady-state and inflammatory setting.24 DCs are potent antigen-presenting cells and are key modulators of the immune response.12 Phenotypically different populations of DCs have been identified in the intestine lamina propria, Peyer’s patches, intestinal lymphoid follicles, MLNs, and ML.6,25 DCs migrate from the intestine to the MLNs through the intestinal lymphatics. Murine intestinal DC populations are defined by expression of the integrins CD103 and CD11c and have been further subdivided according to CD11b expression.26 Moreover, thoracic and ML DCs can be identified by their expression of both CD103 and MHC-II.15,26 CD103+ DCs have been shown in the gut and MLNs in models of chronic gut inflammation.7,27 614
CD103+ DCs are reduced in the gut and MLNs of colitic mice.7 In addition, CD103+ DCs are absent from inflamed area in ileal sections in a rat model of indomethacin-induced enteritis during acute stage of inflammation.28 Of interest, CD103+ DC deficiency contributes to the perpetuation of ileitis, supporting a protective function for this population.29 Therefore, CD103+ DCs could play important roles in the regulation of homeostatic balance between mucosal immunity and tolerance in the gastrointestinal tract. Recent work has demonstrated that migrating gut CD103+ DCs prominently induce the development of Foxp3+ T reg cells in the MLNs.8 Foxp3 is a transcription factor required for the development of T reg cells. Foxp3+ T reg cells function to maintain immune tolerance and prevent inflammatory disease.30 Among DCs, the enzymes that convert vitamin A into retinoic acid are most prominently expressed in a population of CD103+ DCs. Retinoic acid promotes the peripheral differentiation of induced Foxp3+ T reg cell numbers and function during inflammatory responses. Thus, CD103+ DCs are thought to be the key factors involved in the * 2014 Lippincott Williams & Wilkins
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Figure 4. VNS prevents the shock-induced decrease of CD103+ MHC-II+ ML DCs. A, Numbers in the FACS plot show the percent frequency of the CD103+ MHC-II+ DC population. The percent frequency of this cell population was compared in the pre-HS and post-HS phases (120 minutes) from the same animals. B, Bar graphs show the percent frequency of CD103+ MHC-II+ ML DC populations. This cell population was compared between pre-HS and post-HS phases (120 minutes). Values (%) are expressed as mean (SD), n = 4Y5. *p G 0.05, pre-HS versus post-HS (120 minutes). C, The serial analysis of ML established consistent baseline measurements in each animal and allowed us to quantify the relative change in various cell populations after injury. Percent changes in CD103+ MHC-II+ DCs were determined to be compared in the pre-HS phase (baseline) and post-HS phase from the same animals. Data (%) shown as mean (SD). *p G 0.05, pre-HS versus post-HS, †p G 0.05, T/HS versus T/HS + VNS.
induction of T reg cells and promote tolerance to inflammation in the gut.8 In the current study, CD103+ ML DCs and Foxp3+ MLNs T reg cells were significantly decreased after T/HS, suggesting a shift to an inflammatory phenotype in the gut. Previous investigators demonstrated the ability of VNS to prevent intestinal barrier failure, intestinal inflammation, and the production of biologically active ML in models of acute injury.1,10,11,13,31,32 In the present study, we found that performing VNS after acute injury alters T/HS-induced gut injury. This protective effect correlates with our previous published study, showing the effect of VNS after burn injury.11 In addition, we demonstrated that VNS significantly limited injury-induced increased in ML flow. ML flow is known to be modulated by multiple factors, such as gastrointestinal peristalsis, autonomic nerves, and hemorrhage.5 Recent studies have shown that the vagal innervations of the gastrointestinal
tract play a major role in controlling intestinal immune activation;14 however, the mechanism by which VNS exerts its anti-inflammatory effects has not been defined. Work in our laboratory first demonstrated that VNS-induced gut protection is independent of the spleen,33 a departure from previous studies by Tracey et al.34 looking at systemic inflammation where VNS protection is spleen dependent. This finding was recently advanced in a study by Matteoli et al.,35 which found that VNS protection in the gut is independent of T cells and signals through myeloid cells in the gut. Our present results demonstrated that VNS prevents the T/HS-induced decrease in CD103+ MHC-II+ ML DC and Foxp3+ MLNs T reg cell populations in the early phases of injury. To our knowledge, this is the first study directly examining the effects of VNS on the ML DCs and MLNs T reg cells after acute injury. Several immune cells express
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Figure 5. VNS prevents the shock-induced decrease of CD4+ Foxp3+ T reg cells in the MLNs. At 24 hours following resuscitation, MLN cells were isolated and stained with CD4 and Foxp3 antibodies and analyzed by flow cytometry. A, Representative flow cytometry data demonstrate a decrease in CD4+Foxp3+ T regs after T/HS. VNS normalizes the expression of CD4+Foxp3+ T reg in the MLN. B, Graph demonstrating the percentage of CD4+Foxp3+ T regs present in the MLN. Values are expressed as mean (SD), n = 4Y5. *p G 0.05 compared with T/HS group.
various nicotinic acetylcholine receptor (nAChR) subtypes. Mouse DCs are known to express nAChR >2, >5, >6, >7, A2, A4 subunits.36 In vitro studies have shown that nicotine activates DCs and augments their capacity to stimulate T-cell proliferation and cytokines secretion.37 >7 nAChR is also thought to be expressed on CD4+CD25+ T reg cells, and the upregulation of Foxp3 expression on CD4+CD25+ T reg cells occurs in the presence of nicotine stimulation in vitro study.38 These effects of nicotine on the functional properties of DCs and T reg cells might correlate with the results described in our current study regarding the immunomodulatory effect of VNS after T/HS. Finally, CD103j and CD103+ lymph DCs have distinct effects on the differentiation of primed T lymphocytes.39 The balance between CD103j DCs, CD103+ DCs, and macrophages is thought to be critical for maintaining intestinal immune homeostasis.39,40 Therefore, investigating how the functions of CD103j and CD103+ lymph DCs change in response to acute injury of intestinal immune responses will provide essential insights into the development of intestinal immunopathology. 616
In summary, T/HS induced gut injury and depressed ML flow, CD103+ MHC-II+ ML DC, and Foxp3+ MLN T reg cell populations. VNS modulates ML CD103+ DCs trafficking to the MLNs and T reg cell generation in the MLNs, thus altering the gut inflammatory response to acute injury. Treatments that cause signaling via the vagus nerve may represent an ideal strategy to limit the systemic inflammatory response to severe trauma. AUTHORSHIP K.M. performed the surgery and sample collection in the animal experiment. K.M. and B.E. performed the data collection and analysis of flow cytometry. K.M., T.W.C., B.E., V.B., and R.C. conceived the study and participated in its design and coordination. K.M., T.W.C., B.E., and R.C. drafted the manuscript. All authors read and approved the final manuscript. ACKNOWLEDGMENT We thank Ann-Marie Hageny for her technical assistance with the flow cytometry.
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DISCLOSURE This study was funded in part by the American Association for the Surgery of Trauma Research and Education Foundation Scholarship Award (to T.W.C.).
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22. Zellweger R, Ayala A, DeMaso CM, Chaudry IH. Trauma-hemorrhage causes prolonged depression in cellular immunity. Shock. 1995;4:149Y153. 23. De AK, Laudanski K, Miller-Graziano CL. Failure of monocytes of trauma patients to convert to immature dendritic cells is related to preferential macrophage-colony-stimulating factor-driven macrophage differentiation. J Immunol. 2003;170:6355Y6362. 24. Kelsall B. Recent progress in understanding the phenotype and function of intestinal dendritic cells and macrophages. Mucosal Immunol. 2008;1: 460Y469. 25. Iwasaki A. Mucosal dendritic cells. Annu Rev Immunol. 2007;25:381Y418. 26. Farache J, Zigmond E, Shakhar G, Jung S. Contributions of dendritic cells and macrophages to intestinal homeostasis and immune defense. Immunol Cell Biol. 2013;91:232Y239. 27. Rutella S, Locatelli F. Intestinal dendritic cells in the pathogenesis of inflammatory bowel disease. World J Gastroenterol. 2011;17:3761Y3775. 28. Silva MA, Porras M, Jury J, Vergara P, Perdue MH. Characterization of ileal dendritic cell distribution in a rat model of acute and chronic inflammation. Inflamm Bowel Dis. 2006;12:457Y470. 29. Annacker O, Coombes JL, Malmstrom V, Uhlig HH, Bourne T, JohanssonLindbom B, Agace WW, Parker CM, Powrie F. Essential role for CD103 in the T cell-mediated regulation of experimental colitis. J Exp Med. 2005;202:1051Y1061. 30. Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol. 2011;11:119Y130. 31. Krzyzaniak MJ, Peterson CY, Cheadle G, Loomis W, Wolf P, Kennedy V, Putnam JG, Bansal V, Eliceiri B, Baird A, et al. Efferent vagal nerve stimulation attenuates acute lung injury following burn: the importance of the gut-lung axis. Surgery. 2011;150:379Y389. 32. Costantini TW, Krzyzaniak M, Cheadle GA, Putnam JG, Hageny AM, Lopez N, Eliceiri BP, Bansal V, Coimbra R. Targeting alpha-7 nicotinic acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury. Am J Pathol. 2012;181:478Y486. 33. Costantini TW, Bansal V, Krzyzaniak M, Putnam JG, Peterson CY, Loomis WH, Wolf P, Baird A, Eliceiri BP, Coimbra R. Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells. Am J Physiol Gastrointest Liver Physiol. 2010;299: G1308YG1318. 34. Huston JM, Gallowitsch-Puerta M, Ochani M, Ochani K, Yuan R, RosasBallina M, Ashok M, Goldstein RS, Chavan S, Pavlov VA, et al. Transcutaneous vagus nerve stimulation reduces serum high mobility group box 1 levels and improves survival in murine sepsis. Crit Care Med. 2007;35: 2762Y2768. 35. Matteoli G, Gomez-Pinilla PJ, Nemethova A, et al. A distinct vagal antiinflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen. Gut. 2013, Aug 23 [epub ahead of print]. 36. Kawashima K, Yoshikawa K, Fujii YX, Moriwaki Y, Misawa H. Expression and function of genes encoding cholinergic components in murine immune cells. Life Sci. 2007;80:2314Y2319. 37. Aicher A, Heeschen C, Mohaupt M, Cooke JP, Zeiher AM, Dimmeler S. Nicotine strongly activates dendritic cell-mediated adaptive immunity: potential role for progression of atherosclerotic lesions. Circulation. 2003;107:604Y611. 38. Wang DW, Zhou RB, Yao YM, Zhu XM, Yin YM, Zhao GJ, Dong N, Sheng ZY. Stimulation of alpha7 nicotinic acetylcholine receptor by nicotine increases suppressive capacity of naturally occurring CD4+CD25+ regulatory T cells in mice in vitro. J Pharmacol Exp Ther. 2010;335:553Y561. 39. Cerovic V, Houston SA, Scott CL, Aumeunier A, Yrlid U, Mowat AM, Milling SW. Intestinal CD103(j) dendritic cells migrate in lymph and prime effector T cells. Mucosal Immunol. 2013;6:104Y113. 40. Schulz O, Jaensson E, Persson EK, Liu X, Worbs T, Agace WW, Pabst O. Intestinal CD103+, but not CX3CR1+, antigen sampling cells migrate in lymph and serve classical dendritic cell functions. J Exp Med. 2009;206:3101Y3114.
DISCUSSION Dr. William G. Cioffi (Providence, Rhode Island): Dr. Constantini has presented a nice extension of prior work
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from this group advancing our understanding of how vagal nerve stimulation may protect animals from gut barrier failure and remote organ injury following trauma and hemorrhagic shock. They have hypothesized that mesenteric lymph dendritic cells may play a role in the prevention of lung injury and their new finding is that vagal nerve stimulation blunts the loss of dendritic cells in mesenteric lymph. In non-stimulated animals it was a 50% reduction in CD103 MHC2 positive cells, presumably mature dendritic cells. These same animals had a marked increase in lymph flow. So at present they have two observations: one that vagal nerve stimulation protects against gut barrier function and remote organ injury, in this case the lung; and two, that there is a change in dendritic cell populations but these may or may not be related. I have several questions. What was the absolute number of dendritic cells, especially given the marked differences in lymph flow? Since a single dendritic cell may affect hundreds if not thousands of T-cells, are the changes that you observed clinically relevant? What’s the biological activity of these cells in the lymph? Are they mature and functional? Dendritic cells interact when presented with antigen interact with T-cells and help regulate the T-cell cytokine phenotypic response. So do you have any data either distally in the lung or proximally in the gut that indeed with these changes in the dendritic subpopulation you are seeing a different T-cell phenotypic response? And I guess another way to ask the question is, what is your proposed mechanism for dendritic cells protection of lung injury? Finally, can you give us an update on your progress on a clinically-relevant mechanism to provide vagal nerve stimulation at the gut level? This is really a nice paper, a great extension, and bringing a new hypothesis to your findings but at present I’m just not sure that the two are related. Thank you. Dr. Todd W. Constantini (San Diego, California): Dr. Cioffi, thank you very much for your comments. To address the first question, you asked if there was a change in the absolute number of dendritic cells given the differences in mesenteric lymph flow. While there is a an increase in mesenteric lymph flow after hemorrhagic shock, there was no difference in the total number of cells in that lymph fluid. We measured changes in the dendritic cell population by comparing pre-shock and post-shock values for each animal. Using that technique, each animal served as its own control. So, yes we were able to measure a change in the absolute number of dendritic cells. Next you asked whether or not the dendritic cells are mature and functional. Based on two factors it seems at least clear to us that they are mature. Once dendritic cells become activated they won’t leave the gut unless they are mature and functional cells so there is no reason for them to be in the mesenteric lymph unless they are functional. Also, the fact that the dendritic cells display the MHC2 marker suggests also that this is a mature or activated cell.
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Next you asked about the relevance of migration of this dendritic cell population through the mesenteric lymph into the mesenteric lymph node. You also asked whether or not this change in dendritic cells will alter T-cell activation and how it affects the lung. I will answer those two questions together. Based on our knowledge of CD103 positive cells and their role in mediating tolerance to injury, it is believed that CD103 positive cells go on to increase activation of regulatory T-cells or T-regs which has a general anti-inflammatory effect, basically keeping things at homeostasis by restraining effector T cell responses. After injury we see a decrease in the CD103 population which suggests that this tolerance signal is going away and potentially shifting to a more inflammatory phenotype. Part of our current studies are trying to understand what is happening in regards to the balance between tolerance and inflammation after injury. The current thinking is that there is a population of dendritic cells, CD103 negative dendritic cells, which tip the balance to an effector T-cell or a TH-17 response which then sets up for an IL-17 cytokine response and increased systemic inflammation. Interestingly, this IL-17 response is seen at very early time points after injury, generally targets mucosal surfaces, i.e., the gut and the lung, and also can cause neutrophil recruitment. So I think that the migration of cells through the mesenteric lymph may alter the balance between tolerance and immunity, where a decrease in the CD103 population demonstrated here may decrease Treg activation and tip the balance to a TH-17 response and ultimately result in tissue injury. This is the focus of the current studies in our lab. And then, finally, you asked us to update you on the clinical relevance which is a very important question. Obviously, we don’t support the idea that we’re going to directly electrically stimulate the vagus nerve in the neck of patients after injury. We’re really addressing the translational potential of vagal nerve signaling on one main front. We have a relationship with a pharmaceutical group who has a pharmacologic vagal agonist, a vagal-mimetic, for which we are going to use in some preclinical studies in our animal models to see if we can reproduce our findings of direct electrical vagal nerve stimulation. Our initial studies have focused on characterizing the mechanism of action of this drug as it is unclear the exact mechanism through which this vagal agonists exerts its anti-inflammatory effects. I’d say the other interesting possibility on the horizon is the idea of something called an auricular vagal nerve stimulator, something that is being used a little bit in Europe in patients with epilepsy. You can imagine having a device the size of your iPhone with an ear bud on it that goes into your ear and actually stimulates the auricular branch of the vagas nerve which can cause vagal nerve outflow. That’s clearly a more innovative technology that has only recently been implemented in patients but interesting, nonetheless. So stay tuned as we continue this work and hopefully bring this to clinical relevance. Thank you.
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