Characterisation of dendritic cell frequency and phenotype in bovine afferent lymph reveals kinetic changes in costimulatory molecule expression.

The bovine afferent lymphatic cannulation model allows collection of large volumes of afferent lymph and provides an opportunity to study lymphatic cells trafficking from the periphery directly ex-vivo. The technique requires surgical intervention, but influence of the procedure or time post-surgery on cells trafficking in the lymph has not been well documented. Here, we measured the volume of lymph and number of cells/mL collected daily over a two week time-course. Animal to animal variability was demonstrated but no consistent changes in lymph volume or cell density were observed in relation to time post-cannulation. Cell populations (dendritic cells, αß T-cells, γδ T-cells and NK cells) were analysed by flow cytometry at 1, 3 and 10 days post-cannulation (DPC) and a reduced percentage of γδ T-cells in afferent lymph was observed at 1 DPC. In addition, cell surface molecule expression by afferent lymphatic dendritic cells (ALDC) was assessed due to the key role of these cells in initiating an adaptive immune response. Co-stimulatory molecules CD80 and CD86 were upregulated by CD172a+ve ALDC early in the time-course, suggesting that the cannulation procedure and duration of experiment may impact the activation state of DCs in the naïve host. This should be considered when analysing the response of these cells to vaccines or pathogens.

How nanoparticles can induce dimerization and aggregation of cells in blood or lymph.

By analogy with virions, the binding of biologically-inspired nanoparticles (NPs) with ligands to the cellular membrane containing receptors depends on the multivalent ligand-receptor interaction, membrane bending, and cytoskeleton deformation. The interplay of these factors results in the existence of the potential minimum and activation barrier on the pathway towards full absorption of a NP. Herein, I hypothesize and show theoretically that the interaction of a NP, bound to one cell, with another cell can stabilize the potential minimum and increase the corresponding activation barrier, i.e., NPs can mediate the formation of long-living pairs of cells and aggregates containing a few cells inside blood and lymphatic vessels.

Lymph-Derived Neutrophils Primarily Locate to the Subcapsular and Medullary Sinuses in Resting and Inflamed Lymph Nodes.

Neutrophils are the first immune cells to be recruited from the blood to the tissue site of an infection or inflammation. It has been suggested that neutrophils are capable of migrating from the infected tissue via lymphatic vessels to the draining lymph nodes. However, it remains elusive as to which areas within the lymph nodes can be reached by such reversely migrating cells. To address this question, we applied a model for adoptive neutrophil transfer into the afferent lymphatic vessel that drains towards the popliteal lymph node in mice. We showed that resting and in vitro-activated neutrophils did not enter the lymph node parenchyma but localized primarily in the subcapsular and medullary sinuses. Within the medulla, neutrophils show random migration and are able to sense laser-induced sterile tissue injury by massively swarming to the damaged tissue site. Co-injected dendritic cells supported the entry of resting neutrophils into the lymph node parenchyma via the subcapsular sinus. In contrast, in vivo-activated adoptively transferred neutrophils were capable of migrating into the interfollicular areas of the lymph node. Collectively, the data presented here give further insights into the functional behavior of neutrophils within the lymph nodes.

Thoracic duct lymphatic fluid harbors phenotypically naive T cells for use in adoptive T-cell therapy.

BACKGROUND AIMS: Manufacturing of potent chimeric antigen receptor (CAR) T cells requires phenotypically naive and early memory T cells. We hypothesized lymphatic fluid collected from the thoracic duct of children would serve as a unique reservoir for early T cells, which could then be used for CAR T-cell therapy. METHODS: We evaluated lymphatic fluid collected from 25 pediatric patients undergoing thoracic duct cannulation for other clinical indications. RESULTS: Lymphatic fluid in the thoracic duct was rich in T cells, with higher percentage of naive and stem central memory T-cell subsets compared with paired blood samples. T cells from lymphatic fluid showed decreased negative checkpoint regulators on the surface and increased rapid expansion with bead activation. Creation of CD19-directed CAR T cells from blood and lymphatic T cells showed similar lentiviral transduction properties, but CAR T cells generated from lymphatic fluid produced superior cytotoxicity in a murine leukemia model because they were able to achieve equivalent tumor eradication at lower doses. CONCLUSIONS: These results are the first characterization of T cells from the thoracic duct of pediatric patients and suggest an alternative approach for manufacturing of cellular therapy that will improve both expansion and cytotoxic effect.

Efficient homing of T cells via afferent lymphatics requires mechanical arrest and integrin-supported chemokine guidance.

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.

The effect of different treatments of lymph after intestinal ischemia-reperfusion in rats on macrophages in vitro.

BACKGROUND: To observe the effects of different treatments of lymph after intestinal I/R in rats on macrophages in vitro. METHODS: Forty-eight healthy SPF SD rats weighing 300 ± 20 g, were randomly divided into two groups: group A, and group B. The rats in group A were drained of lymph fluid for 180 min; the rats in group B were subjected to 60 min ischemia by clamping the SMA, followed by 120 min reperfusion and 180 min of lymph drainage. The lymph fluid collected was divided into 4 sub-groups: 1. no treatment (A1, Ly, and B1, I/R Ly); 2. protein degradation (A2, Ly PD, and B2 I/R PD); 3. endotoxin removal (A3, Ly ER, and B3, I/R ER); 4. protein degradation plus endotoxin removal (A4, Ly PD+ER, and B4, I/R PD+ER), then used to stimulate a monocyte-macrophage cell line. RESULTS: Compared with group A1, the levels of the inflammatory cytokines, chemokines, HMGB1 concentration, protein and mRNA expression of TLR4, HMGB1 and NF-κBp65 were significantly increased in group B1. There was a significant reduction in proinflammatory cytokines and of the expression of TLR4, NF-κBp65, and chemokines in groups A2, B2, A4, and B4. However, there were no significant decrease of these factors in groups A3 and B3. CONCLUSIONS: The lymph fluid drained after intestinal I/R can cause inflammation in vivo and in vitro. Deproteinization of lymph fluid with proteinase K significantly reduced the concentration of proinflammatory cytokines, chemokines, TLR4 and NF-κBp65 in cell culture supernatant, exerting a protective effect on inflammatory reaction caused by the intestinal I/R. Passage of lymph fluid through an endotoxin removal column did not reduce the levels of active proinflammatory factors produced by macrophages in vitro.

Cellular traffic through afferent lymphatic vessels.

The lymphatic system has long been known to serve as a highway for migrating leukocytes from peripheral tissue to draining lymph nodes (dLNs) and back to circulation, thereby contributing to the induction of adaptive immunity and immunesurveillance. Lymphatic vessels (LVs) present in peripheral tissues upstream of a first dLN are generally referred to as afferent LVs. In contrast to migration through blood vessels (BVs), the detailed molecular and cellular requirements of cellular traffic through afferent LVs have only recently started to be unraveled. Progress in our ability to track the migration of lymph-borne cell populations, in combination with cutting-edge imaging technologies, nowadays allows the investigation and visualization of lymphatic migration of endogenous leukocytes, both at the population and at the single-cell level. These studies have revealed that leukocyte trafficking through afferent LVs generally follows a step-wise migration pattern, relying on the active interplay of numerous molecules. In this review, we will summarize and discuss current knowledge of cellular traffic through afferent LVs. We will first outline how the structure of the afferent LV network supports leukocyte migration and highlight important molecules involved in the migration of dendritic cells (DCs), T cells and neutrophils, i.e. the most prominent cell types trafficking through afferent LVs. Additionally, we will describe how tumor cells hijack the lymphatic system for their dissemination to draining LNs. Finally, we will summarize and discuss our current understanding of the functional significance as well as the therapeutic implications of cell traffic through afferent LVs.

Human MAIT cells exit peripheral tissues and recirculate via lymph in steady state conditions.

Mucosal-associated invariant T cells (MAIT cells) recognize bacterial metabolites as antigen and are found in blood and tissues, where they are poised to contribute to barrier immunity. Recent data demonstrate that MAIT cells located in mucosal barrier tissues are functionally distinct from their blood counterparts, but the relationship and circulation of MAIT cells between blood and different tissue compartments remains poorly understood. Previous studies raised the possibility that MAIT cells do not leave tissue and may either be retained or undergo apoptosis. To directly address if human MAIT cells exit tissues, we collected human donor-matched thoracic duct lymph and blood and analyzed MAIT cell phenotype, transcriptome, and T cell receptor (TCR) diversity by flow cytometry and RNA sequencing. We found that MAIT cells were present in the lymph, despite being largely CCR7- in the blood, thus indicating that MAIT cells in the lymph migrated from tissues and were capable of exiting tissues to recirculate. Importantly, MAIT cells in the lymph and blood had highly overlapping clonotype usage but distinct transcriptome signatures, indicative of differential activation states.

Second generation antipsychotic-induced mitochondrial alterations: Implications for increased risk of metabolic syndrome in patients with schizophrenia.

Metabolic syndrome (MetS) is seen more frequently in persons with schizophrenia than in the general population, and these metabolic abnormalities are further aggravated by second generation antipsychotic (SGA) drugs. Although the underlying mechanisms responsible for the increased prevalence of MetS among patients under SGA treatment are not well understood, alterations in mitochondria function have been implicated. We performed a comprehensive evaluation of the role of mitochondrial dysfunction in the pathophysiology of drug-induced MetS in schizophrenia. We found a downregulation in genes encoding subunits of the electron transport chain complexes (ETC), enzyme activity, and mitochondrial dynamics in peripheral blood cells from patients at high-risk for MetS. Additionally, we evaluated several markers of energy metabolism in lymphoblastoid cell lines from patients with schizophrenia and controls following exposure to antipsychotics. We found that the high-risk drugs clozapine and olanzapine induced a general down-regulation of genes involved in the ETC, as well as decreased activities of the corresponding enzymes, ATP levels and a significant decrease in all the functional parameters of mitochondrial oxygen consumption in cells from patients and controls. We also observed that the medium-risk SGA quetiapine decreased oxygen consumption and respiratory control ratio in controls and patients. Additionally, clozapine and olanzapine induced a downregulation of Drp1 and Mfn2 both in terms of mRNA and protein levels. Together, these data suggest that an intrinsic defect in multiple components of oxidative metabolism may contribute to the increased prevalence of MetS in patients under treatment with SGAs known to cause risk for MetS.

Burn Injury-Associated MHCII+ Immune Cell Accumulation Around Lymphatic Vessels of the Mesentery and Increased Lymphatic Endothelial Permeability Are Blocked by Doxycycline Treatment.

It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII+ immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (∼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.

Migratory capacity and function of dendritic cells from mesenteric afferent lymph nodes after feeding a single dose of vitamin A.

Lamina propria dendritic cells (DCs) have a permanent turnover with constitutive migration to mesenteric lymph nodes and replenishment by progenitors. Luminal bacteria and dietary constituents provide key signals that endow DCs their unique properties in vivo. Taking into account that the intestinal immune system is greatly influenced by retinoids, we evaluated in B6 mice 3, 8, 16 and 24 h after feeding a single dose of vitamin A phenotype and function of cells present in mesenteric afferent lymph nodes as well as signals involved in migration. We studied the frequency of CD11c+MHC-II+CD103+CD86+ and RALDH+ DCs by flow cytometry, we determined CCL-21 and D6 levels in tissue homogenates by Western blot, and we co-cultured cells isolated from afferent lymphatics with sorted CD4+ lymphocytes to assess Foxp-3 induction and homing receptor expression. Sixteen hours after vitamin A administration, DCs isolated from afferent lymphatics were able to induce homing receptors and Foxp3 expression in CD4+ lymphocytes. Our results show that a single dose of vitamin A generated a stream of signals and amplified the tolerogenic activity of DCs migrating to lymphoid tissue.

Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: Unlocking the mystery of post-shock multiple organ failure.

BACKGROUND: Previous studies have shown that mesenteric lymph (ML) has a crucial role in driving the systemic inflammatory response after trauma/hemorrhagic shock (T/HS). The specific mediators in the ML that contribute to its biological activity remain unclear despite decades of study. Exosomes are extracellular vesicles that are shed into body fluids such as serum and urine that can mediate intercellular communication. We hypothesized that exosomes are present in the ML after trauma/shock and are responsible for the biological activity of ML. METHODS: Male rats underwent cannulation of the vessels and mesenteric lymph duct. T/HS was induced by laparotomy and 60 minutes of HS (mean arterial pressure, 35 mmHg), followed by resuscitation. The ML was collected during three distinct time periods (pre-shock, shock, and resuscitation phase) and subsequently separated into exosome and supernatant fractions. Exosomes were characterized by electron microscope, nanoparticle tracking analysis, and immunoblotting. The biological activity of exosomes and supernatant of ML were characterized using a monocyte NF-κB reporter assay and by measuring macrophage intracellular TNF-α production. RESULTS: Exosomes were identified in ML by size and expression of the exosome markers CD63 and HSP70. The number of exosomes present in the ML was 2-fold increased during shock and 4-fold decreased in resuscitation phase compared to pre-shock. However, biological activity of exosomes isolated during the resuscitation phase was markedly increased and caused an 8-fold increase in monocyte NF-κB activation compared to supernatant. Macrophage TNF-α production was also increased after exposure to exosomes harvested in the resuscitation phase. The ML supernatant fraction had no effect on TNF-α production during any phase. CONCLUSIONS: Our findings show that exosomes, and not the liquid fraction of ML, are the major component triggering inflammatory responses in monocytes and macrophages after experimental T/HS.

Effect of Melatonin on Telocytes in the Seminal Vesicle of the Soay Ram: An Immunohistochemical, Ultrastructural and Morphometrical Study.

Telocytes (TCs) are a special type of interstitial cell with characteristic cellular processes that are described in many organs. The current study aimed to investigate TCs in seminal vesicles of the Soay ram responding to melatonin treatment during the nonbreeding season by conventional immunohistochemical stains, and to detect the ultrastructural and morphometrical changes of TCs. TCs in the control group showed a broad range of staining affinity and also reacted positively to CD117/c-kit, CD34, desmin, S-100 protein, and progesterone and estrogen receptors alpha, while after melatonin treatment a strong reaction against these 6 antibodies was recorded. Electron microscopically, TCs in the control group were characterized by a small cell body with distinct long cytoplasmic extensions called telopodes (Tps). Tps had alternation of the thin segment (podomers) and dilated segments (podoms), in which the latter accommodate mitochondria, rough endoplasmic reticulum and caveolae. TCs and their Tps were interconnected by homo- and heterocellular junctions and form a wide network to communicate between different cell types. Tps showed close contact with immune cells, progenitor stem cells, smooth muscle cells and other interstitial cells. Melatonin caused a significant increase in the number of TCs, length of Tps, and number and diameter of secretory vesicles. Also, the melatonin-treated group showed exaggerated secretory activity in the form of a massive release of secretory vesicles from Tps. Moreover, Tps showed an increase in their contact with blood and lymphatic capillaries, nerve endings and Schwann cells. In addition, the shedding of secretory structures (exosomes, ectosomes, and multivesicular bodies) was greater from Tps, which were involved in paracrine signaling in the melatonin-treated group. The length and ramifications of Tps together with the intercellular junctions and the releasing of shed vesicles or exosomes assumed an essential role of TCs in intercellular signaling and coordination. On the basis of their distribution and morphology, we investigated whether the different locations of TCs could be associated with different roles.

A microRNA from infectious spleen and kidney necrosis virus modulates expression of the virus-mock basement membrane component VP08R.

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. Infection of ISKNV is characterized by a unique pathological phenomenon in that the infected cells are attached by lymphatic endothelial cells (LECs). ISKNV mediates the formation of a virus-mock basement membrane (VMBM) structure on the surface of infected cells to provide attaching sites for LECs. The viral protein VP08R is an important component of VMBM. In this study, a novel ISKNV-encoded microRNA, temporarily named ISKNV-miR-1, was identified. ISKNV-miR-1 is complementary to the VP08R-coding sequence and can modulate VP08R expression through reducing its mRNA level. This suggests that formation of VMBM may be under fine regulation by ISKNV.

Investigation of cell death mechanisms in human lymphatic endothelial cells undergoing photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs. METHODS: Verteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT. RESULTS: PDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing <50% cell death PDT induced autophagy rather than apoptosis, as revealed by conversion of LC3-I to the autophagosomal LC3-II and formation of LC3 puncta. Z-VAD-FMK, a caspase inhibitor, prevented cell death induced by high-dose PDT only, while 3-methyladenine, an autophagy suppressor, inhibited cell death induced by low-dose PDT. CONCLUSIONS: Both apoptosis and autophagy are involved in cell death induced by verteporfin-PDT in LECs.

Lympho- and Hemodynamics in Dogs with Acute Experimental Pancreatitis.

Dogs with experimental pancreatitis showed increased lymph fl ow, impaired rheological properties of the lymph and blood plasma, and increased plasma and blood levels of glucose, ALT, and AST.

Vaccination with liposomal poly(I:C) induces discordant maturation of migratory dendritic cell subsets and anti-viral gene signatures in afferent lymph cells.

Vaccine formulations administered in the periphery must activate naive immune cells within the lymph node. In this study, we have directly cannulated the ovine lymphatic vessels to investigate the cellular and molecular mechanisms that transfer information from the periphery into the local draining lymph node via the afferent lymph. Inclusion of poly(I:C) into a liposomal vaccine formulation enhances the neutrophil-associated inflammatory immune response in afferent lymph and increases antigen uptake by migratory dendritic cells (DCs). Interestingly, antigen positive migratory DCs undergo discordant maturation, with peak expression of CD86 at 4 h and CD80 at 48-72 h post vaccination. Afferent lymph monocytes up-regulate expression of genes related to inflammatory and anti-viral immune phenotypes following vaccination however show no differentiation into APCs prior to their migration to the local lymph node as measured by surface MHC II expression. Finally, this study reveals the addition of poly(I:C) increases systemic antigen-specific humoral immunity. These findings provide a detailed understanding of the real time in vivo immune response induced by liposomes incorporating the innate immune agonist poly(I:C) utilising a vaccination setting comparable to that administered in humans.

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Drosophila hematopoiesis: Markers and methods for molecular genetic analysis.

Analyses of the Drosophila hematopoietic system are becoming more and more prevalent as developmental and functional parallels with vertebrate blood cells become more evident. Investigative work on the fly blood system has, out of necessity, led to the identification of new molecular markers for blood cell types and lineages and to the refinement of useful molecular genetic tools and analytical methods. This review briefly describes the Drosophila hematopoietic system at different developmental stages, summarizes the major useful cell markers and tools for each stage, and provides basic protocols for practical analysis of circulating blood cells and of the lymph gland, the larval hematopoietic organ.

Vagal nerve stimulation modulates the dendritic cell profile in posthemorrhagic shock mesenteric lymph.

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 CD4Foxp3 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.