Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

National Clinical Research Center for Digestive Diseases

Capital Medical University

Capital Medical University School of Stomatology

Beijing Clinical Research Institute

Double-negative (DN) T cells could delay the onset and the progression of autoimmune diabetes, yet they were less efficient on reversing autoimmune diabetes. The aim of this study was to investigate whether the combination of DN T cells and anti-thymocyte serum (ATS) could reverse new-onset diabetes in NOD mice.

Combination of double negative T cells and anti-thymocyte serum reverses type 1 diabetes in NOD mice.

Monitoring T lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling. In this study, we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine (EdU) incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model. We found that fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC, PE, PE-Cy7, FITC and PerCP-Cy5.5 when the concentration of the permeabilization reagents was optimized. However, the click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7, and surface staining after the click reaction improved the fluorescence intensity. Thus, an extra step of blocking with PBS with 3% FBS between the click reaction and cell surface staining is needed. Furthermore, the percentage of EdU-positive cells increased in a dose-dependent manner, and the saturated dose of EdU was 20mg/kg. Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo. In addition, T cell proliferation measured by EdU incorporation was comparable to BrdU but was lower than CFSE labelling. In conclusion, we optimized the protocols for EdU administration in vivo and staining in vitro, providing a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.

Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation.

Both innate and adaptive immune cells are involved in the pathogenesis of nonalcoholic steatohepatitis (NASH), but the crosstalk between innate and adaptive immunity is largely unknown. Here we show that compared with WT mice, OX40 mice exhibit decreased liver fat accumulation, lobular inflammation, and focal necrosis after feeding with diets that induce NASH. Mechanistically, OX40 deficiency suppresses Th1 and Th17 differentiation, and OX40 deficiency in T cells inhibits monocyte migration, antigen presentation, and M1 polarization. Soluble OX40 stimulation alone upregulates antigen presentation, chemokine receptor expression, and proinflammatory cytokine secretion by liver monocytes. Furthermore, plasma soluble OX40 levels are positively associated with NASH in humans, suggesting clinical relevance of the findings. In conclusion, we show a mechanism for T cell regulation of innate immune cells. OX40 is a key regulator of both intrahepatic innate and adaptive immunity, generates two-way signals, and promotes both proinflammatory monocyte and macrophage and T cell function, resulting in NASH development.

OX40 Regulates Both Innate and Adaptive Immunity and Promotes Nonalcoholic Steatohepatitis.

 Liver macrophages agitated by Lipopolysaccharide (LPS) can enhance immuno-inflammatory responses in the liver which mediate liver injury and result in dysfunction. Midazolam has been reported to have inhibitory effects on activated immunity and escalated inflammation, however, what the effects of midazolam on the liver injury caused by excessive immuno-inflammatory response in sepsis, and what influence it will exert on inflamed liver macrophages need to be elucidated.  In the present study, LPS and galactosamine-induced acute liver injury mice were used to observe the effect of midazolam . LPS-stimulated bone marrow cells were used to evaluate the influence of midazolam on monocytes .  Midazolam prevented liver tissue injury and decreased serum alanine transaminase (ALT) level in LPS plus galactosamine treated mice. Mechanistically, midazolam suppressed tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) produced by LPS stimulated liver macrophages  and bone marrow monocytes , and reduced the expression of major histocompatibility complex class II (MHC II), cluster of differentiation 40 and 86 (CD40 and CD86) on the cell surface. These results could be reversed by PK-11195, a peripheral benzodiazepine receptor (PBR) blocker.  Midazolam can prevent liver from LPS-induced immune mediated liver injury by inhibiting inflammation and immune activation in liver macrophages.

The Protection of Midazolam Against Immune Mediated Liver Injury Induced by Lipopolysaccharide and Galactosamine in Mice.

The liver is a central immunologic organ with a high density of myeloid and lymphoid immune cells that play important roles in the development and progression of nonalcoholic steatohepatitis (NASH). However, the immune-cell-mediated regulation of NASH and its underlying mechanisms remain obscure. In this study,  mice showed significantly higher plasma alanine transaminase levels, with increased liver fat accumulation, lobular inflammation, and focal necrosis compared with wild-type (WT) mice after 4 wk of feeding on a methionine- and choline-deficient diet (MCD) or 16 wk of feeding on a high-fat diet. Perforin deficiency promoted the M1 polarization of infiltrated monocytes. Moreover, MCD-fed  mice exhibited increased accumulation, survival, activation, and proinflammatory cytokine production of CD8 T cells but not NK cells or CD4 T cells. Adoptive transfer of CD8 T cells or NK cells from WT or  mice, together with non-CD8 cells or non-NK cells from WT mice, indicated that CD8 T-cell-derived perforin participates in the mechanism regulating liver inflammation and thus plays a protective role in the development of NASH. Perforin-deficient CD8 T cells exhibited decreased cytotoxicity toward bone marrow-derived M1 monocytes and macrophages. According to the RNA sequencing data, the perforin deficiency inhibited cell apoptosis and enhanced the activation, migration, and proinflammatory cytokine production of CD8 T cells in mice with NASH. Furthermore, we found higher plasma soluble perforin levels and hepatic perforin expression in NASH patients, suggesting clinical relevance of the findings. We have elucidated an important role for the cytotoxic immune effector molecule perforin from CD8 T cells in restricting hepatic inflammation in mice with NASH and suggest that therapies designed to maximize the function of endogenous perforin in CD8 T cells might have potential benefits as NASH treatments.-Wang, T., Sun, G., Wang, Y., Li, S., Zhao, X., Zhang, C., Jin, H., Tian, D., Liu, K., Shi, W., Tian, Y., Zhang, D. The immunoregulatory effects of CD8 T-cell-derived perforin on diet-induced nonalcoholic steatohepatitis.

The immunoregulatory effects of CD8 T-cell-derived perforin on diet-induced nonalcoholic steatohepatitis.

Allergic asthma is an inflammatory disorder of the airway without satisfactory traditional therapies capable of controlling the underlying pathology. New approaches that can overcome the detrimental effects of immune dysregulation are thus desirable. Here we adoptively transfer ovalbumin (OVA) peptide-primed CD4CD8 double negative T (DNT) cells intravenously into a mouse model of OVA-induced allergic asthma to find that OVA-induced airway hyperresponsiveness, lung inflammation, mucus production and OVA-specific IgG/IgE production are significantly suppressed. The immunosuppressive function of the OVA-specific DNT cells is dependent on the inhibition of CD11b dendritic cell function, T follicular helper cell proliferation, and IL-21 production. Mechanistically, Lag3 contributes to MHC-II antigen recognition and trogocytosis, thereby modulating the antigen-specific immune regulation by DNT cells. The effectiveness of ex vivo-generated allergen-specific DNT cells in alleviating airway inflammation thus supports the potential utilization of DNT cell-based therapy for the treatment of allergic asthma.

Double negative T cells mediate Lag3-dependent antigen-specific protection in allergic asthma.

Neutrophils play critical roles during the initial phase of hepatic ischemia/reperfusion injury (HIRI). However, the regulation of neutrophil activation, infiltration, and proinflammatory cytokine secretion has not been fully elucidated. In this study, we revealed that OX40 was expressed by neutrophils, its expression in neutrophils was time-dependently upregulated following HIRI, and Ox40 knockout markedly alleviated liver injury. Compared with wild-type neutrophils, the adoptive transfer of Ox40-/- neutrophils decreased HIRI in neutrophil-depleted Rag2/Il2rg-/- or Ox40-/- mice. Moreover, consistently, the in vitro experiments showed that Ox40 not only prolonged neutrophil survival but also promoted proinflammatory cytokines, ROS production, and even neutrophil chemotaxis. Further investigation demonstrated that the knockout of Ox40 in neutrophils inhibited NF-κB signaling via the TRAF1/2/4 and IKKα/IKKβ/IκBα pathways. OX40L and OX86 stimulation could enhance neutrophil activation and survival in vitro and in vivo. In conclusion, our study provides a new understanding of OX40, which is expressed not only in adaptive immune cells but also in innate immune cells, i.e., neutrophils, contributing to the activation and survival of neutrophils. These findings provide a novel potential therapeutic target for the prevention of HIRI during liver transplantation or hepatic surgery.

OX40 expression in neutrophils promotes hepatic ischemia/reperfusion injury.

The innate and adaptive immune systems both play important roles in drug-induced liver injury (DILI). However, the crosstalk between the innate and adaptive immunity in DILI is largely unknown. Extensive crosstalk is likely mandated by co-stimulatory interactions between these immune systems. OX40 is a co-stimulatory molecule, but whether it regulates the intrahepatic immune response in DILI remains unknown.

Critical role of OX40 in drug-induced acute liver injury.

Isolating and purifying liver immune cells are crucial for observing the changes in intrahepatic immune responses during the development of liver diseases and exploring the potential immunological mechanisms. Therefore, the aim of this study was to provide an optimal protocol for isolating immune cells with a high yield and less damage. We compared mechanical dissection and collagenase digestion, and the results were represented by the proportion of lymphocytes, Kupffer cells and neutrophils. The apoptosis rates of liver immune cells resulted by different isolation protocols were compared by Annexin V-staining using flow cytometric analysis. Our data indicated that the enzymatic digestion in vitro was more efficient than the mechanical dissection in vitro with a suitable collagenase IV concentration of 0.01%, and the purification of liver immune cells by a one-step density gradient centrifugation in 33% Percoll had the definite advantage of a higher proportion of the target cells. We also provided evidence that enzymatic digestion in vitro method was superior to collagenase digestion in situ for liver T lymphocytes, NK cells and NKT cells isolation and purification. This protocol was also validated in human liver samples. In conclusion, we developed an optimal protocol for isolating and purifying immune cells from mouse and human liver samples in vitro by 0.01% collagenase IV and 33% Percoll density gradient centrifugation with the advantages of higher cell yields and viability. This method provides a basis for further studying liver immune cells and liver immunity with a wide range of applications.

Isolation and purification of immune cells from the liver.

Hepatic inflammation is the driving force for the development and progression of NASH. Treatment targeting inflammation is believed to be beneficial. In this study, adoptive transfer of CD4 T cells converted double negative T cells (cDNT) protects mice from diet-induced liver fat accumulation, lobular inflammation and focal necrosis. cDNT selectively suppress liver-infiltrating Th17 cells and proinflammatory M1 macrophages. IL-10 secreted by M2 macrophages decreases the survival and function of cDNT to protect M2 macrophages from cDNT-mediated lysis. NKG2A, a cell inhibitory molecule, contributes to IL-10 induced apoptosis and dampened suppressive function of cDNT. In conclusion, ex vivo-generated cDNT exert potent protection in diet induced obesity, type 2 diabetes and NASH. The improvement of outcome is due to the inhibition on liver inflammatory cells. This study supports the concept and the feasibility of potentially utilizing this autologous immune cell-based therapy for the treatment of NASH.

CD4 derived double negative T cells prevent the development and progression of nonalcoholic steatohepatitis.

Acetaminophen (APAP) overdose is a major cause of the morbidity of acute liver failure. The current clinically approved treatment for APAP poisoning, N-acetylcysteine (NAC), has a limited therapeutic window, and prolonged treatment with NAC delays liver regeneration. Mesenchymal stem cells (MSCs) also have therapeutic effects on APAP-induced mouse liver failure, but whether the effects are comparable to those of NAC has not been determined, and the mechanism still needs further exploration.

Mesenchymal stem cells protect against acetaminophen hepatotoxicity by secreting regenerative cytokine hepatocyte growth factor.

 Excessive immune activation leads to secondary injury and impedes injured brain recovery after ischemic stroke. However, few effective methods are currently used for equilibrating immune balance. CD3NK1.1TCRβCD4CD8 double-negative T (DNT) cells which do not express NK cell surface markers are unique regulatory cells that maintain homeostasis in several immune-related diseases. However, the therapeutic potential and regulatory mechanism of DNT cells in ischemic stroke are still unknown.  Mouse ischemic stroke is induced by occlusion of the distal branches of the middle cerebral artery (dMCAO). DNT cells were adoptively transferred intravenously into ischemic stroke mice. Neural recovery was evaluated by TTC staining and behavioral analysis. Using immunofluorescence, flow cytometry, and RNA sequencing, the immune regulatory function of DNT cells was investigated at different time points post ischemic stroke.  Adoptive transfer of DNT cells significantly reduces infarct volume and improves sensorimotor function after ischemic stroke. DNT cells suppress peripheral Trem1 myeloid cell differentiation during the acute phase. Furthermore, they infiltrate the ischemic tissue via CCR5 and equilibrate the local immune balance during the subacute phase. During the chronic phase, DNT cells enhance Treg cell recruitment through CCL5, eventually developing an immune homeostatic milieu for neuronal recovery.  DNT cell treatment renders the comprehensive anti-inflammatory roles in specific phases of ischemic stroke. Our study suggests that the adoptive transfer of regulatory DNT cells may be a potential cell-based therapy for ischemic stroke.

Article	Year
Combination of double negative T cells and anti-thymocyte serum reverses type 1 diabetes in NOD mice. Journal of translational medicine\| PubMed ID: 26911290	2016
Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation. International immunopharmacology\| PubMed ID: 27816727	2016
OX40 Regulates Both Innate and Adaptive Immunity and Promotes Nonalcoholic Steatohepatitis. Cell reports\| PubMed ID: 30590049	2018
The Protection of Midazolam Against Immune Mediated Liver Injury Induced by Lipopolysaccharide and Galactosamine in Mice. Frontiers in pharmacology\| PubMed ID: 30670973	2018
The immunoregulatory effects of CD8 T-cell-derived perforin on diet-induced nonalcoholic steatohepatitis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 30951375	2019
Double negative T cells mediate Lag3-dependent antigen-specific protection in allergic asthma. Nature communications\| PubMed ID: 31534137	2019
OX40 expression in neutrophils promotes hepatic ischemia/reperfusion injury. JCI insight\| PubMed ID: 31672934	2019
Critical role of OX40 in drug-induced acute liver injury. British journal of pharmacology\| PubMed ID: 32133635	2020
Isolation and purification of immune cells from the liver. International immunopharmacology\| PubMed ID: 32470880	2020
CD4 derived double negative T cells prevent the development and progression of nonalcoholic steatohepatitis. Nature communications\| PubMed ID: 33510172	2021
Mesenchymal stem cells protect against acetaminophen hepatotoxicity by secreting regenerative cytokine hepatocyte growth factor. Stem cell research & therapy\| PubMed ID: 35246254	2022
TCRαβNK1.1CD4CD8 double-negative T cells inhibit central and peripheral inflammation and ameliorate ischemic stroke in mice. Theranostics\| PubMed ID: 36793857	2023
Erratum to "Isolation and purification of immune cells from the liver" [Int. Immunopharmacol. 85 (2020) 106632]. International immunopharmacology\| PubMed ID: 37121794	2023

Yue Tian

Biography

JoVE Journal Publications

Other Publications