OBJECTIVE: To assess the effect of neutralizing CD96 on natural killer (NK) cell functions in mice with pulmonary infection and explore the possible mechanism. METHODS: Male BALB/c mice were randomly divided into infection group (Cm group), anti-CD96 treatment group (anti-CD96 group) and control group (=5). In the former two groups, was inoculated intranasal administration to establish mouse models of pulmonary infection, and the mice in the control group received intranasal administration of the inhalation buffer. In anti-CD96 group, the mice were injected with anti-CD96 antibody intraperitoneally at the dose of 250 μg every 3 days after the infection; the mice in Cm group received intraperitoneal injections of saline. The body weight of the mice was recorded daily. The mice were sacrificed 5 days after infection, and CD96 expression was detected by quantitative real-time PCR and Western blotting. HE staining and pathological scores were used to evaluate pneumonia of the mice. The inclusion body forming units (IFUs) were detected in the lung tissue homogenates to assess lung tissue chlamydia load. Flow cytometry and ELISA were used to assess the capacity of the lung NK cells to produce interferon-γ (IFN-γ) and regulate macrophages and Th1 cells. RESULTS: infection inhibited CD96 expression in NK cells of the mice. Compared with those in Cm group, the mice in antiCD96 mice showed significantly milder lung inflammation ( < 0.05) and reduced chlamydia load in the lung tissue ( < 0.05). Neutralizing CD96 with anti-CD96 significantly enhanced IFN-γ secretion by the NK cells ( < 0.05) and augmented the immunoregulatory effect of the NK cells shown by enhanced responses of the lung macrophages ( < 0.05) and Th1 cells ( < 0.05). CONCLUSIONS Inhibition of CD96 alleviates pneumonia in -infected mice possibly by enhancing IFN-γ secretion by NK cells and augmenting the immunoregulatory effect of the NK cells on innate and adaptive immunity.
Animals ; Antigens, CD ; metabolism ; Chlamydia Infections ; complications ; immunology ; physiopathology ; Chlamydia muridarum ; Interferon-gamma ; genetics ; metabolism ; Killer Cells, Natural ; metabolism ; Lung Injury ; etiology ; genetics ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

The aim of this paper was to study the influence of triptolide in the immune response pathways of acquired immune deficiency syndrome( AIDS). Target proteins of triptolide and related genes of AIDS were searched in PubChem and Gene databases on line. Molecular networks and canonical pathways comparison analyses were performed by bioinformatics software( IPA). There were 15 targets proteins of triptolide and 258 related genes of AIDS. Close biological relationships of molecules of triptolide and AIDS were established by networks analysis. There were 21 common immune response pathways of triptolide and AIDS,including neuroinflammation signaling pathway,Th1 and Th2 activation pathway and role of pattern recognition receptors in recognition of bacteria and viruses. Triptolide stimulated immune response pathways by the main molecules of IFNγ,JAK2,NOD1,PTGS2,RORC. IFNγ is the focus nodes of triptolide and AIDS,and regulates genes of AIDS directly or indirectly. Triptolide may against AIDS by regulating molecules IFNγ in immune response pathways.
Acquired Immunodeficiency Syndrome ; drug therapy ; immunology ; Computational Biology ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Gene Regulatory Networks ; Humans ; Interferon-gamma ; genetics ; Phenanthrenes ; pharmacology ; Receptors, Pattern Recognition ; immunology ; Signal Transduction ; T-Lymphocytes ; immunology

Objective The aim of this study was to evaluate the diagnostic performance of T-SPOT.TB for tuberculous lymphadenitis. Methods Suspected tuberculous lymphadenitis patients between September 2010 and September 2018 who had both peripheral blood T-SPOT.TB test and lymph node biopsy were retrospectively enrolled in this study. The cutoff value of T-SPOT.TB test for peripheral blood was set as 24 spot forming cell (SFC)/10 ⁶ periphreral blood monocyte cell (PBMC) according to the instruction of testing kits. The gold standard for diagnosis of TBL was the combination of microbiology results, histopathology results and patient's response to anti-TB treatment. Diagnostic efficacy of T-SPOT.TB was evaluated, including sensitivity, specificity, accuracy, predictive values, and likelihood ratio. Results Among 91 patients who met the inclusion criteria, we excluded 8 cases with incomplete clinical information and 6 cases who lost to follow-up. According to the gold standard, there were 37 cases of true TBL (9 confirmed TBL and 28 probable TBL), 30 cases of non-TBL, and 10 cases of clinically indeterminate diagnosis who were excluded from the final analyses. The T-SPOT.TB tests yielded 43 cases of positive response and 24 cases of negative response. The sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR) and negative likelihood ratio (NLR) of peripheral blood T-SPOT.TB for diagnosing TBL were 89.2%, 66.7%, 79.1%, 76.7%, 83.3%, 2.68 and 0.16, respectively. The number of SFCs of T-SPOT.TB in TBL patients [432(134-1264)/10 ⁶ PBMCs] was higher than that in non-TBL patients [0 (0-30) /10 ⁶PBMCs] with a significant difference (Z=-5.306, P <0.001). Conclusion T-SPOT.TB is a rapid and simple diagnostic test for TBL with a high sensitivity and negative predictive value.
Adolescent ; Adult ; Aged ; Female ; Humans ; Interferon-gamma Release Tests ; Male ; Middle Aged ; Mycobacterium tuberculosis/physiology* ; T-Lymphocytes/immunology* ; Tuberculosis, Lymph Node/diagnosis* ; Young Adult

BackgroundImbalance of interferon-gamma (IFN-&gamma;), interleukin (IL)-4, and IL-17 producing by T cells is confirmed to contribute to the pathogenesis of systemic lupus erythematosus (SLE). Autophagy is now emerging as a core player in the development and the function of the immune system. Therefore, we investigated the autophagic behavior in IFN-&gamma;-, IL-4-, and IL-17-producing T cells from patients with SLE.

MethodsThirty patients with SLE and 25 healthy controls matched for gender and age were recruited between September 2016 and May 2017. The autophagic levels in IFN-&gamma; T cells, IL-4 T cells, and IL-17 T cells from patients with newly diagnosed SLE and healthy controls were measured using flow cytometry. The plasma levels of IFN-&gamma; were determined by enzyme-linked immunosorbent assay in SLE patients and healthy controls. Unpaired t-tests and the nonparametric Mann-Whitney U-test were used to compare data from patients with SLE and controls. Spearman's rank correlation coefficient was applied for calculation of the correlation between parallel variables in single samples.

ResultsOur results showed increased percentage of autophagy in IFN-&gamma; T cells from patients with SLE and healthy controls ([8.07 ± 2.72]% vs. [3.76 ± 1.67]%, t = 5.184, P < 0.001), but not in IL-4 T cells or IL-17 T cells (P > 0.05) as compared to healthy donors. Moreover, the plasma levels of IFN-&gamma; in SLE patients were significantly higher than those in healthy controls ([68.9 ± 29.1] pg/ml vs. [24.7 ± 17.6] pg/ml, t = 5.430, P < 0.001). Moreover, in SLE patients, the percentage of autophagy in IFN-&gamma; T cells was positively correlated with the plasma levels of IFN-&gamma; (r = 0.344, P = 0.046), as well as the disease activity of patients with SLE (r = 0.379, P = 0.039).

ConclusionThe results indicate that autophagy in IFN-&gamma; T cells from SLE patients is activated, which might contribute to the persistence of T cells producing IFN-&gamma;, such as Th1 cells, and consequently result in the high plasma levels of IFN-&gamma;, and then enhance the disease activity of SLE.

Adult ; Autophagy ; China ; Female ; Humans ; Interferon-gamma ; metabolism ; Interleukin-17 ; metabolism ; Interleukin-4 ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; Male ; Middle Aged ; Th1 Cells ; physiology

Cisplatin and other platinum-based drugs are used frequently for treatment of lung cancer. However, their clinical performance are usually limited by drug resistance or toxic effects. Carnosic acid, a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), has been reported to have several pharmacological and biological activities. In the present study, the combination effect of cisplatin plus carnosic acid on mouse LLC (Lewis lung cancer) xenografts and possible underlying mechanism of action were examined. LLC-bearing mice were treated with intraperitoneal injection with cisplatin, oral gavage with carnosic acid, or combination with cisplatin and carnosic acid, respectively. Combination of carnosic acid and cisplatin yielded significantly better anti-growth and pro-apoptotic effects on LLC xenografts than drugs alone. Mechanistic study showed that carnosic acid treatment boosted the function of CD8 T cells as evidenced by higher IFN-γ secretion and higher expression of FasL, perforin as well as granzyme B. In the meantime, the proportion of MDSC (myeloid-derived suppressor cells) in tumor tissues were reduced by carnosic acid treatment and the mRNA levels of iNOS2, Arg-1, and MMP9, which are the functional markers for MDSC, were reduced. In conclusion, our study proved that the functional suppression of MDSC by carnosic acid promoted the lethality of CD8 T cells, which contributed to the enhancement of anti-lung cancer effect of cisplatin.
Abietanes ; administration & dosage ; Animals ; Antineoplastic Agents ; administration & dosage ; CD8-Positive T-Lymphocytes ; drug effects ; immunology ; Carcinoma, Lewis Lung ; drug therapy ; genetics ; immunology ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; Drug Synergism ; Humans ; Interferon-gamma ; genetics ; immunology ; Lung Neoplasms ; drug therapy ; genetics ; immunology ; Matrix Metalloproteinase 9 ; genetics ; Mice ; Mice, Inbred C57BL ; Myeloid-Derived Suppressor Cells ; drug effects ; immunology ; Plant Extracts ; administration & dosage ; Rosmarinus ; chemistry

To investigate the effect of bromodomain and extra-terminal (BET) protein inhibitor JQ1 on expression of autoimmune-related genes in CD4+T cells from patients with systemic lupus erythematosus (SLE).
 Methods: Peripheral CD4+T cells were isolated by positive selection with CD4 microbeads. The percentage of CD4+T cells were detected by flow cytometry. CD4+T cells were treated by JQ1 at 100 nm/L for 6, 24, 48 h. The expression of T cell-related genes was measured by quantitative real-time PCR (qPCR). The secretion levels of cytokines in culture supernatant were measured by ELISA at 48 h.
 Results: The percentage of CD4+T cells isolated by CD4 microbeads is 97.2%. Compared with the control group, the mRNA expression levels of IFNG, IL-17F, IL-21, CXCR5 and FOXP3 were down-regulated at 6, 24 and 48 h (P<0.05), and IL-17A mRNA level was decreased at 6 and 24 h (P<0.01); while IL-4 mRNA level was up-regulated at 24, 48 h (P<0.01), and TGF-β1 mRNA level was up-regulated at 6 and 48 h (P<0.05) in SLE CD4+T cells treated with JQ1. The secretion levels of IFN-γ and IL-21 in JQ1-treated group were decreased significantly (P<0.05), while the secretion levels of IL-4 and TGF-β were up-regulated compared with control group (P<0.05).
 Conclusion: JQ1 can reverse the immune dysregulation and improve the immunity homeostasis in CD4+T cells from patients with SLE.
Azepines ; pharmacology ; CD4 Lymphocyte Count ; CD4-Positive T-Lymphocytes ; cytology ; drug effects ; metabolism ; Cytokines ; analysis ; metabolism ; Flow Cytometry ; Humans ; Interferon-gamma ; metabolism ; Lupus Erythematosus, Systemic ; immunology ; metabolism ; Proteins ; antagonists & inhibitors ; RNA, Messenger ; metabolism ; Time Factors ; Transforming Growth Factor beta1 ; Triazoles ; pharmacology

OBJECTIVETo observe the effects of recombinant fusion protein interleukin (IL)-18 on the expression of immune-inflammatory factors in the mice infected with Staphylococcus aureus (SA), and to investigate the mechanism of action of IL-18 in defense of SA infection in vivo.

METHODSA total of 40 specific pathogen-free female BLAB/c mice were randomly divided into four groups: control, SA infection, immunized, and intervention. A mouse model of SA infection was established by nasal inoculation with SA liquid. The immunized group and the intervention group were intranasally given IL-18 before SA modeling, and then the SA infection group and the intervention group received the nasal inoculation with SA liquid; the control group was treated with phosphate buffered saline instead. The levels of IL-4, interferon (IFN)-&gamma;, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), IgM in the serum and bronchoalveolar lavage fluid (BALF) of mice were measured by enzyme-linked immunosorbent assay. The expression of macrophage inflammatory protein (MIP)-1α mRNA and MIP-2β mRNA in the lung tissue of mice were determined by real-time fluorescent quantitative PCR.

RESULTSCompared with the control group, the SA infection group and the immunized group had significantly higher levels of IL-4, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue (P<0.05); the SA infection group had a significantly lower level of IFN-&gamma; and a significantly higher level of TNF in the serum and BALF (P<0.05); the immunized group had a significantly higher level of IFN-&gamma; in the serum and BALF (P<0.05). Compared with the SA infection group, the intervention group had significantly higher levels of IL-4, IFN-&gamma;, G-CSF, and IgM in the serum and BALF and expression of MIP-1α mRNA in the lung tissue. In contrast, the intervention group showed a significantly lower level of TNF in the serum and BALF and expression of MIP-2β mRNA in the lung tissue (P<0.05). All the above indicators in the intervention group were significantly higher than those in the control group (P<0.05), except the serum level of IFN-&gamma;.

CONCLUSIONSIn the mice infected with SA, the recombinant fusion protein IL-18 by mucosal immunity can affect inflammatory factors in the serum and BALF and the expression of MIP-1α mRNA and MIP-2β mRNA in the lung tissue to promote the anti-infective immune response and enhance the ability to clear pathogens.

Animals ; Chemokine CCL3 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; Interferon-gamma ; blood ; Interleukin-18 ; therapeutic use ; Interleukin-4 ; blood ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; pharmacology ; therapeutic use ; Staphylococcal Infections ; drug therapy ; immunology

PURPOSE: To identify new immunogenic HLA-A*33;03-restricted epitopes from the human papillomavirus (HPV) 16 E7 protein for immunotherapy against cervical cancer. MATERIALS AND METHODS: We synthesized fourteen overlapping 15-amino acid peptides and measured intracellular interferon-γ (IFN-γ) production in PBMC and CD8+ cytotoxic T lymphocytes (CTLs) after sensitization with these peptides using flow cytometry and ELISpot assay. The immunogenicity of epitopes was verified using a ⁵¹Cr release assay with SNU1299 cells. RESULTS: Among the fourteen 15-amino acid peptides, E7₄₉₋₆₃ (RAHYNIVTFCCKCDS) demonstrated the highest IFN-γ production from peripheral blood mononuclear cells (PBMCs), and CD8+ CTLs sensitized with E7₄₉₋₆₃ showed higher cytotoxic effect against SNU1299 cells than did CD8+ CTLs sensitized with other peptides or a negative control group. Thirteen 9- or 10-amino acid overlapping peptides spanning E7₄₉₋₆₃, E7₅₀₋₅₉ (AHYNIVTFCC), and E7₅₂₋₆₁ (YNIVTFCCKC) induced significantly higher IFN-γ production and cytotoxic effects against SNU1299 cells than the other peptides and negative controls, and the cytotoxicity of E7₅₀₋₅₉- and E7₅₂₋₆₁-sensitized PBMCs was induced via the cytolytic effect of CD8+ CTLs. CONCLUSION: We identified E7₅₀₋₅₉ and E7₅₂₋₆₁ as novel HPV 16 E7 epitopes for HLA-A*33;03. CD8+ CTL sensitized with these peptides result in an antitumor effect against cervical cancer cells. These epitopes could be useful for immune monitoring and immunotherapy for cervical cancer and HPV 16-related diseases including anal cancer and oropharyngeal cancer.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes/immunology/metabolism ; Epitopes/*immunology/therapeutic use ; Female ; *HLA-A Antigens ; Human papillomavirus 16/*immunology ; Humans ; *Immunotherapy ; Interferon-gamma/analysis/*biosynthesis ; Leukocytes, Mononuclear/immunology/metabolism ; T-Lymphocytes, Cytotoxic/immunology/metabolism ; Uterine Cervical Neoplasms/*therapy

OBJECTIVETo primarily evaluate the effects of ulinastatin on immune function of patients with severe burn injury.

METHODSForty patients with severe burn admitted to our ward from March 2013 to October 2015, conforming to the study criteria, were divided into conventional treatment group (CT, n=20) and ulinastatin treatment group (UT, n=20) according to the random number table and patient's consent. After admission, patients in group CT received antishock treatment, antibiotic treatment, debridement, skin grafting, and nutrition support, etc. On the basis of the above-mentioned treatment, patients in group UT received intravenous drip of ulinastatin from first day after admission twice a day, with a dosage of 8×10(5) U every time, for 7 days in addition. Peripheral venous blood samples were collected from patients in groups CT and UT on post treatment day (PTD) 1, 3, 5 and 7, respectively. Twenty healthy volunteer were selected as health control group (HC), and peripheral venous blood samples were collected on the first day of the study. Percentage of CD4(+) CD25(+) regulatory T lymphocytes (Tregs) was determined by flow cytometer. The proliferative activity of T lymphocytes was detected by microplate reader (denoted as absorbance value). Content of interleukin 2 (IL-2) in culture supernatant of T lymphocytes, and content of IL-4 and &gamma; interferon (IFN-&gamma;) in serum were detected by enzyme-linked immunosorbent assay. Expression of human leukocyte antigen-DR (HLA-DR) on CD14(+) monocytes was determined by flow cytometer. Data were processed with analysis of variance for repeated measurement, chi-square test, and LSD-t test.

RESULTS(1) Compared with that of volunteer in group HC, the percentage of CD4(+) CD25(+) Tregs of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 13.303 to 26.043, P values below 0.01). Compared with that in group CT, the percentage of CD4(+) CD25(+) Tregs of patients in group UT was significantly decreased on PTD 5 and 7 (with t values respectively 8.317 and 15.071, P values below 0.01). (2) The proliferative activity of T lymphocytes of patients in group CT on PTD 1, 3, 5, and 7 was respectively 0.71±0.11, 0.61±0.15, 0.54±0.12, and 0.67±0.17, which was significantly lower than that in group HC (1.21±0.22, with t values from 8.686 to 11.957, P values below 0.01). The proliferative activity of T lymphocytes of patients in group UT on PTD 3, 5, and 7 were respectively 0.81±0.11, 0.85±0.14, and 1.08±0.13, which was significantly higher than that in group CT (with t values from 4.808 to 8.568, P values below 0.01). (3) Compared with those of volunteer in group HC, content of IL-2 in culture supernatant of T lymphocytes of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 8.073 to 9.288, P values below 0.01), content of IL-4 in serum of patients in group CT was significantly increased from PTD 1 to 7 (with t values from 18.926 to 41.451, P values below 0.01), and content of IFN-&gamma; in serum of patients in group CT was significantly decreased from PTD 1 to 7 (with t values from 4.543 to 27.659, P values below 0.01). Compared with those in group CT, content of IL-2 in culture supernatant of T lymphocytes of patients in group UT was significantly increased from PTD 3 to 7 (with t values from 6.507 to 8.869, P values below 0.01), content of IL-4 in serum of patients in group UT was significantly decreased from PTD 3 to 7 (with t values from 6.922 to 8.843, P values below 0.01), and content of IFN-&gamma; in serum of patients in group UT was significantly increased on PTD 5 and 7 (with t values respectively 5.369 and 13.521, P values below 0.01). (4) The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group CT on PTD 1, 3, 5, and 7 were respectively (28±6)%, (25±7)%, (25±7)%, and (39±10)%, which were significantly lower than the percentage of volunteer in group HC [(87±8)%, with t values from 16.323 to 25.645, P values below 0.01]. The percentages of CD14(+) monocytes with positive expression of HLA-DR of patients in group UT on PTD 3, 5, and 7 were respectively (40±6)%, (42±9)%, and (49±10)%, which were significantly higher than those in group CT (with t values from 3.071 to 7.324, P values below 0.01).

CONCLUSIONSOn the basis of CT, additional ulinastatin intervention can decrease CD4(+) CD25(+) Tregs percentage, improve the immune function of T lymphocytes and T helper cells, and increase expression of HLA-DR on CD14(+) monocytes of patients with severe burn injury, thus improve the immune function of patients.

Burns ; drug therapy ; immunology ; Cells, Cultured ; Debridement ; Enzyme-Linked Immunosorbent Assay ; Glycoproteins ; therapeutic use ; Humans ; Interferon-gamma ; blood ; Interleukin-2 ; metabolism ; Interleukin-4 ; blood ; Monocytes ; immunology ; Skin Transplantation ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo study the expression levels of annexin A1 (ANXA1), GATA-3, and T-bet in T lymphocytes of peripheral blood in burned mice with sepsis at early stage, and to analyze their immune regulatory mechanisms.

METHODSSeven-hundred and eighty male mice of clean grade were divided into sham injury group (n=60, sham injured on the back by immersing in 37 ℃ warm water for 10 s), burn group (n=240, inflicted with 20% TBSA deep partial- thickness burn on the back by immersing in 100 ℃ hot water for 10 s), sepsis group (n=240, intraperitoneally injected with 6 mg/kg lipopolysaccharide), and burn+ sepsis group (n=240) according to the random number table. Mice of burn+ sepsis group were treated as that in burn group at first, and then they were treated as that in sepsis group. (1) Immediately after injury, six mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table. According to the random number table, 6 mice of each of the other three groups were respectively selected at post injury hour (PIH) 12, 24, 48, and 72 for the collection of lymphocyte suspension from peripheral blood (1 tube each mouse). Each tube of cell suspension was equally divided into two parts. Fluorescein isothiocyanate (FITC)-labeled human anti-mouse CD4 monoclonal antibody and phycoerythrin (PE)-labeled human anti-mouse interferon-&gamma; monoclonal antibody were added to one part of cell suspension to mark helper T lymphocyte 1 (Th1). FITC-labeled human anti-mouse CD4 monoclonal antibody and PE-labeled human anti-mouse interleukin-4 (IL-4) monoclonal antibody were added to the other part of cell suspension to mark Th2. The percentages of Th1 and Th2 were determined with flow cytometer, and the ratio of Th1 to Th2 was calculated. (2) According to the random number table, 18 mice in sham injury group were selected immediately after injury for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and 18 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 to collect the lymphocyte suspension of peripheral blood (1 tube each mouse). The mRNA expression levels of ANXA1, GATA-3, and T-bet were determined by real-time fluorescent quantitative reverse transcription-PCR. (3) Immediately after injury, 36 mice in sham injury group were selected to collect lymphocyte suspension of peripheral blood (1 tube each mouse) according to the random number table, and then 36 tubes of cell suspension were divided into 6 batches (6 tubes each batch). Each one of 6 kinds of antibody combinations: antibodies for labeling Th1 and Th2 in combination with PE-anthocyanin 7 labeled human anti-mouse ANXA1 monoclonal antibody, PE-anthocyanin 7 labeled human anti-mouse GATA-3 monoclonal antibody, and PE-anthocyanin 7 labeled human anti-mouse T-bet monoclonal antibody was added to 1 tube of cell suspension at each batch. According to the random number table, 36 mice of each of the other 3 groups were respectively selected at PIH 12, 24, 48, and 72 for the collection of lymphocyte suspension of peripheral blood (1 tube each mouse), and then 36 tubes of cell suspension at each time point were divided into 6 batches for marking with 3 kinds of surface markers of Th1 and Th2 (6 tubes each batch). Each one of above-mentioned 6 kinds of antibodies was added to 1 tube of cell suspension at each time point for each batch. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 were determined with flow cytometer. Data were processed with one-way analysis of variance, analysis of variance of factorial design, and SNK test. The relationship between the percentages of ANXA1 positive cell and the percentages of GATA-3 positive cell in Th1 and Th2, and mRNA expression level of ANXA1 and mRNA expression level of GATA-3 in lymphocytes were assessed by linear correlation analysis.

RESULTS(1) Compared with those in sham injury group immediately after injury, the percentages of Th1 and Th2 and the ratio of Th1 to Th2 of mice in burn group were significantly decreased from PIH 24 on, with P values below 0.05; the percentages of Th1 and Th2 and the ratios of Th1 to Th2 of mice in sepsis group and burn+ sepsis group were significantly decreased from PIH 12 on, with P values below 0.05. (2) Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocyte of mice in burn group were significantly decreased from PIH 24 on, with P values below 0.05; the mRNA expression level of T-bet was significantly decreased at PIH 24 but significantly increased at PIH 48 and 72, with P values below 0.05. Compared with those in sham injury group immediately after injury, the mRNA expression levels of ANXA1 and GATA-3 in lymphocytes of mice in sepsis group were significantly decreased from PIH 12 on, and the mRNA expression level of T-bet was increased significantly from PIH 12 on, with P values below 0.05; the mRNA expression levels of ANXA1, GATA-3, and T-bet in lymphocytes of mice in burn+ sepsis group were significantly decreased from PIH 12 on, with P values below 0.05, reaching the nadir at PIH 72 (0.50±0.04, 0.45±0.03, 0.21±0.05, respectively). (3) A significant positive correlation was observed between ANXA1 mRNA expression level and GATA-3 mRNA expression level in lymphocytes of peripheral blood (r=0.862, P<0.05). (4) Compared with those in sham injury group immediately after injury, the percentages of ANXA1 and GATA-3 positive cellsin Th1 and Th2 of mice in burn group were significantly lowered from PIH 24 on, and the percentage of T-bet positive cells was significantly decreased at PIH 24, but it was increased from PIH 48 on, with P values below 0.05. The percentages of ANXA1 and GATA-3 positive cells in Th1 and Th2 of mice in sepsis group were continuously decreased from PIH 12 on, which were lower at most time points than those in sham injury group immediately after injury, with P values below 0.05. The percentages of T-bet positive cells in Th1 and Th2 of mice in sepsis group were significantly increased since PIH 12 as compared with those in sham injury group immediately after injury, with P values below 0.05. The percentages of ANXA1, GATA-3, and T-bet positive cells in Th1 and Th2 of mice in burn+ sepsis group were continuously lowered from PIH 12, with significantly statistical differences at most time points as compared with those in sham injury group immediately after injury, with P values below 0.05. (5) The percentages of GATA-3 positive cells in Th1 and Th2 were significantly positively correlated with those of ANXA1 (with r values respectively 0.747 and 0.787, P values below 0.05).

CONCLUSIONSThe expression levels of ANXA1, GATA-3, and T-bet were continuously lowered in burned mice with sepsis, and it may play an important role in Th1/Th2 balance switching to Th2 bias and immunosuppressive process.

Animals ; Biomarkers ; Burns ; immunology ; metabolism ; GATA3 Transcription Factor ; biosynthesis ; genetics ; Humans ; Interferon-gamma ; biosynthesis ; genetics ; Interleukin-4 ; metabolism ; Male ; Mice ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Sepsis ; blood ; T-Lymphocytes ; metabolism ; Transcription Factors ; biosynthesis ; genetics