| Objectives:The purpose of this study was to reveal the dynamics of plasma fibronectin(pFN)and cytokines of Th1/Th2/Th17 subtypes in the patients with sepsis and severe infections so as to find the diagnostic markers for evaluation of disease development and prognosis.Meanwhile,therapeutic effect of pFN and recombinant human fibronectin polypeptide(rhFNHC-36)was evaluated and its related immunological mechanism was analyzed on septic mice model induced with endotoxin.Methods:1.Plasma samples were collected from 15 patients with acute pancreatitis(AP),34 patients with severe acute pancreatitis(SAP),20 patients with sepsis,and 45 patients with multiple organ dysfunction syndromes(MODS)at admission and several days after therapy.Interleukin-2(IL-2),Interleukin-4(IL-4),Interleukin-6(IL-6),Interleukin-10(IL-10),Tumor Necrosis Factor(TNF),Interferon-γ(IFN-γ),and Interleukin-17A(IL-17A)protein,CRP and pFN levels were detected by cytometric bead array(CBA)and ELISA,respectively.2.Endotoxin shock model was induced in Balb/c mice via intraperitoneal(i.p.)injection with D-galactosamine(D-Gal)and lipopolysaccharide(LPS).3.The therapeutic effects of pFN and rhFNHC-36 were evaluated and the inflammation response was checked.3.1 The mice model was administered with pFN and rhFNHC-36 respectively via i.v.0.5 hours prior,1 hour and 2 hours after LPS induced.PBS was used for control.3.2 The survival rate was recorded.The histological damage in hepatocytes and splenocytes were examined with HE staining.Plasma FN and cytokines(IL-1β、IL-6、IL-10、TNF、IFN-γ、MCP-1)levels were detected by repetitive blood sampling of septic mice.3.3 Peritoneal macrophage were isolated from normal Balb/c mice and stimulated with LPS and pFN or rhFNHC-36 for 4 and 16 hours in vitro,and cDNA was prepared for real-time PCR analysis of M1 and M2 markers.4.The differentiation and functions of T lymphocyte subgroups were identifiedafter pFN and rhFNHC-36 treatment on sepsis mice.4.1 Th1,Th2 and Th17 lymphocytes were identified by the intracellular detectionof TNF-α,IFN-γ,IL-4 and IL-17 respectively with flow cytometry(FCM)on LPSinduced sepsis mice after 6 hours.Cell surface markers(CD25,CD69,CD28 and PD-1)and apoptosis from splenocite T lymphocytes were analysed with PE Annexin and7-AAD by FCM as well.4.2 CD4+ splenocytes were enriched by negative selection from disaggregatedspleens of septic mice,and induced with anti-CD3/CD28 Abs costimulation with orwithout pFN and rhFNHC-36 after 12 hs and 72 hs respectively.The cytokines(IFN-γand IL-4)secretion were analyzed by ELISA.CD44 and CD62 L were detected byFCM,T cells proliferation were detected by FCM with CFSE staining.Results:1.The circulating pFN level was decreased in patients with sepsis(146.88±55.99 μg/ml),MODS(139.6±63.66 μg/ml),SAP(191.7±60.22 μg/ml)compared to healthy control(335.6±89.6 μg/ml)and AP patients(273.9±116.2 μg/ml)(P<0.0001)at the onset of severe infection.The decreased pFN level from patients’ follow-up samples indicated the worsen status of disease development.2.IL-6 was increased in patients with sepsis,MODS and SAP compared to control with great variation with the concentration of 460.7±1260.9 pg/ml、992.6±2587 pg/ml、728.4±2125 pg/ml and 2.98±7.64 pg/ml at the onset of severe infection.Only TNF-α and IFN-γ were increased by 20% and 23% of patients respectively.There were no differences in IL-2,IL-4 and IL-17 A between patients and controls.3.The 72-hour survival rate was markedly increased in the septic mice treated with rhFNHC-36 polypeptide(38%)and pFN(45%)compared to control(0%)(P<0.01).Histopathological data showed that less necrosis and apoptosis occurred on the hepatocytes and splenocyte of endotoxemia mice with the injection of pFN or rhFNHC-36 compared with control.4.Pretreatment with rhFNHC-36 or pFN attenuates increasing plasma concentrations of proinflammatory IL-1β,IL-6,IL-10,TNF,IFN-γ,MCP-1 and decreasing pFN level after LPS challenge.Furthermore,treatment with rhFNHC-36 or pFN in vitro could inhibit M1 but drive M2 polarization in macrophages stimulated with LPS,resulting in downregulating the expression of M1 makers(IL-1β,IL-6,TNF-α,IFN-γ,iNOS and IL-12)and upregulating the expression of M2 makers(Argnase-1 and IL-10).5.The percentage of Th1,Th17 cells was lower in endotoxemia mice,whereas the percentage of Th2 cells was higher.Flow cytometric analysis showed that increased expression of selected inhibitory receptors PD-1 in CD4+T cells were accompanied by decreased expression of CD28 in endotoxemia mice,which were allameliorated by pFN or rhFNHC-36 pretreatment.6.Functional assays revealed impaired secretion of IFN-γ and elevated secretion of IL-4 and decreased proportion of CD44+ CD62L-Th cells following stimulation with anti-CD3/CD28 Abs in vitro,which were reversible by incubation with rhFNHC-36 or pFN.Conclusion:1.The circulating pFN level in severe infecions was lower than in acute pancreatitis and healthy controls.The dynamics of FN may predict the development of SAP and MODS.2.Pre-treatment with pFN or recombinant fibronectin polypeptide(rhFNHC-36)significantly increased survival rate,improved histological damages in liver and spleen,attenuated the decreasing of plasma FN and inhibited the inflammation cytokines expression in LPS-induced sepsis mice model.3.pFN or rhFNHC-36 could inhibit M1 but drive M2 polarization and expression of its related cytokine in macrophages stimulated with LPS in vitro.4.pFN or rhFNHC-36 induced the shift of T lymphocytes differentiation from Th2 to Th1 and Th17,and promoted the cellular activity of T lymphocytes.This study provides the immunological envidences for pFN or rhFNHC-36 in mice models of endotoxic shock. |
