| Nowadays,chronic infectious diseases and malignant tumors have seriously threatened the health of human in the world,and could lead to high mortality.Numerous drugs treated for infectious disease have emerged in recent years,the pathogenesis of tumors has gained a further understanding from the cellular level to the molecular level,and the application of small molecule inhibitors and molecular-targeted therapy has also achieved remarkable effect both in clinic or pre-clinic.However,there is an unmet need for new therapies,and improved understanding of disease.Therefore,achieving the satisfactory clinical outcome has a long way to go.Chronic hepatitis B(CHB)is still widespread in the world,especially in Asia.The current treatments for CHB mainly include interferon-a(IFN-a)and several nucleotides analogues(NAs).It seemed that only IFN-a could make the loss of HBsAg or HBeAg through transferring STAT signaling and inducing IFN-stimulated genes,though with an unsatisfactory clinical outcome.Therefore,it is an important issue that through the studying of the immunological mechanism between sustained response patients and non-response patients to find a strategy that can improve the efficacy of interferon.T cells and NK cells play key roles in the process of anti-virus infection.Interferon can activate both T cells and NK cells,besides,it also acts directly on virus-infected cells.But,during the chronic infection,CD8+T cells and NK cells are functionally exhausted,which results in the persistent infection.IL-2 was recognized to have a role in T cell activation and proliferation,and promoted the cytolytic activity of NK cells,which also play key roles in controlling viral infections.Furthermore,IL-2 could be used directly for patients with renal cell carcinoma and melanoma.It is seldom reported that whether IL-2 could be used after IFN-a therapy.Considering that both IFN-I and IL-2 can transmit signals through STAT1 phosphorylation,we considered that whether sequential IL-2 therapy could synergistically improve the immunity of non-response patients who are failed in IFN-a therapy.What’s more,whether it could be applied in clinical and improve the outcome of patients?Thus,we conduct clinical trials on the above issues.CHB may gradually develop into cirrhosis,and eventually develop to liver cancer.Effective therapy for HCC remains a major challenge,as the tumors are chemotherapy resistant and owing to an inadequate understanding of genomic traits and molecular characteristics,the disease is generally diagnosed at an advanced stage.Neither checkpoint treatment nor sorafenib,it seems hardly to significantly prolong the patient’s survival.Surgical resection remains the most effective treatment and is widely recommended,however,the high recurrence rate remains the most serious challenge,and liver has a strong regenerative capacity after surgical resection.Members of the IL-6 family,including IL-11,can transmit signals via STAT3 phosphorylation.IL-11-STAT3 signaling played a key role during gastrointestinal tumorigenesis and could be targeted therapeutically.In addition,IL-11 has a crucial role in linking compensatory proliferation with recurrence after surgical resection.Therefore,we wonder if the regeneration of liver accompanies or promotes tumor growth?Dose IL-11-STAT3 signaling promote tumor outgrowth,and can we find an effective therapy to inhibit recurrence after surgical resection?This paper mainly conducts the research of the new therapy for non-response patients after IFN-α therapy and of the therapeutic targets for recurrence after surgical resection in HCC.The results as follows:I.Sequential IL-2 therapy orchestrates immune function in non-response patients with refractory hepatitis B after IFN-α treatmentIn the first clinical trial,we recruited 92 HBeAg-positive CHB patients.They received standard treatment with Peg-IFN-α-2b for 48 weeks and with 24 weeks of IFN-αtreatment-free follow-up.After we systematically analyzed the immunology of these patients,we found IFN-α treatment making IL-2R switch mainly to positive immune signaling,which provided the conditions for sequential IL-2 therapy.Then we conducted the second clinical trial that sequential IL-2 therapy was administrated for the patients failed in IFN-α therapy.Through flow cytometry,ELISA,immunofluorescence,western blotting,RTCA,and spearman’s rank correlation coefficient,we found sequential IL-2 therapy could improve clinical outcome for those non-response patients.The results as follows:1.The majority of patients were non-response(NR)patients,and IL-2Rα expression was reduced in CD4+ T cells after Peg-IFN-α-2b therapy.After we systematically analyzed the immunology of these patients during treatment,we found that IL-2R expression was altered;particularly,IL-2Rα(CD25)expression in CD4+ T cells was notably reduced after Peg-IFN-α-2b therapy.In addition,we found a significant increase in IL-2Rβ and IL-2Ra expression on the surface of NK cells in NR patients.Therefore,Peg-IFN-a-2b therapy made IL-2R switch mainly to positive immune signaling and provided the conditions for sequential IL-2 therapy.2.IL-2 induce T cells and NK cells of non-response patients to secrete IFN-γ and TNF-α.We demonstrated the frequency of both multi-functional effector IFN-y+TNF-a+T cells and IFN-y+TNF-a+NK cells got significant increase in PBMC of NR patients,after stimulated with low dose of IFN-a with IL-2.Furthermore,the CD38 and NKG2D expression also got elevated.However,neither PD-1 expression nor Tim-3 expression was increased.Besides,the percentage of Treg cells didn’t change significantly.3.IL-2 promotes lymphocytes of liver biopsy to secrete IFN-y.After stimulated with IL-2 in vitro,we found CD38 and NKp30 expressions in CD8+T cells and NK cells from lymphocytes of liver biopsies were de facto up-regulated.However,NKG2A expression didn’t significantly change.Besides,the IFN-y expression got prominently increased4.IL-2 therapy results in attenuated regulatory T cells and PD-1 expression in non-response patients.We found the frequency of Treg cells and PD-1 expression decreased after sequential IL-2 therapy for twenty-three NR patients,and an significant increase of CD 122 expression in NK cells.5.IL-2 therapy promotes STAT1 activation in non-response patients.After utilizing the PBMCs from NR patients in vitro,we firstly found that stimulation with IL-2,the phosphorylation of STAT5(p-STAT5)was significantly decreased and p-STAT1 expression was significantly up-regulated.What’s more,in vivo,p-STAT5 expression was significantly decreased especially in CD4 T cells after IL-2 therapy.Moreover,analyses using immunofluorescence demonstrated that p-STAT1 expression also prominently increased in vivo of patients after IL-2 therapy.6.IL-2 therapy restores IFN-y production in non-response patients.After systematically test,we demonstrated that the proportion of IFN-γ,TNF-αand CD 107a in CD8+T cells NK cells were significantly elevated.Moreover,IFN-γ,TNF-a as well as IFN-α in serum got markedly increase after IL-2 therapy.However,IL-10 didn’t increase notably.7.IL-2 therapy increases the cytotoxicity and number of HBV-specific CD8+T cells in NR patientsWe found the frequency of pentamer+CD8+T cells was significantly increased after IL-2 therapy at week 24.Moreover,we observed a significantly higher percentage of CD44highCD62Llow effector memory CD8+T cells(TEM),and the increased expression of NKp30.Furthermore,IL-2 therapy also promoted the expression of co-stimulatory molecules,CD86 and CD80,in pDCs.Using a real-time cell analyzer(RTCA),we found that primary CD8+T cells,purified from the PBMCs of patients with CHB following IL-2 therapy,were more efficient at killing the hepatocellular carcinoma(HCC)cell line PLC/PRF/5.8.IL-2 therapy improves outcomes of refractory non-response patients.We found the HBeAg in serum got a significant reduction after IL-2 therapy.What’s more,there was no significant change of HBeAb,AST or ALT.Importantly,there were 5 patients got HBeAg clearance after sequential IL-2 therapy.Other than this,1 patient got HBsAg clearance with anti-HBs development.Besides,the proportion of pentamer+CD8+T and CD107a+IFN-γ+CD8+T cells was negative correlated with HBeAg,while the frequency of Treg cells was positive correlated with HBeAg.Actually,not all patients are eligible for sequential IL-2 treatment,so other treatment strategies should be considered for these patients.Other than above results,we found CD24+CD38hiB cells,which could secret IL-10 and inhibit immune response,increased after IFN-α therapy.Besides,we verified that CD24 was a suitable marker for CD24+CD38hiB cells.Then,after depleting CD24+CD38hiB cells,we found the effectory functions of T cells and NK cells were elevated.At last,through a mouse model of HBV persistence,we found anti-CD24 antibody could improve the therapeutic effect of IFN-α,including the decline of HBeAg and HBsAg.Collectively,we demonstrated that although the majority of patients were non-responders after IFN-α treatment,the expression of IL-2Rα in CD4+T cells showed a decline,making IL-2Ra difficult to convert IL-2Rβγ to the high-affinity receptors,IL-2Rαβγ,which constitutively expressed on Treg cells,and induced FOXP3 expression through STAT5 activation,and finally transferred negative immune signaling.Thus,it seems that IL-2Ra-pSTAT5-FOXP3 signaling pathway was abolished after Peg-IFN-a-2b treatment.The sequential IL-2 therapy increased the expression of intermediate-affinity IL-2 receptor IL-2Rpy,and promoted p-STAT1 expression,meanwhile,inhibited p-STAT5 expression,thus it contributed to positive immune functions,such as CD8+T cell and NK cell effector functions.Besides,sequential IL-2 therapy attenuated regulatory T cell and PD-1 expression in non-responders.Furthermore,we demonstrated that in most of the twenty-three refractory non-response patients,HBeAg level showed a significant decline,including five patients who showed HBeAg seroconversion,and one patient who showed HBsAg clearance,with anti-HBs development.Thus,this proposed therapy is capable of restoring effector functions and improving clinical outcomes.Moreover,for patients who are probably not suitable for IL-2 therapy,anti-CD24 antibody may be another promising candidate.Ⅱ.Hepatectomy promotes recurrence of liver cancer by enhancing IL-11-STAT3 signalingUtilizing the genetic mouse model,orthotropic tumor model,chemically-induced model,and orthotopic allograft model,we demonstrate that IL-11 level increase after surgery and trigger HCC outgrowth.1.HCC recurs after surgical resectionWe compared the recurrence rates of HCC with other widely studied cancers in America,China,and Europe,and found the recurrence rates of HCC seemed higher than those of other tumor types.Then we established a surgical orthotopic HCC resection model,and found postsurgical recurrence.Tumors after surgical resection expressed higher levels of CD31 and PCNA,suggesting that substantial proliferation and angiogenesis accompanied liver generation.2.Surgical wounding triggers HCC outgrowthAfter surgical wounding or resection of liver,reparative responses were significant drivers of tumor development and increased collagen deposition and fibrosis,with the strong CD31 and PCNA staining.Furthermore,through MRI,we showed that handle with mechanical injury significantly promoted the dense degree of primary tumor lesions.3.IL-11 is overexpressed after hepatectomy.Using an oligonucleotide microarray,we identified IL-11 gene expression was significantly upregulated in tumor tissues compared with adjacent tissues from primary HCC patients.By IHC,we demonstrated that IL-11 expression was obviously increased in tumor regions of HCC patients.Besides,we showed that both IL-11 and IL-11Rαwere elevated after surgery in the liver of mice.Using ELISA,we also found that the level of IL-11 increased in liver tissue after resection.At last,we referred to The Cancer Genome Atlas,and demonstrated that IL-11 gene expression was significantly elevated in tumor tissues relative to adjacent tissues from HCC patients.4.IL-11 signaling was required for HCC outgrowth after surgical resection.Through a surgical orthotropic HCC resection model,we found tumors were almost completely absent at autopsy in the Il11ra1KO mice,and overall tumor numbers and burden were significantly reduced.Besides,we verified significant reductions in PCNA,AFP,and pTyr-STAT3 expression in Il11rα1Ko mice compared with control mice.Thus,our results confirm that IL-11 signaling is necessary for recurrence after surgical resection in HCC.5.Inhibition of IL-11 signaling induces tumor cells apoptosis in vitro.We used three-dimensional culture systems and found IL-11 promoted the proliferation of Hepal-6 cells through pTyr-STAT3.However,following the addition of napabucasin,we found less globular cell mass and more 7AAD+cells.Using immunofluorescence,we observed reduced expression of PCNA in cells cultured with napabucasin,irrespective of the supplemental IL-11.What’s more,we confirmed that levels of pTyr-STAT3 increased after stimulation with IL-11,but when napabucasin was used,pTyr-STAT3 expression was strongly inhibited6.Inhibition of STAT3 phosphorylation prevents postsurgical recurrenceIn mice that had been orthotopically injected with Hepal-6 cells,we found that tumor growth was significantly inhibited after administration of napabucasin.The liver tumor burdens were almost complete absence with minor pathological damage and little tumor proliferation.Then we relied on a well-established surgical orthotopic HCC resection model,and found there was almost no tumor recurrence after napabucasin administration,which was also associated with significant reductions in overall tumor numbers and burden,as well as decreased tumorigenesis.7.IL-6 was not required for postsurgical recurrence of HCC.We used Il6KO mice and found that genetic ablation of 116 did not significantly reduce tumor numbers or burden in mice that underwent partial hepatectomy before the injection of Hepal-6 cells.Pathologic and IHC analyses showed no differences between Il6KO mice and WT mice in terms of tumor growth or tumorigenesis.Then,using a surgical orthotopic HCC resection model,we also found that HCC could recur in postsurgical Il6KO mice,and that treatment with napabucasin successfully prevented recurrence with decreased PCNA expression.8.Blocking STAT3 phosphorylation inhibits IL-11-induced HCC outgrowth in vivo.After intravenous injection of IL-11,pathologic analysis revealed increased tumor areas and severe fibrosis,with strong PCNA and AFP staining.However,co-injection of IL-11 and napabucasin led to reduced tumor burden,minor pathologic damage,and little tumor proliferation.Consistent with this,we found significant decreases in PCNA and AFP expression.Besides,through DEN-induced and orthotopic allograft model of HCC,we found postsurgical mice treated with napabucasin displayed significantly reduced recurrence,accompanied by decreased expression of PCNA and pTyr-STAT3.In summary,our current study provides direct evidence that surgery could promote HCC outgrowth through IL-11-STAT3 signaling,and that the inhibition of IL-11-STAT3 signaling could effectively prevent postsurgical recurrence.We demonstrated that recurrence is concomitant with regeneration.Few HCC patients don’t undergo surgical resection,therefore,we suggest that the therapy with napabucasin after surgical resection would substantially contribute to preventing postsurgical recurrence,with potential clinical significance. |
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