Immunoregulation Efficacy Of Th17Cells And Interleukin-17in Acute Graft-versus-host Disease Of Allogeneic Hematopoietic Stem Cell Transplantation

Part: The role of Th17cells and IL-17in acute graft-versus-host diseaseof myeloablative allogeneic hematopoietic stem cell transplantationObject: The current study attempted to investigate the role of Th17cells and IL-17inaGVHD of murine myeloablative allogeneic hematopoietic stem cell transplantation.Methods: The wild type （WT） C57BL/6（H-2^b） mice and IL-17（interleukin-17）knockout (IL-17^-/-) mice in C57BL/6（H-2^b） background were donor mice, and BABL/Cmice were recipient mice. The experiments include three parts. The first part: Therecipients were transplanted with1×10⁷TCD-BMCs from WT donor mice and1×10⁶Th17cells, or transplanted with1×10⁷TCD-BMCs from WT donor mice and1×10⁶CD4+IL-17^-/-T cells after myeloablative conditioning regimen （total irradiation8.5Gy）.Survivals were monitered twice a day and the clinical manifestations of acutegraft-versus-host disease （aGVHD） were evaluated every five days after allogeneic bonemarrow transplantation （allo-BMT）. Histological analysis including liver, lung, gut andskin were obtained from recipients after transplantation and were detected by HE staining.The relative percentages of the state of chimera in the recipients were determined byflowcytometry after harvesting spleens from the recipient. The second part: Aftermyeloablative conditioning regimen （total irradiation8.5Gy）, the recipients weretransplanted with1×10⁷BMCs from WT donor mice and5×10⁶splenic cells （SCs）from WT donor mice （WW）, or transplanted with1×10⁷BMCs from WT donor miceand5×10⁶SCs from IL-17^-/-donor mice （WK）, or transplanted with1×10⁷BMCsfrom IL-17^-/-donor mice and5×10⁶SCs from IL-17^-/-donor mice （KK）. Survivals were monitered twice a day and the clinical manifestations of aGVHD were evaluated every fivedays after allo-BMT. Histological analysis including liver, lung, gut and skin were obtainedfrom recipients after transplantation and were detected by HE staining. The relativepercentages of the state of chimera in the recipients were determined by flowcytometryafter harvesting spleens from the recipient. The third part: The recipients were transplantedwith1×10⁷BMCs from IL-17^-/-donor mice and5×10⁶SCs from IL-17^-/-donor mice（total irradiation8.5Gy）. Then divided the mice into two groups randomly, mice from onegroup were injected with200μL PBS by intraperitoneal injection after allo-BMT and micefrom the other group were injected with2μg per mouse mrIL-17（total volum200μL） byintraperitoneal injection after allo-BMT. Survivals were monitered twice a day and theclinical manifestations of aGVHD were evaluated every five days after allo-BMT.Histological analysis including liver, lung, gut and skin were obtained from recipients aftertransplantation and were detected by HE staining. The relative percentages of the state ofchimera in the recipients were determined by flowcytometry after harvesting spleens fromthe recipient.Results: The majority of the recipients given Th17cells died before15days, but40%of the recipients given IL-17^-/-CD4+T cells survived beyond15days after allo-BMT. Therecipients given IL-17^-/-CD4+T cells lived longer than the recipients given Th17cells（P<0.05）. Compared to the recipients of IL-17^-/-CD4+T cells, the recipients of Th17cellsshowed higher clinical scores and severe aGVHD lesion in liver, lung, gut and skin. Thecomplete chimera derived from donor was observed in recipient mice. Among therecipients of WW cells, WK cells and KK cells, sixty percent of the recipients given WWcells survived beyond30days, thirty percent survived more than50days, twenty percentof the recipients given WK cells survived more than50days, the majority of the recipientsgiven KK cells died within30days after allo-BMT. The recipients given WW cells livedlonger than the recipients given KK cells （P<0.05）. There was no different in survivalbetween the recipients given WW cells and WK cells. No different was found in survivalbetween the recipients given WK cells and KK cells yet. Compared to the recipients ofWW cells, the recipients of KK cells showed higher clinical scores and severe aGVHDlesion in liver, lung, gut and skin. Clinical scores and aGVHD lesion of the recipients givenWK cells located between the recipients given WW cells and KK cells. The completechimera derived from donor was observed in recipient mice. Fifty percent of the recipients given KK cells and IL-17survived beyond18days, but80%of the recipients given KKcells and PBS died before18days after allo-BMT. The recipients given KK cells and IL-17lived longer than the recipients given KK cells and PBS （P<0.05）. Compared to therecipients given KK cells and IL-17, the recipients given KK cells and PBS showed higherclinical scores and severe aGVHD lesion in liver, lung, gut and skin. The complete chimeraderived from donor was observed in recipient mice.Conclusion: Th17cells contribute to but IL-17inhibits acute GVHD in themyeloablative allogeneic hematopoietic stem cell transplantation. Part П: Mechanism of Th17cells promoted aGVHD duringmyeloablative allo-HSCTObject: We carried out experiments to determine the mechanism of Th17cellspromoted aGVHD during myeloablative allo-HSCT model.Methods: Irradiated （8.5Gy） BALB/c mice were transplanted with1×10⁷C57BL/6TCD-BMCs and1×10⁶Th17cells, or1×10⁷C57BL/6TCD-BMCs and1×10⁶CD4+IL-17^-/-T cells. Twelve days after allo-BMT, we harvested spleens, livers, lungsand guts from the recipients, and processed into a single cell suspension. The percent ofNK cell, NKT cell, macrophages, dendritic cell, neutrophilic, CD4+T cell, CD8+Tcell、IFN-γ+cell and so on were measured by fluorescent dye-conjugated mAb. Andserum levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α were determined usingCytometric Bead Array system. The results were analyzed by Graphad Prism5.Results: Compared with the recipients of CD4+IL-17^-/-T cells, higher percentage ofTh1cell was observed from the recipients of Th17cells in the spleen, liver and lung（P<0.05）. The percentage of Th1cells was no different in gut between the recipient ofTh17cells and CD4+IL-17^-/-T cells. The percentage of Th17cells in the live and lung ofthe recipient given Th17cells was higher than the recipient given CD4+IL-17^-/-T cells（P<0.05）, and it was significantly higher in the spleen （P<0.01）. The percentage of Th2cells in the spleen, liver, lung and gut of the recipients given Th17cells was lower than therecipients given CD4+IL-17^-/-T cells. Compared with the recipients of Th17cells, therecipients of CD4+IL-17^-/-T showed higher percentage of CD69+CD4+T cells in the spleen, liver and gut （P<0.05）, it was significantly higher in the lung （P<0.01）. Thepercentage of NK cell, NKT cell, DCs, Mφ and so on was no different in the spleen, liver,lung and gut between the recipient of Th17cells and CD4+IL-17^-/-T cells. We found thatcompared with the recipients of IL-17^-/-CD4+T cell, serum levels of IL-6and IFN-γ wereelevated （P<0.05）, but serum levels of IL-2, IL-4, IL-10, IL-17and TNF-α were nodifference between two kinds of recipients.Conclusion: These data indicate that Th17cells exacerbated aGVHD by promotingTh1cell infiltration in the spleen, liver and gut, and promoting T cell activation, andreducing the number of Th2cells in the spleen, liver, lung and gut during myeloablativeallo-HSCT Part Ⅲ: Mechanism of IL-17inhibited aGVHD during myeloa-blative allo-HSCTObject: To explore the mechanism of IL-17to inhibit aGVHD during myeloablativeallo-HSCT model.Methods: BABL/C mice were divided into three groups after myeloablativeconditioning regimen （total irradiation8.5Gy）, and transplanted with1×10⁷WT BMCsand5×10⁶WT SCs （WW）, or transplanted with1×10⁷WT BMCs and5×10⁶IL-17^-/-SCs （WK）, transplanted with1×10⁷IL-17^-/-BMCs and5×10⁶IL-17^-/-SCs（KK）. Twelve days after allo-BMT, spleen, liver, lung, gut from the recipients wereharvested and processed into a single cell suspension. NK cells, NKT cells, macrophages,DCs, neutrophils, Th1cells, Tc1cells, CD4Treg cells, CD8Treg cells and so on weremeasured by fluorescent dye-conjugated mAb. Serum levels of IL-2, IL-4, IL-6, IL-10,IL-17, IFN-γ and TNF-α were determined using Cytometric Bead Array system. Theresults were analyzed by Graphad Prism5. BABL/C mice were transplanted with1×10⁷IL-17^-/-BMCs and5×10⁶IL-17^-/-SCs after myeloablative conditioning regimen （totalirradiation8.5Gy）, and were divided into two groups randomly. One group was injectedwith PBS and the other group was injected with rmIL-17. Twelve days after allo-BMT,spleen, liver, lung, gut from the recipients were harvested and processed into a single cell suspension. NK cells, NKT cells, macrophages, DCs, neutrophils, Th1cells, Tc1cells,CD4Treg cells, CD8Treg cells and so on were measured by fluorescent dye-conjugatedmAb. Serum levels of IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α were determinedusing Cytometric Bead Array system. The results were analyzed by Graphad Prism5.Results: We analyzed the percentage of Th1cells in the spleen, live, lung and gut ofthe recipients of WW cells, WK cells and KK cells. We observed the percentage of Th1cells in spleen from the recipients of KK cells was higher than the recipients of WK cells（P<0.05）, and was significantly higher than the recipients of WW cells （P<0.01）.Compared with the recipients of WW cells, the recipients of KK cells showed higherpercentage of Th1cells in liver （P<0.05）. Compared with the recipients of WK cells, therecipients of KK cells showed increased Th1cells in liver, but there was no statisticaldifferences （P>0.05）. Compared with the recipients of WK cells and WW cells, therecipients of KK cells showed higher Th1cells in gut （P<0.05）. Percentages of Th1cellswere no difference in the lung from the recipients of WW cells, WK cells and KK cells. Wealso observed that compared with the recipients of WW cells, the recipients of KK cellsshowed higher percentage of Tc1cell in the gut （P<0.05）. Compared with the recipients ofWW cells, the recipients of KK cells showed higher percentage of Mφ in spleen, liver andgut （P<0.05）. The percentage of Mφ was no different in spleen, liver and gut between therecipients of KK cells and WK cells. And it showed no difference between the recipients ofWK cells and WW cells yet. Among the recipients of WW cells, WK cells and KK cells,the percentage of Mφ in lung was no different. Compared with the recipients of WW cells,higher serum level of IFN-γ was seen in the recipients of KK cells measured by CBA（P<0.05）. Serum levels of IFN-γ was no different between the recipients of KK cells andWK cells. Between the recipients of WK cells and WW cells, there was no difference inserum levels of IFN-γ. Compared with the recipients given KK+IL-17, the recipientsgiven KK+PBS showed increased percentage of Th1cells in the spleen, liver and gut（P<0.05）. The percentage of Th1cells was no different in the lung from two kinds of therecipients. Compared with the recipients of KK+IL-17, the recipients of KK+PBSshowed significantly higher percentage of Mφ in the spleen and gut （P<0.01）, and showedhigher percentage of Mφ in liver （P<0.05）. The recipients of KK+IL-17showed higherpercentage of CD8Treg in the spleen than the recipients of KK+PBS （P<0.05）.Compared with the recipients of KK+IL-17, the recipients of KK+PBS showed higher serum levels of IFN-γ measured by CBA （P<0.05）.Conclusion: IL-17inhibits aGVHD by decreasing the Th1cells in the spleen, liverand gut duing myeloablative allo-HSCT. It may reduce the production of IFN-γ and affectthe differentiation of Th1cells by decreasing infiltration of macrophages in the spleen,liver and gut.