Inflammation And T Cells Responses In Interleukin-33 Knock-out Mouse With Toxoplasma Gondii Infection

Objective:Toxoplasma gondii is a strict intracellular protozoan parasite,and T cell response plays an important role in acute toxoplasma infection in which IFN-?produced by Th1 could control the reproduction of parasites and eliminate them.IL-33,a new member of IL-1 family,could reduce Th2-associated response and restrain the secretion of IFN-?.There are no relevant reports about the effect of IL-33 on acute toxoplasma infection.Our study is aimed to reveal the mechanism of IL-33 on inflammation and T cell response in IL-33^-/-mice model with acute toxoplasma infection.Methods:1.IL-33^-/-mice were genotyped by PCR;2.The C57BL/6 wild-type mice and IL-33-deficient mice were infected intraperitoneally with 10²or 10⁴or 10⁶ tachyzoites/0.5ml/mouse of Toxoplasma gondii respectively,and the control ones were injected with normal saline.The menifestations were recorded and survival curve was established;3.The following experiments were performed based on the mice model infected with 10⁴ tachyzoites/mouse:3.1 Histologic examination was carried out on the livers from mice with infection after 4 days by HE staining;3.2 The inflammatory cytokines(IL-12p70?TNF?IFN-??MCP-1?IL-10?IL-6)were detected by cytometric bead array(CBA)from the peripheral serum of infected mice after 2 days and 4 days and IL-4 was detected by enzyme-linked immunosorbent assay(ELISA);4.The parasitic burden was estimated by m RNA level of SAG-1 which was detected by real-time PCR from splenic RNA of the infected mice.The number of parasites were counted from peritoneal lavage fluids of infected mice by Giemsa staining;5.The splenocytes suspension was prepared for checking the subsets of T lymphocytes by flow cytometry from the mice infected after 2 days and 4 days respectively:5.1 T lymphocytes were stained with APC-conjugated anti-CD3 antibody;5.2 CD4⁺/CD8⁺T lymphocytes were stained with APC-conjugated anti-CD3antibody and FITC-conjugated anti-CD4 antibody;5.3 Co-stimulators of CD4⁺T and CD8⁺T lymphocytes were stained with FITC-conjugated anti-CD4 antibody and PE-conjugated anti-CD28 antibody or APC-conjugated anti-PD-1 antibody;5.4 Activated CD4⁺T and CD8⁺T lymphocytes were stained with FITC-conjugated anti-CD4 antibody and PE-conjugated anti-CD25 antibody or APC-conjugated anti-CD69 antibody;5.5 Effector T cells(CD44^high CD62L^low CD4⁺T and CD44^highCD62L^lowCD8⁺T)were stained with FITC-conjugated anti-CD4 antibody,PE-conjugated anti-CD44 antibody and APC-conjugated anti-CD62L antibody;5.6 CD4⁺T-IFN-?⁺/CD4⁺T?CD4⁺T-IL-4⁺/CD4⁺?CD4⁺T-IL-10⁺/CD4⁺T?CD4⁺T-IL-17⁺/CD4⁺T cells were stained with APC-conjugated anti-CD3 antibody,FITC-conjugated anti-CD4 antibody and PE-conjugated anti-IFN-?antibody or PE-conjugated anti-IL-4/IL-10 antibody or PE-conjugated anti-IL-17?antibody,and CD4⁺CD25⁺Foxp3⁺T cells were stained with FITC-conjugated anti-CD4antibody,PE-conjugated anti-CD25 antibody and APC-conjugated anti-Foxp3antibody as well;5.7 CD8⁺T-TNF-?⁺/CD8⁺T cells were stained with APC-conjugated anti-CD3 antibody,FITC-conjugated anti-CD4 antibody and PE-conjugated anti-TNF?antibody.Results:1.The mice used in our study were IL-33-deficient C57BL/6 mice;2.The manifestations of the infected mice were critical with the parasitic intensity in Wt mice and it turned to tenuate in IL-33-deficient mice with longer survival time.3.The SAG-1 m RNA of IL-33-deficient mice(3.99±0.47)infected after 4days was significantly decreased than that in Wt mice(5.60±0.51,P<0.05),while the intraperitoneal parasite burden was increased in IL-33-deficient mice.4.IL-4 were elevated significantly in the IL-33-deficient infected after 2 and 4days(31.67±10.41 pg/ml and 135±15.06 pg/ml respectively),compared with those in Wt mice infected after 2 and 4 days(3.1±0.7 pg/ml and 26.33±7.77 pg/ml,P<0.01).Meanwhile,IFN-?(231.33±64.05 pg/ml)?TNF-?(39.73±18.23 pg/ml)and MCP-1(1049.5±498.05 pg/ml)in IL-33^-/-infected mice after 4 days were decreased compared with those(808.46±10 pg/ml?85.94±52.60 pg/m?1555.1±374.01 pg/ml,P<0.01)in Wt mice infected after 4 days.5.The pathological damages of liver with hydropic degeneration,hepatic blood extravasted,inflammatory cells and lymphocytes accumulation along with point necrosis were developed seriously in wild-type mice with infection,which was tenuated in IL-33-deficient mice with infection.6.The presence of CD28 in CD4⁺T cells from IL-33-deficient mice infected after 2 days was increased(26.43±4.85%)compared with IL-33-deficient mice without infection(19.50±0.71%,P<0.05),while after 4 days,the presence of CD28 in CD4⁺T cells from IL-33-deficient mice was decreased,which was commensurate to uninfected IL-33-deficient mice(18.67±6.67%vs 19.50±0.71%,P>0.05);7.Upon analysis of the presence of PD-1 in CD4⁺T cells over the course of acute infection,no obvious increase was observed in Tg mice,In contrast,IL-33-deficient mice showed a significant increase in numbers 4 days post-infection compared with IL-33-deficient mice without infection(59.10±10.25vs 42.53±4.45%,P<0.05),furthermore,the presence of PD-1 in CD4⁺T cells of IL-33-deficient mice over the course of acute infection were greater than that of Wt mice infected after 2 days and 4 days.(47.8±4.33%vs 32.9±4.62%,59.10±10.25%vs 47.22±5.57%,P<0.05)8.The presence of CD28 in CD8⁺T cells of IL-33-deficient mice infected after 2 days showed a significant decrease compared with the uninfected group(3.67±0.63%vs 7.85±0.43%,P<0.01),but 4 days later,it showed a greater presence than Wt mice infected 4 days(8.83±1.29%vs 4.47±0.59%,P<0.01);9.Upon analysis of the presence of PD-1 in CD8⁺T cells over the course of acute infection,a significant increase at 2 and 4 days were observed in IL-33-deficient mice(7.60±1.14%vs 5.46±0.84%and 7.44±1.54%vs 5.46±0.84%,P<0.01);10.The percentages of CD25⁺CD4⁺T cell in IL-33-deficient mice infected after 2 days was not increased compared with the uninfected group(20.6±1.10%vs20.37±1.19%,P>0.05),while after 4 days,it revealed a significant increase(28.67±3.97%vs 20.37±1.19%,P<0.01),which was still smaller than Wt mice infected after 4 days(28.67±3.97%vs 33.4±3.23%,P<0.01);the percentage of CD69⁺CD4⁺T cells of IL-33-deficient mice showed a significant increase after2 days and 4 days post-infection(43.33±6.60 vs 27.32±4.59%,P<0.05;68.37±4.62%vs 27.32±4.59%,P<0.01);11.The percentage of CD44^highand CD62L^low“effector”CD4⁺T cells in IL-33-deficient mice was greater than that of Wt mice(76.22±3.76%vs69.87±4.18%,P<0.05),there was comparable between Wt and IL-33-deficient mice after 2 days post-infection(76.53±1.80%vs 71.63±7.37%,P>0.05),however,percentages in IL-33-deficient mice infected after 4 days was greater than Wt mice after 4 days(81.87±4.30%vs 56.90±0.65%,P<0.01);12.The percentages of CD44^highand CD62L^low“effector”CD8⁺T cells in IL-33-deficient mice was commensurate to Wt mice without infection and after 2days infection(60.30±3.03%vs 56.07±1.03%and 55.10±10.98%vs 52.15±0.78%,P>0.05),however,the percentages from IL-33-deficient mice showed a significant decrease after 4 days post-infection(44.73±1.63%vs 60.30±3.03%,P<0.05)and but revealed a significantly greater than Wt mice after 4 days(44.73±1.63%?29.23±5.15%,P<0.05)13.The percentages of T lymphocyte in IL-33-deficient infected mice at 2days and 4 days showed a significant decrease(28.01±0.98%vs 49.13±1.72%and49.13±1.72%vs 30.57±1.80%);upon analysis of CD4⁺/CD8⁺radio in IL-33-deficient mice over the course of acute infection,a significant increase in 4days post-infection could be showed(102.51±20.80%vs 73.14±13.18%,P<0.01),and it showed a greater percentages than Wt mice(102.51±20.80%vs50.81±5.45,P<0.01);14.(1)Except the percentages of CD4⁺T-IFN-?⁺/CD4⁺T cell in IL-33-deficient mice had a higher frequency than wild-type mice(26.00±1.05%vs23.36±2.09%,P<0.05),other subsets of CD4⁺T were commensurate to Wt mice(CD4⁺T-IL-4⁺/CD4⁺T for 3.80±0.01%vs 4.17±1.05%,CD4⁺T-IL-10⁺/CD4⁺T for 2.93±0.71%vs 2.68±0.44%,CD4⁺T-IL-17⁺/CD4⁺T for 3.52±0.28%vs3.47±0.21%,and CD4⁺CD25⁺Foxp3⁺T for 10.20±1.06%vs 11.28±1.38%,P>0.05)(2)Upon analysis of the subsets of CD4⁺T cell in IL-33-deficient mice over the course of acute infection,and a down-regulation of CD4⁺T-IFN-?⁺/CD4⁺T(2days:21.11±1.63%vs 26.00±1.05%,4 days:19.75±0.80%vs 26.00±1.05%,P<0.05)and up-regulation of CD4⁺CD25⁺Foxp3⁺T were found(2 days:13.67±0.87%vs 10.20±1.06%and 22.00±2.26%vs 10.20±1.06%,P<0.05).(3)The subsets of CD4⁺T-IFN-?⁺/CD4⁺T(21.11±1.63%)?CD4⁺T-IL-17⁺/CD4⁺T(3.69±0.37%)and CD4⁺CD25⁺Foxp3⁺T(13.67±0.87%)in IL-33-deficient mice 2days after infection showed smaller percentages than that of Wt mice(24.34±0.71%,3.60±0.42%and 17.25±0.92%,P<0.05);(4)The subsets of CD4⁺T-IFN-?⁺/CD4⁺T(19.75±0.80%),CD4⁺T-IL-4⁺/CD4⁺T(4.07±0.84%),CD4⁺T-IL-10⁺/CD4⁺T(3.64±0.23%)and CD4⁺T-IL-17⁺/CD4⁺T(4.08±0.67%)in IL-33-deficient mice 4 days after infection showed greater percentages than that of Wt mice(26.14±2.15%,11.90±2.06%,6.39±2.50%,P<0.05;6.86±1.18%,P<0.01);15.Examination of IL-33-deficient mice revealed a greater percentage of CD8⁺T-TNF⁺/CD8⁺T than Wt mice(60.26±4.58%vs 35.35±8.30%,P<0.01),and a greater percentage after 2 and 4 days infection than Wt mice after 2 and 4days(58.00±3.46%vs 37.33±3.29%and 60.26±4.58%vs 41.12±7.52%).Conclusion:1.Attenuated inflammatory changes were observed in IL-33-deficient mice with acute Toxoplasma Gondii infection.2.Koncking out of IL-33 promoted the activation and multiplication of T cells and amplification of effector T cells3.Knocking out of IL-33 resulted in immune suppression of CD4⁺T cells but promoted the effect of CD8⁺CTL cells in mice with acute Toxoplasma Gondii infection.