The Immunological Functions Of Tim-3 In Early R. Heilongjiangensis Infection

Rickettsiae are obligate intracellular gram-negative bacteria that cause rickettsial diseases, comprising spotted fever, typhus, scrub typhus and so on. Rickettsial diseases are zoonoses with worldwide distribution and natural focus, which have been a serious threat to human health. Spotted fever is a kind of diseases caused by spotted fever group rickettsiae. The usual spotted fever includes Rocky Mountain Spotted Fever(RMSF) caused by R. rickettsii, and Mediterranean Spotted Fever(MSF) caused by R. conorii, Queensland Spotted Fever(QSF) caused by R. australia, and Rickettsial Pox(RP) caused by R. akari. Spotted fever has an incubation period 4-7 days, showed mainly fever, headache, muscular pain,and skin rash. RMSF is the most serious illness, if left untreated, can lead to severe dehydration and death. In China, spotted fever is extensively distrabuted and consists of North Asian Tick-borne Rickettsiosis(NATR) caused by R. sibirica and Far East Spotted Fever(FESF) caused by R. heilongjiangensis. FESF has been considered as an important emerging infectious disease in northeast Asia. Patients with FESF usually have fever, chills, headache, dizziness, myalgia, arthralgia, and anorexia, as well as macular or maculopapular rash, accompanied by hepatomegaly with increased alanine aminotransferase and/or aspartate aminotransferase activity. In a mouse model, R. heilongjiangensis infection caused severe systemic infection complicated by pathological lesions in lungs, liver, spleen, and brain.T-cell immunoglobulin and mucin domain protein 3(Tim-3) was originally recognized as a T-helper type 1(Th1)–specific receptor that regulates Th1 responses and maintains immune homeostasis and tolerance. Many studies have demonstrated that Tim-3 is also expressed in innate immune cells, including macrophages/monocytes, dendritic cells(DCs), and natural killer cells, and that Tim-3 has been believed to contribute to immune homeostasis by maintaining optimal activation of innate immune cells. Additionally, Tim-3 has been found to be expressed in human vascular endothelial cells(ECs). In recent years, there has been increasing research concentrating on the role of Tim-3 in infection and immunity, including viral and bacterial infection and immunity. However, it remains unknown whether Tim-3 expression or Tim-3 signaling plays a role in rickettsial infection and immunity. Vascular ECs are the major host cells for rickettsiae, and the progression of infection is mainly dependent on the growth of the bacteria in these host cells. ECs can produce and react to a wide variety of mediators, including cytokines, growth factors, adhesion molecules, vasoactive substances, and chemokines, and they are also recognized as key immunoreactive cells involved in host defense and inflammation.In the present study, to determine whether rickettsial infection affects Tim-3 expression of host cells in vivo and in vitro and whether the expression of Tim-3 influences the intracellular pathogen fate during rickettsial infection, we investigated the corresponding relations between Tim-3 expression and R. heilongjiangensis infection during the early phase of infection in mouse models and in in vitro ECs.C3H/HeN mice infected with R. heilongjiangensis were euthanized to collect spleens for preparation of RNA and DNA samples on days 0, 1, 2, and 3 after infection. RT-PCR analysis revealed that Tim-3 mRNA expression in mouse spleens was sharply reduced on day 1 and continued to decrease on days 2 and 3 after infection. Similarly, Tim-3 mRNA expression of human vacular endothelial cells(Ea.hy926 cells) was sharply reduced on day 1 and continued to decrease on days 2 and 3 after infection. However, the rickettsial amounts in mice and Ea.hy926 cells continuously increased during the first 3 days after infection.To block the Tim-3 signaling pathway, C3H/HeN mice were intraperitoneally injected with soluble forms of Mouse Tim-3 and Human IgG1 Fc Tail(mTim-3-Ig), and then mice were infected with R. heilongjiangensis. On day 3 after infection, the rickettsial burden in spleens of mice treated with mTim-3-Ig was significantly higher than that formerly treated with control human IgG1-Fc(Ig). Meanwhile, the levels of IFN-γ, TNF-α, IL-2, IL-18, and RANTES in sera from mice treated with mTim-3-Ig were significantly lower than those for mice treated with control Ig.R. heilongjiangensis infection was performed on transgenic C57BL/6 mice overexpressing Tim-3 and on wild-type C57BL/6 mice. On day 3 after infection, wild-type mice showed signs of severe illness, such as decreased activity and ruffled fur, but no mice died, whereas transgenic mice displayed no sign of sickness. Then the mice were euthanized for determination of rickettsial loads and cytokine secretion. The rickettsial loads in the spleens of transgenic mice were significantly lower than those of wild-type mice. In addition, the levels of IFN-γ, TNF-α, IL-2, IL-18, and RANTES in sera from transgenic mice were significantly higher than in sera from wild-type mice.On day 3 after infection with R. heilongjiangensis, the rickettsial amounts in Ea.hy926 cells treated with Recombinant Human Tim-3 Fc Chimera(hTim-3-Ig) or in Ea.hy926 cells with stable expression of Tim-3 siRNA or overexpressed Tim-3 were examined. The rickettsial amount in cells treated with hTim-3-Ig was significantly higher than that in cells treated with control Recombinant Human IgG1-Fc(hIg), and the rickettsial amounts in cells with stable expression of Tim-3 siRNA and overexpressed Tim-3 were significantly higher and lower, respectively, than those in corresponding controls.Human umbilical vascular endothelial cells(HUVECs) treated with a solution containing RANTES, TNF-α, IFN-γ, and IL-1β, with or without the iNOS inhibitor NGMMLA, were infected with R. heilongjiangensis. On day 3 after infection, rickettsial amounts in the cells treated with the solution alone were significantly lower than those treated with the solution plus NGMMLA, while the nitrite level and iNOS mRNA expression of cells treated with the solution alone were significantly higher than those treated with the solution plus NGMMLA. This result suggested that the antirickettsial activity in Endothelial Cells(ECs) is associated with nitric oxide(NO) synthesis. To test whether Tim-3 signaling induces NO-dependent killing of rickettsiae, the mRNA expression of iNOS and IFN-γ was assessed in Ea.hy926 cells treated with Recombinant Human Tim-3 Fc Chimera(hTim-3-Ig) and in Ea.hy926 cells with stable expression of Tim-3 siRNA. On day 3 after infection with R. heilongjiangensis, the iNOS and IFN-γ mRNA levels in both types of cells was significantly lower than that for corresponding controls. In addition, iNOS, IFN-γ, and TNF-α mRNA levels in transgenic mice overexpressing Tim-3 were significantly higher than those in wild-type mice.In summary, during the early phase of R. heilongjiangensis infection, continuously decreased Tim-3 expression and increased rickettsial loads were observed in mice and in in vitro human vascular ECs, suggesting that Tim-3 was involved in rickettsial infection. The rickettsial burdens were significantly higher in mice and in human ECs with the blockage of Tim-3 signaling and in ECs with Tim-3 silencing, while burdens were significantly lower in transgenic mice and transfected ECs with Tim-3 overexpression. These results are strong evidence that enhanced Tim-3 expression could inhibit rickettsial growth during the early phase of rickettsial infection, both in vivo in mice and in vitro in human vascular ECs. Additionally, the expression of iNOS and IFN-γ mRNA in ECs with the blockage of Tim-3 signaling or Tim-3 silencing was significantly lower, while the expression of iNOS, IFN-γ, and TNF-α mRNA in transgenic mice overexpressing Tim-3 was significantly higher, compared with findings for corresponding controls, which suggested that enhanced Tim-3 expression could promote synthesis of NO and thereby facilitate intracellular rickettsial killing. The role of Tim-3 in vascular ECs may not only provide new evidence for understanding the connection between rickettsial infection and host cell responses, but may also lead to the discovery of new therapeutic targets in rickettsial infection.