The Research On The Relationship Between Anti-inflammatory Cytokine IL-1Ra And Brucella Suis S2 Strain Intracelluar Parasitism

Brucella is a Gram-negative intracellular parasitic bacterium thatcauses brucellosis,a zoonnosis that posesa serious threat to human public health safety and the healthy development of animal husbandry.The main members of the interleukin-1family are interleukin-1β(IL-1β)and interleukin-1 receptor antagonist(Interleukin-1Ra,IL-1Ra).IL-1Ra competes with IL-1R(interleukin-1 receptor)on the cell surface and specifically inhibits the biological function of IL-1β to alleviate inflammation.IL-1Ra is a major anti-inflammatory cytokine that is involved in the immune response of the body.Our group found that infection with a weak strain(B.suis S2)and wild strain(B.melitensis)can cause differential expression of two genes,IL-1β and IL-1Ra,and that the ratio of IL-1Ra to IL-1β is indicative of the difference betweennatural Brucella infection and vaccine immunization of animals.However,the role played by IL-1Ra in Brucella pathogenesis is unknown.Therefore,the study analyzed the relationship between IL-1Ra and B.suis S2 intracellular parasitism,which isimportant for further exploring the molecular mechanism of host response to Brucella infection.In this study,we first cloned the nucleic acid sequence encoding mouse IL-1Ra mature protein,and used a prokaryotic expression system to induce the IL-1Ra recombinant protein expression andpurification.The obtained IL-1Ra recombinant protein was demonstrated to be biologically active by IL-1β activity inhibition experiment.Secondly,the intracellular and extracellular IL-1Ra differential expression characteristics were detected in RAW264.7 cells infected by B.suis S2 and other bacteria using Western Blot,real-time fluorescence quantitative PCR and ELISA techniques,and the intracellular bacterial number was also analyzed.The results showed that the expression and secretion of IL-1Ra was significantly increased in RAW264.7 cells infected by S2,while the change of the bacterial number showed a trend of first decreasing and then increasing.The effect of changing IL-1Ra protein content level on B.suis S2 intracellular parasitism was then analyzed by interfering with IL-1Ra expression,IL-1Ra overexpression and extracellular addition of active IL-1Ra recombinant protein,decreasing or increasing IL-1Ra protein content level and monitoring the change in intracellular live bacteria count.The results showed that increasing the protein content level of IL-1Ra inhibited the S2 intracellular parasitism,while decreasing the protein expression level of IL-1Ra favored the S2 intracellular parasitism.Finally,to further analyze whether the effect of IL-1Ra on S2 intracellular parasitism is related to IL-1β.In this study,the nucleic acid sequence encoding the mouse IL-1β mature protein was cloned and active IL-1β recombinant protein was obtained using a prokaryotic expression system.By extracellular addition of active IL-1β recombinant protein and interference with IL-1β expression,the level of IL-1βprotein content was increased or decreased,and changes in intracellular live bacteria count and some cytokines of S2-infected IL-1β or IL-1Ra knockdown RAW264.7 cells were analyzed.The results showed that there was no significant correlation between IL-1β and S2 intracellular parasitism.In IL-1β knockdown RAW264.7 cells infected by S2,the expression of IL-1Ra and TNF significantly decreased and the expression of IL-10 significantly increased;while in IL-1Ra knockdown RAW264.7 cells infected by S2,the expression of TNF and IL-10 significantly decreased and the expression of IL-6 and IL-1β significantly increased.It is possible that the inhibitory effect of IL-1Ra on S2 intracellular parasitism may not be achieved by suppressing IL-1β biological activity,but rather by a combination of cytokines.In conclusion,this study found that IL-1Ra was highly expressed and secreted in RAW264.7 cells infected by B.suis S2 and IL-1Ra was detrimental to the intracellular parasitism of S2.Further analysis revealed that the inhibitory effect of IL-1Ra on the S2 intracellular parasitism was not significantly correlated with its function of inhibiting IL-1β activity.The above findings provide a solid foundation for further research into how IL-1Ra affects Brucella intracellular survival and whether IL-1Ra can be exploited as a target for successful Brucella prevention and treatment.