Wednesday, October 16, 2013

Lack of Endogenous IL-10 Enhances Production of Pro-inflammatory Cytokines and Leads to Brucella abortus Clearance in mice

As we all know cytokines help us fight off pathogens by molecular signaling with the use of IFNs and ILs. IL-10 regulates helps balance pathogen clearance and immune response. Brucella abortus is a chronic inflammatory disease which can be found in humans as well as animals. Previous studies have shown that IL-10 is a critical cytokine for inflammatory response in the host and prevents damage. Another study by Fernandes and Baldwin, showed that anti-IL-10 resulted in up to 10-fold fewer bacteria in the spleen with mice infected with the same strain of Brucella. IL-10 directly affects IL-12 by down regulating it and presents a feedback loop which ensures there is not excessive inflammation. In this current study by Corsetti et. al. published in September 2013 he set out to find out the results of IL-10 in bacterial clearance, inflammatory response, and aftereffects of being infected with Brucella abortus.

In order to test this, two sets of mice were studied, the wild type and the IL-10 KO (knock-out). Blood marrow cells were collected and cultured in DMEM in order to culture bacterial cells. After 10 days, the cells were infected with Brucella abortus and assayed for concentrations of IL-10, IL-12p40, or TNF-alpha. Brucella abortus strain 2308 was grown separately from the laboratory for 3 days and the mice were infected. Five animals from each group were examined at 1,2,3,6, and 14 weeks and spleens were removed. The spleens were plated and colonies were counted for cytokine analysis. To analyze the other factors such as IL-10 and IL-gamma, they stimulated the cells and unstimulated ones were used as negative controls. To test in vivo production of these factors, blood samples were taken and centrifuged, the supernatant was used for cytokine analysis. Real-Time RT-PCR was used on the splenocytes. FACS (Fluorescence activated Cell Sorter) is a type of flow cytometry in which cells in a heterogeneous mixture are sorted out.

A FACS analysis consists of many washes and separation method such as the one presented in the figure. The livers of the mice were also collected at each week period and stained with H and E. Granulomas (inflammation)  were measured using this method.

From this experimentation, we learned that wild-type infected with Brucella abortus presented increased production of IL-10 but none was found in the IL-10 KO mice as expected. IL-10 production was not only found in the dendritic cells but also in the splenocytes showing that it is produced in vivo and in vitro when infected. They determined that there was elevated proinflammatory cytokine production in IL-10 KO dendritic cells. Lack of IL-10 results in increased IL-12 and TNF-alpha production because it usually suppresses it. IL-10 KO cells resulted in less bacteria compared to the WT; by week 14 in the spleen there was no sign of Brucella abortus. All other factors were upregulated in IL-10 KO mice too. IL-10 KO mice has less granuloma in the liver as the infection time increased.

In conclusion, the knockout of IL-10 enhanced the the inflammatory response in Brucella abortus but could have adverse results in a different bacterial infection. The KO IL-10 led to reduction in liver pathology which is regulated by Treg cells and TGF-beta (increased in absence of IL-10). Further studies will be performed on these two key players.

I chose this article because I never thought of a cytokine having a better effect when being knocked out because I thought they all assisted in the immune response. Given this model was performed in mice, it’d be interesting if the same results would come from a human experiment. I believe it’s important to know these effects because it can help in cures of these disease and a key hallmark of this one is inflammation so this may work on other types of infection and even cancers.


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