4. Study of Toll-like Receptors, cont\'d. 4f. In the right part of the left pane
ID: 191619 • Letter: 4
Question
4. Study of Toll-like Receptors, cont'd. 4f. In the right part of the left panel, what do the patterns B « of nitrite production by the 19kD lipoprotein and LPS in TLR2-deficient macrophages reveal? 50 E 40 20 TLR2+- Figure 1. Thioglycollate-elicited macrophages from control (wt) and gene-deleted (TLR2-/-) mice were infected with M tuberculosis at an MOI of 5. 48 hours after the addition of the 19-kD lipoprotein (1 g/ml) or LPS (0.1 g/ml), supematants were assayed for NO production (Griess reaction) and cells were lysed (0.05% Tween) for plate counts of bacterial load. The figure shows the average of triplicate experiments+- SEM 4g. In the right hand panel, macrophages were lysed with Tween (a detergent) before plating. Why is this lysis step necessary? 4h. What can you infer from the wt (wild-type; nonmutant) data in the right panel? 4i. What can you infer from the TLR2-mutant data in the right panel? 4j. The data shown in Figure IB of Thoma-Uszynski et al (above) establishes a correlation between activation of a killing mechanism and elimination of M. tuberculosis, but does not establish a causal relationship. What are two hypotheses that could explain the correlation between these phenomena?Explanation / Answer
4f. Thioglycollate elicited macrophages were isolated from wt and TLR2 deficient mouse. When macrophages from TLR2 mutant mouse were treated with 19Kd lipoprotein and LPS they showed increased nitrite production. Nitrite production was more ( almost 5 fold) when TLR2 deficient macrophages were treated with LPS in comparison to 19Kd lipoprotein.
4g. In this experiment macrophages were lysed with tween. Tween is a detergent which is used for cell lysis. When cell is lysed, cell membrane get disintegrated and the cellular componants get released. This process helps to release the intracellular proteins so they can be further detected by seveal methods.
4h. Macrophages of wt mouse was infected with M.tuberculosis and and treated with 19Kd lipoprotein. After 48 hours the bacterial growth were counted. 19Kd lipoprotein treated wt macropheges shows reduce in number of bactera in comparison to untreated wt macrophage.
4i. macrophages isolated from TLR2 mutatnt were also infected with M.tuberculosis and are treated with 19Kd lipoprotein as wt. However, the TLR2 mutant macrophages showed no difference in bacterial growth in between treated and non treated cell. Thus there is no effect of 19Kd lipoprotein treatment on TLR2 deficient macrophage.
4j, The data showed that -
there is a correlation between nitrite production and elimination of M.tuberculosis.
there is a relation between TLR2 , nitrite production and killing and elimination of M.Tuberculosis.
In the left panel nitrite production were compared. When macrophages were treated with 19Kd lipoprotein, nitrite production increased in wt macropages and also in macrophages isolated from TLR2 mutant. Howerver, macrphages from wt showed increased nitrite production ( 10 times more) in comparison to macrophages from TLR2 deficinet mouse. Thus TLR2 or required for nitrite production in presence of 19Kd lipoprotein.
On the other hand the right pannel showed number of M.tuberculosis after the treatment. Macrophages from wt mouse showed increased killing of macrophages in presence of 19Kd lipoprotein. Whereas in macrophages from TLR2 deficient mouse can not kill the M:Tuberculosis effecintly when they are treated with 19Kd lipoprotein, as there is no difference between cell number of treates and untreated macrophages from TLR2 deficient mouse. TLR2 or toll like receptor 2 is important part of the immune system. When there is an infection occurs, TLR2 can recognize the foreign particle and initiate a signalling cascade, so that the responsible cells like macrophages can kill them.
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