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Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Mechanism of rLcrV and rYopB mediated Immunosuppression in murine peritoneal macrophages
Ajit Sodhi1, Rajesh K Sharma, Harsh V Batra
1School of Biotechnology, BHU, Varanasi, UP 221005, India. ajit.sodhi@lycos.com
Abstract:
Yersinia pestis causative organism of bubonic and pneumonic plague uses type III secretion system for the translocation of effector molecules in to the target host cells. Type III secretion system of Yersinia pestis, secretes Low calcium response V (LcrV) or V antigen and many other Yersinia outer proteins (Yops). LcrV and YopB were initially known as translocator proteins, but recently have been shown to act as effector and regulatory proteins. Macrophages participate actively in immune response by secreting various effector molecules or by phagocytosis in clearing most of the pathogens. We investigated the effect of rLcrV and rYopB (10 microg/ml) on peritoneal macrophages in vitro. It is observed that rLcrV and rYopB inhibited LPS induced TNF-alpha and NO production in murine peritoneal macrophages. rLcrV and rYopB also inhibited expression of phospho-p38, -p42/44, -JNK MAPKs and transcription factors NFkappaB, c-fos and c-jun in LPS treated macrophages. The inhibition in the expression of these signaling molecules has been correlated to the inhibition of TNF-alpha and NO production in macrophages.
Insights
Yersinia pestis effector proteins, LcrV and YopB, were found to inhibit key immune responses in macrophages. These proteins suppressed tumor necrosis factor-alpha and nitric oxide production, impacting the host
Area of Science:
- Bacteriology
- Immunology
- Molecular Biology
Background:
- Yersinia pestis causes plague and utilizes a type III secretion system to deliver effector proteins into host cells.
- Key Yersinia effector proteins, Low calcium response V (LcrV) and Yersinia outer proteins (Yops) like YopB, play roles in host-pathogen interactions.
- Macrophages are critical immune cells involved in pathogen clearance and inflammatory responses.
Purpose of the Study:
- To investigate the in vitro effects of recombinant LcrV (rLcrV) and rYopB on murine peritoneal macrophages.
- To determine if rLcrV and rYopB modulate lipopolysaccharide (LPS)-induced inflammatory mediator production.
- To examine the impact of rLcrV and rYopB on intracellular signaling pathways in macrophages.
Main Methods:
- Treatment of murine peritoneal macrophages with recombinant LcrV and YopB (10 microg/ml).
- Stimulation of macrophages with lipopolysaccharide (LPS).
- Measurement of TNF-alpha and NO production.
- Analysis of the expression of phospho-MAPKs (p38, p42/44, JNK) and transcription factors (NF-kappaB, c-fos, c-jun) using Western blotting or similar techniques.
Main Results:
- Recombinant LcrV and YopB significantly inhibited LPS-induced TNF-alpha and NO production in macrophages.
- rLcrV and rYopB suppressed the expression of key signaling molecules, including phospho-p38, phospho-p42/44, phospho-JNK MAPKs, and transcription factors NF-kappaB, c-fos, and c-jun.
- The observed inhibition of signaling pathways correlated with the reduced production of TNF-alpha and NO.
Conclusions:
- Yersinia pestis effector proteins rLcrV and rYopB possess immunosuppressive properties.
- These proteins interfere with critical macrophage signaling cascades, leading to the suppression of pro-inflammatory mediators.
- Understanding these mechanisms provides insights into Yersinia pathogenesis and potential therapeutic targets.
