Over Expression of ELR+ chemokines , VEGFA and solTNF in M.tb infected Cavia porcellus and their role in the dissemination of infection from lungs to peripheral sites. 1 Kusuma Sai Davuluri , Dr. Ajay Vir Singh, Dharmendra Singh, Dr.Amit Kumar Singh, Dr.D.S.Chauhan1* Department of Microbiology and Molecular biology National JALMA Institute for Leprosy and other mycobacterial diseases, Agra INTRODUCTION
qRT-PCR results
Colony Forming Units
The host disseminates the Mycobacterium tuberculosis (M.tb) from lungs to peripheral sites for antigen specific T-cell mediated immunity, but M.tb itself disseminates to other organs by inducing the angiogenesis that results in extra pulmonary tuberculosis and less penetration of drug. Angiogenesis is also mediated by several cytokines and chemokines including tumor necrosis factor alpha1 (TNFa). ELR+(glutamic acid – leucine – arginine) amino acid motif, are potent inducers of angiogenesis2 Soluble TNF receptors (sTNFR-I and sTNFR-II) circulate in human will neutralize TNFa-induced cytotoxicity3 Our results demonstrate that Vascular endothelial growth factor (VEGFA) and ELR+ chemokine mediated angiogenesis could be responsible for the dissemination of M.tb and might be controlled through soluble TNF (solTNF).
Organ
Week
Inf CFU log avg
XENP-1595 Treated
Lungs
4th week
6.09101
6.055552
Spleen
4th week
4.978424
5.228305
H37Rv infected Chemokines fold Gene change 4th week 8th week CXCL1
0.165
2.14
2.36
20.48
CXCR2
8.30
188.05
1.12
123.8
CXCL6
0.5
73.10
0.34
52.27
CXCL7
29.4
565.15
0.35
355.07
CXCL8
5.78
3.0
1.19
3.2
Table showing decrease in bacterial count at lungs and increase in the bacterial count at spleen during 8th week
CCL-11
0.76
3.19
0.46
3.34
CCL-16
0.64
4.43
0.24
4.8
IMMUNOHISTOCHEMISTRY
VEGFA
0.05
1.37
0.33
2.04
Lungs
8th week
5.321773
5.315837
Spleen
8th week
5.701611
4.821149
25
M.tb 8th week
500 400
20
300
15
200
10 100 5
a
c
b
0
0 1
2
3
4
5
6
7
1
8
25
MATERIALS AND METHODS
2
3
4
5
6
7
8
400
Treated 4th week
20 15
350
Treated 8th week
300 250 200
10
150 100
5
50 0
e
d
1
2
3
4
5
6
7
0
8
1
2
3
4
5
6
7
8
f
CD4 marker stained a. healthy control b.M.tb infected c.Xpro-1595 treated CD8 marker stained d. healthy control e..M.tb infected f..Xpro-1595 treated (Followed DAB method with Hematoxylin as counter stain)
Evaluation of ELR+ chemokines, VEGFR, CXCR-4 expression levels
qRT-PCR
M.tb 4th week
30
To enumerate the bacterial count at different organs during the inhibition of angiogenic factors. To evaluate the drug concentration at the granuloma site and blood in CXCR-4 and VEGFR inhibited M.tb infected guinea pigs.
In-Gel western blotting
600
35
OBJECTIVES
M.tb bacilli suspension containing 1.42×106 CFU/ml of H37Rv is given to guinea pigs through inhalation system. Group-I Naïve Group-2 M.tb Infected Group-3 Xpro-1595 Inhibition two days before infection Group-4 Chemotherapy Group-5 CXCR-4 Inhibition day before the infection Group-6 VEGFR Inhibition day before the infection Group-7 CXCR-4 inhibition along with the chemotherapy Group-8 VEGFR inhibition along with the chemotherapy
M.tb inf Xpro-1595 chemokines fold change 4th week 8th week
IN-GEL WESTERN BLOTTING M
1
2
3
4
5
6
7
8
c
Immunofluorescent histochemistry
c
b
a
a M
1
2
3
4
5
6
7
8
9
10
Evaluation of drug efficiency
d Enumeration of bacterial count at spleen, liver, lung and brain
Evaluation of drug penetration level through HPLC
Fig-2 M.tb infected + Xpro-1595 treated 4th week lung and spleen, 8th week lung and spleen respectively
f
CXCR-4 marker stained a. healthy control b.M.tb infected c.Xpro-1595 treated VEGFA marker stained d. healthy control e.M.tb infected f.Xpro-1595 treated (Stained with FITC conjugated antibody and DAPI counter staining)
RESULTS Efficient immune response at lungs but failed Fig-1 to regulate dissemination Fig-2 Fig-1 M.tb infected 4th week lung and spleen, 8th week lung and spleen respectively
e
Fite-Faracco staining
a
b
b
a. CXCR-4 binding is seen at 46kDa Protein is extracted from BAL samples of M.tb infected guinea pig and Xprotreated guinea pigs during 8th week infection M-Molecular marker Lane-1 – M.tb infected Lane-2 – M.tb infected Lane-3 – M.tb infected Lane-4 – M.tb infected Lane-5 - Xpro treated Lane-6- Xpro treated Lane-7 – Xpro treated Lane-8 – Healthy control
CONCLUSION
c
There is a subsequent increase in the expression of ELR+ chemokines from 4th week to 8th week infection. solTNF involves in the migration of CD4+ and CD8+cells during the infection and chemokines synthesis. Bacterial count is not found in the liver and lymph nodes of XENP-1595 treated guinea pigs. By regulating the overexpression of solTNF the dissemination and inflammation were regulated maintaining the normal morphology of cells. VEGFA and ELR+ chemokines might be the main factors of dissemination of infection. Note- From Group-4 to Group-8 work has not been done yet
d
M.tb infected a.spleen b. lymph node showing bacteria Xpro treated a.spleen d.lymph node without bacteria
Hematoxylin & Eosin staining
Fig showing granuloma with necrotic area at 4th week and normal morphology at 8th week
b. VEGFA binding seen at 44KDa Protein is extracted from BAL samples of M.tb infected guinea pig and Xprotreated guinea pigs during 4th and 8th week respectively M-Molecular marker Lane-1 – M.tb infected 4t week Lane-2 – M.tb infected 4th week Lane-3 – M.tb infected 4th week Lane-4 – Xpro treated 4th week Lane-5 - Xpro treated 8th week Lane-6 – M.tb infected 8th week Lane-7– M.tb infected 8th week Lane-8 – M.tb infected 8th week Lane-9 – Xpro treated 8th week Lane 10 - Xpro treated 8th week
REFERENCES 1. Alexander HS et al., Inflammatory cytokines in vascular dysfunction and vascular disease. Biochem Pharmacol 2009;78:539– 52. Strieter R.M. et al., Chemokines as Mediators of Neovascularization (Arterioscler Thromb Vasc Biol. 2008;28:1928-1936. 3. LOWRY S.F. et al., Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor a in vitro and in vivo Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 4845-4849, June 1992 Biotech Online Poster Presentation Competition By KITS, ABLE, IISc, IBAB
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ACKNOWLEDGEMENT We are thankful to the technical staff and MTS of Animal experimentation and histopathology department