There is one research paper published in the PNAS science journal about role of Aquaporin 3 mediated H2O2 transport in colonic epithelia. Here I try to explain it in easy way with slides. Enjoy it.

1. Aquaporin-3 mediates hydrogen
peroxide-dependent
responses to environmental stress
in colonic epithelia
Presenter :- Vaishnav Jayvadan
Biochemistry (P.)
Thiagarajah et al.,
January 2017.
PNAS.

2. Introduction
Lance W. et al.,2014

3. H2O2
Murderer
Antimicrobial defence
Mediator
Regulation of gene
expression
Messenger
Chemotactic attractant
of immune cells
Albert van der Vliet et al.,2014

4. Aquaporin
(AQP)
Classical AQP
AQP1,
AQP2,
AQP4,
AQP5
Aquaglyceroporin
AQP3,
AQP7,
AQP9,
AQP10.
Umberto Laforenza 2011

5. Previous studies
In the intestine, the generation of extracellular H2O2 at the
mucosal or serosal surface is carried out by NADPH oxidase
(NOX) enzymes, such as NOX1 and dual oxidase 2 (DUOX2).
(Rada B et al., 2008)
H2O2 is a stable reactive oxygen species (ROS) molecule that acts
as an extracellular and intracellular signal that mediates pleiotropic
effects in tissues, including recruitment of immune cells to
damaged areas and cell migration. (Niethammer P et al., 2009)
mice lacking AQP3 experienced impaired recovery after chemical-
induced colitis (Basuroy S et al., 2010)

6. Experiment-1
AQP3 renders the colonic epithelium
responsive to extracellular H2O2

7. Experiment-1(a)
To check AQP3 mediated entry of
extracellular H2O2 in Caco-2 cells

8. Caco-2 cells expressing HyPer
H2O2 conc.=100µM
AgNO3=5µM
n=6
*P<0.05
Result of H2O2 Fluorescence Ratio imaging

9. AQP3 mediates entry of extracellular
H2O2 into the Caco-2 cells
Conclusion

10. Experiment-1 (b)
To check AQP3 mediated entry of
extracellular H2O2 in isolated colonic
crypts

11. H2O2 conc.=100µM
**P<0.01
Result of CM-H2-DCFDA fluorescence
Chloromethyl derivative of 2',7'-
dichlorodihydrofluorescein diacetate

12. AQP3 mediates entry of extracellular
H2O2 into the colonic crypts.
Conclusion

13. To check AQP3-dependent H2O2
transport underlies signal transduction
in the intestine.
Experiment - 2

14. H2O2 PI-3-kinase c-Scr
Focal adhesion
complex
FAK Paxillin
Induction of actine
driven lamellopodia
Cell migration
Wound Closure
S. Basuroy et al.,2010

15. Experiment – 2 (a)
Effect of AQP3 dependent H2O2
transport on wound closure.

16. Result of scratch wound assay
n=4
AgNO3 = 5 µM
Auphen = 10 µM
DPI = 20 µM
Catalase = 200 U
n=4
**P<0.01
*P<0.05
*P<0.05

17. Wound closure is facilitated by H2O2
transport via AQP3.
Conclusion

18. Experiment – 2(b)
Effect of AQP3 dependent H2O2
transport in induction of lamellopodia

19. Result of lamellopodia imaging by actin staining
in wound associated Caco-2 cells
Rhodamine Phalloidin n=4
**P<0.01

20. Induction of lamellopodia depends
upon H2O2 transport via AQP3.
Conclusion

21. Experiment – 2(c)
Effect of AQP3 mediated transport
of H2O2 on paxillin

22. Result of Immunocytochemistry
n=4
**P<0.01
Alexa flour 488
Alexa flour 647

23. AQP3 play role in phosphorylation of
paxillin
Conclusion

24. Experiment – 2(d)
To test role of AQP3 mediated transport
of H2O2 in wound healing (In vivo)

25. Result of In Vivo colonoscopy
n=6 mice **P<0.01

26. AQP3 facilitated H2O2 entry underlies
H2O2 dependent epithelial response to
injury and barrier repair
Conclusion

27. Experiment – 3
Whether AQP3 dependent H2O2 transport
modulate signal transduction in host
defense against C. rodentium infection

29. Experiment – 3(A)
To check the effect of microbial peptide
fMLF on Caco-2 cells.

30. Addition of fMLF in Caco-2 cells
**P<0.01 *P<0.05 n=5
fMlF = N-Formyl-L-methionyl-L-leucyl-L-phenylalanine

31. Experiment – 3(B)
To test whether colonic pathogen C.
rodentium can induce production of
extracellular H2O2.

32. n=3*P<0.05
Result of colonoid microinjection

33. Infection of C. rodentium cause
accumulation of ROS in colonoids.
Conclusion

34. Experiment – 3(C)
To test whether AQP3 mediates
mucosal inflammatory response after
Citrobacter rodentium infection.

35. Measure of body weight and crypt length
12 day
*P<0.05*P<0.05
n=6 n=6

36. Relative expression of IL-6 and TNF-
m-RNA in isolated ECs
n = 6
At 5 day post infection
**P<0.01

37. At 5 day post infection At 25 day post infection
n=5
n=4
Relative expression of IL-6 and TNF- m-RNA
in isolated ECs from AQP3+/+ and AQP3-/-
**P<0.01
**P<0.01
***P<0.001
NAC : n-acetyl cysteine

38. AQP3 is involved in mucosal inflammatory
response required for subsequent bacterial
clearance and recovery
Conclusion

39. BACKUP

40. Dose responsive studies