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dc.contributor.advisorLiu, Lixin
dc.creatorHao, Li
dc.date.accessioned2020-07-16T19:56:55Z
dc.date.available2021-07-16T06:05:10Z
dc.date.created2020-07
dc.date.issued2020-07-16
dc.date.submittedJuly 2020
dc.identifier.urihttp://hdl.handle.net/10388/12925
dc.description.abstractB cell adaptor molecule of 32 kDa (Bam32), also known as dual adapter for phosphotyrosine and 3-phosphoinositides 1 (DAPP1), is an adaptor protein downstream of phosphoinositide-3-kinase (PI3K) signaling pathway. Although the roles of Bam32 have been revealed in some subsets of leukocytes during the past two decades, its role in neutrophils remains completely unknown. In my study, I respectively explored the roles of Bam32 and its upstream PI3K subunits in intermediary chemoattractant CXCL2-induced and end-target chemoattractant Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm)-induced neutrophil recruitment, neutrophil reactive oxygen species (ROS) production, and microvascular hyperpermeability in mice. Firstly, I revealed that PI3Kγ, an upstream regulator of Bam32, plays a critical role in neutrophil-driven microvascular leakage induced by WKYMVm. Using intravital microscopy in mice, I found a PI3Kγ-specific mechanism in WKYMVm-induced but not CXCL2-induced microvascular hyperpermeability. The increased microvascular permeability triggered by WKYMVm was not entirely due to neutrophil adhesion and emigration in mouse cremasteric microvasculature. The PI3Kγ-specific hyperpermeability was neutrophil-mediated as this was reduced after depletion of neutrophils in mouse circulation. Chimeric mice with PI3Kγ-deficient neutrophils but wild-type (WT) endothelium also showed reduced microvascular hyperpermeability. Furthermore, I found that the catalytic function of PI3Kγ was required for reactive oxygen species (ROS) generation in neutrophils stimulated with WKYMVm. Pharmacological scavenging PI3Kγ-dependent ROS in the tissue eliminated the discrepancy in hyperpermeability between different PI3K transgenic mice and alleviated WKYMVm-induced microvascular leakage in all mouse strains tested. Secondly, I uncovered a role for Bam32 in regulating CXCL2-induced neutrophil recruitment through small GTPase Rap1. Using intravital microscopy in mouse cremaster muscle, I found that transmigrated neutrophil number, neutrophil chemotaxis velocity and total neutrophil chemotaxis distance were increased in Bam32-/- mice when compared with WT mice. In CXCL2-induced mouse peritonitis, the total emigrated neutrophils were increased in Bam32-/- mice at 2 h but not 4 h. CXCL2-induced chemotaxis distance and migration velocity of isolated Bam32-/- neutrophils in vitro were increased. I examined the activation of small GTPases Rac1 and Rap1, the expression levels of phospho-Akt2 and total Akt2 and their crosstalk with Bam32 in neutrophils. The deficiency of Bam32 resulted in the elevation of Rac1-GTP binding and suppression of Rap1-GTP binding. Pharmacological inhibition of Rac1 by NSC23766 markedly reduced activation of Rap1 in WT neutrophils and reduced Bam32-/- neutrophil chemotaxis, whereas inhibition of Rap1 by GGTI298 increased WT neutrophil chemotaxis. In addition, deficiency of Bam32, as well as inhibition of Rap1 activation, increased the levels of CXCL2-induced Akt1/2 phosphorylation at Thr308/309 in neutrophils. Inhibition of Akt by SH-5 attenuated CXCL2-induced adhesion and emigration in Bam32-/- mice. Thirdly, I unveiled a role for Bam32 in modulating WKYMVm-induced formation of microvascular hyperpermeability through the production of neutrophil ROS. I observed significantly reduced WKYMVm-induced microvascular hyperpermeability accompanied by markedly decreased neutrophil emigration only in Bam32-/- mice. The Bam32-specific decrease in WKYMVm-induced hyperpermeability was neutrophil-dependent as this was verified in bone marrow transplanted Bam32 chimeric mice. I also discovered that Bam32 was critically required for WKYMVm-induced intracellular and extracellular production of ROS in neutrophils. Pharmacological scavenging of ROS eliminated the differences in WKYMVm-induced hyperpermeability between Bam32-/- and WT mice. In addition, the deficiency of Bam32 reduced WKYMVm-induced ERK1/2 but not p38 or JNK phosphorylation in neutrophils. Inhibition of ERK1/2 signaling cascade suppressed WKYMVm-induced ROS generation in WT neutrophils and microvascular hyperpermeability in WT mice. Taken together, my study shows both PI3Kγ and PI3K downstream adaptor Bam32 regulate WKYMVm-induced microvascular hyperpermeability through modulating ROS production in neutrophils. Moreover, Bam32 suppresses CXCL2-induced neutrophil recruitment through Rap1 activation. My study provides insights regarding PI3K signaling pathway for regulating neutrophil recruitment, neutrophil ROS production, and microvascular barrier function in acute inflammation.
dc.format.mimetypeapplication/pdf
dc.subjectBam32
dc.subjectNeutrophil recruitment
dc.subjectROS
dc.subjectMicrovascular permeability
dc.subjectSmall GTPases
dc.subject
dc.titleThe role of PI3Kγ and PI3K adaptor protein Bam32 in neutrophil recruitment and microvascular hyperpermeability
dc.typeThesis
dc.date.updated2020-07-16T19:56:55Z
thesis.degree.departmentPharmacology
thesis.degree.disciplinePharmacology
thesis.degree.grantorUniversity of Saskatchewan
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)
dc.type.materialtext
dc.contributor.committeeMemberDesai, Kaushik
dc.contributor.committeeMemberGordon, John R.
dc.contributor.committeeMemberTaghibiglou, Changiz
dc.contributor.committeeMemberBardal, Stanley
local.embargo.terms2021-07-16


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