Microbial exposure alters HIV-1-induced mucosal CD4+ T cell death pathways Ex vivo.
BACKGROUND
Early HIV-1 infection causes massive CD4+ T cell death in the gut and translocation of bacteria into the circulation. However, the programmed cell death (PCD) pathways used by HIV-1 to kill CD4+ T cells in the gut, and the impact of microbial exposure on T cell loss, remain unclear. Understanding mucosal HIV-1 triggered PCD could be advanced by an ex vivo system involving lamina propria mononuclear cells (LPMCs). We therefore modeled the interactions of gut LPMCs, CCR5-tropic HIV-1 and a commensal gut bacterial species, Escherichia coli. In this Lamina Propria Aggregate Culture (LPAC) model, LPMCs were infected with HIV-1BaL by spinoculation and cultured in the presence or absence of heat killed E.coli. CD4+ T cell numbers derived from flow cytometry and viable cell counts were reported relative to mock infection. Viable cells were identified by viability dye exclusion (AqVi), and intracellular HIV-1 Gag p24 protein was used to identify infected cells. Annexin V and AqVi were used to identify apoptotic versus necrotic cells. Caspase-1 and Caspase-3 activities were blocked using specific inhibitors YVAD and DEVD, respectively.


RESULTS
CD4+ T cell depletion following HIV-1 infection was reproducibly observed by 6 days post infection (dpi). Depletion at 6 dpi strongly correlated with infection frequency at 4 dpi, was significantly blocked by Efavirenz treatment, and was primarily driven by p24-negative cells that were predominantly necrotic. HIV-1 infection significantly induced CD4+ T-cell intrinsic Caspase-1 activity, whereas Caspase-1 inhibition, but not Caspase-3 inhibition, significantly blocked CD4+ T cell depletion. Exposure to E.coli enhanced HIV-1 infection and CD4+ T depletion, and significantly increased the number of apoptotic p24+ cells. Notably, CD4+ T cell depletion in the presence of E.coli was partially blocked by Caspase-3, but not by Caspase-1 inhibition.


CONCLUSIONS
In the LPAC model, HIV-1 induced Caspase-1 mediated pyroptosis in bystander CD4+ T cells, but microbial exposure shifted the PCD mechanism toward apoptosis of productively infected T cells. These results suggest that mucosal CD4+ T cell death pathways may be altered in HIV-infected individuals after gut barrier function is compromised, with potential consequences for mucosal inflammation, viral dissemination and systemic immune activation.
Amanda K
AK
Steele
Eric J
EJ
Lee
Jennifer A
JA
Manuzak
Stephanie M
SM
Dillon
John David
JD
Beckham
Martin D
MD
McCarter
Department of Medicine, University of Colorado Denver, Mail Stop B-168, 12700 E, 19th Avenue, Aurora, CO 80045, USA. mario.santiago@ucdenver.edu.
Mario L
ML
Santiago
Cara C
CC
Wilson
D017209:Apoptosis; D018791:CD4 Lymphocyte Count; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D020170:Caspase 1 / Q000378:metabolism; D002470:Cell Survival; D004926:Escherichia coli / Q000276:immunology*; D005434:Flow Cytometry; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D018928:Immunity, Mucosal; D066298:In Vitro Techniques; D009092:Mucous Membrane / Q000276:immunology
D020170:Caspase 1