(c) Gating strategy for detection of HIV-HSA-infected macrophages

(c) Gating strategy for detection of HIV-HSA-infected macrophages. kinase-1 (RIPK1) degradation which in concert with?IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo. LCL 1?M) but not in U937 cells (Fig.?1a). Like the effect of SM on undifferentiated U1 cells, LCL161 induced significant cell death in PMA-differentiated primary macrophage-like U1 cells (LCL 1?M) but not in PMA-differentiated U937 cells (Fig.?1b). Apoptosis of HIV-infected U1 cells was further confirmed by showing cleavage of caspase-3 in U1 but not in U937 cells by immunoblotting (Fig.?1c). Open in a separate window Physique 1 SM induces cell death of HIV-infected myeloid cells. (a) U937 (technical replicate (tn)?=?9) and chronically infected counterpart U1 cells (5.0??105) (tn?=?10) were treated with LCL161 at 1, 2, and 4?M for 48?h. (b) ML 7 hydrochloride PMA differentiated U937 (tn?=?7) and U1 cells (tn?=?11) were treated with SM LCL161 at 1, 2, and 4?M for 48?h. Cell death was assessed by intracellular PI staining. The LCL 1?M) MDMs but not in mock-infected MDMs (Fig.?2b). Representative histograms of the intracellular PI staining are shown (Fig.?2c). The p24 values in MDMs infected with HIV-1 CS204 for 7 d are shown in Fig.?2d. SM-induced cell death experiments were conducted at 48?h post-treatment with LCL161 as higher % of cell death was observed at 48?h compared to 24?h post treatment (Supp Fig. 1a). To determine whether SM-induced cell death in in vitro HIV-infected MDM was due to apoptosis, caspase activation ML 7 hydrochloride was quantified based on the fluorescent signal of cleaved caspase substrates. Treatment of HIV-1 CS204-infected MDM with LCL161 showed activation of caspases 3, 8, and 9 in contrast to the mock-infected MDM (Fig.?2e). A representative histogram for the induction of caspase 3, 8 and 9 is usually shown (Supp Fig. 2). Open in a separate window Physique 2 SM induces cell death of HIV-infected MDMs (a). MDMs were treated with increasing concentration of ML 7 hydrochloride LCL161 for 48?h and cytosolic fractions were collected. 30?g of total proteins were subjected to western immunoblotting to probe for cIAP1 and cIAP2. (b) Human MDMs were in vitro infected with HIV-1 CS204 (100?ng p24/well) for 7?days (n?=?4). The cells were then treated with LCL161 for 48? h and cell death was assessed by PI staining and flow cytometry. The representative histograms are ML 7 hydrochloride shown in (c). (d) After 7?days of contamination, supernatants were analyzed for p24 by ELISA (n?=?6). (e) Mock and HIV-infected MDMs treated with SM for 48?h and caspase-3 (n?=?3), -8 (n?=?4), and -9 (n?=?6) positive cells were analyzed by flow cytometry. MDMs generated from ML 7 hydrochloride na?ve (f) and ART-treated (g) HIV-individuals were treated with LCL161 for 48?h. Cell death was assessed by PI staining and flow cytometry. The LCL 1?M) and ART-treated patients (LCL 1?M) showed a significantly increased susceptibility to LCL161-induced cell death in a dose-dependent manner (Fig.?2f, g). Moreover, ART treatment did not affect the expression of cIAP1 and cIAP2 in uninfected or HIV-infected macrophages (Supp Fig. 1b) suggesting that ART treatment did not affect the IAP pathway in uninfected or HIV-infected macrophages. SMs specifically kill HIV-infected MDMs The frequency of HIV-infected macrophages in in vitro HIV-infected macrophages may not exceed 10%. However, the HIV-infected myeloid cells are rarely detected in the blood of HIV-infected individuals41; hence the frequency of HIV-infected macrophages in Mouse monoclonal to CD62L.4AE56 reacts with L-selectin, an 80 kDaleukocyte-endothelial cell adhesion molecule 1 (LECAM-1).CD62L is expressed on most peripheral blood B cells, T cells,some NK cells, monocytes and granulocytes. CD62L mediates lymphocyte homing to high endothelial venules of peripheral lymphoid tissue and leukocyte rollingon activated endothelium at inflammatory sites ex vivo derived macrophages from the HIV-infected patients may be extremely low. But the above results show killing of 20C30% macrophages (Fig.?2b, f, g) following treatment with SMs. Therefore, it was important to ascertain whether SMs were killing HIV-infected cells alone, uninfected bystander cells alone or both. To examine this, we employed a R5 laboratory strain of HIV-1, HIV-Bal-HSA (HIV-HSA), expressing mouse HSA (CD24). Expression of HSA by HIV-infected cells can be used to quantify infected cells by flow cytometry using FITC-conjugated anti-mouse HSA antibody42. HIV-HSA contamination rates in MDMs ranged from 5 to 30% over 15 d post contamination depending upon the donor variability (Fig.?3a). Flow cytometric analysis of HIV-HSA-infected macrophages revealed two distinct HSA-positive (HSA?+) and HSA-negative (HSA??) populations of macrophages (Fig.?3b)..