Limits...
Type I interferon responses are impaired in latently HIV infected cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: The latent HIV-1 reservoir represents the primary barrier to the eradication of HIV-1 infection. The design of novel reservoir-clearance strategies, however, is impeded in part by the inability to distinguish latently HIV-infected cells from uninfected cells. Significant impairment of the type I interferon (IFN-I) response is observed during productive HIV-1 infection. Although this remains poorly described in the context of latent HIV-1 infection, presence of potential defects may serve as a novel therapeutic target. Therefore, IFN-I pathways were characterized using two latently HIV-1-infected cell lines, U1 and OM10.1, in comparison to their respective uninfected parental U937 and HL60 cell lines.

Findings: Constitutive expression and induction of important mediators of IFN-I signaling including IFNα/β cytokines, IFNAR1, MHC-I, ISG15, and PKR were evaluated following exogenous IFNα or poly(I:C) treatment. Differences in basal expression of IFNAR1, MHC-I, and PKR were observed between the latently HIV-1 infected and uninfected cell lines. In parallel, significant impairments in the induction of MHC-I, ISG15 and PKR, as well as secretion of IFNα/β cytokines were observed in response to appropriate exogenous stimulation within the two latently HIV-infected U1 and OM10.1 cells, relative to their HIV-uninfected parental cells.

Conclusions: In comparison to the HIV-uninfected U937 and HL60 cell lines, widespread defects in IFN-I responsiveness were observed within the latently HIV-infected U1 and OM10.1 cells. These impairments represent novel therapeutic targets, which may be amenable to strategies currently employed in cancer therapy.

Electronic supplementary material: The online version of this article (doi:10.1186/s12977-016-0302-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Responsiveness to poly(I:C) was defective in U1 cells, when compared to U937 cells. U937 and U1 cells were transfected (Lipofectamine®2000) with media alone or 0.1, 1, or 10ug/mL of poly(I:C) for 48 h. a Minimal constitutive secretion of of IFNα/β (n = 5) was observed in U937 and U1 cells, as quantified using using the HEK-Blue™ IFNα/β biologic assay (dashed line denotes lower limit of detection of assay = 0.15 U/mL). b Secretion of IFNα/β by U937 cells, but not latently HIV-infected U1 cells was detected following poly(I:C) transfection (n = 5). c Poly(I:C) induced ISG15 expression (n = 5), as measured by flow cytometry, was observed only in the HIV-uninfected U937 cells. d Similarly, poly(I:C) induced expression of PKR (n = 5) was impaired in U1 cells, but not in U937 cells. Representative histogram and cumulative summary of poly(I:C)-induced ISG expression is shown. †p = 0.03; ‡p = 0.0004 as measured by one-way ANOVA and p < 0.05 by pairwise Dunnett’s Test compared to unstimulated cells. *p < 0.01; **p < 0.001, ***p < 0.05 by Two-way ANOVA with Bonferroni post-test for multiple comparisons
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5017046&req=5

Fig5: Responsiveness to poly(I:C) was defective in U1 cells, when compared to U937 cells. U937 and U1 cells were transfected (Lipofectamine®2000) with media alone or 0.1, 1, or 10ug/mL of poly(I:C) for 48 h. a Minimal constitutive secretion of of IFNα/β (n = 5) was observed in U937 and U1 cells, as quantified using using the HEK-Blue™ IFNα/β biologic assay (dashed line denotes lower limit of detection of assay = 0.15 U/mL). b Secretion of IFNα/β by U937 cells, but not latently HIV-infected U1 cells was detected following poly(I:C) transfection (n = 5). c Poly(I:C) induced ISG15 expression (n = 5), as measured by flow cytometry, was observed only in the HIV-uninfected U937 cells. d Similarly, poly(I:C) induced expression of PKR (n = 5) was impaired in U1 cells, but not in U937 cells. Representative histogram and cumulative summary of poly(I:C)-induced ISG expression is shown. †p = 0.03; ‡p = 0.0004 as measured by one-way ANOVA and p < 0.05 by pairwise Dunnett’s Test compared to unstimulated cells. *p < 0.01; **p < 0.001, ***p < 0.05 by Two-way ANOVA with Bonferroni post-test for multiple comparisons

Mentions: Using the Lipofectamine®2000 reagent (ThermoFisher Scientific, Waltham, MA, USA), cells were transfected with various concentrations of poly (I:C) (InvivoGen, San Diego, CA, USA) for 48 h. Similar levels of transfection efficiency were confirmed in all cell lines using Rhodamine labelled poly(I:C) (Invivogen). Following stimulation, IFNα/β secretion was assessed using the HEK-Blue™ IFNα/β (InvivoGen) biologic assay as described [26], and intracellular ISG15 and PKR expression were assessed as before. Low levels of endogenous IFNα/β were detectable in culture supernatant of unstimulated U937 and U1 cells (Fig. 5a). However, upregulation of IFNα/β production in response to poly(I:C) transfection was only observed in U937 cells (Fig. 5b). While poly(I:C) caused a dose-dependent increase in intracellular ISG15 expression in U937 cells, no effect was observed in the latently HIV-infected U1 cells (Fig. 5c). Similarly, poly(I:C)-induced PKR expression was impaired in the U1 cells, when compared to U937 cells (Fig. 5d). Similar defects in IFN-I pathway induction following poly(I:C) stimulation were also quantified within HL60 and OM10.1 cells (Additional file 3).Fig. 5


Type I interferon responses are impaired in latently HIV infected cells
Responsiveness to poly(I:C) was defective in U1 cells, when compared to U937 cells. U937 and U1 cells were transfected (Lipofectamine®2000) with media alone or 0.1, 1, or 10ug/mL of poly(I:C) for 48 h. a Minimal constitutive secretion of of IFNα/β (n = 5) was observed in U937 and U1 cells, as quantified using using the HEK-Blue™ IFNα/β biologic assay (dashed line denotes lower limit of detection of assay = 0.15 U/mL). b Secretion of IFNα/β by U937 cells, but not latently HIV-infected U1 cells was detected following poly(I:C) transfection (n = 5). c Poly(I:C) induced ISG15 expression (n = 5), as measured by flow cytometry, was observed only in the HIV-uninfected U937 cells. d Similarly, poly(I:C) induced expression of PKR (n = 5) was impaired in U1 cells, but not in U937 cells. Representative histogram and cumulative summary of poly(I:C)-induced ISG expression is shown. †p = 0.03; ‡p = 0.0004 as measured by one-way ANOVA and p < 0.05 by pairwise Dunnett’s Test compared to unstimulated cells. *p < 0.01; **p < 0.001, ***p < 0.05 by Two-way ANOVA with Bonferroni post-test for multiple comparisons
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5017046&req=5

Fig5: Responsiveness to poly(I:C) was defective in U1 cells, when compared to U937 cells. U937 and U1 cells were transfected (Lipofectamine®2000) with media alone or 0.1, 1, or 10ug/mL of poly(I:C) for 48 h. a Minimal constitutive secretion of of IFNα/β (n = 5) was observed in U937 and U1 cells, as quantified using using the HEK-Blue™ IFNα/β biologic assay (dashed line denotes lower limit of detection of assay = 0.15 U/mL). b Secretion of IFNα/β by U937 cells, but not latently HIV-infected U1 cells was detected following poly(I:C) transfection (n = 5). c Poly(I:C) induced ISG15 expression (n = 5), as measured by flow cytometry, was observed only in the HIV-uninfected U937 cells. d Similarly, poly(I:C) induced expression of PKR (n = 5) was impaired in U1 cells, but not in U937 cells. Representative histogram and cumulative summary of poly(I:C)-induced ISG expression is shown. †p = 0.03; ‡p = 0.0004 as measured by one-way ANOVA and p < 0.05 by pairwise Dunnett’s Test compared to unstimulated cells. *p < 0.01; **p < 0.001, ***p < 0.05 by Two-way ANOVA with Bonferroni post-test for multiple comparisons
Mentions: Using the Lipofectamine®2000 reagent (ThermoFisher Scientific, Waltham, MA, USA), cells were transfected with various concentrations of poly (I:C) (InvivoGen, San Diego, CA, USA) for 48 h. Similar levels of transfection efficiency were confirmed in all cell lines using Rhodamine labelled poly(I:C) (Invivogen). Following stimulation, IFNα/β secretion was assessed using the HEK-Blue™ IFNα/β (InvivoGen) biologic assay as described [26], and intracellular ISG15 and PKR expression were assessed as before. Low levels of endogenous IFNα/β were detectable in culture supernatant of unstimulated U937 and U1 cells (Fig. 5a). However, upregulation of IFNα/β production in response to poly(I:C) transfection was only observed in U937 cells (Fig. 5b). While poly(I:C) caused a dose-dependent increase in intracellular ISG15 expression in U937 cells, no effect was observed in the latently HIV-infected U1 cells (Fig. 5c). Similarly, poly(I:C)-induced PKR expression was impaired in the U1 cells, when compared to U937 cells (Fig. 5d). Similar defects in IFN-I pathway induction following poly(I:C) stimulation were also quantified within HL60 and OM10.1 cells (Additional file 3).Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: The latent HIV-1 reservoir represents the primary barrier to the eradication of HIV-1 infection. The design of novel reservoir-clearance strategies, however, is impeded in part by the inability to distinguish latently HIV-infected cells from uninfected cells. Significant impairment of the type I interferon (IFN-I) response is observed during productive HIV-1 infection. Although this remains poorly described in the context of latent HIV-1 infection, presence of potential defects may serve as a novel therapeutic target. Therefore, IFN-I pathways were characterized using two latently HIV-1-infected cell lines, U1 and OM10.1, in comparison to their respective uninfected parental U937 and HL60 cell lines.

Findings: Constitutive expression and induction of important mediators of IFN-I signaling including IFN&alpha;/&beta; cytokines, IFNAR1, MHC-I, ISG15, and PKR were evaluated following exogenous IFN&alpha; or poly(I:C) treatment. Differences in basal expression of IFNAR1, MHC-I, and PKR were observed between the latently HIV-1 infected and uninfected cell lines. In parallel, significant impairments in the induction of MHC-I, ISG15 and PKR, as well as secretion of IFN&alpha;/&beta; cytokines were observed in response to appropriate exogenous stimulation within the two latently HIV-infected U1 and OM10.1 cells, relative to their HIV-uninfected parental cells.

Conclusions: In comparison to the HIV-uninfected U937 and HL60 cell lines, widespread defects in IFN-I responsiveness were observed within the latently HIV-infected U1 and OM10.1 cells. These impairments represent novel therapeutic targets, which may be amenable to strategies currently employed in cancer therapy.

Electronic supplementary material: The online version of this article (doi:10.1186/s12977-016-0302-9) contains supplementary material, which is available to authorized users.

No MeSH data available.