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. 2023 Nov 1;15(11):2206.
doi: 10.3390/v15112206.

MiR-155 Negatively Regulates Anti-Viral Innate Responses among HIV-Infected Progressors

Puja Pawar  1 , Jyotsna Gokavi  1 , Shilpa Wakhare  1 , Rajani Bagul  2 , Ujjwala Ghule  2 , Ishrat Khan  3 , Varada Ganu  1 , Anupam Mukherjee  3 , Ashwini Shete  1 , Amrita Rao  2 , Vandana Saxena  1
Affiliations

MiR-155 Negatively Regulates Anti-Viral Innate Responses among HIV-Infected Progressors

Puja Pawar et al. Viruses. .

Abstract

HIV infection impairs host immunity, leading to progressive disease. An anti-retroviral treatment efficiently controls viremia but cannot completely restore the immune dysfunction in HIV-infected individuals. Both host and viral factors determine the rate of disease progression. Among the host factors, innate immunity plays a critical role; however, the mechanism(s) associated with dysfunctional innate responses are poorly understood among HIV disease progressors, which was investigated here. The gene expression profiles of TLRs and innate cytokines in HIV-infected (LTNPs and progressors) and HIV-uninfected individuals were examined. Since the progressors showed a dysregulated TLR-mediated innate response, we investigated the role of TLR agonists in restoring the innate functions of the progressors. The stimulation of PBMCs with TLR3 agonist-poly:(I:C), TLR7 agonist-GS-9620 and TLR9 agonist-ODN 2216 resulted in an increased expression of IFN-α, IFN-β and IL-6. Interestingly, the expression of IFITM3, BST-2, IFITM-3, IFI-16 was also increased upon stimulation with TLR3 and TLR7 agonists, respectively. To further understand the molecular mechanism involved, the role of miR-155 was explored. Increased miR-155 expression was noted among the progressors. MiR-155 inhibition upregulated the expression of TLR3, NF-κB, IRF-3, TNF-α and the APOBEC-3G, IFITM-3, IFI-16 and BST-2 genes in the PBMCs of the progressors. To conclude, miR-155 negatively regulates TLR-mediated cytokines as wel l as the expression of host restriction factors, which play an important role in mounting anti-HIV responses; hence, targeting miR-155 might be helpful in devising strategic approaches towards alleviating HIV disease progression.

Keywords: HIV; LTNPs; TLRs; disease progression; host restriction factors; innate; miR-155.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Altered innate immune response in HIV-infected individuals. PBMCs from HIV-infected (including LTNPs shown as squares and progressors shown as filled circles) and HIV-uninfected individuals (shown as filled triangles) were used to assess the gene expression profile of various TLRs such as TLR2 (a), TLR3 (b), TLR4 (c), TLR7 (d), TLR8 (e), TLR9 (f) and innate immune cytokines such as IFN-α (g), IFN-β (h), TNF α (i), IL-6 (j). The relative gene expression of these genes was normalized to β-actin. Statistical analysis was performed by using the Mann–Whitney test. * indicates p value < 0.05 and ** indicates p value < 0.01.
Figure 2
Figure 2
Impaired innate immune response during HIV disease progression. PBMCs isolated from LTNP and progressor study groups were used to analyse the gene expression of TLRs 3 (a), 7 (b) and 9 (c) and innate immune cytokines, i.e., IFN-α (d), IFN-β (e), TNF α (f), IL-6 (g). Fold change expression was calculated as 2−ΔΔCt over the HIV-uninfected individuals, denoted as healthy control (HC), which is indicated as a dotted line. Statistical analysis was performed by using Mann–Whitney test. ** indicates p value < 0.01 and *** indicates p value < 0.001.
Figure 3
Figure 3
Correlation between innate immune cytokines and HIV-1 viral load/CD4 count in HIV-infected individuals. Correlation between IFN-α, IFN-β, TNF α and IL-6 mRNA levels with CD4 count (a), between CD4 count with viral load (b), between IFN-α, IFN-β mRNA levels with viral load (c) and between TNF α, IL-6 mRNA levels with viral load (d). The relative gene expression of these genes was normalized to β-actin. Correlation analysis was performed by using Spearman’s test.
Figure 4
Figure 4
Differential expression of host restriction factors in LTNPs and progressors. PBMCs isolated from LTNPs and progressors study groups were used to analyse the gene expression of host restriction factors such as APOBEC-3G (a), IFTIM-1 (b), IFTIM-3 (c), IFI-16 (d) and BST-2 (e). Fold change expression was calculated as 2−ΔΔCt over the HIV-uninfected individuals, denoted as healthy control (HC), which is indicated as a dotted line. Statistical analysis was performed by using Mann–Whitney test. * indicates p value < 0.05 and ** indicates p value < 0.01.
Figure 5
Figure 5
TLR stimulation in PBMCs restores the expression of innate cytokines and the anti-viral host restriction factors in HIV-infected progressors. PBMCs from progressors stimulated with Poly(I:C) (TLR3 agonist (1 uM)) were used to assess the gene expression of innate immune cytokines such as IFN-α (a), IFN-β (b), IL-6 (c) and TNF-α (d). Similarly, the gene expression analysis was performed by using PBMCs stimulated with GS-9620 (TLR7 agonist (1 uM)) (IFN-α (e), IFN-β (f), IL-6 (g), TNF-α (h)) and ODN 2216 (TLR9 agonist (3 uM))) IFN-α (i), IFN-β (j), IL-6 (k), TNF-α (l)), respectively. In addition, mRNA expression levels of host restriction factors such IFITM-1 (m), IFITM-3 (n), IFI-16 (o), BST-2 (p) and APOBEC-3G (q) in PBMCs stimulated with poly(I:C) (TLR3 agonists (1 uM)) and IFITM-1 (r), IFITM-3 (s), IFI-16 (t), BST-2 (u) and APOBEC-3G (v) in PBMCs stimulated with GS-9620 (TLR7 agonist (1 uM)) were quantified by RT-PCR. Fold change expression was calculated as 2−ΔΔCt over non-stimulated control (NS). Statistical analysis was performed by using the Wilcoxon test. * indicates p value < 0.05, ** indicates p value < 0.01 and *** indicates p value < 0.001.
Figure 6
Figure 6
Inhibition of miR-155 increases the expression of transcription factors, innate immune cytokines and host restriction factors. MiR-155 (a) mRNA expression analysis was performed in HIV-infected study groups, i.e., LTNPs and progressors. Further, PBMCs of progressors transfected with miR-155 inhibitor or mock scrambled miR inhibitor control (denoted as NC) were used to examine the mRNA expression of genes encoding transcription factors such as NF-κB (b) and IRF3 (c), innate immune cytokines such as IFN-α (d), IFN-β (e), TNF-α (f) and IL-6 (g) and host restriction factors such as IFTIM-3 (h), IFI-16 (i), BST-2 (j), APOBEC-3G (k) and IFITM-1 (l). Fold change expression was calculated as 2−ΔΔCt over control (NC) condition. Intracellular cytokine staining was performed to identify the frequency of IFN-α+ (m) and TNF-α+ (n) cells post transfection with miR-155 inhibition. Statistical analysis was performed using Wilcoxon test. * indicates p value < 0.05, ** indicates p value < 0.01 and *** indicates p value < 0.001.
Figure 7
Figure 7
A graphical representation showing that miR-155 negatively regulates the anti-viral immune responses in progressors at multiple stages during the innate immune pathway. Increased expression of miR-155 in the PBMCs of progressors targets the expression of TLRs, in particular TLR3, transcription factors such as NF-κB and IRF-3, resulting in decreased expression of innate cytokines such as IFN-α, IFN-β. IL-6 and TNF-α, as well as host restriction factors viz. APOBEC-3G, IFITM-3, IFI-16 and BST-2, which could result in rapid HIV disease progression in such individuals. Pink arrow denoted increased or decreased expression levels while red arrow indicate inhibition. This image is generated by using BioRender.
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