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Intact type I Interferon production and IRF7 function in sooty mangabeys.

Bosinger SE, Johnson ZP, Folkner KA, Patel N, Hashempour T, Jochems SP, Del Rio Estrada PM, Paiardini M, Lin R, Vanderford TH, Hiscott J, Silvestri G - PLoS Pathog. (2013)

Bottom Line: We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles.Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans.These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

View Article: PubMed Central - PubMed

Affiliation: Divison of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, United States of America. sbosing@emory.edu

ABSTRACT
In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-α) responses to TLR7/9 ligands in vitro, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that "muted" IFN-α responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-α responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-α promoter. Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans. These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

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SIVmac239 and TLR7 agonists induce IFN-β production by SMs.PBMCs were stimulated with CL097 (A) or SIVmac239 (B), RNA was harvested at indicated time points and quantitated by qPCR. Fold-changes were normalized by GAPDH, and are expressed as relative to unstimulated replicates. Values are averages of three animals; values for individual animals were averaged from triplicate wells. (C) PBMCs from RMs and SMs were incubated for CL097 and supernatants were harvested at the times indicated and IFN-β was assessed by ELISA.
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ppat-1003597-g003: SIVmac239 and TLR7 agonists induce IFN-β production by SMs.PBMCs were stimulated with CL097 (A) or SIVmac239 (B), RNA was harvested at indicated time points and quantitated by qPCR. Fold-changes were normalized by GAPDH, and are expressed as relative to unstimulated replicates. Values are averages of three animals; values for individual animals were averaged from triplicate wells. (C) PBMCs from RMs and SMs were incubated for CL097 and supernatants were harvested at the times indicated and IFN-β was assessed by ELISA.

Mentions: During the acute phase of SIV infection, SMs produce high levels of type I IFNs and show massive upregulation of ISGs, which can be induced by any type I IFN (α,β,ω,λ) [10], [21]. To test the possibility that SMs can produce other non-α type I IFNs in response to TLR7 stimulation, we measured the level of IFN-β gene expression at the RNA level in PBMCs derived from both SMs and RMs after in vitro stimulation with the TLR7 ligand CL097 as well as SIVmac239. It should be noted that, in these experiments, we could not link IFN-β production to pDCs due to lack of an IFN-β-specific monoclonal antibody that can be used for flow cytometric analysis. As shown in Figure 3, both the TLR7 ligand CL097 and SIVmac239 induced a marked upregulation of IFN-β gene expression. Concurrently, we also observed robust upregulation of IFN-β protein in the supernatants of PBMCs stimulated with CL097. While multiple cell types may be contributing to IFN-β production, these data are consistent with previously published results showing high levels of IFN-β production by pDCs in the lymph nodes of SIV-infected SMs during the acute phase of infection [21], and indicate that, in SMs, the ability to produce additional type I IFNs after TLR7 stimulation is largely intact.


Intact type I Interferon production and IRF7 function in sooty mangabeys.

Bosinger SE, Johnson ZP, Folkner KA, Patel N, Hashempour T, Jochems SP, Del Rio Estrada PM, Paiardini M, Lin R, Vanderford TH, Hiscott J, Silvestri G - PLoS Pathog. (2013)

SIVmac239 and TLR7 agonists induce IFN-β production by SMs.PBMCs were stimulated with CL097 (A) or SIVmac239 (B), RNA was harvested at indicated time points and quantitated by qPCR. Fold-changes were normalized by GAPDH, and are expressed as relative to unstimulated replicates. Values are averages of three animals; values for individual animals were averaged from triplicate wells. (C) PBMCs from RMs and SMs were incubated for CL097 and supernatants were harvested at the times indicated and IFN-β was assessed by ELISA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757038&req=5

ppat-1003597-g003: SIVmac239 and TLR7 agonists induce IFN-β production by SMs.PBMCs were stimulated with CL097 (A) or SIVmac239 (B), RNA was harvested at indicated time points and quantitated by qPCR. Fold-changes were normalized by GAPDH, and are expressed as relative to unstimulated replicates. Values are averages of three animals; values for individual animals were averaged from triplicate wells. (C) PBMCs from RMs and SMs were incubated for CL097 and supernatants were harvested at the times indicated and IFN-β was assessed by ELISA.
Mentions: During the acute phase of SIV infection, SMs produce high levels of type I IFNs and show massive upregulation of ISGs, which can be induced by any type I IFN (α,β,ω,λ) [10], [21]. To test the possibility that SMs can produce other non-α type I IFNs in response to TLR7 stimulation, we measured the level of IFN-β gene expression at the RNA level in PBMCs derived from both SMs and RMs after in vitro stimulation with the TLR7 ligand CL097 as well as SIVmac239. It should be noted that, in these experiments, we could not link IFN-β production to pDCs due to lack of an IFN-β-specific monoclonal antibody that can be used for flow cytometric analysis. As shown in Figure 3, both the TLR7 ligand CL097 and SIVmac239 induced a marked upregulation of IFN-β gene expression. Concurrently, we also observed robust upregulation of IFN-β protein in the supernatants of PBMCs stimulated with CL097. While multiple cell types may be contributing to IFN-β production, these data are consistent with previously published results showing high levels of IFN-β production by pDCs in the lymph nodes of SIV-infected SMs during the acute phase of infection [21], and indicate that, in SMs, the ability to produce additional type I IFNs after TLR7 stimulation is largely intact.

Bottom Line: We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles.Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans.These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

View Article: PubMed Central - PubMed

Affiliation: Divison of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center, Atlanta, Georgia, United States of America. sbosing@emory.edu

ABSTRACT
In contrast to pathogenic HIV/SIV infections of humans and rhesus macaques (RMs), natural SIV infection of sooty mangabeys (SMs) is typically non-pathogenic despite high viremia. Several studies suggested that low immune activation and relative resistance of CD4+ central memory T-cells from virus infection are mechanisms that protect SMs from AIDS. In 2008 it was reported that plasmacytoid dendritic cells (pDCs) of SMs exhibit attenuated interferon-alpha (IFN-α) responses to TLR7/9 ligands in vitro, and that species-specific amino acid substitutions in SM Interferon Regulatory Factor-7 (IRF7) are responsible for this observation. Based on these findings, these authors proposed that "muted" IFN-α responses are responsible for the benign nature of SIV infection in SMs. However, other studies indicated that acutely SIV-infected SMs show robust IFN-α responses and marked upregulation of Interferon Stimulated Genes (ISGs). To investigate this apparent disparity, we first examined the role of the reported IRF7 amino acid substitutions in SMs. To this end, we sequenced all IRF7 exons in 16 breeders, and exons displaying variability (exons 2,3,5,6,7,8) in the remainder of the colony (177 animals). We found that the reported Ser-Gly substitution at position 191 was a sequencing error, and that several of the remaining substitutions represent only minor alleles. In addition, functional assays using recombinant SM IRF7 showed no defect in its ability to translocate in the nucleus and drive transcription from an IFN-α promoter. Furthermore, in vitro stimulation of SM peripheral blood mononuclear cells with either the TLR7 agonist CL097 or SIV(mac239) induced an 500-800-fold induction of IFN-α and IFN-β mRNA, and levels of IFN-α production by pDCs similar to those of RMs or humans. These data establish that IFN-α and IRF7 signaling in SMs are largely intact, with differences with RMs that are minor and unlikely to play any role in the AIDS resistance of SIV-infected SMs.

Show MeSH
Related in: MedlinePlus