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BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses.

Dutta D, Dutta S, Veettil MV, Roy A, Ansari MA, Iqbal J, Chikoti L, Kumar B, Johnson KE, Chandran B - PLoS Pathog. (2015)

Bottom Line: The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β).The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production.These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

View Article: PubMed Central - PubMed

Affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.

ABSTRACT
The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

No MeSH data available.


Related in: MedlinePlus

PLA and knockdown studies demonstrating specificity of BRCA1 interactions with IFI16-inflammasome components and relocalization to the cytoplasm during de novo KSHV infection.(A) PLA detecting IFI16 in uninfected and KSHV infected HMVEC-d cells. Red dots are indicative of PLA reactions representing the subcellular distribution of IFI16. Red arrows: nuclear IFI16. Yellow arrows: cytoplasmic IFI16. (B) Specificity control for PLA using only secondary antibodies. (C-G) PLA (red dots) detecting various complexes in uninfected (left panels) and de novo KSHV infected HMVEC-d cells at 24 h p.i. (right panels). Red arrows: nuclear PLA. Yellow arrows: cytoplasmic PLA. (C) IFI16 and BRCA1 complexes. (D) IFI16 and ASC complexes. (E) BRCA1 and ASC complexes. (F) BRCA1 and caspase-1 complexes. (G) IFI16 and Caspase-1 complexes. Boxed areas are enlarged. Quantitative analysis of the average number of PLA spots per cell and that of nucleus vs cytoplasm of infected cells is presented in the rightmost columns. ***: p<0.001, **: p<0.01, and ns: not-significant. (H) Effect of IFI16 knockdown on BRCA1-ASC association during KSHV infection. HMVEC-d cells treated with control Si-RNA (Si C) or IFI16-Si-RNA (Si IFI16) were infected with KSHV and lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-ASC antibodies. Blots were stripped and probed with anti-BRCA1 antibodies to detect the presence of BRCA1. WCLs were used as input controls for WBs to show IFI16 knockdown, presence of ASC and cleavage of procaspase-1. (I) BRCA1 does not interact with AIM2 and NLRP3. Uninfected or KSHV infected HMVEC-d cell lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-NLRP3 or AIM2 antibodies. Equal inputs for IPs were assessed by NLRP3, AIM2 and BRCA1 WBs. β-actin was used as an equal loading control. HC and LC: IgG heavy and light chains, respectively.
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ppat.1005030.g004: PLA and knockdown studies demonstrating specificity of BRCA1 interactions with IFI16-inflammasome components and relocalization to the cytoplasm during de novo KSHV infection.(A) PLA detecting IFI16 in uninfected and KSHV infected HMVEC-d cells. Red dots are indicative of PLA reactions representing the subcellular distribution of IFI16. Red arrows: nuclear IFI16. Yellow arrows: cytoplasmic IFI16. (B) Specificity control for PLA using only secondary antibodies. (C-G) PLA (red dots) detecting various complexes in uninfected (left panels) and de novo KSHV infected HMVEC-d cells at 24 h p.i. (right panels). Red arrows: nuclear PLA. Yellow arrows: cytoplasmic PLA. (C) IFI16 and BRCA1 complexes. (D) IFI16 and ASC complexes. (E) BRCA1 and ASC complexes. (F) BRCA1 and caspase-1 complexes. (G) IFI16 and Caspase-1 complexes. Boxed areas are enlarged. Quantitative analysis of the average number of PLA spots per cell and that of nucleus vs cytoplasm of infected cells is presented in the rightmost columns. ***: p<0.001, **: p<0.01, and ns: not-significant. (H) Effect of IFI16 knockdown on BRCA1-ASC association during KSHV infection. HMVEC-d cells treated with control Si-RNA (Si C) or IFI16-Si-RNA (Si IFI16) were infected with KSHV and lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-ASC antibodies. Blots were stripped and probed with anti-BRCA1 antibodies to detect the presence of BRCA1. WCLs were used as input controls for WBs to show IFI16 knockdown, presence of ASC and cleavage of procaspase-1. (I) BRCA1 does not interact with AIM2 and NLRP3. Uninfected or KSHV infected HMVEC-d cell lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-NLRP3 or AIM2 antibodies. Equal inputs for IPs were assessed by NLRP3, AIM2 and BRCA1 WBs. β-actin was used as an equal loading control. HC and LC: IgG heavy and light chains, respectively.

Mentions: To further confirm the association of IFI16, ASC and procaspase-1 with BRCA1, we performed PLA in uninfected HMVEC-d cells and cells infected with KSHV for 24 h using anti-IFI16, anti-BRCA1, anti-ASC and anti-procaspase-1 antibodies. Specificity of the assay was confirmed by using secondary antibodies linked to probes alone (Fig 4B) and also by using only one primary antibody plus both secondary antibodies linked to probes (S2 Fig, A to E). The lack of detection of any signal following PLA in all cases confirmed the specificity of all the antibodies tested (Fig 4B and S2 Fig, A to E). When we used rabbit and mouse anti-IFI16 antibodies against different IFI16 epitopes in PLA for detecting cellular localization of IFI16 during infection, we detected IFI16 in the nucleus of uninfected cells (Fig 4A, UI, red arrows). In contrast, as we have demonstrated previously [3], at 24 h p.i., IFI16 was detected in both the nucleus and the cytoplasm of infected cells (Fig 4A, KSHV 24 h, red and yellow arrows).


BRCA1 Regulates IFI16 Mediated Nuclear Innate Sensing of Herpes Viral DNA and Subsequent Induction of the Innate Inflammasome and Interferon-β Responses.

Dutta D, Dutta S, Veettil MV, Roy A, Ansari MA, Iqbal J, Chikoti L, Kumar B, Johnson KE, Chandran B - PLoS Pathog. (2015)

PLA and knockdown studies demonstrating specificity of BRCA1 interactions with IFI16-inflammasome components and relocalization to the cytoplasm during de novo KSHV infection.(A) PLA detecting IFI16 in uninfected and KSHV infected HMVEC-d cells. Red dots are indicative of PLA reactions representing the subcellular distribution of IFI16. Red arrows: nuclear IFI16. Yellow arrows: cytoplasmic IFI16. (B) Specificity control for PLA using only secondary antibodies. (C-G) PLA (red dots) detecting various complexes in uninfected (left panels) and de novo KSHV infected HMVEC-d cells at 24 h p.i. (right panels). Red arrows: nuclear PLA. Yellow arrows: cytoplasmic PLA. (C) IFI16 and BRCA1 complexes. (D) IFI16 and ASC complexes. (E) BRCA1 and ASC complexes. (F) BRCA1 and caspase-1 complexes. (G) IFI16 and Caspase-1 complexes. Boxed areas are enlarged. Quantitative analysis of the average number of PLA spots per cell and that of nucleus vs cytoplasm of infected cells is presented in the rightmost columns. ***: p<0.001, **: p<0.01, and ns: not-significant. (H) Effect of IFI16 knockdown on BRCA1-ASC association during KSHV infection. HMVEC-d cells treated with control Si-RNA (Si C) or IFI16-Si-RNA (Si IFI16) were infected with KSHV and lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-ASC antibodies. Blots were stripped and probed with anti-BRCA1 antibodies to detect the presence of BRCA1. WCLs were used as input controls for WBs to show IFI16 knockdown, presence of ASC and cleavage of procaspase-1. (I) BRCA1 does not interact with AIM2 and NLRP3. Uninfected or KSHV infected HMVEC-d cell lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-NLRP3 or AIM2 antibodies. Equal inputs for IPs were assessed by NLRP3, AIM2 and BRCA1 WBs. β-actin was used as an equal loading control. HC and LC: IgG heavy and light chains, respectively.
© Copyright Policy
Related In: Results  -  Collection

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ppat.1005030.g004: PLA and knockdown studies demonstrating specificity of BRCA1 interactions with IFI16-inflammasome components and relocalization to the cytoplasm during de novo KSHV infection.(A) PLA detecting IFI16 in uninfected and KSHV infected HMVEC-d cells. Red dots are indicative of PLA reactions representing the subcellular distribution of IFI16. Red arrows: nuclear IFI16. Yellow arrows: cytoplasmic IFI16. (B) Specificity control for PLA using only secondary antibodies. (C-G) PLA (red dots) detecting various complexes in uninfected (left panels) and de novo KSHV infected HMVEC-d cells at 24 h p.i. (right panels). Red arrows: nuclear PLA. Yellow arrows: cytoplasmic PLA. (C) IFI16 and BRCA1 complexes. (D) IFI16 and ASC complexes. (E) BRCA1 and ASC complexes. (F) BRCA1 and caspase-1 complexes. (G) IFI16 and Caspase-1 complexes. Boxed areas are enlarged. Quantitative analysis of the average number of PLA spots per cell and that of nucleus vs cytoplasm of infected cells is presented in the rightmost columns. ***: p<0.001, **: p<0.01, and ns: not-significant. (H) Effect of IFI16 knockdown on BRCA1-ASC association during KSHV infection. HMVEC-d cells treated with control Si-RNA (Si C) or IFI16-Si-RNA (Si IFI16) were infected with KSHV and lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-ASC antibodies. Blots were stripped and probed with anti-BRCA1 antibodies to detect the presence of BRCA1. WCLs were used as input controls for WBs to show IFI16 knockdown, presence of ASC and cleavage of procaspase-1. (I) BRCA1 does not interact with AIM2 and NLRP3. Uninfected or KSHV infected HMVEC-d cell lysates were IP-ed with anti-BRCA1 antibodies followed by WB with anti-NLRP3 or AIM2 antibodies. Equal inputs for IPs were assessed by NLRP3, AIM2 and BRCA1 WBs. β-actin was used as an equal loading control. HC and LC: IgG heavy and light chains, respectively.
Mentions: To further confirm the association of IFI16, ASC and procaspase-1 with BRCA1, we performed PLA in uninfected HMVEC-d cells and cells infected with KSHV for 24 h using anti-IFI16, anti-BRCA1, anti-ASC and anti-procaspase-1 antibodies. Specificity of the assay was confirmed by using secondary antibodies linked to probes alone (Fig 4B) and also by using only one primary antibody plus both secondary antibodies linked to probes (S2 Fig, A to E). The lack of detection of any signal following PLA in all cases confirmed the specificity of all the antibodies tested (Fig 4B and S2 Fig, A to E). When we used rabbit and mouse anti-IFI16 antibodies against different IFI16 epitopes in PLA for detecting cellular localization of IFI16 during infection, we detected IFI16 in the nucleus of uninfected cells (Fig 4A, UI, red arrows). In contrast, as we have demonstrated previously [3], at 24 h p.i., IFI16 was detected in both the nucleus and the cytoplasm of infected cells (Fig 4A, KSHV 24 h, red and yellow arrows).

Bottom Line: The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β).The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production.These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

View Article: PubMed Central - PubMed

Affiliation: H. M. Bligh Cancer Research Laboratories, Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.

ABSTRACT
The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1β, IL-18 or interferon β (IFN-β). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1β generation. IFI16 also induces IFN-β during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1β production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-β production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1β formation and the induction of IFN-β via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.

No MeSH data available.


Related in: MedlinePlus