Virus binding to a plasma membrane receptor triggers interleukin-1 alpha-mediated proinflammatory macrophage response in vivo

Abstract

The recognition of viral components by host pattern-recognition receptors triggers the induction of the antiviral innate immune response. Toll-like receptor 9 (TLR9) and NLRP3 inflammasome were shown to be the principal specific sensors of viral double-stranded DNA. Here we present evidence that macrophages in vivo activated an innate immune response to a double-stranded DNA virus, adenovirus (Ad), independently of TLR9 or NLRP3 inflammasome. In response to Ad, macrophage-derived IL-1 alpha triggered IL-1RI-dependent production of a defined set of proinflammatory cytokines and chemokines. The IL-1 alpha-mediated response required a selective interaction of virus arginine-glycine-aspartic acid (RGD) motifs with macrophage beta(3) integrins. Thus, these data identify IL-1 alpha-IL-1RI as a key pathway allowing for the activation of proinflammatory responses to the virus, independently of its genomic nucleic acid recognition.

Figure 1. CD169- and MARCO-positive splenic marginal zone macrophages trap adenovirus and initiate an IL-1RI-dependent innate immune response in vivo.

(A) Distribution of Ad particles in the spleen 30 min after virus injection. RP- red pulp; GC-germinal center; MZ-marginal zone. Anti-Ad antibody stain is red. Co-localization of Ad particles (red) with (B) CD169- or (C) MARCO-positive macrophages in the splenic MZ. (D) mRNA levels for IL-1α, IL-1β, KC and MIP-2 in the spleen 30 min after Ad injection. N=4. * - P < 0.01. Except for the columns indicated by the star, no statistically significant differences between experimental groups and WT group were identified (P > 0.05). C –mock-infected mice, treated with saline. AU – arbitrary units. (E, F) Kinetics of the transcriptional activation of IL-1α, IL-1β, KC and MIP-2 in wild type (E) or Il-1r^−/− (F) mice. Individual mice were injected with Ad and sacrificed at indicated time points. (G) Protein levels for pro-inflammatory cytokines and chemokines in spleen of wild type (WT) and Il-1r^−/− mice 1 h after Ad injection. N=4. As a positive control for inflammatory cytokine and chemokine induction, mice were injected with LPS. (H) The amounts of pro-inflammatory cytokines and chemokines in the spleens of mice 1 h after Ad injection. N=3. Mock – negative control mice injected with saline * - P < 0.01.

Figure 2. IL-1α is the dominant mediator of an innate immune response to Ad in vivo

(A) The mRNA levels for IL-1α, IL-1β, KC and MIP-2 in spleens of mice 30 min after Ad injection. Biological duplicates are shown. C – mock-infected mice, injected with saline. (B) Quantitative representation of mRNA levels from the gel shown in (A) after phosphorimager analysis. N=6. n.s. – not statistically significant. * - P < 0.01. AU – arbitrary units. (C) IL-1α, IL-1β, and MIP-2 mRNA levels in livers of mice 30 min after Ad injection. N=3. (D) Quantitative representation of mRNA levels from the gel shown in (C) after phosphorimager analysis. N=6. n.s. – not statistically significant. * - P < 0.01. (E) Protein levels of inflammatory cytokines and chemokines in spleens 1 h after Ad injection. Pos-C are dots that show the manufacturer’s internal positive control samples on the membrane. Control – the spleen protein sample of a mouse injected with saline. (F) The amounts of pro-inflammatory cytokines and chemokines in the spleens of mice 1 hour after Ad injection. N=4. Mock – negative control mice injected with saline. n.s. – not statistically significant. * - P < 0.01.

Figure 3. Ad induces expression of IL-1α and its translocation to the nuclei in marginal zone macrophages

Immunofluorescent and confocal microscopy analysis of IL-1α expression on spleen sections of mock-injected (A) or Ad-injected mice at 1 h (B) or 3 h (C) post injection. The physical border of the germinal centers are depicted by punctuate lines. Confocal microscopy analysis confirms co-localization of IL-1α positive staining with marginal zone macrophage staining (D-F). Arrows indicate CD169-positive cells in the marginal zone, co-localized with IL-1α-positive staining. Representative pictures are shown. N=5. Confocal microscopy analysis shows the co-localization of nuclei of splenic marginal zone cells stained with DAPI (blue, G), IL-1α (red, H), and CD169 macrophage marker (I). Macrophage cells with co-localized DAPI and IL-1α staining are shown by arrows. (J). Semi-quantitative presentation of the proportion of IL-1α-expressing marginal zone cells with IL-1α-positive nuclei in mock-injected mice and Ad injected mice. The nuclear IL-1α-positive staining was analyzed in two hundred IL-1α-positive cells on spleen sections of the Ad-injected group. No IL-1α-positive nuclei were found on spleen sections of mock-injected animals. N=6. * - P < 0.01.

Figure 4. Activation of an IL-1α-mediated innate response to Ad does not depend on NALP3-inflammasome, MyD88, TRIF, or TRAF6

(A) Mice were injected with Ad and mRNA levels for IL-1α, IL-1β, KC and MIP-2 were analyzed 30 min p.i. N=4. Except for the columns indicated by the star, no statistically significant differences between experimental groups and WT group were identified (P > 0.05). * - P < 0.01. C – mock-infected mice, treated with saline. AU – arbitrary units. (B) Analysis of proteins in spleens of mice 1 hour after Ad injection. N=4. Mock – negative control mice injected with saline. Pos-C are dots that show the manufacturer’s internal positive control samples on the membrane. (C) The amounts of pro-inflammatory cytokines and chemokines in the spleens of mice 1 hour after Ad injection. N=3. Mock – negative control mice injected with saline. n.s. – no statistically significant difference were identified between WT and gene knockout animals (P > 0.05). (D) IL-1α, MIP-2 and KC mRNA levels in spleens of mice 30 min after Ad injection. N=4. * - P < 0.01. C – mock-infected mice, treated with saline. n.s. - not statistically significant.

Figure 5. The engagement of β₃ integrins by Ad is critical for initiation of the innate immune response

(A) The mRNA levels for IL-1α, IL-1β, KC and MIP-2 in spleens of wild type mice (WT) and mice knockout for β ₃-, β₅-, or conditionally knockout for β₁-integrin in hematopoietic cells (β₁^−/−), as well as WT mice injected with Ad mutant lacking an RGD motif within its penton protein (AdΔRGD) 30 min after virus injection. N=3. C – mock-infected mice, injected with saline. (B) Quantitative representation mRNA levels from the gel shown in (A) after phosphorimager analysis. N=6. Statistically significant differences between experimental groups and mock-injected controls [C] or WT injected with Ad are indicated by the star. * - P < 0.01. WT mice injected with AdΔRGD are indicated by the arrow. AU – arbitrary units. (C) IL-1α, IL-1β, and MIP-2 mRNA levels in livers of mice shown in (A). N=3. (D) Quantitative representation mRNA levels from the gel shown in (C) after phosphorimager analysis. N=6. Statistically significant differences between experimental groups and WT mice injected with Ad are indicated by the star. * - P < 0.01. (E) Protein levels of cytokines and chemokines in spleens of mice knockout for integrins β₁, β₂, β₃, or WT mice injected with Ad or AdΔRGD 1 hour after virus injection. N=4. Pos-C are dots that show the manufacturer’s internal positive control samples on the membrane. Control – the spleen protein sample of a mouse injected with saline. (F) The amounts of cytokines and chemokines in the spleens of mice 1 hour after Ad injection. N=4. Mock – negative control mice injected with saline. Statistically significant differences between experimental groups and WT mice injected with Ad are indicated by the star. * - P < 0.01.

Figure 6. Confocal microscopy analysis of IL-1α translocation into the nuclei of marginal zone macrophages in WT mice and β₃ integrin knockout mice injected with Ad, or WT mice injected with AdΔRGD mutant

Mice were injected intravenously with a high dose of the indicated viruses (10¹¹ virus particles per mouse), and 3 hours later spleens were harvested and sections were prepared and stained with DAPI (blue) to detect nuclei of splenocytes, as well as Abs specific for CD169 (green) or IL-1α (red). Confocal images were obtained using a Zeiss 510 Meta Confocal microscope. The physical border of splenic germinal centers are indicated by punctuate lines. Marginal zone macrophages expressing IL-1α are indicated by arrows. Representative pictures are shown. N=4.

Figure 7. Transcriptional and functional activation of IL-1α in response to ts1 mutant virus and the model of IL-1α-mediated activation of the innate immune response to Ad by macrophages in vivo

(A) Mice were injected with Ad or ts1 mutant and mRNA levels for IL-1α were analyzed 30 min p.i. N=4. * - P < 0.01. C – mock-infected mice, treated with saline. AU – arbitrary units. (B) Analysis of proteins in spleens of mice 1 hour after Ad or ts1 injection at indicated doses. N=4. Mock – negative control mice injected with saline. Pos-C are dots that show the manufacturer’s internal positive control samples on the membrane. (C) The amounts of pro-inflammatory cytokines and chemokines in the spleens of mice 1 hour after Ad or ts1 injection. N=3. Mock – negative control mice injected with saline.* - P < 0.01. (D) Model of Ad induction of IL-1α that triggers the activation of macrophage innate immune and inflammatory responses in vivo. 1. Ad interaction with a macrophage receptor induces transcription and synthesis of pre-IL-1α. 2–4. β₃ integrin interaction with virus RGD motifs triggers intracellular signaling that promotes virus internalization into the cell and endosome rupture, thus, leading to the amplification of IL-1α gene transcription, pre-IL-1α processing, translocation of IL-1α -NTP to the nucleus, and enabling mature IL-1α to initiate IL-1RI signaling. 5. IL-1α -mediated IL-1RI signaling leads to the activation and production of pro-inflammatory cytokines and chemokines, including KC, MIP-2, MCP-1, and IL-6.