DC isoketal-modified proteins activate T cells and promote hypertension

Abstract

Oxidative damage and inflammation are both implicated in the genesis of hypertension; however, the mechanisms by which these stimuli promote hypertension are not fully understood. Here, we have described a pathway in which hypertensive stimuli promote dendritic cell (DC) activation of T cells, ultimately leading to hypertension. Using multiple murine models of hypertension, we determined that proteins oxidatively modified by highly reactive γ-ketoaldehydes (isoketals) are formed in hypertension and accumulate in DCs. Isoketal accumulation was associated with DC production of IL-6, IL-1β, and IL-23 and an increase in costimulatory proteins CD80 and CD86. These activated DCs promoted T cell, particularly CD8+ T cell, proliferation; production of IFN-γ and IL-17A; and hypertension. Moreover, isoketal scavengers prevented these hypertension-associated events. Plasma F2-isoprostanes, which are formed in concert with isoketals, were found to be elevated in humans with treated hypertension and were markedly elevated in patients with resistant hypertension. Isoketal-modified proteins were also markedly elevated in circulating monocytes and DCs from humans with hypertension. Our data reveal that hypertension activates DCs, in large part by promoting the formation of isoketals, and suggest that reducing isoketals has potential as a treatment strategy for this disease.

Figure 10. Increased isoprostanes and isoketals in hypertensive humans.

(A) Plasma levels of F2-isoprostanes in normotensive subjects (NT) as well as well-controlled hypertension (HT) and resistant hypertensive (RH) patients (*P < 0.05 vs. NT; ^#P < 0.05 vs. HT). (B) Gating strategy for flow cytometric analysis of isoketals in mononuclear cells obtained from the buffy coats of humans with or without hypertension. (C) Isoketal adducts in human monocytes, as detected by surface staining for CD14. (D) Quantification of isoketals in CD14⁺ cells (***P < 0.001). (E) Isoketal adducts in human CD83⁺ cells. (F) Quantification of isoketals in CD83⁺ cells(*P < 0.05). (G) Proposed pathway for DC activation. Hypertensive stimuli increase ROS production and formation of isoketals and protein isoketal adducts in DCs. These adducts enhance DC immunogenicity and promote DC cytokine production. These activated DCs promote T cell proliferation and cytokine production. Activated T cells infiltrate target tissues, including the kidney and vasculature, and release cytokines, such as IL-17, TNF-α, and IFN-γ, that promote hypertension.

Figure 9. Transfer of hypertension by DCs.

(A) DCs were obtained from either sham or angiotensin II–infused mice, and 1 × 10⁶ cells were adoptively transferred to recipient WT or Rag1^–/– mice, and blood pressure was monitored using telemetry. (B) Systolic blood pressure in response to low-dose angiotensin II infusion (140 ng/kg/min) 10 days after DC adoptive transfer (n = 5–7, **P < 0.01). DCs were treated with 1 mM t-BHP, and isoketals were measured by flow cytometry using Alexa Fluor 488–tagged D11 antibody in (C) CD11b⁺/CD11c^–, (D) CD11b⁺/CD11c⁺, and (E) CD11b^–/CD11c⁺ cells. (F) Expression of CD86 in DCs. (G) Coculture of t-BHP–treated DCs with T cells promoted survival of CD8⁺ T cell. (H) Adoptive transfer of t-BHP–treated DCs in mice increased systolic blood pressure (n = 4–7, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 7. Oxidant stress and isoketals alter DC gene expression in hypertension.

(A) Microarray analysis of CD11c⁺ cells from spleens of WT mice infused with either sham or angiotensin II with or without 2-HOBA treatment or cotreatment with a combination of hydralazine and hydrochlorothiazide as well as cells from spleens of Nox2^–/– mice. The gene expression profiles for the various treatment groups. (B) Genes altered by angiotensin II (blue), genes reversed by Nox2^–/– (pink), genes reversed by 2-HOBA treatment (green), and a Venn diagram summarizing altered genes, including those normalized in both Nox2^–/– and 2-HOBA–treated groups. (C) Normalization of angiotensin II–induced hypertension by cotreatment with a combination of hydralazine and hydrochlorothiazide. (D) Volcano plot showing the 505 genes altered by angiotensin II compared with sham and colored by P value. Red represents high and blue represents low P values in WT angiotensin II vs. WT sham. Color codes for each gene in D were used in E–G. The genes altered by angiotensin II in C but plotted based on how these genes behaved (E) after 2-HOBA treatment, (F) in Nox2^–/– mice, and (G) after treatment with a combination of hydralazine and hydrochlorothiazide, when compared with WT sham (n = 6, **P < 0.01)

Figure 6. DCs from angiotensin II–hypertensive mice promote T cell survival, proliferation, and cytokine production.

(A) Experimental approach for the immunization assay. Flow cytometry was used to determine percentages of live cells using (B) 7-AAD, (C) CD45⁺ cells, and (D) CD3⁺ cells. (E) Total number of live cells, CD45⁺ cells, and CD3⁺ cells. (F–H) Representative flow cytometry profiles and graph showing percentages and numbers of CD4⁺ and CD8⁺ T cells. Proliferation of (G) CD8⁺ T cells and (H) CD4⁺ T cells, as reflected by CSFE dilution assays. (I) Cytokines IFN-γ, IL-17a, and TNF-α released by T cells stimulated with sham, angiotensin II, or angiotensin II and 2-HOBA DCs (n = 6–12, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4. Accumulation of isoketals in activated DCs.

Mice were made hypertensive with angiotensin II, as in Figure 1. (A) Gating strategy to identify CD11c⁺/CD11b⁺ cells. (B and E) Intracellular staining of isoketals in CD11b⁺/CD11c^–, (C and F) CD11b⁺/CD11c⁺, and (D and G) CD11b⁺/CD11c^– cells expressing CD80 and CD86 (n = 6, *P < 0.05, **P < 0.01 ***P < 0.001).

Figure 8. Effect of isoketal protein modification on DC immunogenicity.

(A) MHCI was immunoprecipitated from DCs of sham and angiotensin II–treated mice, and Western blot using D11 antibody was performed. Mouse kidney homogenates (100 μg total protein) were exposed to 100 μmol/liter of isoketal, hydroxynonenal (HNE), MDA, or methylglyoxal (MGO) for 30 minutes. One million DCs were then pulsed with these modified proteins for 1 hour, washed, and exposed to T cells prelabeled with CFSE at a ratio of 1 DC to 10 T cells for 7 days. (B) Effect of DCs pulsed with lipid-modified proteins on CD8⁺ T cells. (C) Effect of DCs pulsed with lipid-modified proteins on CD4⁺ T cells. The left and middle panels of B and C show proliferation of T cells that were obtained from sham or angiotensin II–treated mice, respectively. The right panels of B and C show the effects of the lipid adducts directly added to DCs. (D) Proliferation of memory and naive T cells from angiotensin II–treated mice in response to DCs pulsed with isoketal-adducted proteins.

Figure 5. Isoketals contribute to DOCA-salt hypertension.

(A and B) Intracellular staining of isoketal adducts in CD11c⁺ cells from sham mice and mice with DOCA-salt hypertension, treated or not with 2-HOBA. (C) Systolic pressures from radiotelemetry recordings showing the effect of 2-HOBA on DOCA-salt hypertension (n = 6, **P < 0.01).

Figure 3. DC accumulation of isoketals in hypertension.

(A) Intracellular staining of isoketals using Alexa Fluor 488–tagged D11 in CD11b⁺/CD11c^–, (B) CD11b⁺/CD11c⁺, and (C) CD11b^–/CD11c⁺ cells from sham and angiotensin II–infused mice with or without 2-HOBA treatment. Mean data are shown in the bar graphs (n = 6, *P < 0.05, **P < 0.01, ***P < 0.001). (D) Analysis of isoketals in various DC subtypes from sham or angiotensin II–infused mice (shown are representative data of 6 mice per group). (E) Stable isotope dilution multiple reaction monitoring mass spectrometry analysis of isoketal-lysine-lactam adduct in DCs. Representative LC/MS chromatographs from pooled samples for each group are shown. The top row shows multiple reaction monitoring chromatographs for isoketal-lysine-lactam (IsoK-Lys) in sample, while the bottom row shows multiple reaction monitoring chromatograph for [¹³C₆¹⁵N₂] internal standard for same samples.

Figure 2. Isoketals contribute to angiotensin II hypertension and kidney damage.

(A) Effect of isoketal scavengers 2-HOBA, 5-methyl-2-HOBA (5-Me-2-HOBA), and pentylpyridoxamine (PnPM) and control compounds N-methyl-2-HOBA (N-Me-2-HOBA) and 4-HOBA on the hypertensive response to angiotensin II. (B) Masson’s Trichrome immunohistochemistry showing fibrosis (green arrows) in kidney section from mice with angiotensin II–induced hypertension (scale bar: 100 μm). (C) Quantification of total renal fibrosis. (D) Collagen IV staining in kidney sections (scale bar: 10 μm). (E) Anti-CD3 staining showing T cell infiltration in kidney sections from angiotensin II–infused mice (scale bar: 10 μm). (F) Quantification of collagen IV staining. (G) Nephrin and (H) albumin concentrations were measured in urine using an ELISA-based assay (n = 6–7, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 1. Angiotensin II–induced hypertension increases O₂^η– production in DCs and isoketal formation in tissues.

(A) WT and Nox2^–/– mice received angiotensin II or sham infusions for 2 weeks, and CD11c⁺ cells were isolated from the spleens. O₂^η– was determined using HPLC to monitor conversion of dihydroethidium to the O₂^η– oxidation adduct 2-hydroxyethidium, as indicated by the first peak (arrows) of the HPLC tracings. (B) Quantification of O₂^η– in WT and Nox2^–/– mice (n = 6). (C) O₂^η– was measured in DCs at either baseline or following 24 hours of angiotensin II treatment in vitro (100 nM) (n = 7). (D) Pathway illustrating lipid peroxidation and formation of isoketals. The isoketals react with lysine residues on proteins forming lactam adducts that cross-linked proteins. This process is prevented by scavenging with 2-HOBA. (E) Immunohistochemistry of heart and aortic sections showing accumulation of isoketals in hypertension using a single-chain antibody that recognizes isoketal-lysine adducts on all proteins (scale bar: 100 μm). (F) Quantification of isoketals in the hearts and aortas (n = 6–7, *P < 0.05).