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. 2013 Mar;43(3):655-66.
doi: 10.1002/eji.201242765. Epub 2013 Jan 31.

Genetic deletion of the HIF-1α isoform I.1 in T cells enhances antibacterial immunity and improves survival in a murine peritonitis model

Peter Georgiev  1 , Bryan G Belikoff, Stephen Hatfield, Akio Ohta, Michail V Sitkovsky, Dmitriy Lukashev
Affiliations

Genetic deletion of the HIF-1α isoform I.1 in T cells enhances antibacterial immunity and improves survival in a murine peritonitis model

Peter Georgiev et al. Eur J Immunol. 2013 Mar.

Abstract

Hypoxia-adenosinergic suppression and redirection of the immune response has been implicated in the regulation of antipathogen and antitumor immunity, with hypoxia-inducible factor 1α (HIF-1α) playing a major role. In this study, we investigated the role of isoform I.1, a quantitatively minor alternative isoform of HIF-1α, in antibacterial immunity and sepsis survival. By using the cecal ligation and puncture model of bacterial peritonitis, we studied the function of I.1 isoform in T cells using mice with total I.1 isoform deficiency and mice with T-cell-targeted I.1 knockdown. We found that genetic deletion of the I.1 isoform resulted in enhanced resistance to septic lethality, significantly reduced bacterial load in peripheral blood, increased M1 macrophage polarization, augmented levels of proinflammatory cytokines in serum, and significantly decreased levels of the anti-inflammatory cytokine IL-10. Our data suggest a previously unrecognized immunosuppressive role for the I.1 isoform in T cells during bacterial sepsis. We interpret these data as indicative that the activation-inducible isoform I.1 hinders the contribution of T cells to the antibacterial response by affecting M1/M2 macrophage polarization and microbicidal function.

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Conflict of interest statement

CONFLICT OF INTERESTS

The authors have declared that no competing interests exist

Figures

Figure 1
Figure 1. Expression of HIF-1α I.1 isoform in TCR-activated T cells and its effect on pro-inflammatory cytokine production
(A) Scheme of the differential expression of alternative isoform I.1 and ubiquitous isoform I.2 of murine HIF-1α. Exons are represented by squares. Dashed lines indicate alternative splicing. Horizontal arrows represent promoters. (B) I.1 mRNA expression in splenic T cells after TCR activation in vitro. T cells were isolated from spleen of C57BL/6 mice and activated by anti-CD3/anti-CD28 magnetic beads for 24 hrs under either normoxic (21 % O2) or hypoxic (1 % O2) conditions. Total RNA were subjected to RT-qPCR analysis of HIF-1α I.1 isoform. Data are shown as a mean ± SEM and representative of three independent experiments. Statistical significance between untreated ex vivo control and activated samples was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: **, p<0.01. (C) I.1 mRNA expression in splenic T cells after in vivo TCR activation. C57BL/6 mice were i.p. injected with 50 µg bacterial superantigen (SEB) for 3hrs. T cells were purified from spleens by anti-CD4/anti-CD8 magnetic beads and total RNA were subjected to RT-qPCR. Data are expressed as a mean ± SEM of 3 animals and representative of two independent experiments. Statistical significance between groups was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: *, p<0.05. (D) Effect of I.1 isoform deletion on cytokine production. Mice with total HIF-1α I.1 isoform knock-out were injected i.p with 50µg SEB. Plasma was collected 3 hrs after injection and serum levels of TNF-α, IL-2, and IL-6 were determined by ELISA. Data are expressed as a mean ± SEM of 3 mice per group and representative of three independent experiments. Statistical significance between groups was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: *, p<0.05; **, p<0.01.
Figure 2
Figure 2. Effect of genetic deletion of I.1 isoform of HIF-1α on CLP-induced sepsis in mice
(A) Effect of the I.1 isoform gene knockout on sepsis survival. Polymicrobial sepsis was induced in C57Bl/6 male mice by CLP using a single puncture 18 gauge needle and mice were observed for 15 days. Control sham surgeries were performed in three WT and I.1-KO mice. Survival data are pooled from three independent experiments using I.1-KO mice (36 total) to WT controls (36 total). Significance between groups was calculated using log-rank test, *p < 0.05. (B) Bacterial load measured as CFU from plated 25µL of peripheral blood drawn 24 hrs post CLP induced sepsis, CFU from 5mL of peritoneal lavage fluid collected 72 hrs after CLP, and CFU in homogenized spleen collected 72hrs post CLP. Results in are expressed as a mean ± SEM of 36 mice pooled from three independent experiments. Significance between groups was calculated using non-paired Student t-test. Two-tailed levels of significance are indicated by: *, p<0.05 (C) Cytokine levels in blood of septic mice after CLP. Plasma collected 6, 24 and 48 hours after CLP and cytokine levels were determined by ELISA. Results are expressed as a mean ± SEM of 12 animals per group and are representative of three independent experiments. For IL-6 and IL-10 significance between two groups (I.1KO vs. WT) was calculated using two-way ANOVA analysis. Levels of significance are indicated by asterisk: **, p<0.01. For MIP-2 ANOVA comparison return P-value >0.05, therefore non-paired Student t-test was used to compare 6h time points. Two-tailed levels of significance are indicated by: **, p<0.01
Figure 3
Figure 3. Flow cytometry analysis of immune cells after CLP
(A) Analysis of peritoneal lavage cells. Peritoneal lavage was collected from mice, which were sacrificed after 72 hours post CLP procedure (single puncture 18 gauge needle). Neutrophils were stained as GR1-positive/F4/80-negative, macrophages as F4/80-positive/GR1-low, B cells as B220-positive. (B) FACS analysis of splenic T lymphocyte subsets. Splenocytes were collected from septic mice after 72h post CLP and stained for CD4+, CD8+, NK1.1+ (NK cells), CD4+CD25+Foxp3+ (Tregs), CD4+ Foxp3+CD39+, CD4+Foxp3+CD73+. Data are expressed as a mean ± SEM of 5 mice per group and representative of two independent experiments. Statistical significance between groups was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: *, p<0.05.
Figure 4
Figure 4. Effect of conditional knockdown of I.1 isoform in T cells
(A) Evaluation of efficiency of I.1 exon deletion by RT-qPCR. Measurements of I.1 mRNA amounts in T cells. T cells were isolated by autoMACS from spleens of I.1-floxed (WT) and I.1-floxed_CD4Cre+/− (I.1CD4) mice and activated using beads carrying anti-CD3 and anti-CD28 mAb at either hypoxic (1% O2) or normoxic (21% O2) conditions for 48 hours. Total RNA was extracted and subjected to RT-PCR. (B) RT-qPCR analysis of RNA from splenic macrophages and B cells. Macrophages and B cells were isolated from spleen by autoMACS using CD11b–FITC and B220-FITC and anti-FITC magnetic beads. Data are expressed as a mean ± SEM of 3 mice per group and representative of two independent experiments. Statistical significance between groups was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: **, p<0.01. (C) Flow cytometry analysis of intracellular IFN-γ production by I.1 deficient CD4+ and CD8+ T lymphocytes activated in vitro with soluble anti-CD3 mAb for 24h, followed by PMA/ionophore for 2h. Results are depicted as a mean ± SEM of two independent experiments. Significance between two groups was calculated using two-way ANOVA. Asterisks indicates level of significance ** p<0.01%.
Figure 5
Figure 5. Effect of T cell-specific I.1 deficiency on polymicrobial sepsis survival and bacteremia after CLP
(A) 15-day survival analysis of I.1-lox/lox_CD4Cre+ mice (I.1CD4) after CLP-induced sepsis. CLP was performed using a single puncture 18 gauge needle (n=16 per group). Control sham surgeries were performed in three WT and I.1CD4 mice. Presented survival data are pooled from three independent experiments. *p<0.05 when comparing WT to I.1CD4 mice by log rank test. (B) Evaluation of bacterial burden in blood. A 25ul aliquot of blood was collected 24 hrs post CLP to check for levels of circulating bacteria. Results are expressed as a mean ± SEM and representative of three independent experiments. Significance between two groups was calculated using non-paired Student t-test. Two-tailed levels of significance are indicated by: *, p<0.05 (C) IL-6 and IL-10 production in I.1CD4 mice. Plasma was collected at 6, 24 and 48 hours post CLP and serum cytokine levels were determined by ELISA. Results are expressed as a mean ± SEM of 5–10 animals per group and are representative of three independent experiments. Significance between two groups (I.1CD4 vs. WT) was calculated using two-way ANOVA analysis. Levels of significance are indicated by asterisk: **, p<0.01.
Figure 6
Figure 6. Neutrophils and macrophage functions in mice with T cell-specific I.1 deficiency
(A) Concentration of IL-10 in peritoneum after LPS activation. Five WT vs. I.1-KO mice were injected with 2ml of 3% thioglycollate i.p. for 2 days followed by 4 hours of 5mg/kg LPS i.p. Results are expressed as a mean ± SEM and representative of two independent experiments. Significance between two groups was calculated using non-paired Student t-test. Two-tailed levels of significance are indicated by: *, p<0.05 (B) Production of IL-10 by T cells in vitro. T cells were isolated from spleens of three WT and three I.1-KO mice and activated by beads coated with anti-CD3/anti-CD28 mAb for 48h. Results are expressed as a mean ± SEM and representative of three independent experiments. Significance between two groups was calculated using non-paired Student t-test. Two-tailed levels of significance are indicated by: **, p<0.01 (C) IL-10 mRNA expression in T cells activated in vivo. I.1KO and WT mice were injected i.p. with 25–100 µg of SEB for 3 hours. Data are expressed as a mean ± SEM of 3 mice per group and representative of two independent experiments. Statistical significance between groups was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: *, p<0.05; **, p<0.01. (D) Macrophage polarization in WT vs. I.1CD4 mice. Peritoneal macrophages obtained from mice with T cell specific HIF-1α I.1 isoform deficiency 72hrs after CLP-induced sepsis. Polarization states were determined by expression assessment of specific chemokine and cytokine mRNA with the use of RT-qPCR: M1-specific (iNOS) and M2-specific (CCL17). Data are expressed as a mean ± SEM of 3 mice per group and representative of two independent experiments. Statistical significance between groups was determined using non-paired Student t-test. Two-tailed levels of significance are indicated by asterisk: *, p<0.05. (E) The phagocytic capacity of macrophages from mice with T-cell conditional knockdown of I.1 isoform. Peritoneal macrophages were collected from WT and I.1CD4 mice after 5 days post CLP and their phagocytic functions were measured using in vitro system of pH-sensitive fluorogenic E. coli cells. F4/80-positive macrophages with ingested labeled E.coli were analyzed by FACS. Data are expressed as a mean ± SEM of 5 mice in two independent experiments. Significance between two groups was calculated using non-paired Student t-test. Two-tailed levels of significance are indicated by *, p<0.05; **, p<0.01
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