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Keywords:

  • Animal models;
  • Cytokines;
  • Hypoxia-inducible factor;
  • Sepsis;
  • T lymphocytes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

Sepsis is a complex clinical syndrome representing a major healthcare obstacle with an unacceptably high mortality rate [1]. Despite massive efforts, the absence of effective therapies remains a critical barrier to improving survival, and failures in sepsis therapies point to the critical need for new treatment paradigms [2]. Sepsis includes two major stages: systemic inflammatory response syndrome (SIRS) characterized by a hyperinflammatory response to infection, and compensatory anti-inflammatory response syndrome (CARS) accompanied by production of anti-inflammatory cytokines that limits tissue damage associated with the inflammatory response [2-5]. The resulting period of immune suppression is characterized by inhibited macrophage function, diminished proinflammatory cytokine production, and sustained production of anti-inflammatory cytokines such as IL-10 [6, 7]. During the immunocompromized phase of sepsis, patients become increasingly susceptible to secondary hospital-acquired infections, which play a decisive role in the lethal outcome [8-12]. In humans, the inhibition of proinflammatory cytokines leads to increased subsequent mortality due to opportunistic infections accompanied by macrophage deactivation, which was shown to be mediated by anti-inflammatory cytokines such as IL-10 [13-15].

Although macrophages and neutrophils play a central role in eliminating bacteria, T cells are required for efficient protection against bacterial infection via production of proinflammatory cytokines [16-20]. Within the first 24 h of infection, T cells can produce proinflammatory cytokines that can enhance the early innate immune response against bacterial infection [15, 21, 22], and inadequate activation of T cells results in an insufficient innate immune response leading to decreased bacterial clearance and survival [17]. Stimulation of macrophages through Toll-like receptors (TLR) in combination with Th1 cytokines leads to M1 polarization of proinflammatory and cytotoxic macrophages [23], while Th2 cytokines such as IL-10 create the alternatively activated macrophages of M2 phenotype, which are anti-inflammatory and poorly microbicidal [23-25].

Hypoxia-inducible factor 1α (HIF-1α) is stabilized by hypoxia, which is associated with inflammatory tissue damage during sepsis [19, 26]. HIF-1α protein can be encoded by two alternative mRNA isoforms [27] (Fig. 1A). The difference between these isoform is due to a difference in the first exons, which results from alternative splicing (Fig. 1A). The conventional mRNA isoform contains the I.2 exon and is ubiquitously expressed using the constitutive I.2 promoter in various tissues [27, 28] and is indispensable for cell survival, glycolysis, and angiogenesis [29]. The alternative mRNA isoform starts with exon I.1 and is predominantly expressed in immune organs and testis [29, 30]. The I.1 mRNA isoform encodes an HIF-1α protein lacking the first 12 N-terminal amino acids, however this “short” I.1 isoform retains DNA-binding bHLH domain and has similar transcriptional activities as the ubiquitous “full-size” I.2 isoform [30]. Importantly, the expression of the I.1 promoter is strongly induced in antigen receptor-activated T cells as an immediate-early response gene [30]. Therefore, the I.1 isoform was dubbed the “activation-inducible isoform” [31]. Previously, we demonstrated that genetic deletion of the I.1 isoform results in higher proinflammatory cytokine production by T cells [31], and macrophages in vitro [32].

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Figure 1. Expression of the hypoxia-inducible factor 1α (HIF-1α) I.1 isoform in TCR-activated T cells and its effect on proinflammatory 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 h under either normoxic (21% O2) or hypoxic (1% O2) conditions. Total RNA was subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of HIF-1α I.1 isoform. Data are shown as a mean + SEM of three animals and are from one experiment representative of three independent experiments. Statistical significance between untreated ex vivo control and activated samples was determined using nonpaired 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 3 h. 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 three animals and are from one experiment representative of two independent experiments. Statistical significance between groups was determined using nonpaired 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 knockout were injected i.p with 50 μg SEB. Plasma was collected 3 h 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 three mice per group and are from one experiment representative of three independent experiments. Statistical significance between groups was determined using nonpaired Student t-test. Two-tailed levels of significance are indicated by asterisk: *p < 0.05; **p < 0.01.

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In the present study, we demonstrate a connection between the T-cell-orchestrated antibacterial response and the activation-inducible I.1 isoform of HIF-1α. We show that activation-inducible I.1 isoform of HIF-1α negatively regulates T-cell contributions into the antibacterial immune response during polymicrobial sepsis by affecting M1/M2 macrophage polarization.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

Anti-inflammatory effects of TCR activation-inducible I.1 isoform in vivo

Our previous in vitro studies established that the I.1 isoform of HIF-1α is expressed as an immediate-early response gene in T cells after activation through TCR stimulation [30, 31, 33-35] (Fig. 1A). We confirmed and extended these findings by showing that the I.1 isoform of HIF-1α is not only induced by TCR stimulation in vitro (Fig. 1B), but also in vivo when T cells are activated by bacterial superantigens (SAgs) (Fig. 1C), which can activate large number of T cells by cross-linking their TCR with MHC class II molecules of antigen presenting cells; thereby, causing rapid polyclonal T-cell proliferation and cytokine production [22]. We found that i.p. injection of Staphylococcus aureus enterotoxin B (SEB) strongly induces I.1 isoform expression in T cells as early as 3 h after SEB injection. Previously, we demonstrated that I.1-deficient T cells produce more proinflammatory cytokines in vitro after TCR stimulation either by cross-linking mAb or by allogenic MHC in mixed lymphocyte culture [31]. However, the alternative isoform I.1 is a quantitatively minor isoform, which contributes to 10–15% of total HIF-1α mRNA [31]. It was not clear whether this minor alternative isoform plays a significant role in vivo by producing sufficient immunosuppression and affecting the antipathogen immune response. We reasoned that if the alternative HIF-1α isoform I.1 indeed regulates the overall intensity of the antipathogen immune response in vivo, then the elimination of this isoform in I.1 gene deficient mice would lead to reduced immunosuppression, higher proinflammatory cytokine production, stronger antibacterial response, and improved survival in mouse models of sepsis. Our previous findings established that T cells can potentially contribute to the antibacterial immune response by secreting proinflammatory cytokines, which activate microbicidal properties of macrophages during sepsis [19]. Therefore, it is important to investigate whether elimination of the I.1 isoform in T cells leads to enhanced production of proinflammatory cytokines in vivo after TCR activation with bacterial SAgs. Indeed, we show that proinflammatory cytokines are strongly induced in I.1-deficient mice (I.1-KO) after SEB injection (Fig. 1D). These results further extended our in vitro observations that the I.1 isoform acts as an immunosuppressive factor in activated T cells.

In addition, activation of peritoneal macrophages in vivo by LPS also induced expression of I.1 mRNA isoform (Supporting Information Fig. 1A), which comes in agreement with previous reports of in vitro upregulation of I.1 isoform after TLR-mediated activation [32]. However, we did not detect significant changes in proinflammatory cytokine production in response to TLR stimulation in vivo in I.1-KO as compared with that of WT mice (Supporting Information Fig. 1B).

Improved resistance of I.1-KO to bacterial sepsis

To study the effect of I.1-deficiency in T cells on the antibacterial immune response during sepsis, we adopted the cecal ligation and puncture (CLP) model. This murine model of bacterial sepsis creates a polymicrobial infection resulting from the leakage of enteric content as a result of intestinal perforation [36]. To study the contribution of T cells into the antibacterial immune response during sepsis, we had to avoid early lethal outcomes during the first 24 h of systemic infection. This would allow sufficient time for T cells to not only get recruited and activated, but also to contribute to the antibacterial immune response to such an extent that prevention of T-cell inhibition by genetic deletion of I.1 would be discernible. Therefore, we adopted a CLP model of long-lasting sepsis with approximately 50% mortality because it would allow enough time for T cells to contribute to the antibacterial immune response [19] and because it closely mimics the mortality rate observed in human patients [1, 36]. In this model, severity and sepsis mortality can be adjusted by changing the size of the needle used or the number of punctures applied [19, 37].

While deficiency in conventional HIF-1α results in early embryonic mortality due to failure in vascularization [38], naïve gene-deficient I.1−/− mice did not show apparent differences when compared with the WT C57BL/6 mice [31]. Furthermore, analysis of immune organs from untreated WT and I.1-KO by flow cytometry revealed no significant differences in immune cells subsets in lymph nodes, thymus, and spleen. In addition, the T-cell repertoire in the spleen showed no significant differences in the proportions of CD4+ T cells, CD8+ T cells, natural killer T (NKT) cells, and Treg cells (data not shown). The likely explanation of this apparent lack of phenotypic differences is that, since the I.1 isoform is TCR-activation dependent, it is not expressed in naïve T cells. Thus, one can expect no significant differences in I.1-KO versus WT mice in the absence of infection or inflammation [31, 32]. However, after developing CLP-induced polymicrobial sepsis, mice deficient in the I.1 isoform (I.1-KO) demonstrated significantly higher survival rates when compared with control HIF-1α-expressing mice (Fig. 2A). Accordingly, I.1-KO mice had a significantly decreased bacterial burden in peripheral blood, peritoneal cavity, and spleen (Fig. 2B).

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Figure 2. Effect of genetic deletion of I.1 isoform of hypoxia-inducible factor 1α (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-deficient mice (I.1-KO). Survival data are pooled from three independent experiments using I.1-KO mice (36 in total) to WT controls (36 in total). Significance between groups was calculated using log-rank test, *p < 0.05. (B) Bacterial load measured as CFU from plated 25 μL of peripheral blood drawn 24 h post CLP-induced sepsis, CFU from 5 mL of peritoneal lavage fluid collected 72 h after CLP, and CFU in homogenized spleen collected 72 h post CLP. Results in are expressed as mean ± SEM of 36 mice pooled from three independent experiments. Significance between groups was calculated using nonpaired 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 h after CLP and cytokine levels were determined by ELISA. Results are expressed as a mean ± SEM of 12 animals per group and are from one experiment representative of three independent experiments. For IL-6 and IL-10 significance between two groups (I.1-KO versus 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, nonpaired Student t-test was used to compare 6-h time points. Two-tailed levels of significance are indicated by **p < 0.01.

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Deficiency in I.1 isoform was also associated with higher serum concentrations of proinflammatory cytokines such as IL-6 and chemokines, such as MIP-2 (Fig. 2C). In addition, the concentrations of the anti-inflammatory cytokine IL-10 were found to be significantly lower in I.1-KO (Fig. 2C).

Flow cytometry analysis of peritoneal lavage (PT) after 3 days of CLP-induced sepsis revealed that I.1 deficiency results in several changes in population of immune cells (Fig. 3A). I.1-KO had higher percentages of neutrophils and lower numbers of macrophages, CD4+, and CD8+ T cells as compared with those in WT mice. Lymphocyte repertoire in spleens of septic mice showed higher percentages of natural killer (NK) cells in I.1-KO mice (Fig. 3B). Interestingly, I.1 deficiency was also associated with higher expression of Foxp3 in Treg cells, which is in agreement with a recently published study indicating HIF-1α as a factor, which downregulates Foxp3 expression and affects Th17/Treg-cell polarization [39]. In contrast, Treg cells demonstrated lower levels of surface expression of ecto-5′-nucleotidase CD73, which creates extracellular adenosine (Fig. 3B). It is known that hypoxia-induced adenosine is increased during sepsis, and adenosine receptors negatively regulate the immune response to pathogen [36, 39-41]. The observed downregulation of CD73 can potentially increase proinflammatory response and increase neutrophil chemotaxis.

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Figure 3. Flow cytometry analysis of immune cells after CLP. (A) Analysis of peritoneal lavage cells. Peritoneal lavage was collected from mice that were sacrificed after 72 h 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) Flow cytometric analysis of splenic T lymphocyte subsets. Splenocytes were collected from septic mice after 72 h post CLP and stained for CD4+, CD8+, NK1.1+ (NK cells), CD4+CD25+Foxp3+ (Treg cells), CD4+Foxp3+CD39+, CD4+Foxp3+CD73+. Data are expressed as a mean + SEM of five mice per group and are from one experiment representative of two independent experiments. Statistical significance between groups was determined using nonpaired Student t-test. Two-tailed levels of significance are indicated by asterisk: *p < 0.05.

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Development of mice with T-cell-specific knockdown of I.1 isoform

To discriminate between the effects of the I.1 isoform in cells of the innate and adaptive immune systems, we decided to study the mice with T-cell-specific I.1-knockdown. We created I.1CD4 conditional knockdown mice where I.1 exon is surrounded by loxP sites and Cre recombinase is expressed via the CD4 promoter [42]. This results in Cre-mediated excision of the I.1 exon in thymocytes during their double-positive stage (Supporting Information Fig. 2). Since the alternative isoform I.1 is shorter than the ubiquitous isoform I.2, but otherwise has the same amino acid sequence, the development of I.1-specific antibody for detection is impossible. Therefore, the efficiency of I.1 deletion in T cells was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RT-qPCR confirmed that while I.1 mRNA is strongly induced by TCR-activation in WT T cells, the I.1 isoform is efficiently deleted in I.1CD4 mice (Fig. 4A), while no difference in I.1 mRNA was detected in splenic B cells and macrophages (Fig. 4B). Untreated I.1CD4 mice did not show significant phenotypic differences in immune cell populations of thymus, spleen, or lymph nodes (data not shown). Our initial hypothesis that the I.1 isoform of murine HIF-1α acts as an immunosuppressive factor in activated T cells in vitro was further supported by data showing that T cells isolated form I.1CD4 mice produce more IFN-γ when stimulated by anti-CD3/anti-CD28 mAb in vitro (Fig. 4C).

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Figure 4. Effect of conditional knockdown of I.1 isoform in T cells (A) Evaluation of efficiency of I.1 exon deletion by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Measurements of I.1 mRNA amounts in T cells. T cells were isolated by auto-MACS 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 h. 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 auto-Macs using CD11b-FITC and B220-FITC and anti-FITC magnetic beads. Data are expressed as a mean ± SEM of three mice per group and are from one experiment representative of two independent experiments. Statistical significance between groups was determined using nonpaired 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 24 h, followed by PMA/ionophore for 2 h. Results are depicted as a mean ± SEM of three mice per group and are from one experiment representative of two independent experiments. Significance between two groups was calculated using two-way ANOVA. Asterisks indicates level of significance **p < 0.01%.

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T-cell-specific knockdown of I.1 isoform results in better resistance to bacterial sepsis

We predicted that the T-cell-specific knockdown of the I.1 isoform would improve the antibacterial immune response and overall sepsis survival due to enhanced production of macrophage-stimulating proinflammatory cytokines by T cells. Indeed, the survival of I.1CD4 mice was significantly higher as compared with that of WT (i.e. I.1-lox/lox_CD4Cre−/−) littermate controls (Fig. 5A). These results were similar to those obtained by using mice with total I.1 isoform knockout. Accordingly, during CLP-induced sepsis I.1CD4 mice showed lower levels of circulating bacteria (Fig. 5B) accompanied with higher IL-6 plasma levels and lower IL-10 levels as compared with those of WT controls (Fig. 5C).

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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 with I.1CD4 mice by log-rank test. (B) Evaluation of bacterial burden in blood. A 25-μL aliquot of blood was collected 24 h post CLP to check for levels of circulating bacteria. Results are expressed as a mean ± SEM of ten mice per group and are from one experiment representative of three independent experiments. Significance between two groups was calculated using nonpaired 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 h 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 from one experiment representative of three independent experiments. Significance between two groups (I.1CD4 versus WT) was calculated using two-way ANOVA analysis. Levels of significance are indicated by asterisk: **p < 0.01.

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Effect of I.1 isoform in T cells on polarization and antimicrobial functions of macrophages

The observation of IL-10 downregulation in I.1-KO during sepsis prompted further investigation of its mechanism. IL-10 can be secreted by Th2, Treg, NK cells, and also by macrophages and neutrophils [43]. Interestingly, LPS activation of peritoneal macrophages resulted in significantly lower IL-10 levels in PT (Fig. 6A and Supporting Information Fig. 3), which implies that I.1-deficient macrophages may also contribute in IL-10-mediated regulation of the antibacterial response. To test whether activation-inducible isoform I.1 regulates IL-10 production in T cells, splenic T cells were activated with anti-CD3/anti-CD 28-coated magnetic beads. We found, that I.1-deficient T cells produce significantly less IL-10 when compared to WT T cells (Fig. 6B).

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Figure 6. Neutrophils and macrophage functions in mice with T-cell-specific I.1 isoform deficiency. (A) Concentration of IL-10 in peritoneum after LPS activation. Five WT versus I.1-KO mice were injected with 2 mL of 3% thioglycollate i.p. for 2 days followed by 4 h of 5 mg/kg LPS i.p. Results are expressed as a mean + SEM of five mice per group and are from one experiment representative of two independent experiments. Significance between two groups was calculated using nonpaired 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 48 h. Results are expressed as a mean + SEM of three mice per group and are from one experiment representative of three independent experiments. Significance between two groups was calculated using nonpaired 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 h. Data are expressed as a mean + SEM of three mice per group and representative of two independent experiments. Statistical significance between groups was determined using nonpaired Student t-test. Two-tailed levels of significance are indicated by asterisk: *p < 0.05; **p < 0.01. (D) Macrophage polarization in WT versus I.1CD4 mice. Peritoneal macrophages obtained from mice with T-cell-specific HIF-1α I.1 isoform deficiency 72 h after CLP-induced sepsis. Polarization states were determined by expression assessment of specific chemokine and cytokine mRNA with the use of reverse transcription-quantitative polymerase chain reaction (RT-qPCR): M1-specific (iNOS) and M2-specific (CCL17). Data are expressed as a mean + SEM of three mice per group and representative of two independent experiments. Statistical significance between groups was determined using nonpaired 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 Escherichia coli cells. F4/80-positive macrophages with ingested labeled E. coli were analyzed by flow cytometry. Data are expressed as a mean + SEM of five mice per group and are from one experiment representative of two independent experiments. Significance between two groups was calculated using nonpaired Student t-test. Two-tailed levels of significance are indicated by *p < 0.05; **p < 0.01.

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Since HIF-1α is a major transcriptional factor, it was rational to examine whether I.1 deficiency leads to changes in gene expression. However, I.1 is a minor isoform, and no significant changes were observed in the expression of HIF-1-dependent genes including VEGF, GLUT1, LDH etc. in T cells activated in vitro by anti-CD3 mAb or in vivo by SEB (data not shown). On the other hand, RT-qPCR measurement of cytokine gene expression after SEB injection revealed a dramatic reduction of IL-10 mRNA in I.1-KO T cells (Fig. 6C) suggesting that the I.1 isoform may be involved in the regulation of IL-10 expression.

The observation of diminished IL-10 production by I.1-deficient T cells pointed to a possibility that M1/M2 polarization of macrophages may be affected by I.1 isoform in T cells during sepsis [14, 44-46]. Microbicidal M1 and anti-inflammatory M2 polarization states of macrophages can be assessed by expression of specific chemokines and cytokines [47]. To investigate whether I.1 deficiency in T cells would affect antibacterial properties of macrophages and their development during sepsis, peritoneal macrophages were isolated from T-cell-specific I.1-knockdown mice (I.1CD4) and I.1-expressing WT mice, which underwent CLP-induced sepsis for 5 days. We found that macrophages derived from mice with T cell-specific I.1 knockdown were more M1-polarized as assessed by mRNA expression of iNOS (M1 phenotype [47]), and CCL17 (M2 phenotype [47]) (Fig. 6D) when compared with macrophages derived from WT septic mice. Furthermore, we analyzed phagocytic macrophages from T-cell-specific I.1-KO mice for their capacity to engulf bacteria. In vitro phagocytosis assay using peritoneal macrophages from 5-day-septic mice revealed significantly higher rates of phagocytosis in macrophages isolated from I.1CD4 mice as compared with those from WT mice (Fig. 6E).

Taken together, our data indicate that I.1 isoform deficiency in T cells leads to higher proinflammatory cytokine production and lower IL-10 production, which may enhance M1 polarization and augment the phagocytic capacity of macrophages, thereby improving the antibacterial immune response during sepsis.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

This study extends our previous findings that the I.1 isoform of HIF-1α inhibits proinflammatory T-cell functions [31-35]. The data presented herein point to an immunosuppressive role of the I.1 isoform in T cells during bacterial sepsis and implicate this isoform in hindering the antibacterial immune response.

Despite its disproportionately strong effects on T-cell functions in vitro, the quantitatively minor alternative isoform I.1 of HIF-1α has not been studied for its effect on the antipathogen response and survival in vivo. Since the HIF-1α isoform I.1 contains a different first exon than the ubiquitous isoform I.2, and is expressed from its own promoter [28, 30] (Fig. 1A), it is rational to suggest that the N-terminal difference between I.1 isoform and conventional HIF-1α isoform I.2 may result in different transcriptional partners and/or changes in sequence recognition. Furthermore, the phenomenon of rapid activation-induced upregulation of I.1 expression suggests a critical role of I.1 as an immunosuppressive “emergency brake,” which starts to apply immediately after T cells become activated [30, 31, 34]. Data presented in this study suggest that the I.1 isoform may be involved in regulation of expression of cytokine genes such as IL-10, which leads to attenuation of proinflammatory functions of immune cells. Likewise, other studies demonstrated that overexpression of HIF-1α induces IL-10 production [48, 49]. The role of IL-10-dependent inhibition of the antibacterial response is well established [14, 46]. Our study points to the involvement of the I.1 isoform of HIF-1α in regulation of IL-10 expression, which may represent the physiological regulation of the innate antibacterial response. Induction of activation-inducible isoform I.1 may lead to attenuation of the proinflammatory functions of T cells and macrophages, which may negatively affect M1 macrophage polarization, inhibit microbicidal functions of macrophages, and result in suppression of the antibacterial immune response.

It is now established that HIF-1α is essential for the efficient antibacterial functions of macrophages, which include migration to inflamed tissues and production of cytotoxic molecules such as NO [50-52]. Therefore, it is important to discriminate the differential roles of the alternative isoforms of HIF-1α. It is possible, that the indispensable role of HIF-1α in macrophages is due to the demand of the conventional isoform I.2, which is required for glycolytic metabolism in inflamed hypoxic tissues [51, 53]. On the other hand, it was shown that the alternative isoform I.1 of HIF-1α can attenuate TLR-activated macrophages [32] and TCR-activated T cells [31]. Therefore, it is plausible to suggest that while conventional HIF-1α isoform I.2 is required for metabolism and free radical production in macrophages, the I.1 isoform serves as a suppressor of proinflammatory responses. Further comparative studies using tissue-specific gene-knockdowns of HIF-1α isoforms can help to resolve the differential roles of HIF-1α in innate and adaptive immune responses.

The role of the activation-inducible HIF-1α isoform I.1 as an inhibitory factor in activated T cells can be potentially exploited for modulation of the inflammatory response and to prevent excessive collateral tissue injury. The present study also suggests that selective targeting of the I.1 isoform, which has restricted tissue expression, may prevent the unwanted side effects of elimination of HIF-1α. The targeted inhibition of the I.1 isoform may unleash powerful antibacterial functions of T cells that contribute into macrophage activation and bacterial clearance.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

Mice

All animal experiments were conducted in accordance with IACUC guidelines of Northeastern University and the National Institutes of Health guidelines on the use of laboratory animals (IACUC Permit # 11–0720R). Male mice with 8–12 weeks of age were used for experiments. HIF-1α- I.1-KO were generated as previously described [31]. Mice with T-cell-targeted deletion of HIF-1α (I.1-lox/lox_CD4Cre+/−) were generated by breeding homozygous I.1-floxed mice [31] with CD4-Cre transgenic mice [42]. Mice that were I.1-floxed and heterozygous for CD4-Cre had I.1-deficient T cells; control WT mice were their CD4-Cre-negative/I.1-floxed siblings. Mice were genotyped for the presence of CD4-Cre allele.

Surgical procedure of CLP

CLP was performed as previously described [19, 37]. Briefly, mice were anesthetized with 5% isoflourane for induction and 3% for maintenance. Fifty percent of the cecum was ligated distal to the ileocecal valve in order to prevent bowel obstruction. The cecum was perforated with a single puncture using an 18-gauge needle (BD Biosciences, San Jose, CA, USA). A small amount of feces (5 mm) was manually extruded from the perforation site into the peritoneal cavity. Lactated Ringers solution plus 5% dextrose for fluid resuscitation were administered by subcutaneous injection in all studies to create a more clinically relevant sepsis model as this is standard care for human patients. Fluid resuscitation was initiated 1.5 h after CLP and received twice daily for 5 days. Antibiotic therapy was withheld. Sham-treated controls underwent the same surgical procedures (i.e. laparotomy and resuscitation), but the cecum was neither ligated nor punctured.

Blood collection, peritoneal content, and aerobic bacteria quantification

Blood, PL, and organ collection were performed as previously described [37]. Bacteremia was determined from whole blood taken from the facial vein. Briefly, the mandible was shaven and cleansed with 70% ethanol to prevent contamination of cultures from bacteria present on the skin surface. The submandibular vein was punctured using a sterile 25-gauge needle for blood collection. A total of 25 μL undiluted whole blood was immediately plated for culture. The local bacterial load in CLP mice was determined by diluting 100 μL of PL with sterile PBS in 10-fold increments to a maximum dilution of 1/105. The peritoneum was lavaged with a total of 5 mL of PBS collected in 1 mL intervals. To remove cellular debris, each milliliter collected was strained using a 70 μm-nylon filter (BD Biosciences). Bacterial counts were also performed on aseptically harvested livers and spleens. Tissues were weighted, homogenized, and debris pelleted. Supernatants were then serially diluted and plated for culture. All samples were plated on sheep blood agar 5% tryptic soy agar monoplates (Remel, Lenexa, KS, USA) and incubated overnight at 37°C under aerobic conditions. CFUs were determined by manual counting and then multiplied by their dilution factor.

Reagents and antibodies

Cytokine levels were determined using IL-6, IL-2, IL-4, IL-10, MIP-2, TNF-α, IFN-γ DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA). All antibodies were from BD Biosciences (Franklin Lakes, NJ, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM HEPES, and 50 μM 2-ME. SEB and LPS were purchased from Sigma-Aldrich (St. Loius, MO, USA).

Lymphocytes isolation and activation

T cells were isolated as described previously [31]. After in vivo activation with SEB, T cells were separated from splenocytes by auto-MACS separator (Miltenyi Biotec, Auburn, CA, USA) using FITC-conjugated anti-CD4, anti-CD8 antibodies (BD Biosciences) and anti-FITC magnetic beads (Miltenyi Biotec) according to manufacturer's protocol. For in vitro activation, T lymphocytes were enriched by Petri dish adhesion and activated by Dynabeads T-cell activator anti-CD3/anti-CD28 magnetic beads according to manufacturer's manual (Invitrogen, Oslo, Norway). After 48 h of activation, T cells were gently separated using Dynamag-2 magnet separator (Invitrogen). For in vivo activation, mice (3–5 per group) were injected i.p. with 100 μL of SEB solution in HBSS (25, 50, or 100 μg per mouse). Bleeding and spleen harvesting were performed either at 3 or 24 h.

Flow cytometry gating strategy

Signals were acquired on FACS calibur flow cytometer (BD Biosciences) using CellQuest Pro Software. Acquisition threshold was set such that platelets, dead cells, and debris were not recorded. Lymphocytes were sequentially defined by gating on forward scatter (FSC)-high/side scatter (SSC)-low, then by staining with anti-CD4 or anti-CD8 antibodies to determine T cells, anti-B220 mAb to determine B cells. NK cells and NKT cells were differentiated using anti-NK1.1 and anti-CD3 mAb. Treg cells were stained with Foxp3 staining kit (eBioscience). Treg cells were defined as CD4+CD25+Foxp3+. For CD39 and CD73 expression, Treg cells were stained as CD4+Foxp3+CD39+CD73+. Macrophages and neutrophils were first gated by FSC-high/SSC-high and then stained with mAb for granulocyte marker GR1 and macrophage marker F4/80. Macrophages were determined as F4/80-positive/GR1-low, neutrophils as F4/80-negative/ GR1-high. FACS was used to analyze cell surface marker expression and determine the phagocytic capabilities of macrophages obtained from the peritoneal cavity of septic mice. PL cells were washed and blocked with CD16/32 for 10 min before staining.

Measurement of intracellular IFN-γ production

Intracellular IFN-γ production was measured by flow cytometry as previously described [31]. Briefly, splenocytes were incubated with 0.01–0.1 μg/mL of soluble anti-CD3 mAb for 24 h, followed by addition of 20 ng/mL PMA (Sigma) and 200 ng/mL A23187 (Sigma) for 2 h. BD Cytofix/Cytoperm Plus kit with Golgi plug (BD Biosciences) was used for intracellular IFN-γ staining.

Phagocytosis assay

To access macrophage-mediated phagocytosis, we used heat-killed fluorescein-conjugated pHrodo Escherichia coli (Invitrogen, Molecular Probes, Carsbad, CA, USA) as previously described [37]. Briefly, pHrodo E. coli were opsonized with purified polyclonal IgG antibodies specific for the E. coli particles according to the manufacturer's instructions. We collected approximately 7.5 × 105 PL cells 5 days post-CLP. Cells were then incubated with opsonized pHrodo E. coli for 45 min at a ratio of 1:20 (PL cell:bacteria). Phagocytosis was carried out according to the manufacturer's instructions in serum-free media to avoid artificial alteration due to the presence of exogenous factors.

LPS activation of peritoneal phagocytes

Peritoneal macrophages and neutrophils were activated as described [54]. Briefly, phagocytic cells were recruited by i.p. injection of 2 mL 3% thioglycollate for 2 days. 5 mg/kg LPS in 1 mL PBS was injected i.p. for 4 h.

RNA isolation, reverse transcription, and qPCR analysis

RNA was extracted using RNA-STAT-60 (Tel-Test, Friendswood, TX, USA) according to manufacturer's protocol, and cDNA was synthesized using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Real-time PCR was performed using SYBR Green mix (ABI Prism) and L32 mRNA as a housekeeping gene reference. Relative amounts of I.1 mRNA were evaluated using comparative Ct analysis as previously described [31]. Analysis of IL-2, IL-4, IL-10, TNF-α, CCL17, and iNOS mRNA was performed using RT2 qPCR Primer Assay (SA Biosciences, Frederick, MD, USA).

Statistical analysis

Comparison of survival curves was estimated using log-rank (Mantel-Cox) test. Significance between two groups was calculated using nonpaired Student t-test or two-way ANOVA (GraphPad Prism 5.0). All values are expressed in mean ± SEM. Two-tailed levels of significance are indicated by * p < 0.05; ** p < 0.01.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

This work was supported by NIH grant No. R21 AI068816–01A1. We thank Susan Ohman and Tehya Johnson for helpful suggestion in the preparation of this manuscript.

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information
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Abbreviations
CLP

cecum ligation and puncture

HIF-1α

hypoxia-inducible factor 1α

I.1CD4

I.1-lox/lox_CD4Cre+/− mice

I.1-KO

I.1-deficient mice

PL

peritoneal lavage

RT-qPCR

reverse transcription-quantitative polymerase chain reaction

SAg

superantigen

SEB

Staphylococcus aureus enterotoxin B

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Conflict of interests
  9. References
  10. Supporting Information

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eji2515-sup-0001-Figures.pdf941K

Figure S1. Expression of I.1 isoform in peritoneal cells.

Figure S2. Scheme of T-cell specific knockout of I.1 isoform.

Figure S3. Intraperitoneal cytokine concentrations in I.1- CD4 T-cell conditional knockdown vs. WT mice after CLP.

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