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Interleukin ‐ 23 Secreted by Activated Macrophages Drives γ δ T Cell Production of Interleukin ‐ 17 to Aggravate Secondary Injury After Intracerebral Hemorrhage

View Article: PubMed Central - PubMed

ABSTRACT

Background: Neuroinflammation plays a key role in intracerebral hemorrhage (ICH)–induced secondary brain injury, but the specific roles of peripheral inflammatory cells such as macrophages and lymphocytes remain unknown. The purpose of this study was to explore the roles of macrophages, T lymphocytes, and the cytokines they secrete as potential targets for treating secondary brain injury after ICH.

Methods and results: Our results showed that peripheral macrophages and T lymphocytes successively infiltrated the brain, with macrophage counts peaking 1 day after ICH and T‐lymphocyte counts peaking after 4 days. These peaks in cellular infiltration corresponded to increases in interleukin (IL)‐23 and IL‐17 expression, respectively. We found that hemoglobin from the hematoma activated IL‐23 secretion by infiltrating macrophages by inducing the formation of toll‐like receptor (TLR) 2/4 heterodimer. This increased IL‐23 expression stimulated γδT‐cell production of IL‐17, which increased brain edema and neurologic deficits in the model mice as a proinflammatory factor. Finally, we found that sparstolonin B (SsnB) could ameliorate brain edema and neurologic deficits in ICH model mice via inhibition of TLR2/TLR4 heterodimer formation, and notably, SsnB interacted with myeloid differentiation factor 88 Arg196.

Conclusions: Together, our results reveal the importance of the IL‐23/IL‐17 inflammatory axis in secondary brain injury after ICH and thus provide a new therapeutic target for ICH treatment.

No MeSH data available.


Related in: MedlinePlus

γδT‐cell production of interleukin (IL)‐17 after intracerebral hemorrhage (ICH). A, IL‐17+ T lymphocytes identified by fluorescence‐activated cell sorting (FACS) staining at 1, 4, and 7 days after ICH. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times, *P<0.05 vs sham. B, Immunofluorescence staining of CD3+ and IL‐17+‐positive cells in the perihematoma area of wild‐type (WT) mice at 4 days after ICH (scale bars=20 μm). C, Flow cytometric analysis of γδT cell receptor (γδT) and CD4 expression on the CD3+IL‐17+ T cells. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. D and E, Percentages of IL‐17+ cells among infiltrating T lymphocytes of WT, IL‐17−/−, and IL‐23−/− mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs WT. F, Neurologic deficit score (NDS) for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. Two‐way ANOVA reported significant difference in main effects of genotype (P<0.05) but not of time points (P>0.05), there was no interaction between genotype and time points (P>0.05). G, Brain water content for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. H, IL‐1β and tumor necrosis factor‐α (TNF‐α) mRNA levels in infiltrating CD45+ cells of WT and γδT−/− mice at day 4 after ICH. *P<0.05, **P<0.01 vs WT, n=4.
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jah31823-fig-0005: γδT‐cell production of interleukin (IL)‐17 after intracerebral hemorrhage (ICH). A, IL‐17+ T lymphocytes identified by fluorescence‐activated cell sorting (FACS) staining at 1, 4, and 7 days after ICH. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times, *P<0.05 vs sham. B, Immunofluorescence staining of CD3+ and IL‐17+‐positive cells in the perihematoma area of wild‐type (WT) mice at 4 days after ICH (scale bars=20 μm). C, Flow cytometric analysis of γδT cell receptor (γδT) and CD4 expression on the CD3+IL‐17+ T cells. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. D and E, Percentages of IL‐17+ cells among infiltrating T lymphocytes of WT, IL‐17−/−, and IL‐23−/− mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs WT. F, Neurologic deficit score (NDS) for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. Two‐way ANOVA reported significant difference in main effects of genotype (P<0.05) but not of time points (P>0.05), there was no interaction between genotype and time points (P>0.05). G, Brain water content for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. H, IL‐1β and tumor necrosis factor‐α (TNF‐α) mRNA levels in infiltrating CD45+ cells of WT and γδT−/− mice at day 4 after ICH. *P<0.05, **P<0.01 vs WT, n=4.

Mentions: IL‐23 has been shown to induce CD4+ T‐cell differentiation into Th17 cells, and this IL‐23/IL‐17 axis plays a central role in several inflammatory diseases.17, 18, 19, 35, 36 Thus, we examined the role of IL‐17 in ICH‐induced brain injury by intracellular cytokine staining and found enrichment of IL‐17+ cells in the perihematoma tissue at day 4 after ICH (Figure 5A), which corresponds to the peak in T‐lymphocyte infiltration. To examine the source of IL‐17 in the injured brain, we performed immunostaining for various surface markers along with intracellular cytokine staining and found that IL‐17 was colocalized with CD3 in the perihematoma area at 4 days after ICH (Figure 5B), but absent in CD11b+, βIII tubulin+, and GFAP+ cells (data were not shown), indicating that T lymphocytes were likely the source of IL‐17 in ICH brain, which was further confirmed by FACS data (Figure S4). Flow cytometric analysis of γδT and CD4 expression on IL‐17+ CD3+ T cells indicated that most IL‐17–producing T lymphocytes were CD4− γδT+ cells (Figure 5C).


Interleukin ‐ 23 Secreted by Activated Macrophages Drives γ δ T Cell Production of Interleukin ‐ 17 to Aggravate Secondary Injury After Intracerebral Hemorrhage
γδT‐cell production of interleukin (IL)‐17 after intracerebral hemorrhage (ICH). A, IL‐17+ T lymphocytes identified by fluorescence‐activated cell sorting (FACS) staining at 1, 4, and 7 days after ICH. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times, *P<0.05 vs sham. B, Immunofluorescence staining of CD3+ and IL‐17+‐positive cells in the perihematoma area of wild‐type (WT) mice at 4 days after ICH (scale bars=20 μm). C, Flow cytometric analysis of γδT cell receptor (γδT) and CD4 expression on the CD3+IL‐17+ T cells. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. D and E, Percentages of IL‐17+ cells among infiltrating T lymphocytes of WT, IL‐17−/−, and IL‐23−/− mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs WT. F, Neurologic deficit score (NDS) for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. Two‐way ANOVA reported significant difference in main effects of genotype (P<0.05) but not of time points (P>0.05), there was no interaction between genotype and time points (P>0.05). G, Brain water content for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. H, IL‐1β and tumor necrosis factor‐α (TNF‐α) mRNA levels in infiltrating CD45+ cells of WT and γδT−/− mice at day 4 after ICH. *P<0.05, **P<0.01 vs WT, n=4.
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jah31823-fig-0005: γδT‐cell production of interleukin (IL)‐17 after intracerebral hemorrhage (ICH). A, IL‐17+ T lymphocytes identified by fluorescence‐activated cell sorting (FACS) staining at 1, 4, and 7 days after ICH. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times, *P<0.05 vs sham. B, Immunofluorescence staining of CD3+ and IL‐17+‐positive cells in the perihematoma area of wild‐type (WT) mice at 4 days after ICH (scale bars=20 μm). C, Flow cytometric analysis of γδT cell receptor (γδT) and CD4 expression on the CD3+IL‐17+ T cells. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. D and E, Percentages of IL‐17+ cells among infiltrating T lymphocytes of WT, IL‐17−/−, and IL‐23−/− mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs WT. F, Neurologic deficit score (NDS) for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. Two‐way ANOVA reported significant difference in main effects of genotype (P<0.05) but not of time points (P>0.05), there was no interaction between genotype and time points (P>0.05). G, Brain water content for WT and γδT−/− mice as analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT, n=5. H, IL‐1β and tumor necrosis factor‐α (TNF‐α) mRNA levels in infiltrating CD45+ cells of WT and γδT−/− mice at day 4 after ICH. *P<0.05, **P<0.01 vs WT, n=4.
Mentions: IL‐23 has been shown to induce CD4+ T‐cell differentiation into Th17 cells, and this IL‐23/IL‐17 axis plays a central role in several inflammatory diseases.17, 18, 19, 35, 36 Thus, we examined the role of IL‐17 in ICH‐induced brain injury by intracellular cytokine staining and found enrichment of IL‐17+ cells in the perihematoma tissue at day 4 after ICH (Figure 5A), which corresponds to the peak in T‐lymphocyte infiltration. To examine the source of IL‐17 in the injured brain, we performed immunostaining for various surface markers along with intracellular cytokine staining and found that IL‐17 was colocalized with CD3 in the perihematoma area at 4 days after ICH (Figure 5B), but absent in CD11b+, βIII tubulin+, and GFAP+ cells (data were not shown), indicating that T lymphocytes were likely the source of IL‐17 in ICH brain, which was further confirmed by FACS data (Figure S4). Flow cytometric analysis of γδT and CD4 expression on IL‐17+ CD3+ T cells indicated that most IL‐17–producing T lymphocytes were CD4− γδT+ cells (Figure 5C).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Neuroinflammation plays a key role in intracerebral hemorrhage (ICH)&ndash;induced secondary brain injury, but the specific roles of peripheral inflammatory cells such as macrophages and lymphocytes remain unknown. The purpose of this study was to explore the roles of macrophages, T lymphocytes, and the cytokines they secrete as potential targets for treating secondary brain injury after ICH.

Methods and results: Our results showed that peripheral macrophages and T lymphocytes successively infiltrated the brain, with macrophage counts peaking 1&nbsp;day after ICH and T&#8208;lymphocyte counts peaking after 4&nbsp;days. These peaks in cellular infiltration corresponded to increases in interleukin (IL)&#8208;23 and IL&#8208;17 expression, respectively. We found that hemoglobin from the hematoma activated IL&#8208;23 secretion by infiltrating macrophages by inducing the formation of toll&#8208;like receptor (TLR) 2/4 heterodimer. This increased IL&#8208;23 expression stimulated &gamma;&delta;T&#8208;cell production of IL&#8208;17, which increased brain edema and neurologic deficits in the model mice as a proinflammatory factor. Finally, we found that sparstolonin B (SsnB) could ameliorate brain edema and neurologic deficits in ICH model mice via inhibition of TLR2/TLR4 heterodimer formation, and notably, SsnB interacted with myeloid differentiation factor 88 Arg196.

Conclusions: Together, our results reveal the importance of the IL&#8208;23/IL&#8208;17 inflammatory axis in secondary brain injury after ICH and thus provide a new therapeutic target for ICH treatment.

No MeSH data available.


Related in: MedlinePlus