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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

Hemoglobin (Hb)‐induced toll‐like receptor (TLR) 2/4 heterodimer formation on infiltrating macrophages. A, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in F4/80+ cells of perihematomal tissues at 1 day after intracerebral hemorrhage (ICH; scale bars=20 μm). B, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in cultured bone marrow (BM)–derived dendritic cells (BMDCs) after stimulation with Hb (5 μmol/L) for 3 hours (scale bars=10 μm). C, Coprecipitation of TLR2 and TLR4 on cultured BMDCs stimulated with components of red blood cells including Hb, hemin, bilirubin (Bili), Fe2+, and Fe3+, each at a concentration of 5 μmol/L for 3 hours (n=3). D, Coprecipitation of TLR2 and TLR4 on brain infiltrating macrophages or macrophages separated from spleen at 1 day after ICH. Data were obtained for samples pooled from 10 mice, and the experiments were repeated 3 times. E, Neurologic deficit score (NDS) for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. TLR2−/−/TLR4−/−BM→WT represents transfer of TLR2/TLR4 double‐knockout BM cells into wild‐type (WT) mice. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. Two‐way ANOVA reported significant difference in main effects of BM‐chimeric (P<0.05) but not of time points (P>0.05), there was no interaction between BM‐chiemeric and time points (P>0.05). F, Brain water content for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. G, Representative immunofluorescence staining images showing βIII tubulin and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) double‐positive cells in the perihematoma area. Brain sections obtained on day 4 after ICH (red=tubulin, green=TUNEL, blue=4′‐6‐diamidino‐2‐phenylindole [DAPI], scale bars=20 μm). H, Quantification of βIII tubulin and TUNEL double‐positive cells at 4 days after ICH. *P<0.05 vs WT, ##P<0.01 vs sham, n=5.
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jah31823-fig-0003: Hemoglobin (Hb)‐induced toll‐like receptor (TLR) 2/4 heterodimer formation on infiltrating macrophages. A, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in F4/80+ cells of perihematomal tissues at 1 day after intracerebral hemorrhage (ICH; scale bars=20 μm). B, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in cultured bone marrow (BM)–derived dendritic cells (BMDCs) after stimulation with Hb (5 μmol/L) for 3 hours (scale bars=10 μm). C, Coprecipitation of TLR2 and TLR4 on cultured BMDCs stimulated with components of red blood cells including Hb, hemin, bilirubin (Bili), Fe2+, and Fe3+, each at a concentration of 5 μmol/L for 3 hours (n=3). D, Coprecipitation of TLR2 and TLR4 on brain infiltrating macrophages or macrophages separated from spleen at 1 day after ICH. Data were obtained for samples pooled from 10 mice, and the experiments were repeated 3 times. E, Neurologic deficit score (NDS) for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. TLR2−/−/TLR4−/−BM→WT represents transfer of TLR2/TLR4 double‐knockout BM cells into wild‐type (WT) mice. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. Two‐way ANOVA reported significant difference in main effects of BM‐chimeric (P<0.05) but not of time points (P>0.05), there was no interaction between BM‐chiemeric and time points (P>0.05). F, Brain water content for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. G, Representative immunofluorescence staining images showing βIII tubulin and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) double‐positive cells in the perihematoma area. Brain sections obtained on day 4 after ICH (red=tubulin, green=TUNEL, blue=4′‐6‐diamidino‐2‐phenylindole [DAPI], scale bars=20 μm). H, Quantification of βIII tubulin and TUNEL double‐positive cells at 4 days after ICH. *P<0.05 vs WT, ##P<0.01 vs sham, n=5.

Mentions: Our previous research showed that hemoglobin (Hb) promotes microglial activation by inducing the formation of TLR2/TLR4 heterodimers and then exacerbates brain injury following ICH by amplifying the production of inflammatory cytokines.22 To determine whether Hb has a similar effect on infiltrating macrophages, we observed the colocalization of TLR2 and TLR4 on F4/80+ cells perihematomal in ICH mouse brain tissue by immunohistochemical staining (Figure 3A). However, F4/80+ cells included not only macrophages but also microglia. To clarify whether TLR2 and TLR4 colocalized on macrophages, we stimulated BMDCs with Hb (5 μmol/L, 3 hours) and observed colocalization of TLR2 and TLR4 on these cells (Figure 3B). Furthermore, to investigate the link between TLR2 and TLR4 on macrophages, we cultured BMDCs from WT mice and separated macrophages from the brain or spleen of WT mice at day 1 after ICH. Then immunoprecipitation assays were performed to detect the coprecipitation of TLR2 and TLR4 in those cells. Among the red blood cell components Hb, hemin, bilirubin, Fe2+, and Fe3+, only Hb was found to induce coprecipitation of TLR2 and TLR4 (Figure 3C). Immunoprecipitation assays also revealed that Hb induced coprecipitation of TLR2 and TLR4 on brain‐derived macrophages of ICH mice, but not on spleen‐derived macrophages (Figure 3D). These findings suggest that Hb induces TLR2/TLR4 heterodimer formation on macrophages located specifically in the brain but not in the peripheral region.


Interleukin ‐ 23 Secreted by Activated Macrophages Drives γ δ T Cell Production of Interleukin ‐ 17 to Aggravate Secondary Injury After Intracerebral Hemorrhage
Hemoglobin (Hb)‐induced toll‐like receptor (TLR) 2/4 heterodimer formation on infiltrating macrophages. A, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in F4/80+ cells of perihematomal tissues at 1 day after intracerebral hemorrhage (ICH; scale bars=20 μm). B, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in cultured bone marrow (BM)–derived dendritic cells (BMDCs) after stimulation with Hb (5 μmol/L) for 3 hours (scale bars=10 μm). C, Coprecipitation of TLR2 and TLR4 on cultured BMDCs stimulated with components of red blood cells including Hb, hemin, bilirubin (Bili), Fe2+, and Fe3+, each at a concentration of 5 μmol/L for 3 hours (n=3). D, Coprecipitation of TLR2 and TLR4 on brain infiltrating macrophages or macrophages separated from spleen at 1 day after ICH. Data were obtained for samples pooled from 10 mice, and the experiments were repeated 3 times. E, Neurologic deficit score (NDS) for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. TLR2−/−/TLR4−/−BM→WT represents transfer of TLR2/TLR4 double‐knockout BM cells into wild‐type (WT) mice. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. Two‐way ANOVA reported significant difference in main effects of BM‐chimeric (P<0.05) but not of time points (P>0.05), there was no interaction between BM‐chiemeric and time points (P>0.05). F, Brain water content for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. G, Representative immunofluorescence staining images showing βIII tubulin and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) double‐positive cells in the perihematoma area. Brain sections obtained on day 4 after ICH (red=tubulin, green=TUNEL, blue=4′‐6‐diamidino‐2‐phenylindole [DAPI], scale bars=20 μm). H, Quantification of βIII tubulin and TUNEL double‐positive cells at 4 days after ICH. *P<0.05 vs WT, ##P<0.01 vs sham, n=5.
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jah31823-fig-0003: Hemoglobin (Hb)‐induced toll‐like receptor (TLR) 2/4 heterodimer formation on infiltrating macrophages. A, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in F4/80+ cells of perihematomal tissues at 1 day after intracerebral hemorrhage (ICH; scale bars=20 μm). B, Representative immunofluorescence staining images showing colocalization of TLR2 and TLR4 in cultured bone marrow (BM)–derived dendritic cells (BMDCs) after stimulation with Hb (5 μmol/L) for 3 hours (scale bars=10 μm). C, Coprecipitation of TLR2 and TLR4 on cultured BMDCs stimulated with components of red blood cells including Hb, hemin, bilirubin (Bili), Fe2+, and Fe3+, each at a concentration of 5 μmol/L for 3 hours (n=3). D, Coprecipitation of TLR2 and TLR4 on brain infiltrating macrophages or macrophages separated from spleen at 1 day after ICH. Data were obtained for samples pooled from 10 mice, and the experiments were repeated 3 times. E, Neurologic deficit score (NDS) for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. TLR2−/−/TLR4−/−BM→WT represents transfer of TLR2/TLR4 double‐knockout BM cells into wild‐type (WT) mice. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. Two‐way ANOVA reported significant difference in main effects of BM‐chimeric (P<0.05) but not of time points (P>0.05), there was no interaction between BM‐chiemeric and time points (P>0.05). F, Brain water content for BM‐chimeric mice analyzed at 1, 4, and 7 days after ICH. *P<0.05 vs WT BM→TLR2−/−/TLR4−/− mice, n=4. G, Representative immunofluorescence staining images showing βIII tubulin and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) double‐positive cells in the perihematoma area. Brain sections obtained on day 4 after ICH (red=tubulin, green=TUNEL, blue=4′‐6‐diamidino‐2‐phenylindole [DAPI], scale bars=20 μm). H, Quantification of βIII tubulin and TUNEL double‐positive cells at 4 days after ICH. *P<0.05 vs WT, ##P<0.01 vs sham, n=5.
Mentions: Our previous research showed that hemoglobin (Hb) promotes microglial activation by inducing the formation of TLR2/TLR4 heterodimers and then exacerbates brain injury following ICH by amplifying the production of inflammatory cytokines.22 To determine whether Hb has a similar effect on infiltrating macrophages, we observed the colocalization of TLR2 and TLR4 on F4/80+ cells perihematomal in ICH mouse brain tissue by immunohistochemical staining (Figure 3A). However, F4/80+ cells included not only macrophages but also microglia. To clarify whether TLR2 and TLR4 colocalized on macrophages, we stimulated BMDCs with Hb (5 μmol/L, 3 hours) and observed colocalization of TLR2 and TLR4 on these cells (Figure 3B). Furthermore, to investigate the link between TLR2 and TLR4 on macrophages, we cultured BMDCs from WT mice and separated macrophages from the brain or spleen of WT mice at day 1 after ICH. Then immunoprecipitation assays were performed to detect the coprecipitation of TLR2 and TLR4 in those cells. Among the red blood cell components Hb, hemin, bilirubin, Fe2+, and Fe3+, only Hb was found to induce coprecipitation of TLR2 and TLR4 (Figure 3C). Immunoprecipitation assays also revealed that Hb induced coprecipitation of TLR2 and TLR4 on brain‐derived macrophages of ICH mice, but not on spleen‐derived macrophages (Figure 3D). These findings suggest that Hb induces TLR2/TLR4 heterodimer formation on macrophages located specifically in the brain but not in the peripheral region.

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