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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

Roles of macrophages and T lymphocytes in intracerebral hemorrhage (ICH)–induced inflammation. A, Absolute numbers of infiltrating macrophages on day 1 and day 4 after ICH. B, Absolute numbers of infiltrating T lymphocytes on day 4 after ICH in clodronate liposomes (CLP)–treated or liposome (vehicle)‐treated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs vehicle. C, Neurologic deficit score (NDS) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=6 per group. D, Brain water content (BWC) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=4 per group. Two‐way ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). E and F, Absolute numbers of infiltrating macrophages and T lymphocytes on day 4 after ICH in fingolimod‐treated or untreated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. *P<0.05 vs vehicle. G, NDS for vehicle (dimethyl sulfoxide [DMSO])‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=6 per group. Two‐ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). H, BWC for vehicle (DMSO)‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=4 per group. I, NDS for wild‐type (WT), Rag1−/−, and Rag1−/− mice that received CD3+ T lymphocytes from WT mice (WT CD3→Rag1−/−) at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, 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 genotypes and time points (P>0.05). J, BWC for WT, Rag1−/−, and WT CD3→Rag1−/− mice at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, n=5.
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jah31823-fig-0002: Roles of macrophages and T lymphocytes in intracerebral hemorrhage (ICH)–induced inflammation. A, Absolute numbers of infiltrating macrophages on day 1 and day 4 after ICH. B, Absolute numbers of infiltrating T lymphocytes on day 4 after ICH in clodronate liposomes (CLP)–treated or liposome (vehicle)‐treated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs vehicle. C, Neurologic deficit score (NDS) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=6 per group. D, Brain water content (BWC) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=4 per group. Two‐way ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). E and F, Absolute numbers of infiltrating macrophages and T lymphocytes on day 4 after ICH in fingolimod‐treated or untreated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. *P<0.05 vs vehicle. G, NDS for vehicle (dimethyl sulfoxide [DMSO])‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=6 per group. Two‐ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). H, BWC for vehicle (DMSO)‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=4 per group. I, NDS for wild‐type (WT), Rag1−/−, and Rag1−/− mice that received CD3+ T lymphocytes from WT mice (WT CD3→Rag1−/−) at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, 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 genotypes and time points (P>0.05). J, BWC for WT, Rag1−/−, and WT CD3→Rag1−/− mice at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, n=5.

Mentions: Because the absolute numbers of macrophages peaked on day 1 after ICH, we first explored whether macrophage infiltration of the brain is required for ICH‐induced injury by depleting peripheral macrophages using CLPs. In these liposomes, clodronate is encapsulated at a concentration of 7 mg/mL, and systemic administration with a dose of 0.2 mL/20 to 25 g has been demonstrated to achieve efficient depletion of macrophages within 24 to 36 hours.32, 33 We confirmed depletion of 76.2% of F4/80+ macrophages in the spleen of ICH mice at 4 days after the first CLP injection (Figure S2). Intraperitoneal injection of CLPs also significantly reduced the number of infiltrating macrophages in the brain at 1 day and 4 days after ICH (Figure 2A), without influencing the infiltration of T lymphocytes (Figure 2B). Moreover, we found that CLP injection significantly reduced the NDS (Figure 2C) and BWC (Figure 2D) of WT mice with ICH. These findings that macrophage depletion alleviated ICH‐induced brain damage in mice suggest that macrophage infiltration plays a key role in ICH‐induced brain injury.


Interleukin ‐ 23 Secreted by Activated Macrophages Drives γ δ T Cell Production of Interleukin ‐ 17 to Aggravate Secondary Injury After Intracerebral Hemorrhage
Roles of macrophages and T lymphocytes in intracerebral hemorrhage (ICH)–induced inflammation. A, Absolute numbers of infiltrating macrophages on day 1 and day 4 after ICH. B, Absolute numbers of infiltrating T lymphocytes on day 4 after ICH in clodronate liposomes (CLP)–treated or liposome (vehicle)‐treated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs vehicle. C, Neurologic deficit score (NDS) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=6 per group. D, Brain water content (BWC) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=4 per group. Two‐way ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). E and F, Absolute numbers of infiltrating macrophages and T lymphocytes on day 4 after ICH in fingolimod‐treated or untreated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. *P<0.05 vs vehicle. G, NDS for vehicle (dimethyl sulfoxide [DMSO])‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=6 per group. Two‐ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). H, BWC for vehicle (DMSO)‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=4 per group. I, NDS for wild‐type (WT), Rag1−/−, and Rag1−/− mice that received CD3+ T lymphocytes from WT mice (WT CD3→Rag1−/−) at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, 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 genotypes and time points (P>0.05). J, BWC for WT, Rag1−/−, and WT CD3→Rag1−/− mice at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, n=5.
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jah31823-fig-0002: Roles of macrophages and T lymphocytes in intracerebral hemorrhage (ICH)–induced inflammation. A, Absolute numbers of infiltrating macrophages on day 1 and day 4 after ICH. B, Absolute numbers of infiltrating T lymphocytes on day 4 after ICH in clodronate liposomes (CLP)–treated or liposome (vehicle)‐treated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. **P<0.01 vs vehicle. C, Neurologic deficit score (NDS) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=6 per group. D, Brain water content (BWC) at 1, 4, and 7 days after ICH in the CLP‐ and vehicle‐treated mice. *P<0.05 vs vehicle, n=4 per group. Two‐way ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). E and F, Absolute numbers of infiltrating macrophages and T lymphocytes on day 4 after ICH in fingolimod‐treated or untreated mice. Data were obtained for samples pooled from 5 mice, and the experiments were repeated 3 times. *P<0.05 vs vehicle. G, NDS for vehicle (dimethyl sulfoxide [DMSO])‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=6 per group. Two‐ANOVA reported a significant difference in main effects of all treatment groups (P<0.05) but not of time points (P>0.05), there was no interaction between treatments and time points (P>0.05). H, BWC for vehicle (DMSO)‐, fingolimod‐, and fingolimod+R‐treated mice at 1, 4, and 7 days after ICH. *P<0.05 vs vehicle, n=4 per group. I, NDS for wild‐type (WT), Rag1−/−, and Rag1−/− mice that received CD3+ T lymphocytes from WT mice (WT CD3→Rag1−/−) at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, 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 genotypes and time points (P>0.05). J, BWC for WT, Rag1−/−, and WT CD3→Rag1−/− mice at 1, 4, and 7 days after ICH. #P<0.05 vs WT mice, *P<0.05 vs Rag1−/− mice, n=5.
Mentions: Because the absolute numbers of macrophages peaked on day 1 after ICH, we first explored whether macrophage infiltration of the brain is required for ICH‐induced injury by depleting peripheral macrophages using CLPs. In these liposomes, clodronate is encapsulated at a concentration of 7 mg/mL, and systemic administration with a dose of 0.2 mL/20 to 25 g has been demonstrated to achieve efficient depletion of macrophages within 24 to 36 hours.32, 33 We confirmed depletion of 76.2% of F4/80+ macrophages in the spleen of ICH mice at 4 days after the first CLP injection (Figure S2). Intraperitoneal injection of CLPs also significantly reduced the number of infiltrating macrophages in the brain at 1 day and 4 days after ICH (Figure 2A), without influencing the infiltration of T lymphocytes (Figure 2B). Moreover, we found that CLP injection significantly reduced the NDS (Figure 2C) and BWC (Figure 2D) of WT mice with ICH. These findings that macrophage depletion alleviated ICH‐induced brain damage in mice suggest that macrophage infiltration plays a key role in ICH‐induced brain injury.

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