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Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation.

Cao M, Cortes M, Moore CS, Leong SY, Durosier LD, Burns P, Fecteau G, Desrochers A, Auer RN, Barreiro LB, Antel JP, Frasch MG - Front Cell Neurosci (2015)

Bottom Line: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure.We identified a unique HMOX1 down and FBP (up) phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways.Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynaecology, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada ; Department of Neurosciences, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada.

ABSTRACT

Objective: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood.

Methods: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.

Results: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.

Discussion: We identified a unique HMOX1 down and FBP (up) phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

No MeSH data available.


Related in: MedlinePlus

Purity validation of fetal sheep brain primary microglia in cultures and LPS second hit. (A) Photomicrographs (ICC) confirming cell purity. Iba1+ staining in microglia vs. undetectable GFAP signal in microglia indicating no contamination with astrocytes in the culture. Microglia were cultured in eight-well chamber slide with DMEM +5% FCS for 4–7 days. Microglia were collected from the floating fraction and stained for Iba1 and GFAP. Scale bar = 50 μm. Magnification 40× for both images. (B) Purity of fetal sheep brain primary microglia cultures was verified by flowcytometry. After several days in culture, fetal sheep microglia were scraped from the wells using a cell scraper and blocked for 30 min using normal mouse IgG and 10% human serum. Cells were then stained using a FITC-conjugated monoclonal bovine anti-CD11b (1:40, Bio-Rad) on ice for 20 min. Cells were washed in FACS buffer and analyzed using a BD FACSCalibur and FlowJo software. (C) Microglia from in vivo LPS exposed brain appear more aggregated in vitro than microglia derived from in vivo controls. Microglia were cultured in 24-well plates with DMEM +5% FCS for 4–7 days, when images were taken. Cells were extracted from a twin control fetal brain and an in vivo LPS exposed fetal brain (Magnification 20× for both images). (D) Effect of “second-hit” in vitro LPS treatment on microglial phenotype. BOTTOM: IL-1β concentration in conditioned media of microglia derived from fetal sheep brain that were exposed to LPS vs. saline in vivo (***P < 0.0001). Cultures from in vivo LPS-exposed (SH), n = 4, cultures from in vivo Control (Naïve), n = 10. Cell culture media supernatant was obtained by centrifugation upon cell culture termination. A sheep specific IL-1β ELISA was performed to measure cytokine levels in cell culture media. In vitro, at baseline, microglia secreted more IL-1β in the in vivo LPS group (SHC) than in naïve Control (NC). LPS re-exposure (SHL) further increased IL-1β vs. naïve LPS (NL) by ~4.6-fold.
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Figure 2: Purity validation of fetal sheep brain primary microglia in cultures and LPS second hit. (A) Photomicrographs (ICC) confirming cell purity. Iba1+ staining in microglia vs. undetectable GFAP signal in microglia indicating no contamination with astrocytes in the culture. Microglia were cultured in eight-well chamber slide with DMEM +5% FCS for 4–7 days. Microglia were collected from the floating fraction and stained for Iba1 and GFAP. Scale bar = 50 μm. Magnification 40× for both images. (B) Purity of fetal sheep brain primary microglia cultures was verified by flowcytometry. After several days in culture, fetal sheep microglia were scraped from the wells using a cell scraper and blocked for 30 min using normal mouse IgG and 10% human serum. Cells were then stained using a FITC-conjugated monoclonal bovine anti-CD11b (1:40, Bio-Rad) on ice for 20 min. Cells were washed in FACS buffer and analyzed using a BD FACSCalibur and FlowJo software. (C) Microglia from in vivo LPS exposed brain appear more aggregated in vitro than microglia derived from in vivo controls. Microglia were cultured in 24-well plates with DMEM +5% FCS for 4–7 days, when images were taken. Cells were extracted from a twin control fetal brain and an in vivo LPS exposed fetal brain (Magnification 20× for both images). (D) Effect of “second-hit” in vitro LPS treatment on microglial phenotype. BOTTOM: IL-1β concentration in conditioned media of microglia derived from fetal sheep brain that were exposed to LPS vs. saline in vivo (***P < 0.0001). Cultures from in vivo LPS-exposed (SH), n = 4, cultures from in vivo Control (Naïve), n = 10. Cell culture media supernatant was obtained by centrifugation upon cell culture termination. A sheep specific IL-1β ELISA was performed to measure cytokine levels in cell culture media. In vitro, at baseline, microglia secreted more IL-1β in the in vivo LPS group (SHC) than in naïve Control (NC). LPS re-exposure (SHL) further increased IL-1β vs. naïve LPS (NL) by ~4.6-fold.

Mentions: Complete brain was taken from the fetus during necropsy after perfusion and immediately immersed in 4% PFA for 48–72 h. The tissue sample was then washed and stored in 1× PBS buffer changed daily for 3 days. Finally, the brain was stored in 70% ethanol until further processing. All the brain tissue samples were kept at 4°C when they were in liquid. The fetal brains were cut into two equal halves of left and right hemispheres, and then sliced coronally and placed into cassettes to be processed with Leica TP 1020 Automatic Tissue Processor (Leica Instruments, Mussloch, Germany). The tissues were embedded in paraffin with Leica EG 1160 Paraffin Embedding Center (Leica Instruments, Mussloch, Germany). Five-micrometer slices were obtained from slicing the embedded tissue samples with the Leica RM2145 Rotary Microtome (Leica Instruments, Mussloch, Germany), and mounted on the Fisherbrand Colorfrost Plus microscope slides (Fischer Scientific). The sectioned brain tissue samples went through de-paraffinization with CitroSolv (Fischer Scientific), 100, 95, 70, and 50% ethanol at room temperature, and antigen retrieval with 10 mM citrate buffer at pH 6 before being washed with water and 1× PBS, and blocked by Background Sniper Blocking Reagent (Biocare Medical, Cat. No BS966JJ). Then the sections were incubated with the primary antibody (Iba1, rabbit polyclonal antibody 4, 1:250 dilution, Wako, Cat No. 019-19741) for 1 h, followed by washing with 1× PBS and incubation with secondary antibody (Alexa Fluor 568 goat anti-rabbit IgG, 1:400 dilution, Life Technologies, Cat no A-11011) for 30 min in the dark. After that, the sections were washed again with 1× PBS, and the nuclei were counterstained with DAPI (1:4000 dilution, Sigma D-9564). Finally, the sections were cover-slipped with Fisherfinest Premium Cover glass (22 × 50-1, Fisher Scientific) and Fluoromount-G (SouthernBiotech, Cat no 0100-01) mounting medium, and viewed after 24 h of drying. Widefield fluorescence microscopy was performed on the stained brain tissue samples with a Zeiss Axiovert 200 M inverted microscope (Jena, Germany), at the magnification of 40× using a HBO100 mercury-arc lamp as a light source. The images were captured using a Zeiss Axiocam HRm (high-resolution monochrome) CCD (charged-coupled device) camera. Six high power field (HPF) images at 40× magnification were obtained for each animal. Multichannel imaging was used with the Iba1 channel and the DAPI channel for obtaining the pictures used for macrophage quantification. Appropriate ranges of color were selected showing positive contiguous cytoplasmic staining as a criterion for microglia cell count scoring which were then applied uniformly to calibrated images for all brain regions (Figure 2). Scoring was performed in a blinded fashion to experimental groups. To normalize for cell density Iba1+ signal over the whole area measured (100 sq micron) was divided by the respective optical intensity values for each HPF according to Lin et al. (2000).


Fetal microglial phenotype in vitro carries memory of prior in vivo exposure to inflammation.

Cao M, Cortes M, Moore CS, Leong SY, Durosier LD, Burns P, Fecteau G, Desrochers A, Auer RN, Barreiro LB, Antel JP, Frasch MG - Front Cell Neurosci (2015)

Purity validation of fetal sheep brain primary microglia in cultures and LPS second hit. (A) Photomicrographs (ICC) confirming cell purity. Iba1+ staining in microglia vs. undetectable GFAP signal in microglia indicating no contamination with astrocytes in the culture. Microglia were cultured in eight-well chamber slide with DMEM +5% FCS for 4–7 days. Microglia were collected from the floating fraction and stained for Iba1 and GFAP. Scale bar = 50 μm. Magnification 40× for both images. (B) Purity of fetal sheep brain primary microglia cultures was verified by flowcytometry. After several days in culture, fetal sheep microglia were scraped from the wells using a cell scraper and blocked for 30 min using normal mouse IgG and 10% human serum. Cells were then stained using a FITC-conjugated monoclonal bovine anti-CD11b (1:40, Bio-Rad) on ice for 20 min. Cells were washed in FACS buffer and analyzed using a BD FACSCalibur and FlowJo software. (C) Microglia from in vivo LPS exposed brain appear more aggregated in vitro than microglia derived from in vivo controls. Microglia were cultured in 24-well plates with DMEM +5% FCS for 4–7 days, when images were taken. Cells were extracted from a twin control fetal brain and an in vivo LPS exposed fetal brain (Magnification 20× for both images). (D) Effect of “second-hit” in vitro LPS treatment on microglial phenotype. BOTTOM: IL-1β concentration in conditioned media of microglia derived from fetal sheep brain that were exposed to LPS vs. saline in vivo (***P < 0.0001). Cultures from in vivo LPS-exposed (SH), n = 4, cultures from in vivo Control (Naïve), n = 10. Cell culture media supernatant was obtained by centrifugation upon cell culture termination. A sheep specific IL-1β ELISA was performed to measure cytokine levels in cell culture media. In vitro, at baseline, microglia secreted more IL-1β in the in vivo LPS group (SHC) than in naïve Control (NC). LPS re-exposure (SHL) further increased IL-1β vs. naïve LPS (NL) by ~4.6-fold.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Purity validation of fetal sheep brain primary microglia in cultures and LPS second hit. (A) Photomicrographs (ICC) confirming cell purity. Iba1+ staining in microglia vs. undetectable GFAP signal in microglia indicating no contamination with astrocytes in the culture. Microglia were cultured in eight-well chamber slide with DMEM +5% FCS for 4–7 days. Microglia were collected from the floating fraction and stained for Iba1 and GFAP. Scale bar = 50 μm. Magnification 40× for both images. (B) Purity of fetal sheep brain primary microglia cultures was verified by flowcytometry. After several days in culture, fetal sheep microglia were scraped from the wells using a cell scraper and blocked for 30 min using normal mouse IgG and 10% human serum. Cells were then stained using a FITC-conjugated monoclonal bovine anti-CD11b (1:40, Bio-Rad) on ice for 20 min. Cells were washed in FACS buffer and analyzed using a BD FACSCalibur and FlowJo software. (C) Microglia from in vivo LPS exposed brain appear more aggregated in vitro than microglia derived from in vivo controls. Microglia were cultured in 24-well plates with DMEM +5% FCS for 4–7 days, when images were taken. Cells were extracted from a twin control fetal brain and an in vivo LPS exposed fetal brain (Magnification 20× for both images). (D) Effect of “second-hit” in vitro LPS treatment on microglial phenotype. BOTTOM: IL-1β concentration in conditioned media of microglia derived from fetal sheep brain that were exposed to LPS vs. saline in vivo (***P < 0.0001). Cultures from in vivo LPS-exposed (SH), n = 4, cultures from in vivo Control (Naïve), n = 10. Cell culture media supernatant was obtained by centrifugation upon cell culture termination. A sheep specific IL-1β ELISA was performed to measure cytokine levels in cell culture media. In vitro, at baseline, microglia secreted more IL-1β in the in vivo LPS group (SHC) than in naïve Control (NC). LPS re-exposure (SHL) further increased IL-1β vs. naïve LPS (NL) by ~4.6-fold.
Mentions: Complete brain was taken from the fetus during necropsy after perfusion and immediately immersed in 4% PFA for 48–72 h. The tissue sample was then washed and stored in 1× PBS buffer changed daily for 3 days. Finally, the brain was stored in 70% ethanol until further processing. All the brain tissue samples were kept at 4°C when they were in liquid. The fetal brains were cut into two equal halves of left and right hemispheres, and then sliced coronally and placed into cassettes to be processed with Leica TP 1020 Automatic Tissue Processor (Leica Instruments, Mussloch, Germany). The tissues were embedded in paraffin with Leica EG 1160 Paraffin Embedding Center (Leica Instruments, Mussloch, Germany). Five-micrometer slices were obtained from slicing the embedded tissue samples with the Leica RM2145 Rotary Microtome (Leica Instruments, Mussloch, Germany), and mounted on the Fisherbrand Colorfrost Plus microscope slides (Fischer Scientific). The sectioned brain tissue samples went through de-paraffinization with CitroSolv (Fischer Scientific), 100, 95, 70, and 50% ethanol at room temperature, and antigen retrieval with 10 mM citrate buffer at pH 6 before being washed with water and 1× PBS, and blocked by Background Sniper Blocking Reagent (Biocare Medical, Cat. No BS966JJ). Then the sections were incubated with the primary antibody (Iba1, rabbit polyclonal antibody 4, 1:250 dilution, Wako, Cat No. 019-19741) for 1 h, followed by washing with 1× PBS and incubation with secondary antibody (Alexa Fluor 568 goat anti-rabbit IgG, 1:400 dilution, Life Technologies, Cat no A-11011) for 30 min in the dark. After that, the sections were washed again with 1× PBS, and the nuclei were counterstained with DAPI (1:4000 dilution, Sigma D-9564). Finally, the sections were cover-slipped with Fisherfinest Premium Cover glass (22 × 50-1, Fisher Scientific) and Fluoromount-G (SouthernBiotech, Cat no 0100-01) mounting medium, and viewed after 24 h of drying. Widefield fluorescence microscopy was performed on the stained brain tissue samples with a Zeiss Axiovert 200 M inverted microscope (Jena, Germany), at the magnification of 40× using a HBO100 mercury-arc lamp as a light source. The images were captured using a Zeiss Axiocam HRm (high-resolution monochrome) CCD (charged-coupled device) camera. Six high power field (HPF) images at 40× magnification were obtained for each animal. Multichannel imaging was used with the Iba1 channel and the DAPI channel for obtaining the pictures used for macrophage quantification. Appropriate ranges of color were selected showing positive contiguous cytoplasmic staining as a criterion for microglia cell count scoring which were then applied uniformly to calibrated images for all brain regions (Figure 2). Scoring was performed in a blinded fashion to experimental groups. To normalize for cell density Iba1+ signal over the whole area measured (100 sq micron) was divided by the respective optical intensity values for each HPF according to Lin et al. (2000).

Bottom Line: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure.We identified a unique HMOX1 down and FBP (up) phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways.Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynaecology, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada ; Department of Neurosciences, Faculty of Medicine, CHU Ste-Justine Research Centre, Université de Montréal Montréal, QC, Canada.

ABSTRACT

Objective: Neuroinflammation in utero may result in life-long neurological disabilities. The molecular mechanisms whereby microglia contribute to this response remain incompletely understood.

Methods: Lipopolysaccharide (LPS) or saline were administered intravenously to non-anesthetized chronically instrumented near-term fetal sheep to model fetal inflammation in vivo. Microglia were then isolated from in vivo LPS and saline (naïve) exposed animals. To mimic the second hit of neuroinflammation, these microglia were then re-exposed to LPS in vitro. Cytokine responses were measured in vivo and subsequently in vitro in the primary microglia cultures derived from these animals. We sequenced the whole transcriptome of naïve and second hit microglia and profiled their genetic expression to define molecular pathways disrupted during neuroinflammation.

Results: In vivo LPS exposure resulted in IL-6 increase in fetal plasma 3 h post LPS exposure. Even though not histologically apparent, microglia acquired a pro-inflammatory phenotype in vivo that was sustained and amplified in vitro upon second hit LPS exposure as measured by IL-1β response in vitro and RNAseq analyses. While NFKB and Jak-Stat inflammatory pathways were up regulated in naïve microglia, heme oxygenase 1 (HMOX1) and Fructose-1,6-bisphosphatase (FBP) genes were uniquely differentially expressed in the second hit microglia. Compared to the microglia exposed to LPS in vitro only, the transcriptome of the in vivo LPS pre-exposed microglia showed a diminished differential gene expression in inflammatory and metabolic pathways prior and upon re-exposure to LPS in vitro. Notably, this desensitization response was also observed in histone deacetylases (HDAC) 1, 2, 4, and 6. Microglial calreticulin/LRP genes implicated in microglia-neuronal communication relevant for the neuronal development were up regulated in second hit microglia.

Discussion: We identified a unique HMOX1 down and FBP (up) phenotype of microglia exposed to the double-hit suggesting interplay of inflammatory and metabolic pathways. Our findings suggest that epigenetic mechanisms mediate this immunological and metabolic memory of the prior inflammatory insult relevant to neuronal development and provide new therapeutic targets for early postnatal intervention to prevent brain injury.

No MeSH data available.


Related in: MedlinePlus