LPS-induced complex polarization of astrocytes and microglia in rats

doi:10.21203/rs.3.rs-1522218/v1

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LPS-induced complex polarization of astrocytes and microglia in rats

https://doi.org/10.21203/rs.3.rs-1522218/v1

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A1 astrocytes are activated through M1 microglial secretion, which often be induced via 0.1 mg/kg systemic lipopolysaccharides (LPS). While a larger dose of 5 mg/kg LPS, similar to the dose used for liver injury or sepsis model, was adopted to induce A1. This study was designed to investigate a safer and effective LPS dose to induce A1, and the synchronized changes of other glial phenotypes since both astrocytes and microglia can be activated by LPS. Gene expression changes in 72 specific phenotypic markers of astrocytes and microglia were tested following the administration of systemic LPS ranging from 0.1 to 5 mg/kg, after exposure for 24 h in the cortex, hippocampus and spinal cord, using methods including RT-PCR, co-immunofluorescence, and single-cell RNA sequence. The results showed that anti-inflammatory A2 and M2 phenotypes were also activated by LPS, not the pro-inflammatory A1, M1, and A-pan phenotypes alone. But more A1 than A2 were activated even with low-dose LPS exposure. With respect to microglia, LPS induced a mild and equal level of M1 and M2 activation, indicating 24 h exposure is not proper to stimulate M1. Only several activated markers like C3, Marco and Ym1 showed good consistency among tissues with various dose of LPS. Collectively, systemic LPS induced a rather complex polarization of A1, A2, A-pan, M1, and M2. A smaller LPS dose like 1 or 3 mg/kg could be available to induce A1, and selection of phenotypic markers should be optimized by considering the LPS dose and target tissue.

Systemic lipopolysaccharide

Glial phenotype

Phenotypic marker

A1 astrocytes

M1 microglia

Activated astrocytes and microglia play a crucial role in neuroinflammation known to be involved in neurodegenerative diseases, neuropsychiatric disorders, and brain trauma [1, 2]. Similar to microglia, astrocytes have recently been found to play a dual role too. Lipopolysaccharides (LPS) promotes neurotoxic astrocytic polarization termed the A1 phenotype, while ischemia induced by middle cerebral artery occlusion (MCAO) leads to neuroprotective A2 astrocytic polarization [3]. Among the few methods available to induce A1 astrocytes, systemic administration of LPS at 5 mg/kg is often employed in mice [4, 5], but over 60% fatality rate was observed 24 h after injection, with the pretreatment before LPS ineffective. Thus, we speculated whether the maximum lethal LPS often used to create liver failure and sepsis model [6, 7], was optimal in inducing A1 polarization.

Microglia are more sensitive to pathogens or damage than astrocytes. They can be stimulated by LPS or interferon (IFN)-γ to an M1 phenotype expressing pro-inflammatory cytokines, or by interleukin (IL-4) or IL-13 to an M2 phenotype to resolve inflammation and for tissue repair [8–10]. Furthermore, A1 astrocytes induced by LPS were found to be activated through microglial secretion of neuroinflammatory cytokines such as tumor necrosis factor (TNF)-α, IL-1α, and complement component C1q [4]. Interestingly, the dose of LPS used to induce M1 activation was often much smaller than 5 mg/kg, although it ranged widely [11–13]. Whether a smaller LPS dose is effective to induce A1 astrocytes, followed M1 activation remains to be determined.

Each phenotype of astrocytes or microglia has multiple markers, and the reported markers often vary [9, 14, 15]. In a study performed by Xu et al., TNF-α and IL-1β were used as M1 markers, and Cd206, Ym1/2, and Arg1 were used as M2 markers in the cerebral cortex [14]. In a study by Wang et al., Cd16, TNF-α, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein (MCP)-1 were used as M1 markers, and Cd206, transforming growth factor (TGF)-β, Arg1, and Ym1 as M2 characteristic markers in the hippocampus [15]. It is unclear whether these phenotypic markers are universal in LPS-induced microglial activation, if there are differences in tissues, and if there is a dose-response relationship between markers and level of LPS-induced inflammation.

Therefore, we included 72 phenotypic markers in 3 central nervous tissues (CNTs), stimulated with 6 doses of systemic LPS to recognize the polarization of astrocytes and microglia phenotypes. By comparing the similarities and differences in three dimensions, we expect to obtain more specific information about phenotypic polarization and marker selection.

Animals

Adult male Sprague-Dawley rats weighting 250–300 g used in this study were provided by the Experimental Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). Rats were housed at 23 ± 1°C with a 12 h light-dark cycle. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences (Beijing, China), and were performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Rats were randomly divided into 6 groups (0, 0.1, 0.33, 1, 3, and 5 mg/kg LPS), n = 8.

Lps Treatment

LPS from Escherichia coli 055:B5 (dissolved in endotoxin-free PBS) was purified by phenol extraction (L2880, Sigma-Aldrich, St. Louis, MO). The animals were administered with 0.1, 0.33, 1, 3 and 5 mg/kg LPS intraperitoneally. LPS volume was diluted to 500 µl with endotoxin-free PBS for each injection. In the control group, 500 µl endotoxin-free PBS was injected intraperitoneally. Rats were sacrificed with an overdose of anesthetics 24 h after LPS treatment, and the cerebral cortex, hippocampus and spinal cord were extracted.

RT-PCR

The cerebral cortex from the same group were pooled and total RNA was extracted with Trizol regent (15596026, Invitrogen, Carlsbad, CA) using ultrasonic cracking, and reverse transcribed with Prime Script RT Master Mix (RR036, Takara, Otsu, Japan) according to the manufacturer's instructions. RT-PCR was performed using SYBR Premix Ex Taq (RR820, Takara) and Step One Real Time PCR System (Applied Biosystems, Foster City, CA). The sequence of primers used are presented in Supplementary Table 1. The same procedure was used for the hippocampus and spinal cord samples.

Immunofluorescence Assay

The spinal cord was collected from rats (n = 3) in the 3 mg/kg LPS group and control group. Rats were transcardially perfused with PBS followed by fresh 4% paraformaldehyde. Frozen sections were permeated in 0.3% Triton-X-100 for 30 min, and incubated with 10% bovine serum albumin for 60 min, followed by overnight incubation with rabbit anti-C3 (1:20, Abcam), rabbit anti-S100a10 (1:100, Proteintech) or mouse anti-GFAP (1:200, Cell Signaling Technology) primary antibodies at 4°C. The sections were incubated with the appropriate secondary antibodies (anti-rabbit IgG Alexa Flour 488, 1:400, Abcam; anti-mouse IgG Alexa Flour 594, 1:400, Abcam) for 1 hour at room temperature, and visualized using a CCD spot camera (Olympus DP71).

Single-cell Rna Sequence

Rat 87455 spinal cord were dissociated by enzyme digestion, and single cells were performed using 10x Genomics Chromium Single Cell 3’ Reagent Kits v3 by Shanghai Majorbio Bio-pharm Technology Co.,Ltd. The detailed procedure was presented in Supplementary method description. More than 360 million reads were obtained, and the average number of genes detected per cell was 1723. Reads mapped confidently to Genome was 86.3%. Raw sequencing data was processed with the cell ranger pipeline (version 3.0.1, 10X Genomics).

Statistical analysis

Results are presented as mean ± SEM, and statistical analyses were performed using GraphPad Prism 8.0 software package (version 8 for Windows, San Diego, CA). RT-PCR results were analyzed using one-way ANOVA followed by Tukey HSD test. Statistical significance was set at p < 0.05.

Systemic LPS exposure for 24 h induced comprehensive polarization of astrocytes and microglia in the CNTs

As a classical reagent used to induce A1 and M1 activation, systemic LPS exposure was expected to predominantly polarize pro-inflammatory A1 and/or M1. However, in addition to reactive A1 and M1 (Fig. 1A, 1C), we also observed activation of A2 (Fig. 1B), especially M2 (Fig. 1D) in the cerebral cortex, hippocampus, and spinal cord. Compared with the diverse variation (equivalent amount of red and blue) of M1 markers (Fig. 1C), A1 presented a more consistent change (increased amount of red than blue) with increasing LPS (Fig. 1A). Less markers (light red) of A2 were activated compared with A1 (deep red) when exposed to LPS in the 3 CNTs, and similar activation between M2 and M1 (similar color). With respect to reactive markers of A-pan, increased level in the hippocampus was less (less red) than in the cortex and spinal cord (Fig. 5A). Several activated markers such as Gbp2, Psmb8, Serping1, and C3 of A1; Il1β, Cxcl1, Cxcl10, and Marco of M1; Ym1 of M2; Lcn2 of A-pan showed good consistency (almost all red) among tissues with various dose of LPS.

Maximum Lps Dose Did Not Increase Polarization Of Astrocytes And Microglia

For further quantitative analysis of the complex heatmap results, the number of genes upregulated more than 2-fold were determined to compare the differences (Fig. 2A). A 2-fold increase was selected as a criteria since several previous studies have reported a 2-fold upregulation by PCR [4]. Most A1 and A-pan (Fig. 5B) markers were activated when the LPS dose was larger than 0.33 mg/kg, especially in the cortex and spinal cord, while those of A2 markers presented a relatively lower activation, exhibiting a preferential activation of A1 and A-pan than A2, under LPS stimulation. Markers of M1 and M2 phenotypes were also upregulated with LPS, but there no difference between M1 and M2, both being equally activated.

With respect to the number of phenotypic markers activated more than 2-fold, there was no difference between 3 and 5 mg/kg LPS doses (Fig. 2A). The survival curve showed a 40% survival rate induced by 5 mg/kg LPS, and higher than 70% with decreasing LPS (Fig. 5C). We further analyzed whether 5 mg/kg LPS-induced A1 activation was higher than the other doses. To completely compare the dose differences, we adopted the violin plot analyses (Fig. 2B-C). For A1 astrocytes, LPS doses varying from 0.33 to 5 mg/kg, induced a similar activation level (similar distribution) in the cortex; the 5 mg/kg dose presented no increase in activation (short and thin pattern) in the hippocampus and spinal cord except for outliers like Psmb8, Gbp2, Serping1, and C3. For the A2 phenotype, a relatively mild upregulation (small y-coordinate) was detected with any LPS dose exposure in the hippocampus and spinal cord (Fig. 2B). Regarding the microglial activation, there was no increase in activation of M1 markers in the 5 mg/kg group than the 1 or 3 mg/kg LPS group; there was no increase in M2 activation among the 5 doses except that in the cortex (Fig. 2C). For A-pan phenotype, the 5 mg/kg group was activated more only in the cortex (Fig. 5D).

Classical Phenotypic Markers

We determined whether the reported markers are all suitable as classical markers for LPS. C3 which is a marker of A1 astrocytes showed a good dose response with LPS in the 3 CNTs, while the A2 phenotypic marker S100a10 presentd a mild increase (Fig. 3A). With respect to the microglial phenotypic markers, both Cd86 and Cd206 were unchanged in the spinal cord, while they were activated slightly in the cortex and hippocampus (Fig. 3A). Interestingly, a commonly used A-pan marker, GFAP, only showed a significant increase in the cortex (Fig. 3B). Considering that the microglial secreted C1q, TNF-α, and IL-1α promote C3 activation, changes in these cytokines were also tested. No corresponding increase in C3 with LPS was observed except for C1qa and TNF-α in the spinal cord (Fig. 3B). Phenotypic markers showing a good dose response with LPS are presented, including the 3 common markers in the 3 CNTs: Marco for M1, Ym1 for M2, and C3 for A1 (Fig. 3C).

To confirm the PCR results, we further performed co-immunofluorescence in the spinal cord. Using the classical C3 and S100a10 as an example, our RT-PCR results showed a 7.9-fold in upregulation of C3, a 2.3-fold increase in S100a10, and a 2.2-fold increase in GFAP in the 3 mg/kg LPS group. Consistent with this, the co-location of C3 with GFAP (Fig. 3D) and S100a10 with GFAP (Fig. 3E) were also increased in the 3 mg/kg LPS group.

Enrichment Of Phenotypic Genes Of Microglia And Astrocytes

To obtain specific and in-depth information about the phenotypic markers, single-cell RNA sequence of T4-T5 spinal cord was tested. The top 3 genes in the top 4 clusters were presented, and they did not overlap with the classical phenotypic markers (Fig. 4A). Although several have been labeled as markers, the expression and distribution of microglial phenotypic genes showed a large difference, like the most enriched C1qa, C1qb, and the less enriched Cd40, Cd86, Ccl2 of M1 markers (Fig. 4B), among which, only C1qa, Cd40, and Ccl2 were upregulated by LPS. For M2 phenotypic genes, the expression of Tgfβ1, Stat3, Stat6, and Il33 were enriched, while the commonly used Arg1, Il10 and Il4 showed much less expression (Fig. 4C). For A1 phenotypic genes, the expression of C3, Srgn, RT1-S3, Ggta1, and Gbp2 were enriched (Fig. 4D), and all were upregulated by LPS. For A2 phenotypic genes, only S100a10 was enriched (Fig. 4E).

The detrimental role of A1 astrocytes in neurological disorders like neurodegenerative diseases, spinal cord injury, and pain has been recently proven, and its inhibition shows promise in ameliorating the disease progression [4, 5, 16, 17]. The polarization of A1 can be strongly induced by systemic LPS. Studies by Liddelow et al. [4], Zamanian et al. [9], and Kano et al. [18] used 5 mg/kg dose of systemic LPS exposure to activate A1. Our results also proved that LPS-induced A1 activation, moreover, fewer A2 than A1 markers were activated by any dose of LPS, indicating that it is a good inducer for activating A1. Besides, even a small intraperitoneal LPS dose (0.1 mg/kg) could activate more than half of A1 markers. A majority of A1 markers were activated in the CNTs when the LPS dose was larger than 0.33mg/kg, but the maximum dose presented no increase in activation. Considering the high mortality of animals exposed to 5 mg/kg of LPS, 1 or 3 mg/kg with a higher survival rate and similar degree of A1 activation could be more suitable.

Genes induced by both neuroinflammation and ischemia are classified as pan-reactive (A-pan) genes [5], different from the specific genes (A1) induced by neuroinflammation and A2 induced by ischemia. Interestingly, with increasing LPS, A-pan markers presented a similar trend to A1, and almost all were activated at high doses (≥ 1 mg/kg), especially in the cerebral cortex and spinal cord. It should be noted that the dichotomy of A1, A2, and A-pan is oversimplified, and the status of astrocytes may include a battery of different, but overlapping, functional phenotypes. Since A-pan were largely activated by LPS and showed similar tendency with A1, we infer that selective blocking of A1 formation may be discounted by functionally similar A-pan genes. Further studies are required to clarify this.

LPS-induced M1 polarization has been broadly validated in rodents, but doses of LPS vary between studies [10–13]. Fenn et al. [12] and Liao et al.’s [13] showed that injection of 0.33 mg/kg of LPS could increase the expression of M1 markers such as IL-1β, IL-6, TNF-α, and iNOS. Liu et al. treated mice with a lower dose of LPS (0.1 mg/kg) for 24 h to induce M1 activation, and they detected upregulation of TNF-α, IL-1β, IL-6, Cd86, Cd16, and Cd32 in the brain [11]. In our experiments, we show that systemic LPS with doses ranging from 0.1 to 5 mg/kg for 24 h activate M1, and with an increase in LPS dose, the number of activated M1 markers increased slowly. However, only half of the M1 genes were activated. Besides, M2 markers presented a similar increasing trend and degree as M1. Thus, we infer that systemic LPS lasting 24 h with a dosage ranging from 0.1 to 5 mg/kg may not be a suitable to induce M1 polarization. A likely explanation is that the duration of LPS incubation for microglial activation may be more critical than the dosage. In a study comparing 4 h and 24 h of LPS treatment, a weaker activation of M1 markers Cd86, iNOS, and IL-1β was detected at 24 h compared to 4 h [12]. It is not clear whether a shortened stimulation of LPS contributes more to M1 than M2 polarization. Contrary to this, in studies by Wang et al. and Zhao et al. [15, 19], small doses of LPS (0.75 and 0.25 mg/kg, respectively) were administered intraperitoneally for 7 days to induce cognitive deficits by activating M1.

Besides the upregulation of pro-inflammatory A1 and M1 genes by LPS, we also detected increased anti-inflammatory A2 and M2 markers, demonstrated by co-immunofluorescence of elevated S100a10 with GFAP. Chhor et al. found that the majority of M1 markers (10/13) and M2b markers (8/13, an immunoregulatory phenotype) were maximally expressed after 12 h of exposure to LPS. Moreover, M2c genes (IL-10, IL-4Rα, and SOCS3, an acquired-deactivating phenotype) were also increased in enriched microglia 4 h after LPS injection [20]. Similarly, we observed that more than half of M2 genes especially M2a (IL-4, IL-1Ra, CD206, Arg-1, Ym1, a repair and regeneration phenotype) were activated when the dose of LPS was larger than 0.1 mg/kg, but did not increase with an increase in LPS. Identically, A2 markers, which were more sensitive to MCAO, could also be activated by LPS, though it was milder than A1. This reflects that protective response often coexists with noxious stress. Moreover, with a boost in LPS inflammatory stimuli, the protective anti-inflammatory response did not show a corresponding increase. Besides, when trying to produce a homogeneous phenotype by LPS, the mixture of A2 or M2 cannot be ignored.

In addition to the various LPS doses, gene changes in different tissues were also determined. Consistent with the study by Clarke et al. that Serpina3n, C3 genes were differentially up-regulated in cortical, hippocampal, and striatal astrocytes [5], we found a 143-fold increase of Serpina3n in the cortex, 1.2-fold in hippocampus, and 205-fold in spinal cord. A similar trend with a 28, 44, and 27-fold increase of C3 in the cortex, hippocampus and spinal cord, respectively was observed with 5 mg/kg LPS, indicating region-specific transcriptional identities induced by LPS. Interestingly, aged hippocampal astrocytes showed increased up-regulated of reactive astrocyte genes compared with cortical astrocytes [5], and our maximal LPS dose induced increased activation of genes in the cortex than hippocampus. This suggests that hippocampus is more vulnerable to diseases of aging compared with the cortex, while the condition was opposite under LPS stimulation. With the increase in LPS dose, the differentially expressed genes except that from A2 astrocytes in each region were consistent, when the intersection of the three tissues increased while other subsets decreased (Supplementary Fig. 1). This indicates that the tissue difference between reactive phenotypic markers narrows with LPS increasing, which helps in selecting the target tissue for varied doses of LPS.

It has been reported that microglial-secreted C1q, TNF-α, and IL-1α activates A1 astrocytes [4]. Our RT-PCR results showed an inconsistent expression of C1qa, TNF-α, IL-1α and C3. Furthermore, LPS-induced upregulation of C1q, TNF-α and IL-1α protein has been tested in purified microglia [4]. The diversity of vivo and vitro studies, mRNA and protein levels, and LPS exposure time may explain the observed discrepancy. Our single-cell RNA sequence results further showed a relatively enriched expression of C1qa in the spinal cord. Whether the LPS-induced A1 astrocytes in any region were activated by the interaction of C1q, TNF-α, and IL-1α remains to be further studied.

It is unclear if inflammatory degree is aggravated with increasing LPS, and whether 24 of the 72 phenotypic markers presenting a good dose-dependent activation with LPS reflects the inflammation level, or are involved in inducing inflammation, or glial cell polarization. All these aspects remain to be elucidated. Meanwhile, 3 markers (Marco, Ym1, and C3) showed a dose-response relationship with LPS in all the 3 CNTs. There were also markers like Gbp2, Psmb8, Serping1 of A1; Il1β, Cxcl1, Cxcl10 of M1; Lcn2 of A-pan which were largely activated among the 3 CNTs with all doses of LPS. The variation of dose related markers between tissues may reflect a distinct tissue vulnerability with LPS stimulation. On the other hand, selection of these markers may be more appropriate to reflect the synchronous change caused by interventions. Interestingly, the top 3 genes in top 4 clusters (belonging to microglia) were not classical phenotypic markers. It is possible that subsets of microglia are more specific in cell polarization.

There were several limitations to our study. We employed three variables-doses, phenotypes, and tissues-in our study, and did not include time variants. Considering that astrocytes are often activated following microglia, polarization changes over time need to be determined in future studies. Our focus was on the dose relationship between LPS and phenotypes, therefore we did not include the MCAO model to compare the specificity of the opposite phenotypic markers.

In conclusion, our results show that systemic LPS not only activates the proinflammatory A1 and M1, but also anti-inflammatory A2 and M2 phenotypes. Systemic LPS with a 24 h exposure activates a similar degree of M2 and M1, thus may not be suitable for inducing M1. An LPS dose smaller than 5 mg/kg can be adopted to polarize A1. Selection of the phenotypic markers should be made according to the tested tissue and LPS dosage.

Acknowledgements

We thank Prof. Chao Ma in the Department of Anatomy, Histology and Embryology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences for his kindness help in immunohistochemistry.

Author contributions

Afang Zhu drafted the manuscript, and performed the RT-PCR procedures. Huan Cui performed the immunofluorescence staining. Wenliang Su performed the intrathecal and intraperitoneal injection. Chaoqun Liu performed the tissues extraction. Le Shen helped to draft the manuscript. Xuerong Yu performed the data analysis and study design. Yuguang Huang conceived of the study and participated in its design. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China #81901148 (Afang Zhu), #82071252 (Yuguang Huang), and the Postdoctoral Research Foundation of China #2019M650566 (Afang Zhu).

Declaration of competing interest

None.

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No competing interests reported.

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LPS-induced complex polarization of astrocytes and microglia in rats

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Version 1

Abstract

Figures

Introduction

Materials And Methods

Animals

Lps Treatment

RT-PCR

Immunofluorescence Assay

Single-cell Rna Sequence

Statistical analysis

Results

Maximum Lps Dose Did Not Increase Polarization Of Astrocytes And Microglia

Classical Phenotypic Markers

Enrichment Of Phenotypic Genes Of Microglia And Astrocytes

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

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