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. , Zamanian et al. , and Kano et al.  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 , 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.  and Liao et al.’s  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 . 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 . 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 . 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 , 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 , 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 . 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 . 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.