*In vivo* studies support the anti-inflammatory effect of 17βestradiol in several neurodegenerative diseases (Vegeto et al., 2006, 2008). However, relatively recent studies strongly suggest that 17β-estradiol promotes proinflammatory response to bacterial LPS by enhancing the expression of such proinflammatory genes as *TNF-*α and *IL-1*β (Calippe et al., 2008, 2010) likely by enhancing the microglial expression of TLR4 (Loram

et al., 2012). The present study shows that 17β-estradiol has no significant effect on LPS-activated microglia in OVX rats. It also does not affect the proinflammatory response to LPS as assessed by the activity of the NFκB signaling pathway and the levels of TNF-α produced at the site of brain inflammation. The lack of a 17β-estradiol effect on several key elements of brain inflammation is in line with our previous observation in which an HRT based on 17β-estradiol alone did not blunt neuroimmune responses to systemically injected LPS. In contrast, an HRT consisting of 17β-estradiol and progesterone was able to dampen LPS-induced fever and LPS-activated COX-2 expression in fever controlling area of the hypothalamus (Mouihate and Pittman, 2003). Similar to TNF-α, COX-2 is also under the control of LPS-activated NFκB signaling pathway (Mouihate et al., 2005; Wu, 2005). Thus, it seems that the dampening effect of the combination of hormonal treatment with 17β-estradiol and progesterone on the brain immune response operates in similar fashion regardless of the route of the immune challenge.

While 17β-estradiol alone did not significantly affect markers of brain inflammation such as microglial activation, NFκB signaling pathway and the levels of TNF-α, we have noted that this hormone invariably enhanced the expression levels of COX-2. Owing to the important role of COX-2 in the formation of such proinflammatory prostaglandin as PGE2 (Rivest, 2010), it is possible that 17β-estradiol has the potential to exacerbate PGE2 mediated brain inflammation. It is noteworthy that activation of COX-2 can also lead to synthesis of prostaglandins endowed with antiinflammatory properties such as PGD2 and its derivative PGJ2 (Gilroy et al., 1999; Petrova et al., 1999; Mouihate et al., 2004). The synthesis of such anti-inflammatory prostaglandins is unlikely as LPS-induced activation of microglia and the NFκB signaling were not affected by 17β-estradiol.

Collectively, our results do not support the idea that hormonal treatment based on 17β-estradiol alone is neuroprotective, at least in this brain inflammation model.

In a series of preliminary data, a group of OVX rats were given progesterone alone. Progesterone treatment did not elicit any significant anti-inflammatory response within the brains of OVX rats given intra-cerebral LPS (see **Figure A1** in Appendix) probably because progesterone effect is more apparent when the OVX rats are primed with 17β-estradiol. Indeed, 17β-estradiol administration increases the expression of progesterone receptors within female rat brains (Simerly et al., 1996; Scott et al., 2002; Quadros and Wagner, 2008).

#### **DIFFERENTIAL EFFECT ON TNF-α AND IL-6**

The inhibitory effect of combinatory HRT on TNF-α production was not extended to IL-6. This observation is very peculiar as both *TNF-*α and *IL-6* genes are under the control of NFκB signaling pathway, the activity of which was significantly depressed. These data are akin to our *in vivo* studies and other's *in vitro* observations where a phytoestrogen (resveratrol compound) inhibited LPS activated production of TNF-α but not that of IL-6 (Richard et al., 2005; Mouihate et al., 2006). The mechanism underlying this selective inhibition of TNF-α is not clear yet. It is possible that LPS activated IL-6 is mediated through activation of transcription factors other than NFκB. These transcriptional factors, which include ERK1/2, p38 MAPKs, and NF-IL6 were probably not affected by the combined HRT (Matsusaka et al., 1993; Zhang et al., 1994; Rego et al., 2011). In some circumstances, 17βestradiol alone or in combination with progesterone has been shown to stimulate IL-6 production (Verthelyi, 2001; Brooks-Asplund et al., 2002; Isse et al., 2010), adding more complexity to the mechanism through which *IL-6* gene is affected by ovarian hormones.

#### **OVARIAN HORMONES AND NEUROGENESIS**

Brain inflammation hampers neurogenesis (Monje et al., 2003) likely via microglia derived TNF-α (Lafortune et al.,1996; Hanisch, 2002; Lambertsen et al., 2009; Nimmervoll et al., 2013). In the present study, we confirmed that the brain inflammatory response, as illustrated by microglial activation and TNF-α protein expression, resulted in decreased number of newly born neurons. This observation is in line with previous studies demonstrating the deleterious effect of TNF-α on the survival of neural precursor cells (Ekdahl et al., 2003; Iosif et al., 2006; Keohane et al., 2010; Ekdahl, 2012).  Such effect was absent when progesterone was omitted from the HRT regimen.

However, ovarian hormones can also affect neurogenesis through a sensitization/desensitization to LPS effects. Indeed, TLR4 receptors are expressed on neural stem cells and play a major role in neurogenesis (Rolls et al., 2007; Shechter et al., 2008). There are indications that ovarian hormones can affect the expression levels of TLR4 in immune competent cells. For example, 17β-estradiol enhances TLR4 expression in macrophages (Rettew et al., 2009), while progesterone depresses its expression in the brain of mice with experimental autoimmune encephalomyelitis (Garay et al., 2012) or that of rats subjected to subarachnoid hemorrhage (Wang et al., 2011). Whether ovarian hormones alter the expression of TLR4 on neural precursor cells and thus prime these newly born cells to the deleterious effect of LPS is still an open question.

### **CONCLUSION**

LPS-induced brain inflammation resulted in activated microglial cells and enhanced levels of molecular markers of inflammation such as NFκB signaling pathway and its proinflammatory target proteins (TNF-α and COX-2). HRT based on 17β-estradiol alone was devoid of anti-inflammatory properties in TLR4-induced brain inflammation. In contrast, both LPS-activated microglia and the resulting activated molecular proinflammatory machinery were significantly reduced in OVX rats given an HRT regimen containing 17β-estradiol and progesterone. Interestingly, the anti-inflammatory effect of complete HRT created conducive environment for the survival of newly born neurons.

### **AUTHOR CONTRIBUTION**

Abdeslam Mouihate designed the research, performed research, analyzed data, and wrote the manuscript.

### **ACKNOWLEDGMENTS**

This work was supported by Kuwait University Research Grant No. MY01/10. Confocal imaging was performed in The Research Unit for Genomics, Proteomics and Cellomics Sciences supported by Research Project No. SRUL02/13. The author thanks Dr. Sarah J. Spencer for the critical reading of this manuscript, Mrs. H. Mehdawi and Mrs. B. Sebastian for the technical help and animal care and husbandry and Ms. S. Kalakh for blind-counting microglia.

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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 13 April 2014; accepted: 07 May 2014; published online: 27 May 2014. Citation: Mouihate A (2014) TLR4-mediated brain inflammation halts neurogenesis: impact of hormonal replacement therapy. Front. Cell. Neurosci. 8:146. doi: 10.3389/fncel.2014.00146*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2014 Mouihate. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

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