The inflammatory lipidome consists of pro-inflammatory LMs and also of LMs that end and resolve inflammation while promoting restoration and regeneration of the tissue, i.e., healing (Serhan et al. 2007). We have previously shown decreased levels of pro-resolving LMs in the human AD brain (Lukiw et al. 2005; Wang et al. 2015; Zhu et al. 2016). Two of these LMs, LXA4 and RvD1, were present in lower levels in the CSF of AD patients compared to those without clinical evidence of memory deficits, i.e., diagnosed with SCI, and were positively correlated to the scores from the MMSE test (Wang et al. 2015). A decrease in RvD1 in the CSF of AD patients was not seen in the present study, possibly due to the greater specificity of LC-MS/MS compared to immunochemical assays in which signals may consist of several compounds with molecular similarities. The LC-MS/MS analysis showed that the levels of RvD4 and NPD1 were lower in both AD and MCI patients compared to SCI patients, while RvE4 and MaR1 were lower in MCI patients only. The differences between the diagnostic groups in RvD4 were also seen in a gender-separated comparison, but this was not evident for NPD1. The difference in MaR1 levels was statistically significant only in men, thus contributing most to the difference seen for all cases. Further analysis within male and female groups showed some additional differences in pro-resolving LMs, such as for RvD3, which was lower in women with MCI than with SCI, for RvE1 that was lower in men with AD than with SCI, and for LXA4 that was reduced in men with MCI compared to men with SCI. Moreover, DHA levels in CSF were reduced in men with AD. Both intermediate precursors, 17-HDHA and 15-HETE were higher in AD, either compared to MCI or to SCI.
Several factors may influence the levels of lipids and give rise to the different results seen in men and women, including diet, age, sex hormones, and the ability to synthesize lipids. Indeed, the ability to synthesize long-chain fatty acids was shown to be higher for women than men, as suggested by a higher conversion rate of α-linoleic acid (ALA) to DHA and EPA (Burdge and Wootton 2002). In a study on mice (Rodriguez-Navas et al. 2016), females had higher brain levels of PUFAs than males, both after a Western-style high fat diet and regular chow diet, while plasma levels were similar.
Although analyses of LTB4 in CSF have been performed since the eighties (Westcott et al. 1987), the significance of its presence in CSF in the context of AD is not known. We show that LTB4 in CSF of both AD and MCI patients was slightly higher but statistically significant than in SCI and positively correlated to the levels of Aβ42 in AD patients. In studies on multiple sclerosis (MS) (Neu et al. 1992), higher levels of LTB4 were found in the CSF of MS patients compared to controls, suggesting LTB4 as an indicator of inflammation in the brain. LTB4 increased the production of Aβ in neurons in culture (Joshi et al. 2014), providing a direct link to the molecular pathology in AD. In addition, we found that the CSF levels of 15-HETE, an intermediary in the synthesis of LTB4, were slightly higher but statistically significant in women with AD compared to women with SCI diagnosis and negatively correlated to MMSE scores. Yao et al. previously detected increased levels of 15-HETE in CSF from AD patients (Yao et al. 2005).
The pro-inflammatory LMs PGD2 and PGE2 were lower in the CSF of MCI patients compared to SCI, and in the case of PGE2, also reduced in AD patients compared to SCI. Analysis of human post mortem entorhinal cortex showed higher levels of PGD2 in AD compared to non-demented controls (Zhu et al. 2016), and studies on CSF samples showed higher levels of PGE2 in patients with probable AD (Montine et al. 1999) and in MCI patients but lower in AD (Combrinck et al. 2006). PGD2 synthetase and the PGD2 receptor DP1 were upregulated in plaque-associated glia in post mortem AD brains and an AD mouse model (Mohri et al. 2007). PGD2 mediated neuronal cell death in in vitro cocultures of neurons and microglia exposed to Aβ42 (Bate et al. 2006). PGE2 is increased in the CSF of patients with severe MS (Prüss et al. 2013). In a mouse model of AD, PGE2 was shown to mediate TNF-α- and presenilin (PS)1/2-dependent deposition of Aβ (Guan et al. 2019). The literature thus suggests that PGD2 and PGE2 play harmful roles in AD. The explanation for our findings of lower levels of these factors in the CSF of AD patients will need further studies. However, in addition to the role of PG’s at the initiation of an inflammatory response (Serhan and Savill 2005), it is hypothesized that there is a post-resolution immunological activity during which PGE2 may exert modulatory and anti-inflammatory effects (Feehan and Gilroy 2019). The reduced levels of PGE2 in CSF from AD and MCI patients could thus be seen as a deficit for the post-resolution stage.
Our novel finding of the presence of several bioactive LMs in human CSF highlight their abundance, and the present data on decreased levels of MaR1, NPD1, RvD3, RvD4, RvE1, RvE4, and LXA4 in patients with cognitive dysfunction are in line with our previous research showing impaired resolution in AD. Of these, NPD1 is the most well-studied, and beneficial effects in the brain have been shown (Lukiw et al. 2005; Bazan 2009; Stark and Bazan 2011), as well as direct protection on human neuronal cells (Zhu et al. 2016). NPD1 here showed a positive correlation to the CSF levels of Aβ42, known to be decreased in AD patients.
The decreased levels of MaR1 in CSF of MCI cases can mainly be attributed to reduced levels in men with MCI. We previously found decreased levels of MaR1 in the hippocampus (Wang et al. 2015) and entorhinal cortex (Zhu et al. 2016) of AD patients and beneficial effects of MaR1 in several cellular models (Zhu et al. 2016; Wang et al. 2021). Surprisingly, there was a negative correlation of MaR1 to the MMSE scores in MCI patients, indicating a more complex nature of immune regulation in this heterogeneous group of patients than previously thought. However, in general, the correlative relationships between the lipids and cognition and AD CSF biomarkers indicated a positive role, where the levels of AA, DHA, and EPA all showed a comparatively strong positive correlation to cognition in AD cases, while within the group of SCI cases the LMs derived from DHA and EPA showed such a relationship. Of note, we, along with other researchers, consistently detect the presence of pro-resolving LMs in pathological as well as healthy tissues, which adds credibility to an evolving concept of the resolution pathway as an ever-present “care-taker-guardian” of the tissue rather than a response that is elicited only on demand. Studies in animal models of cancer (Sulciner et al. 2018; Panigrahy et al. 2019; Fishbein et al. 2021) provide a fascinating perspective on resolution as a defender of the tissue, adding further support to this concept in which future therapies for disorders that today are hard to treat may be found.
Our results uncovered alterations in the pro-resolving CSF lipidome during the dysfunctions of inflammatory resolution in AD. Our data demonstrate that it is possible to detect bioactive lipids in CSF samples and to show that pro-resolving LMs such as RvD4, NPD1, MaR1, and RvE1 are reduced in CSF samples from patients with cognitive dysfunction, supporting the disturbance of the resolution of inflammation in the brain. Some of these LMs and the PUFA precursors show positive correlations with MMSE test scores, indicating their relevance for cognitive function.
This is an explorative study, original in that it uses LC-mass-spectrometry to analyze the pro-inflammatory and pro-resolving lipidome in samples from cases of AD and MCI as well as SCI in a reasonably large cohort considering the analysis method. However, further studies on a larger cohort are necessary. Although we suggest the use of the CSF lipidomic profile as a biomarker of cognitive decline, its usefulness as a novel biomarker must be determined in replication studies, including longitudinal observations of cognitive decline. We are in the process of performing such studies and hope that the results from the present study motivate other researchers to explore and hopefully confirm the association of alteration in CSF LMs that we believe can be seen in our data. Age and gender were included in our analyses, and the influence of gender on the abundance of LMs is also of importance and requires further investigation.
The majority of the differences observed reach the threshold of P-values 0.005 or 0.001, and in some cases even 0.0001. Considering the explorative nature and novel findings in our study, the analyses resulting in a P-value of > 0.005 should be interpreted with caution and as an impetus for further investigation rather than hard evidence. Although we did not perform a sensitivity power analysis prior to our investigation, we believe that the sample size in our cohort is large enough for an explorative study. Therefore, the fact that MaR1 and RvE4 were decreased only in MCI compared to SCI, whereas NPD1 and RvD4 were reduced in both AD and MCI, may be a reflection of a limited sample size.