Chronic low-grade inflammation is a hallmark feature of AD, as evidenced by increased expression of proinflammatory cytokines in postmortem AD brains (24–26), yet outside of examining known CSF biomarkers, few studies have examined the contribution of elevated inflammatory levels on future incidence of clinical conversion. In the present study, we evaluated inflammatory markers at baseline to determine differences between those who progressed to MCI or AD and those that remained stable over time. Evaluating individuals before conversion to AD retroactively evaluates early features of AD risk and can provide an outlook on time to conversion. Therefore, the goal of this study was to address whether: i) inflammatory cytokines are involved in incident MCI or AD as compared to canonical CSF AD biomarkers, ii) sex or APOE4 impact these relationships, and iii) there is a relationship between the inflammatory markers via mediation analysis.
As expected, CSF biomarkers Aβ42, p-tau, and t-tau predicted conversion to MCI or AD. This was especially true among APOE4 carriers for Aβ42 and p-tau, which has been previously confirmed (27,28). In line with the primary goal of the study, inflammatory CSF markers TNF-α, IL-9, and IL-12p40 were associated with conversion to MCI/AD with sex and APOE4 interactions.
While we know that neural degeneration and the chronic presence of pathology can stimulate inflammation, evidence suggests that inflammation itself can both generate and increase pathology. For instance, modulatory proteins associated with innate immunity are directly connected to amyloid-beta production (29) indicating the importance of neuroimmune actions on AD. Therefore, it is possible that inflammatory markers may be important indicators of MCI and AD risk, possibly accelerating decline. Additionally, several interactions involving sex and APOE4 status among these inflammatory markers were observed, suggesting these genetic factors may influence the relationship between early inflammation and future AD risk, providing key insight for precision medicine approaches. Lastly, the association between these markers suggests a common tie to the NF-kB inflammatory pathway, which may be activated in AD.
CSF TNF-α, one of the inflammatory markers most consistently implicated in AD, was associated with conversion in individuals who were cognitively normal at baseline. Previous work has shown increased levels of TNF-α in both the brains and plasma of patients with clinical AD (30). This may be partially related to Aβ, which can directly stimulate microglial production of TNF-α through activation of the transcription factor NF-κB (31). In addition, early increases in TNF-α can increase Aβ burden through the upregulation of β-secretase production and increase of γ-secretase activity (32,33), demonstrating how TNF-α and Aβ can perpetuate a vicious cycle of increasing pathology and chronic inflammation. In corroboration of a role for TNF-α in AD, a strong association between elevated TNF-α levels and decreased functional connectivity in an AD susceptible cohort has been observed at a whole-brain level (34). Aside from TNF-α as a consistently elevated proinflammatory marker in AD, its regulation of the NF-κB inflammatory cascade is well characterized (35,36).
When decomposing the association between TNF-α levels in cognitively normal individuals and clinical converters, the effect was driven by conversion to AD, not conversion to MCI, indicating that elevated levels of TNF-α resulted in accelerated onset of Alzheimer’s dementia. Given that TNF-α both promotes and is regulated by NF-κB, it could be that TNF-α accumulates at a faster rate than other inflammatory factors. In AD patients who experienced systemic inflammatory events (SIE), rapid cognitive decline was observed over 6 months creating a 2-fold decrease in cognitive function. Additionally, SIE AD patients also exhibited an association between elevated plasma TNF-α levels and a 10-fold greater rate of cognitive decline over a 6-month observation period (37).
Importantly, a sex*TNF-α interaction was observed in which women with higher TNF-α levels had shorter time to conversion to MCI or AD. There is precedence for this result with recent evidence suggesting important sex differences in immune parameters. This includes brain responses to inflammatory mediators and markers of microglial disruption which may contribute to higher risk of AD among women (38). For instance, given an acute endotoxin challenge, females responded with higher levels of pro-inflammatory plasma cytokines like TNF-α, compared to males (39). This may be due to a putative link between levels of TNF-α and sex hormones. A significant positive correlation between luteinizing hormone and TNF-α levels has been observed (albeit only in males) providing some evidence that sex hormones exert their influence on AD by modulating systemic TNF-α levels (40). There are also sex differences among function, morphology, and volume of microglia, with evidence suggesting that women later in life have higher numbers of microglia than men, creating an exaggerated immune response (41). Interestingly, our finding that females with higher TNF-α levels have a shorter time to conversion to MCI/AD suggests that elevated inflammation is associated with AD risk. It has been suggested that elevated inflammatory factors, including TNF-α, are associated with lower brain levels of amyloid-beta and tau in cognitively normal individuals (42), suggesting inflammation may be protective for a time before eventually exhausting this compensatory effect and converting to AD at an accelerated rate. Studies have started showing prevention of cognitive loss with anti-TNF-α therapy in a mouse model of compromised cognition (43) lending additional evidence to an important role of TNF-α in the initiation and amplification of the inflammatory cascade.
Baseline elevated IL-9 levels were associated with conversion to MCI or AD, but in contrast to TNF-α, the results were driven by conversion to MCI and not AD. Additionally, no interaction with sex was observed. IL-9 is an understudied pro-inflammatory cytokine that promotes cell proliferation and whose expression is driven by NF-κB (44). Its pleiotropic effects have been implicated in both neurological (i.e., multiple sclerosis ) and systemic disorders (i.e., psoriasis ). Additionally, neurons are thought to possess the ability to induce T cells to release IL-9 . Thus, IL‐9 may represent a critical link in the crosstalk between neurons and perivascular T cells in the brain. Changes related to IL‐9 have not been consistently reported in AD (48,49), but there is mouse APOE4 knock-in models showing greater IL‐9 production than the wild‐type ε3 allele (50). We add to this evidence by reporting an interaction between APOE4 and IL‐9 levels, such that APOE4 carriers with higher IL-9 levels at baseline had higher rates of conversion.
Lastly, higher baseline levels of the proinflammatory cytokine IL-12p40 were also significantly associated with conversion to AD. IL-12, which has different subunits, including p40 as investigated here, is involved in the differentiation of naive T cells into Th1 cells  and plays an important role in the activities of natural killer (NK) cells and T lymphocytes, enhancing their activity. IL-12 also stimulates production of TNF-α from T and NK cells (52). Its role in AD remains unclear, but recent evidence indicates a role for IL-12 in amyloid induced neurodegeneration (53). The p40 subunit of IL-12 is produced by microglia and was increased in transgenic mouse models demonstrating a significant correlation between cognitive performance and CSF IL-12p40 levels (52,53). More support of its role in AD was shown by elevated CSF IL-12p40 levels in AD patients (54). Interestingly, the potential association of plasma IL-12p40 levels with the diagnosis of MCI or AD was also highlighted by a recent plasma multianalyte profiling study (55). Together, these findings imply that IL-12p40 is involved in AD progression.
To investigate interrelationships between cytokines showing significant associations with conversion to MCI/AD, mediation analyses using a priori knowledge of the markers and their role in the NF-κB pathway were conducted. As mentioned previously, IL-12p40 has been shown to stimulate the production of TNF-α (23), a canonical promotor of the NF-κB inflammatory pathway (33,34), and evidence shows that IL-9 production is mediated by NF-κB (22). TNF-α was tested as a significant mediator between IL12-p40 and IL-9, and mediation analysis results suggest that TNF-α mediated 89% of the association between IL12-p40 and IL-9. Based on the available literature, this study is the first showing that these cytokines may be involved in the same NF-κB inflammatory pathway, thereby further implicating the inflammatory cascade’s importance in AD detection.
Current limitations of this study include an inability to test specific demographic variables, including race, which we know play a significant role in inflammation and AD and yet have not been prioritized in ADNI recruitment. The investigation of longitudinal cognitive data from this sample and other datasets could help determine if differences exist between converters and non-converters, and shed light on specific cognitive domains that may be more closely tied to inflammation. We also note that because this was interval censored data, we are not able to know the actual time of conversion and are restricted to examining data at discrete time points. Competing risks were not discussed (i.e., death occurring prior to follow-up). Additionally, because we only had baseline CSF inflammatory markers, we were restricted to a cross-sectional mediation analysis that limits our confidence in temporality of events, but as more data become available, longitudinal analyses of change in inflammatory markers can be evaluated. Lastly, the analyses include adjusted p-values to explore interactions, so the results should be carefully considered. Future studies aimed at replication of these findings in other independent cohorts, including those which are more nationally representative of the AD population, would help validate these results.