Research ArticleEnduring and Sex-specific Changes in Hippocampal Gene Expression after a Subchronic Immune Challenge
Introduction
The neuroimmune system, and its regulation by peripheral immune states, plays an important regulatory role for behavioral, affective, and cognitive functions (Raison et al., 2006, Tchessalova et al., 2018). In addition to their central role in behavioral and physiological changes during illness and injury (Dantzer et al., 1998), neuroimmune cells and cytokines are activated as a consequence of physical and psychological stressors (Bekhbat and Neigh, 2017), and are critical for normal neural functions including synaptic plasticity and memory (Avital et al., 2003, Yirmiya and Goshen, 2011, del Rey et al., 2013, Adamsky et al., 2018).
Activation of the neuroimmune system as a consequence of surgery, injury, or illness also has long-lasting consequences for memory, affective behaviors, and cognition. In rodent models of sepsis, persistent changes in memory and affective processes have been observed after cecal ligation and puncture (CLP) (Chavan et al., 2012, Huerta et al., 2016) and after high-dose lipopolysaccharide (LPS) treatment (Weberpals et al., 2009, Anderson et al., 2015). We have recently demonstrated that a lower-dose subchronic systemic immune challenge results in striking memory deficits that emerge and persist at least three months after the end of the immune challenge (Tchessalova and Tronson, 2019). These long-lasting memory deficits are likely mediated by changes in neuroimmune and neuronal function that persist long after immune activation and likely contribute to cognitive decline, affective dysregulation, and increased risk of dementia and Alzheimer’s disease long after recovery from major illnesses including sepsis and heart attack (Semmler et al., 2007, Gharacholou et al., 2011, Marra et al., 2018).
That transient illness or immune challenge can result in long-lasting memory deficits is now well established, but it is less clear what is persistently changed in the brain to mediate changes in cognition and memory that persist long after resolution of the immune response. One possibility is that neuroimmune signaling lingers long after a peripheral immune challenge. Indeed, some sepsis models observe that persistent microglial activation and neuroimmune signaling correlate with memory impairments at least one month after surgery or LPS injection (Weberpals et al., 2009, Fenn et al., 2014, Anderson et al., 2015). In contrast, other studies observe memory deficits in the absence of ongoing neuroimmune activation in both CLP and LPS models, suggesting that persistent neuroimmune activation is not required for late-emerging memory impairments (Ming et al., 2015, Huerta et al., 2016, Singer et al., 2016, Tchessalova and Tronson, 2019). In these studies, structural changes in the brain after transient immune challenge, including dysregulation of dendritic spine turnover or density and neuronal loss have been implicated as potential mechanisms for persistent memory impairments (Semmler et al., 2007, Liu et al., 2008, Kondo et al., 2011, Volpe et al., 2015). Additionally, recent work has demonstrated changes in function of seemingly quiescent microglia months after prior immune challenge (Wendeln et al., 2018). Thus prior immune experience may result in “priming” of microglia (Norden et al., 2015), or “training” of the innate immune system (Netea and van der Meer, 2017), in which immune cells respond in an exaggerated way to subsequent immune, stress, or other experiences.
If ongoing neuroimmune signaling is not necessary for persistent changes in memory, then how are such long-lasting changes in synaptic function, neuroimmune function, and memory processes maintained long after the immune challenge? One possibility is that these persistent changes in memory are mediated and maintained by enduring alterations of gene expression and associated transcriptional regulatory mechanisms (Tchessalova et al., 2018). Changes in transcriptional regulation as a result of chromatin and DNA modifications (Cholewa-Waclaw et al., 2016) occur after a variety of environmental exposures and life experience, including stress and exposure to addictive drugs, and mediate persistent changes in cellular function and behavior (Gray et al., 2014, Nestler, 2014, Tafet and Nemeroff, 2016).
We have previously demonstrated that a subchronic LPS challenge results in memory impairments that emerge and persist months after the last injection (Tchessalova and Tronson, 2019). Here we examined whether the same protocol also causes changes in hippocampal gene expression that persist long after an immune challenge in males and in females. We used an unbiased approach, RNA-sequencing, to identify both immune- and non-immune-related gene expression changes long after a systemic immune challenge in both males and females. Since changes in gene expression after experiences such as stress (Gray et al., 2014) are often more evident in response to a subsequent stressor, we determined gene expression both months after a subchronic immune challenge, and in response to a subsequent immune challenge. We found that a transient, subchronic LPS protocol (Tchessalova and Tronson, 2019) resulted in persistent and sex-specific dysregulation of gene expression in the hippocampus. Males and females differed in both number and patterns of gene expression, where males show more changes in baseline gene expression and females show greater changes in response to an additional, acute challenge. These data have important implications for identifying the precise mechanisms by which a prior illness or injury can impact on cognitive function and vulnerability to cognitive decline and dementia.
Section snippets
Animals
Twenty four male and female 8–9 week old C57BL/6N mice were purchased from Envigo (Indianapolis, IN, USA). The sample size for these experiments was n = 6 per immune challenge condition (Vehicle, Long-Term, Long-term+Acute, Acute), with 3 males and 3 females per group. All mice were individually housed with ad libitum access to standard mouse chow and water as individual housing in mice prevents fighting-induced stress in males (Meakin et al., 2013) and is ethologically appropriate for males
Subchronic immune challenge induces persistent changes in gene expression
We observed striking changes in gene expression in the hippocampus 12 weeks after subchronic immune challenge in males, with fewer changes observed in females. Of over 20,000 genes detected, there were 230 DEGs in the hippocampus of males and 26 DEGs in the hippocampus of females (Fig. 1). In males, 183 genes were significantly upregulated and 47 significantly downregulated (see https://doi.org/10.7302/7zz3-jh58 for Supp. Table 1 for a full list of DEGs). In females, 7 genes were significantly
Discussion
Here we demonstrated long-lasting consequences of subchronic immune challenge, and striking sex differences, on gene expression in the hippocampus. In males, we observed enduring dysregulation of gene expression months after the end of a two-week immune challenge. In contrast, females showed few persistent changes at baseline, but striking changes in gene expression in response to an additional acute LPS injection three months after the subchronic immune challenge. These different
Acknowledgements
Thanks to University of Michigan Bioinformatics core for their expertise and data analysis of differentially expressed genes and the University of Michigan DNA sequencing core for their processing of samples; to Dr. Shigeki Iwase and Dr. Christina Vallianatos for assistance with prepping of the RNA samples; Dr. Dave Bridges for helpful discussions on RNAseq and analyses; Ms. Brynne Raines, Ms. Melanie Gil, and Ms. Caitlin Posillico for their extensive feedback on this manuscript.
Funding and disclosures
NIH/NIMH R00 MH0934509 to NCT. The authors declare no conflicts of interest.
Data availability
This data is also publicly available on the following databases:
Gene Expression Omnibus (GEO), GEO accession number GSE126678; Sequence Read Archive (SRA), with SRA number SRP186132 and BioProject number PRJNA522922. Supplemental tables are available at Deep Blue Data https://doi.org/10.7302/7zz3-jh58.
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