Full-length Article
Peripheral viral infection induced microglial sensome genes and enhanced microglial cell activity in the hippocampus of neonatal piglets

https://doi.org/10.1016/j.bbi.2016.02.010Get rights and content

Highlights

  • Postnatal infection up-regulated microglial sensome genes in piglet hippocampus.

  • Microglia isolated from hippocampus of infected piglets had increased activity.

  • Bipolar rod-like microglia were evident in hippocampus of infected piglets.

  • The microglial sensome may foretell an exaggerated neuroinflammatory response.

Abstract

Although poorly understood, early-life infection is predicted to affect brain microglial cells, making them hypersensitive to subsequent stimuli. To investigate this, we assessed gene expression in hippocampal tissue obtained from a previously published study reporting increased microglial cell activity and reduced hippocampal-dependent learning in neonatal piglets infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus that induces interstitial pneumonia. Infection altered expression of 455 genes, of which 334 were up-regulated and 121 were down-regulated. Functional annotation revealed that immune function genes were enriched among the up-regulated differentially expressed genes (DEGs), whereas calcium binding and synaptic vesicle genes were enriched among the down-regulated DEGs. Twenty-six genes encoding part of the microglia sensory apparatus (i.e., the sensome) were up-regulated (e.g., IL1R1, TLR2, and TLR4), whereas 15 genes associated with the synaptosome and synaptic receptors (e.g., NPTX2, GABRA2, and SLC5A7) were down-regulated. As the sensome may foretell microglia reactivity, we next inoculated piglets with culture medium or PRRSV at PD 7 and assessed hippocampal microglia morphology and function at PD 28 when signs of infection were waning. Consistent with amplification of the sensome, microglia from PRRSV piglets had enhanced responsiveness to chemoattractants, increased phagocytic activity, and secreted more TNFα in response to lipopolysaccharide and Poly I:C. Immunohistochemical staining indicated PRRSV infection increased microglia soma length and length-to-width ratio. Bipolar rod-like microglia not evident in hippocampus of control piglets, were present in infected piglets. Collectively, this study suggests early-life infection alters the microglia sensome as well as microglial cell morphology and function.

Introduction

Neuroimmune factors are thought to play important roles in the etiology of psychiatric disorders such as autism spectrum disorders and schizophrenia. The supposition is predicated on the prenatal and early postnatal brain being vulnerable to infectious insults that, if present, affect development and increase risk for behavioral disorders later (Andersen, 2003, Rees and Inder, 2005). Pro-inflammatory cytokines produced by immune cells, rather than the pathogen per se are strongly implicated (Dantzer et al., 2008). Therefore, an important role for microglia seems likely as they are resident immune cells in the brain, produce pro-inflammatory cytokines in response to infection, and play a key role in development by phagocytizing neural progenitors and engaging in synaptic pruning. Indeed, untimely activation of microglia has been shown to reduce prenatal cortical neurogenesis, resulting in fewer cortical neurons in adulthood (Cunningham et al., 2013). Furthermore, rat pups infected with Escherichia coli to induce acute neuroinflammation, showed less cognitive resilience as adults when exposed to another immune stressor (Bilbo et al., 2006, Bilbo and Schwarz, 2009). A working hypothesis is that early-life infection alters microglial cells in a manner that renders them hypersensitive to subsequent stimuli (Meyer et al., 2011). However, there is a gap of knowledge regarding the effects of early postnatal viral infection on microglia activity and their sensitivity to subsequent insults.

Recently, we have conducted studies using a neonatal domestic piglet model. The piglet immune system is similar to human as is growth and development of its gyrencephalic brain (Conrad and Johnson, 2015). In our previous studies with neonatal piglets, infection with porcine reproductive and respiratory syndrome virus (PRRSV) resulted in reduced neurogenesis and altered pyramidal neuron structure in hippocampus (Conrad et al., 2015), reduced gray and white matter in several brain regions (Conrad et al., 2014), and compromised performance in a hippocampal-dependent spatial T-maze task (Elmore et al., 2014). In addition, peripheral infection was associated with robust activation of microglia in the hippocampus (Elmore et al., 2014). These studies indicated PRRSV infection induced a neuroinflammatory response and affected brain development.

To significantly extend these findings and to address the gap of knowledge regarding infection and microglia activity and subsequent sensitivity, the present study first assessed gene expression patterns in hippocampal tissue obtained from our previously published study reporting increased microglial cell activation and reduced hippocampal-dependent learning in piglets infected with PRRSV (Elmore et al., 2014). Results of this analysis suggested infection up-regulated a number of genes that are part of the newly defined microglia sensome (Hickman et al., 2013). As this sensory apparatus may foretell microglia reactivity, we subsequently assessed the effects of peripheral infection on hippocampal microglia morphology and function. The important findings suggest early-life infection alters the sensome, rendering microglia more responsive to subsequent immune stimuli.

Section snippets

Animals, housing, virus inoculation and detection

All animal care and experimental procedures were in accordance with the NRC Guide for the Care and Use of Laboratory Animals and approved by the University of Illinois at Urbana-Champaign Institutional Animal Care and Use Committee. A detailed description of animals and housing can be found in Elmore et al. (2014) and Conrad et al. (2015). Briefly, naturally farrowed crossbred piglets obtained at PD 2 were assigned to either control (control, sham-inoculated) or PRRSV treatment balancing for

Respiratory viral infection affects the hippocampal transcriptome in neonatal piglets

We first assessed gene expression patterns in hippocampal tissue obtained from our previously published study (Elmore et al., 2014). A total of 455 DEGs were revealed in the hippocampus in response to PRRSV (P < 0.05, Benjamini-Hochberg adjusted for multiplicity; Fig. 1A), of which 334 were up-regulated and 121 were down-regulated. The hierarchical clustering heatmap of the overall DEG pattern suggested a consistent effect of PRRSV infection on hippocampal transcriptomic profiles (Fig. 1B). The

Discussion

The hippocampal transcriptome induced by PRRSV revealed a gene expression pattern indicative of (a) neuroinflammation as the up-regulated DEGs were predominantly involved in the immune response; and (b) reduced plasticity as synaptic genes were mostly down-regulated. This was evident 21 d after inoculation at a time piglets showed signs of recovery and in the absence of compelling evidence that virus entered the brain. By isolating microglia from adult mice and direct RNA sequencing, Hickman et

Acknowledgments

This work was supported by grants from the NIH (R01 HD069899) to R.W.J.; Jeju National University of Rural Development of Administration of the Republic of Korea (538 JNU Korea 2012-06052) to L.B.S. (for RNA-sequencing associated analysis). We thank Dr. William Van Alstine for provision of PRRS virus, and Jennifer Rytych for her help in microglial cell isolation.

References (31)

  • S.D. Bilbo et al.

    A lifespan approach to neuroinflammatory and cognitive disorders: a critical role for glia

    J. Neuroimmune Pharmacol.

    (2012)
  • M.S. Conrad et al.

    The domestic piglet: an important model for investigating the neurodevelopmental consequences of early life insults

    Annu. Rev. Anim. Biosci.

    (2015)
  • M.S. Conrad et al.

    An in vivo three-dimensional magnetic resonance imaging-based averaged brain collection of the neonatal piglet (Sus scrofa)

    PLoS One

    (2014)
  • C.L. Cunningham et al.

    Microglia regulate the number of neural precursor cells in the developing cerebral cortex

    J. Neurosci.

    (2013)
  • R. Dantzer et al.

    From inflammation to sickness and depression: when the immune system subjugates the brain

    Nat. Rev. Neurosci.

    (2008)
  • Cited by (27)

    • Transcriptome-wide analysis reveals core sets of transcriptional regulators of sensome and inflammation genes in retinal microglia

      2021, Genomics
      Citation Excerpt :

      The sensome genes are essential for the activation of microglia in physiological and pathological conditions. The sensome genes involved in neuroinflammation are up-regulated while the down-regulared sensome genes are involved in neuroplasticity [14]. Hickman et al., 2013 and 2019 [12,44] established the transcriptomes of microglia between healthy adult and aged mice and found transcripts encoding proteins for sensing endogenous ligands and microbes.

    • Microglia in the human infant brain and factors that affect expression

      2020, Brain, Behavior, and Immunity - Health
      Citation Excerpt :

      Regardless of aetiology, a potentially reduced territory of surveillance, due to fewer microglia in the hippocampus may result in a diminished ability to detect and respond to irregularities in synapses and perturbations to homeostasis (Kettenmann et al., 2011; Nimmerjahn et al., 2005). Porcine models suggest peripheral viral infection during development may alter the sensitivity of microglia in the hippocampus to future stimuli (Ji et al., 2016). Interestingly, we found the presence of an URTI to be independently associated with a smaller area covered by microglia in the same implicated hippocampus regions.

    • Microglia Biology: One Century of Evolving Concepts

      2019, Cell
      Citation Excerpt :

      In general, microglia in aged mice show a shift toward an alternative priming state with downregulation of inflammasome genes (Hickman et al., 2013). Conversely, early-life peripheral viral infection in piglets induced the expression of sensome genes in hippocampal microglia (Ji et al., 2016). As the primary immune effector cells of the CNS, microglia rapidly respond to changes in the CNS environment and orchestrate neuroinflammation by secreting important immune mediators.

    • Herring roe oil supplementation alters microglial cell gene expression and reduces peripheral inflammation after immune activation in a neonatal piglet model

      2019, Brain, Behavior, and Immunity
      Citation Excerpt :

      Pigs have gyrencephalic brains with similar developmental trajectories compared to humans (Dobbing and Sands, 1979; Rice and Barone, 2000), and can be transitioned to formula diets within days of birth, making them ideal for dietary studies. Our lab has previously shown that in a neonatal piglet model of early life viral infection, microglial sensome genes, which allow microglia to express receptors to sense microenvironmental stimuli (Hickman et al., 2013), were upregulated in infected piglets (Ji et al., 2016). Two separate studies were conducted to determine if 1) supplementing sow milk replacer with HRO at a concentration that provided twice the recommended level of DHA for infants (Fats, 1994) would alter the fatty acid profile of the neonatal brain, and 2) HRO supplementation would attenuate inflammation in the periphery and brain.

    • Environment matters: microglia function and dysfunction in a changing world

      2017, Current Opinion in Neurobiology
      Citation Excerpt :

      Notably, infection at a later stage of development, postnatal day (P)30, does not have the same persistent impact, implicating a critical window [42]. Early-life viral infection of piglets, who demonstrate similar brain growth trajectories and time course as humans, similarly induces a long-term priming effect on microglia [43•]. Specifically, microglia collected at P28 from piglets infected with virus at P7, display enhanced phagocytic and migratory activity, increased sensitivity to a second challenge with LPS and Poly I:C, as well as increased soma size compared to controls, closely supporting our findings in rodents [20,44].

    View all citing articles on Scopus
    View full text