Maternal immune activation in late gestation increases neuroinflammation and aggravates experimental autoimmune encephalomyelitis in the offspring
Introduction
Multiple sclerosis (MS), the most prevalent inflammatory demyelinating disease of the central nervous system (CNS), affects approximately 0.1% of the global population, mostly young adults in Europe and North America (Hauser and Oksenberg, 2006). Both MS and its murine model, experimental autoimmune encephalomyelitis (EAE), are characterized by an autoimmune response against CNS proteins driven by autoreactive T cells, which infiltrate the CNS, causing gliosis, demyelination, axon degradation and neuronal death (Brown and Sawchenko, 2007, Murphy et al., 2010, Neumann, 2003, Rodriguez, 2007).
Many studies have employed the EAE model to analyze the cell types involved in the pathogenesis of the disease, such as T CD4+ (Kroenke and Segal, 2007) and CD8+ (Goverman et al., 2005) cells as well as astrocytes and microglia (Glass et al., 2010, Murphy et al., 2010). Prior to the onset of the disease, antigen-presenting cells (APC) polarize T cells toward Th1 and Th17 subtypes in response to myelin antigens, initiating the peripheral immune response that culminates in massive cell infiltration into the CNS. In the target organ, those infiltrating cells interact with glial cells to orchestrate the immune response to myelin proteins during the progression of the disease.
Despite all the knowledge that has been gathered on MS pathogenesis, the underlying cause and risk factors for the autoimmunity remain poorly understood. The genetic background seems to influence, but not determine, susceptibility to MS (Sospedra and Martin, 2005). Environmental factors, such as sunlight exposure (Handel et al., 2010) and previous infections (Sospedra and Martin, 2005), could play a role in predisposing an organism to MS. However, the involvement of the prenatal period in this phenomenon and particularly the impact of acute maternal immune activation in the offspring’s autoimmunity are currently unknown.
A recent body of evidence from our laboratory has shown that prenatal immune stimulation by lipopolysaccharides (LPS) impacts behavior and neurochemistry in later life (Zager et al., 2014, Zager et al., 2012). Recent findings demonstrated that adult offspring of LPS-stimulated mice presented enhanced peripheral Th1 cytokines and increased delayed-type hypersensitivity responses (Zager et al., 2013). Additionally, it has been shown that prenatal immune activation by the viral mimetic polyriboinosinic–polyribocytidylic acid (PolyI:C) induces preferential development of Th17 cells by the offspring (Mandal et al., 2010a, Mandal et al., 2010b), supporting the hypothesis of the presence of altered adaptive immunity in the offspring of immune-stimulated dams.
Based on previous findings, disruption of the maternal cytokine balance during critical gestational periods influences the development of the offspring’s immune response (Mandal et al., 2010a, Mandal et al., 2010b, Zager et al., 2013). Thus, the aim of the present study was to investigate the impact of prenatal immune activation by LPS during late gestation on neuroinflammatory responses in primary mixed glial cultures from offspring as well as on EAE progression in adult offspring.
Section snippets
Animals
Male and female C57BL/6J mice from our own colony, weighing 20–30 g each, were used. The animals were housed in standard polypropylene cages (4 animals/cage, except where indicated) at a controlled room temperature (22 ± 2 °C) and humidity level (65–70%), with artificial lighting (12 h light/12 h dark cycle) and with free access to Nuvilab® rodent chow (Nuvital Company, São Paulo, Brazil) and filtered water. Sterilized and residue-free wood shavings were used as animal bedding. The animals used in
Experiment 1: in vitro production of inflammatory mediators by mixed glial cells
In this experiment, cells were isolated from the brains of neonates and then restimulated in vitro with LPS. The immunofluorescence microscopy revealed the major cell types involved in the mixed culture: neurons, astrocytes and microglia, as evidenced by positive staining for MAP-2, GFAP and Iba-1, respectively (Fig. 2A). The two-way ANOVA showed an effect of prenatal treatment [F (1,24) = 6.08, p = 0.01], of in vitro stimulus [F (1,56) = 793.69, p = 0.01] and of the interaction treatment × stimulus [F
Discussion
Our results show that maternal immune activation during mice late gestation influences the development of offspring’s immune system, an effect that persists until adulthood. Specifically, the offspring of the LPS-treated dams showed increased CNS cytokine production in a primary culture of glial cells and aggravated clinical manifestations and immune responses during the course of EAE.
We have previously shown that the offspring of dams challenged during late gestation presented an increase in
Author contributions
A.Z., J.P.P., G.M. and J.P.N. conceived and designed the experiments. A.Z., J.P.P., G.M., S.C.R. and T.P.A. performed the experiments. A.Z., J.P.P. and G.M. analyzed the data. J.P.N., J.P.P. and G.M. contributed reagents, materials, and analysis tools. A.Z. and J.P.N. wrote the paper.
Funding
This work was supported by Grants from the CNPq and FAPESP (Thematic Awards #09/51886-3 to J.P.N., #09/51998-6 to A.Z.).
Competing interests
The authors have declared that no competing interests exist.
Acknowledgements
The authors would like to express their gratitude to Nicolle Queiroz for assistance with the CBA analysis and to Adriana de Siqueira and Prof. Paulo Maiorka for their valuable suggestions during this project.
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