Neonatal corticosterone mitigates autoimmune neuropsychiatric disorders associated with streptococcus in mice

Abstract

Increased glucocorticoid concentrations have been shown to favor resilience towards autoimmune phenomena. Here, we addressed whether experimentally induced elevations in circulating glucocorticoids mitigate the abnormalities exhibited by an experimental model of Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). This is a pathogenic hypothesis linking repeated exposures to Group-A-beta-hemolytic streptococcus (GAS), autoantibodies targeting selected brain nuclei and neurobehavioral abnormalities. To persistently elevate glucocorticoid concentrations, we supplemented lactating SJL/J mice with corticosterone (CORT; 80 mg/L) in the drinking water. Starting in adolescence (postnatal day 28), developing offspring were exposed to four injections - at bi-weekly intervals - of a GAS homogenate and tested for behavioral, immunological, neurochemical and molecular alterations. GAS mice showed increased perseverative behavior, impaired sensorimotor gating, reduced reactivity to a serotonergic agonist and inflammatory infiltrates in the anterior diencephalon. Neonatal CORT persistently increased circulating glucocorticoids concentrations and counteracted these alterations. Additionally, neonatal CORT increased peripheral and CNS concentrations of the anti-inflammatory cytokine IL-9. Further, upstream regulator analysis of differentially expressed genes in the striatum showed that the regulatory effect of estradiol is inhibited in GAS-treated mice and activated in GAS-treated mice exposed to CORT. These data support the hypothesis that elevations in glucocorticoids may promote central immunomodulatory processes.

Figure 1

Experimental design: Timing of the neonatal treatment and of the injections, expressed in weeks, and the experimental procedures performed with two different and independent batches of mice. After corticosterone administration during the neonatal phase (postnatal days, PND, 1–10), mice received 5 injections of GAS homogenate or Phosphate Buffer Saline (PBS), formulated with the indicated adjuvants (CFA = Complete Freund’s adjuvant; IFA = Incomplete Freund’s adjuvant). At sacrifice, we collected: in Batch I, plasma samples for antibody and cytokine determination and brain samples for immunohistochemistry; in Batch II, we collected brain areas for monoamine determinations and RNA sequencing.

Figure 2

(a) Hair corticosterone (CORT) concentrations measured at weaning in control (WATER) and CORT-treated subjects (n = 11 per group); (b) Plasma CORT concentrations measured at sacrifice (i.e. two weeks after the fifth GAS injection; n = 8–12 per group): log-transformed data were used for the statistical analysis; actual concentration data are reported for the sake of clarity. CORT-treated subjects showed increased concentrations of CORT both in the short- (weaning) and long-term (adulthood). ^*p < 0,05 compared to respective WATER controls; (c) Western Blot analysis of GAS extracts probed with pooled sera from mice treated with GAS homogenates or adjuvant alone as Control, neonatal CORT and GAS homogenates or neonatal CORT and adjuvant. Lane 1, Western Blot results after probing with sera of control mice injected with four doses of adjuvant alone; lane 2, Western Blot results after probing with sera of mice injected with four doses of GAS homogenates; lane 3, Western Blot results after probing with sera of mice injected with four doses of neonatal CORT and adjuvant; lane 4, Western Blot results after probing with sera of mice injected with four doses of neonatal CORT and GAS homogenates. Lanes have been cropped from different parts of the same gel (see Supplementary Fig. 2).

Figure 3

(a) Behavioral response (head twitch) to challenge with DOI (5 mg/kg, i.p., week 13, fifth injection) during a single 10-min session. Data are expressed as mean + SEM (n = 8–12 per group); (b) Sensorimotor gating measured through PPI (week 11, fourth injection). Average inhibition of the startle reflex to a 120-dB stimulus following the presentation of a pre-pulse of 67, 70, 73, and 76 dB. Values are expressed as mean percentage PPI (%PPI) + SEM (n = 8–12 per group); (c) Perseverative behavior in a T-maze (week 11, fourth injection) measured as the percentage of spontaneous alternations (circular symbol) with 95% CI (whiskers). The dashed lines represent chance level; (a) CI intersecting the dashed line indicates that percentage of alternations was not statistically different from chance.

Figure 4

Neuropathology assessment of microglia activity in mice brains. H&E staining shows the presence of inflammatory infiltrates in the white matter of the rostral diencephalon of a representative CORT-PBS mouse (B) and a WATER-GAS mouse (C) not detected in WATER-PBS mice (A) and CORT-GAS mice (D). The presence of Iba1+ microglia activity - diffusely present in WATER-GAS mice (G)- was rarely observed in CORT-PBS mice (F) and CORT-GAS mice (H), and was mainly detected associated with small, periventricular inflammatory infiltrates. Iba1+ microglia activity was not detected in WATER-PBS mice (E). A considerable number of IL-9+ cells were observed in the inflammatory infiltrates persisting in different regions of the brains of CORT-GAS mice (I,L). The arrow in H indicates the area in serial brain sections where increased density of IL-9+ cells was detected. (Original magnification: 5x (B,D), 10x (A,C,E,F,G,L), 20x (H,I).

Figure 5

Schematic recapitulating the main topics addressed in the current study: etiological hypothesis (a), modulatory role of neonatal corticosterone administration (b), and potential molecular mechanisms involved (c). Specifically, in panel (a) we exemplified the etiological hypothesis linking streptococcal immunizations, antibody response and behavioral abnormalities. In panel (b) we sketched a candidate mechanism through which neonatal corticosterone may mitigate the PANDAS-like neurobehavioural abnormalities. In (c) we visually represented the main findings from the upstream regulator analysis. The predicted effects of GAS and CORT+ GAS are shown for signaling involving estradiol and the other signaling molecules from the analyses that are regulated by estradiol.