Mapping the impact of exposure to maternal immune activation on juvenile Wistar rat brain macro- and microstructure during early post-natal development.

Maternal immune activation is consistently associated with elevated risk for multiple psychiatric disorders in the affected offspring. Related to this, an important goal of our work is to explore the impact of maternal immune activation effects across the lifespan. In this context, we recently reported the effects of polyriboinosinic-polyribocytidylic acid-induced maternal immune activation at gestational day 15, immediately prior to birth, at gestational day 21 and again at post-natal day 21, providing a systematic assessment of plasma interleukin 6, body temperature and weight alterations in pregnant rats and preliminary evidence for gross morphological changes and microglial neuropathology in both male and female offsprings at these time points. Here, we sought to complement and extend these data by characterising in more detail the mesoscale impact of gestational polyriboinosinic-polyribocytidylic acid exposure at gestational day 15 on the neuroanatomy of the juvenile (post-natal day 21) rat brain using high-resolution, ex vivo anatomical magnetic resonance imaging in combination with atlas-based segmentation. Our preliminary data suggest subtle neuroanatomical effects of gestational polyriboinosinic-polyribocytidylic acid exposure (n = 10) relative to saline controls (n = 10) at this time-point. Specifically, we found an increase in the relative volume of the diagonal domain in polyriboinosinic-polyribocytidylic acid offspring (p < 0.01 uncorrected), which just failed to pass stringent multiple comparisons correction (actual q = 0.07). No statistically significant microstructural alterations were detectable using diffusion tensor imaging. Further studies are required to map the proximal effects of maternal immune activation on the developing rodent brain from foetal to early post-natal life and confirm our findings herein.

On-target and off-target effects of novel orthosteric and allosteric activators of GPR84.

Many members of the G protein-coupled receptor family, including examples with clear therapeutic potential, remain poorly characterised. This often reflects limited availability of suitable tool ligands with which to interrogate receptor function. In the case of GPR84, currently a target for the treatment of idiopathic pulmonary fibrosis, recent times have seen the description of novel orthosteric and allosteric agonists. Using 2-(hexylthiol)pyrimidine-4,6 diol (2-HTP) and di(5,7-difluoro-1H-indole-3-yl)methane (PSB-16671) as exemplars of each class, in cell lines transfected to express either human or mouse GPR84, both ligands acted as effective on-target activators and with high co-operativity in their interactions. This was also the case in lipopolysaccharide-activated model human and mouse immune cell lines. However in mouse bone-marrow-derived neutrophils, where expression of GPR84 is particularly high, the capacity of PSB-16671 but not of 2-HTP to promote G protein activation was predominantly off-target because it was not blocked by an antagonist of GPR84 and was preserved in neutrophils isolated from GPR84 deficient mice. These results illustrate the challenges of attempting to study and define functions of poorly characterised receptors using ligands that have been developed via medicinal chemistry programmes, but where assessed activity has been limited largely to the initially identified target.

Evolution of a maternal immune activation (mIA) model in rats: Early developmental effects.

Maternal immune activation (mIA) in rodents is rapidly emerging as a key model for neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Here, we optimise a mIA model in rats, aiming to address certain limitations of current work in this field. Specifically, the lack of clear evidence for methodology chosen, identification of successful induction of mIA in the dams and investigation of male offspring only. We focus on gestational and early juvenile changes in offspring following mIA, as detailed information on these critical early developmental time points is sparse. Following strain (Wistar, Lister Hooded, Sprague Dawley) comparison and selection, and polyriboinosinic-polyribocytidylic acid (poly I:C) dose selection (2.5-15 mg/kg single or once daily for 5 days), mIA was induced in pregnant Wistar rats with 10 mg/kg poly I:C i.p. on gestational day (GD) 15. Early morphometric analysis was conducted in male and female offspring at GD21 and postnatal day (PD) 21, eight dams for each treatment at each time point were used, 32 in total. Subsequent microglia analysis was conducted at PD21 in a small group of offspring. Poly I:C at 10 mg/kg i.p. induced a robust, but variable, plasma IL-6 response 3 h post-injection and reduced body weight at 6 h and 24 h post-injection in two separate cohorts of Wistar rats at GD15. Plasma IL-6 was not elevated at PD21 in offspring or dams. Poly I:C-induced mIA did not affect litter numbers, but resulted in PD21 pup, and GD21 placenta growth restriction. Poly I:C significantly increased microglial activation at PD21 in male hippocampi. We have identified 10 mg/kg poly I:C i.p on GD15 as a robust experimental approach for inducing mIA in Wistar rats and used this to identify early neurodevelopmental changes. This work provides a framework to study the developmental trajectory of disease-relevant, sex-specific phenotypic changes in rats.

Acid-dependent Interleukin-1 (IL-1) Cleavage Limits Available Pro-IL-1ß for Caspase-1 Cleavage.

Noncommunicable diseases such as cardiovascular disease (stroke and heart attack), cancer, chronic respiratory disease, and diabetes are a leading cause of death and disability worldwide and are worsened by inflammation. IL-1 is a driver of inflammation and implicated in many noncommunicable diseases. Acidosis is also a key feature of the inflammatory microenvironment; therefore it is vital to explore IL-1 signaling under acidic conditions. A HEK-IL-1 reporter assay and brain endothelial cell line were used to explore activity of mature IL-1α and IL-1ß at pH 7.4 and pH 6.2, an acidic pH that can be reached under inflammatory or ischemic conditions, alongside cathepsin D-cleaved 20-kDa IL-1ß produced under acidic conditions. We report that mature IL-1 signaling at IL-1 receptor type 1 (IL-1R1) is maintained at pH 6.2, but the activity of the decoy receptor, IL-1R2, is reduced. Additionally, cathepsin D-cleaved 20-kDa IL-1ß was minimally active at IL-1R1 and was not further cleaved to highly active 17-kDa IL-1ß. Therefore formation of the 20-kDa form of IL-1ß may prevent the generation of mature bioactive IL-1ß and thus may limit inflammation.

Release of interleukin-1α or interleukin-1ß depends on mechanism of cell death.

The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1α and IL-1ß, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1ß. Conversely, necroptosis caused the processing and release of IL-1α, and this was independent of IL-1ß processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.

Acidosis drives damage-associated molecular pattern (DAMP)-induced interleukin-1 secretion via a caspase-1-independent pathway.

The proinflammatory cytokine IL-1ß is a key mediator of inflammatory responses that contribute to and exacerbate brain injury. IL-1ß is synthesized by microglia in the brain as an inactive precursor (pro-IL-1ß). Cleavage of pro-IL-1ß to a mature form is stimulated by damage-associated molecular patterns (DAMPs). These DAMPs are sensed by a pattern recognition receptor called NLRP3, which forms an inflammasome, resulting in the activation of caspase-1 and cleavage of pro-IL-1ß. To date, regulation of the inflammasome in culture has been studied under normal culture conditions, and it is not known how DAMPs signal under disease relevant conditions such as acidosis. Given the presence of acidosis in pathological states, our objective was to test the hypothesis that acidic conditions modify DAMP-induced IL-1ß release from cultured primary mouse glial cells. When LPS-primed glial cells were stimulated with DAMPs under acidic conditions (pH 6.2), the predominant IL-1ß form secreted was the 20-kDa rather than the 17-kDa caspase-1-dependent species. Lactic acidosis, induced by the addition of 25 mm lactic acid, also induced the release of 20-kDa IL-1ß. This 20-kDa product was produced independently of NLRP3 and caspase-1 but was inhibited by the cathepsin D inhibitor pepstatin A. These data suggest that under disease relevant acidosis, DAMPs and lactic acid induce the secretion of IL-1ß independently of the inflammasome. Therapeutic strategies directed to the inhibition of IL-1ß processing should therefore consider alternative processing of IL-1ß in addition to caspase-1-dependent processing.