A Proof-of-Concept Study of Maternal Immune Activation Mediated Induction of Toll-Like Receptor (TLR) and In ammasome Pathways Leading To Neuroprogressive Changes and Schizophrenia-Like Behaviors in Offspring

Pinku Mani Talukdar National Institute of Mental Health and Neurosciences: National Institute of Mental Health and Neuro Sciences Fazal Abdul National Institute of Mental Health and Neuro Sciences Michael Maes Deakin University Geelong Waterfront Campus Michael Berk Deakin University Geelong Waterfront Campus Ganesan Venkatasubramanian National Institute of Mental Health and Neurosciences: National Institute of Mental Health and Neuro Sciences Bindu M Kutty National Institute of Mental Health and Neurosciences: National Institute of Mental Health and Neuro Sciences MONOJIT DEBNATH (  monozeet@gmail.com ) National Institute of Mental Health and Neuro Sciences https://orcid.org/0000-0002-5843-072X


Introduction
Schizophrenia is a heterogenous and one of the most debilitating neuropsychiatric disorders with a complex etiopathology. A multitude of risk factors may induce different neurotoxic adverse outcome pathways which contribute to the phenome of schizophrenia including neurocognitive impairments and the symptomatome.
These include genetic variants and changes in gut permeability leading to increased translocation of amongst other Gram-negative bacteria, coupled with de cits in innate immune responses [1]. Among the environmental factors, microbial infection at various life stages, beginning with prenatal, early postnatal, and adolescence, has been reported to confer a signi cant risk of schizophrenia [2,3]. The innate immune system is primarily involved in recognizing danger molecules derived from the host proteins and microorganisms and subsequently driving the downstream signaling processes to mount an appropriate immune response against them. The innate immune system achieves this function through sensors and receptors present on the cell surface or intracellular vesicles and such as Toll-like receptors (TLRs) and in cytosol like retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) [4]. A family of TLRs (TLR1-TLR13) has been identi ed and these recognize a variety of ligands such as glycans, nucleic acids, proteins, lipids, and lipoproteins [5]. Amongst the NLRs, the NLR family pyrin domain containing 3 (NLRP3) responds to a wide array of endogenous and exogenous stimuli and forms a multi-protein complex called NALP3 in ammasome. This in ammasome has a sensor protein, in ammatory caspases, and adapter protein in some cases. In ammasome activation leads to canonical caspase-1 (cas1), noncanonical caspase-11 (or the equivalent caspase-4 and caspase-5 in humans), or caspase-8; this promotes secretion of IL-1β and IL-18 and subsequently leads to apoptotic and pyroptotic cell death [6].
It is noteworthy that TLRs and NLRs cross-talk and collaborative actions are instrumental in mediating immunein ammatory responses against microbes [7,8]. Transcriptional activation of pro-IL-1β and pro-IL- 18 and their processing to an active form by the in ammasome require TLR signaling, further implying their role in downstream immune signaling processes. An emerging body of research suggests an important role of an impaired TLR-in ammasome cross-talk in many diseases of immune origin [9]. The activation and cross-talk between TLR-in ammasome in CNS infection and during CNS injury and neurodegeneration are increasingly evident [10,11]. The elements of TLR and in ammasome pathways have a wide expression in the brain and regulate key processes of synaptic plasticity. Uncontrolled activation of TLRs and in ammasomes in the brain induces microglial activation and promotes neuroin ammation [12].
An activated innate immune system has consistently been reported in patients with schizophrenia. Schizophrenia is accompanied by a breakdown of the paracellular and vascular pathways (increased gut permeability) and consequent translocation of Gram-negative bacteria, which is associated with neurocognitive impairments including executive functions, sematic and episodic memory, and psychosis, excitation, and negative symptoms [13]. Furthermore, rst episode schizophrenia is often accompanied by a moderate cytokine-storm with activation of M1 macrophage, T helper (Th)-1 and Th-2 phenotypes, indicating a bacterial or viral infection [14]. Moreover, there is evidence of levels of complement proteins, aberrant expressions of TLR molecules as well as the core components of in ammasome and altered activity innate immune cells in schizophrenia [15][16][17][18]. Altered expressions and activity of TLR molecules have been reported in the peripheral blood and postmortem brain of patients with schizophrenia [15,19]. A recent study has demonstrated increased gene expression of CASP1, NLRP3, PYCARD, and IL1β in peripheral blood cells, indicating increased in ammasome activity in patients with psychiatric disorders, including schizophrenia [20]. Another postmortem brain study of schizophrenia patients reported increased expression of caspase 1 and IL-1β [21]. These studies provide empirical evidence of peripheral in ammation and neuroin ammation in schizophrenia, driven by TLR and the in ammasome. Schizophrenia has been proposed to be a neuroprogressive disorder, and immune pathways seem to play a pivotal role in neuroprogression [22]. TLRs and the in ammasome mediate cell death pathway as well as neurodegeneration [10]. However, a mechanistic link between immune activation and neuroprogression in schizophrenia is yet to be validated through clinical and/or pre-clinical studies.
Maternal immune activation (MIA) models of schizophrenia in rodents have been used predominantly to delineate the impact of prenatal infection on immunological and neurobiological sequelae relevant to schizophrenia pathophysiology [23][24][25][26]. In a recent study, we have demonstrated the effect of MIA on schizophrenia-like-behaviors through activation of the immuno-in ammatory, oxidative stress and apoptotic pathways [27]. Acute in utero exposure to LPS increased the expressions of TLR2 and TLR4 in fetal brains and the postnatal amygdala [28]. Besides, prenatal LPS infection upregulated the expression of in ammasome and IL-1β in the brain of the offspring mice. Offspring also displayed schizophrenia-like behaviors [29]. However, it is unknown whether MIA leads to schizophrenia-like behavioral abnormalities in the offspring through a concerted action of TLR and in ammasome. Further, whether such a concerted action of TLRs and in ammasome drives the neuroprogressive changes in schizophrenia has also not been explored. To address this knowledge gap, we conducted a study to discern the effects of poly (I:C) and LPS induced MIA on TLR and in ammasome pathway in the hippocampus of the offspring and their impact on schizophrenia-like behaviors during peri-adolescent and adult periods. Further, the TLR and in ammasome pathway data were correlated with the signatures of apoptotic pathway to gain insights into the mechanism(s) leading to neuroprogression in schizophrenia. This study for the rst time provides mechanistic insight into the pathways underlying neuroprogression driven by immune aberrations in schizophrenia.

Animals
All experiments were performed on Sprague-Dawley rats (Rattus norvegicus) after obtaining approval from the Institutional Animal Ethics Committee (AEC/65/389/HG). 13-15 weeks old rats (both sex) weighing approximately 250-300 gms were housed in standard cages (22.5 × 35.5 × 15 cm) with corncob bedding and maintained on a 12-hour light/dark cycle with ad libitum access to food and water. Female and male rats were housed together overnight for mating. Following this, female rats were examined for the presence of a vaginal plug in the morning, and the presence of which was marked as Gestational day 0 (GD0). Additionally, body weights of putative pregnant female rats were checked periodically to con rm pregnancy. The pregnant rats were then subjected to MIA studies.
Establishment of the MIA model Three different groups of pregnant rats (n=15 rats / group) received either poly (I:C) (20 mg/kg) or LPS (1.5 mg/kg) or an equivalent volume of saline (0.9%) injections intraperitoneally on GD12. Both the poly (I:C) and LPS models were used to compare the effects of viral and bacterial agonists speci c to the TLRs (TLR3-virus-speci c and TLR2/LTR4-bacteria -speci c) on behavioral de cits. After 24 hours of injection, maternal blood was collected from each group of rats (n=5/group) by cardiac puncture. Plasma was separated and stored at -80 0 C. Plasma levels of proin ammatory cytokines, namely IL-1β, IL-6, TNF-α, and IL-17A, were analyzed by multiplex suspension assay in a Bio-plex 200 platform using commercially available rat Milliplex map kit (RECYTMAG-65K) following the manufacturer's guidelines (RECYTMAG-65K, Millipore, France).
The remaining pregnant rats from each group (n=10/group) could have a normal delivery. Offspring were weaned at postnatal day 21 (PD21); male and female pups were housed separately, 3 pups per cage. The offspring obtained from poly (I:C) (n=12-14), LPS (n=12-14) and saline (n=12-14)-treated dams were allowed to grow in normal conditions and subjected to various behavioral assessments such as open eld test (OFT), social interaction test (SIT) and prepulse inhibition (PPI) during the peri-adolescent (39-44 days) and adult (60-70 days) periods. After the completion of behavioral assessments during the adult period, hippocampal tissues were collected from these offspring for the quanti cation of gene expression.

Assessments in the Offspring rats
Three behavioral tests, the Open Field Test (OFT), Social Interaction Test (SIT), and Prepulse Inhibition (PPI) test, were carried out on three different sets of offspring rats, each set comprising 12-14 rats. In the present study, we have used 12-14 offspring rats for each behavioral test, and this was calculated based on our previous research on similar behavioral assessments [30]. The sample size required for gene expression studies was 5, based on a study that examined the expression of cerebellar cytokines in the offspring of poly (I:C) treated mice [18]. The behavioral and molecular experiments were performed on the same number of both the male and female offspring.
Behavioral assessments in the offspring rats Schizophrenia-like behavioral assessments such as anxiety-like and exploratory behaviors, social behaviors, and prepulse inhibition (PPI) tests were carried out in the offspring during peri-adolescent and adult periods. Rats (n=12-14/group) were subject to i) open eld test (OFT) to assess anxiety-like and exploratory behaviors, ii) social interaction test (SIT) to examine social behaviors (both the sociability and social novelty), as described in a previous study [30] and iii) prepulse inhibition (PPI) using the Coulbourn instruments startle apparatus (Holliston, Massachusetts) during the peri-adolescent (39-44 days) and adult (60-70 days) periods. The details of the methods followed for assessing behavioural abnormalities in the offspring are available in our recently published paper [27].
Quanti cation of expressions of genes of TLR and in ammasome pathways in the hippocampal tissue of offspring rats Hippocampal tissue (approximately 100 mg) was collected from offspring rats (n = 5) during the adult period and stored in RNAlater solution to quantify the expression of TLR2, TLR3 and TLR4, and in ammasome (NLRP3, caspase-1, IL-1β, and IL-18) pathway-related genes. Hippocampal brain tissue was homogenized and the total RNA was extracted using a commercially available RNeasy lipid tissue mini-Kit (Qiagen, Germantown, Maryland). The mRNA was converted to cDNA using a high-capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems, USA), and the cDNA was diluted to a nal concentration of 10 ng/µl. The quantitative PCR (qPCR) was performed in a Quant Studio 6 platform (Applied Biosystems, USA) using SYBR Green assays. The details of the primer sequences and their accession IDs are provided in were run in triplicate for each target gene. The qPCR reaction was performed with a commercially available SYBR Green master mix (TB Green® Premix Ex Taq™ II, Takara, CA, USA). Reactions were performed in a nal volume of 10 μl containing SYBR green Master Mix, and primers of each gene as well as 1μl of cDNA. All ampli cation batches included no template control (NTC), and cDNA of a control rat (not from the treatment groups) was used as a calibrator (reference sample). It was run in each plate to assess intra-and inter-assay variability, which was <0.5 Ct. The 2 −ΔΔCt method of relative quanti cation was used. The result is presented as the fold change of target gene expression relative to a reference sample, normalized to the endogenous control (GAPDH).

Statistical Analyses
GraphPad Prism 8.0.1 (Graphpad Software, La Jolla, CA, USA) was used for statistical analyses. For analyzing SIT and PPI data, two-way ANOVA was used to nd the effect of treatment vs. chambers and treatment vs trials across the whole session, followed by Dunnett's multiple comparison tests. For the analysis of the behavioral tests and gene expression data, one-way ANOVA followed by Dunnett's multiple comparison tests were performed. Pearson's correlation matrix was used to assess the association of behavioral parameters with gene expression data.

Results
Poly (I:C) and LPS treatment induce MIA Signi cantly higher levels of IL-6, IL-17A and TNF-α were observed in both the poly (I:C) and LPS-treated pregnant rats, the levels of IL-1β were greater only in LPS-treated rats. The details of the MIA assessments in the pregnant rats are described in our previous study [27].
Behavioral abnormalities in the MIA offspring The details of the results of three behavioral tests like anxiety-like behaviors, social behaviors and PPI in the offspring of poly (I:C) and LPS-treated dams were described in our previous paper [27] and are summarized below.

Poly (I:C) and LPS-treated offspring exhibit anxiety-like behaviors
The rats born to the poly (I:C) and LPS-treated dams showed signi cant anxiety-like behaviors in the OFT. They spent more time in the periphery and not in the center of the chamber compared to rats born to saline-treated dams during both the peri-adolescent and adult periods. The ambulatory behaviors, especially the ambulation rate were comparable across all three groups indicating normal locomotor behaviors.
Poly(I:C) and LPS-treated offspring rats display de cits in social behaviors Offspring of both the poly (I:C) and LPS-treated dams displayed signi cant social behavioral de cits in terms of both sociability and social novelty aspects of social cognition during peri-adolescent and adult periods.

MIA offspring exhibits altered prepulse inhibition
There was no signi cant difference in percentage PPI in offspring of both the treated and control groups during the peri-adolescent period. However, LPS offspring had a decreased percentage of PPI at both 80 dB and 85 dB, but poly (I:C) offspring showed diminished PPI only at 85dB during the adult period.

Assessments of TLR and in ammasome pathways in MIA offspring
Among the TLR pathway genes, signi cantly elevated expression of TLR3 was observed in offspring of only poly (I:C) (MD of 0.43 with 95% CI 0.074 to 0.78; P=0.02) and TLR4 in both the poly (I:C) (MD of 0.92 with 95% CI 0.14 to 1.4; P=0.002) and LPS (MD of 0.66 with 95% CI 0.14 to 1.2 P=0.01) treated rats, while the expression of TLR2 did not vary across the offspring of the treated and control groups (Fig. 1a). Besides this, hippocampal gene expressions of IL-1β, IL-18 and NLRP3 were signi cantly upregulated in the offspring of both the poly (I:C) and LPS treated dams. In contrast, the expression of Cas1 was signi cantly elevated only in the offspring of the LPS treated dams (Fig. 1b).
Correlation between TLR, in ammasome pathway-related genes, and apoptotic pathway-related molecules expressions in the offspring: Pearson correlation was performed to determine the relationship between the hippocampal TLR gene expression and in ammasome pathway-related parameters in offspring rats. As TLR and in ammasome pathway activation leads to apoptotic changes, a correlation analysis among the markers of these three pathways was performed.
The gene expression data of the apoptotic pathway were taken from our previous study on the same hippocampal tissue samples [27]. The ndings from correlations among TLR, in ammasome and apoptotic pathway related genes have been summarized in Table- Correlation between TLR and in ammasome pathway-related genes expression, and schizophrenia-like behavior de cits in the offspring: Pearson correlation was performed to determine the relationship between hippocampal gene expression and behavioral parameters in offspring rats. None of the studied genes showed correlation with anxiety-like behaviors; however, signi cant correlations were observed with social behaviors and PPI de cits.

Discussion
In this study, the MIA status was established by injecting the rats with poly (I:C) and LPS on GD12 and indexed by heightened levels of in ammatory cytokines, such as IL-1β, IL-6, IL-17A and TNF-α in the maternal plasma, twenty-four hours after injection. In the offspring, the MIA signi cantly altered anxiety-like, social interactions and sensorimotor gating as measured by examining prepulse inhibition, which are models of human schizophrenia symptoms. Anxiety-like behaviors and socio-cognitive de cits were observed in the offspring of both the poly (I:C) and LPS-treated dams during the adolescent and adult periods. Reduced PPI during the adult period was also observed in the offspring of both the poly (I:C) and LPS-treated dams. Sex was not found to have any effect on the behavioral measures studied here.
In a recent study using MIA model, we demonstrated an important role of immune-in ammatory, oxidative stress and apoptotic pathway in MIA-induced schizophrenia-like behaviors in the offspring [27]. However, there are no studies pertaining to the role and interactions of immune initiators such as TLR and in ammasome and their effect on apoptotic pathway and subsequently to schizophrenia-like behaviors.

Impact of MIA on TLR and in ammasome and schizophrenia-like behaviors in the offspring
In this study, upregulated expressions of TLR3 in the offspring of poly (I:C) and TLR4 in the offspring of both the poly (I:C) and LPS-treated dams. Importantly, all three TLRs (TLR2, TLR3 and TLR4) studied here exhibited associations with schizophrenia-like behavioral abnormalities in offspring, suggesting a pivotal role of increased TLR signaling in behavioral de cits akin to schizophrenia in the offspring. To the best of our knowledge, this is the rst study to demonstrate the long-lasting effects of MIA on three TLR molecules in the hippocampus of the offspring and their impact on schizophrenia-like behaviors, employing both the poly (I:C) and LPS models. In a previous study, poly (I:C) injection during gestation impaired neonatal locomotor development, inhibited cortical neurogenesis and behavioral disturbances, including abnormal sensorimotor gating in the offspring during the adult period, and these were found to be mediated by TLR3 [31]. Besides poly (I:C), LPS-induced MIA led to upregulation of TLR2 and TLR4 in the fetal brain and the amygdala of the adult offspring; however, the authors did not examine their impact on behavioral de cits [28]. TLR activation seems to induce neuroin ammation and lead to increased production of reactive oxygen and nitrogen species ROS/RNS) and activates oxidative and nitrosative stress (O&NS) pathway in the brain and subsequently result to schizophrenia psychopathology. Taken together, ours and other studies suggest important implications of TLRs in the neuronal functions and behavioral de cits.
The other salient ndings were upregulated expression of NLRP3, IL-1β, and IL-18 in the hippocampus of the offspring of both the poly (I:C) and LPS-treated offspring and Cas1 only in the LPS offspring. It is interesting to note that the elevated levels of the in ammasome elements had signi cant impact on schizophrenia-like behavioral de cits in the offspring. There is a lack of data on the role of in ammasome in MIA induced schizophrenia-like behaviors. The only study on LPS induced MIA mediated schizophrenia-like behaviors through in ammasome activity showed increased expression of NLRP3 in ammasome and IL-1β in the hippocampus of the offspring mice [29]. Our ndings on four core components of the in ammasome provide further support towards the role of the in ammasome in MIA induced schizophrenia-like behaviors in the offspring. It is noteworthy that both poly (I:C) and LPS induced MIA led to similar changes in the behavioral de cits and hippocampal expressions of TLR and in ammasome in the offspring rats. However, a previous study reported differential effects of Poly (I:C) and LPS on the in ammatory responses, based on IL-2, IL-5 and IL-6 levels as well as behaviors, such as anxiety-like, and sensorimotor development in the offspring [32]. Since the design and concept of this and our current study are different, the ndings between these two studies cannot be compared.
We suggest that the effect of bacterial infection on the immune and behavioral sequelae could be more than the viral infection. LPS induced activation of TLR molecules, especially TLR4 leads to induction of all the core elements of in ammasome pathway (NLLRP3, cas1, IL-1β and IL-18) and this may induce a radical TLR4 cycle.
Besides, recent advances in the eld suggest a moderate cytokine storm in rst episode schizophrenia patients, indicating a bacterial rather than viral infection. Further studies are required to gain insights into this complex cascade of events.
Collaborative action of TLR and in ammasome and schizophrenia-like behavior in the offspring A combined action of the TLR and in ammasome is increasingly recognized to play a crucial role in triggering immune-in ammatory responses in many diseases, including CNS diseases [9,11]. There are no studies on the collaborative action of TLR and in ammasome in any neuropsychiatric disorders. The most salient nding of this study was the positive correlation between the TLR genes and the components of the in ammasome pathway. It is noteworthy that NLRP3 acts synergistically with TLR2/TLR4 and TLR3 to modulate in ammatory responses against bacterial and viral pathogens, respectively [8,33]. The synergistic interactions of TLR and NLRP3 and subsequent activation of IL-1β and induction of neuroin ammation has been reported in neurodegenerative diseases [11,34]. The activation of TLRs leads to activation of NLRP3 and Cas1 and this subsequently leads to induction of IL-1β, and IL-18. The combined action of these molecules may lead to pyroptosis, an in ammatory form of cell death [35]. In the current study, the expressions of both the TLR2 and TLR3 genes were found to signi cantly correlate with the expressions of the core elements of the in ammasome pathway, including the effector cytokines such as IL-1β and IL-18. This implies that in the face of viral and bacterial infection, TLRs and in ammasome activation and interactions lead to neuroin ammation due to elevated production of IL-1β and IL-18, and in turn, might contribute to schizophrenia pathogenesis. This observation reports for the rst time synergistic action of TLR and in ammasome in the hippocampal tissue of the offspring rats, showing schizophrenia-like behavior.
5.3 TLR and in ammasome activation and pyroptosis in the brain of the offspring rats TLR molecules also showed a positive correlation with the genes of the apoptotic pathway. Another interesting nding was the positive correlation between the expressions of the effector cytokines (IL-1β and IL-18) of in ammasome and genes of the apoptotic pathway in the hippocampus of the offspring rats of the poly (I:C) and LPS-treated dams. Increased expressions of IL-1β and IL-18 are linked with greater expressions of proapoptotic markers and decreased expression of neuroprotective genes. It is now well-documented that the NLRP3 in ammasome activates cell death pathways. However, studies showing such a link are currently not available for any psychiatric conditions. Our novel ndings suggest that MIA, through activation of TLR and in ammasome, might lead to pyroptosis in the brain of the offspring and thus result in schizophrenia-like behavioral abnormalities. We propose that prenatal infection and subsequent MIA drive schizophrenia-like behavioral changes through neurodevelopmental aberrations and long-lasting neuroprogressive changes. This process occurs through a persistent and complex chain of events involving a collaboration of multiple elements of the innate immune system, especially TLR, and in ammasome as the primary drivers of the entire process.
These data provide direct evidence towards a mechanistic link between MIA and schizophrenia-like behaviors by driving neuroprogressive changes. These molecules could emerge as the potential targets of pharmacological therapy. In a recent study on the MIA model of schizophrenia, paliperidone was shown to revert activation of TLR3, elicited by poly (I:C) injection and restore cognitive de cits [36].

Conclusion
Increased activation as well as expressions of TLRs and in ammasome promote in ammation and drive pathogenetic pathways in many autoin ammatory diseases of the central nervous system [37]. The ndings of the current study highlight that a collaborative action of TLR and in ammasome in mediating the effect of prenatal infection-induced MIA on schizophrenia-like behavioral de cits. Further, the signatures of these pathways seem to potentially interact with the mediators of the apoptotic pathway and in uence their expression in the hippocampus of the offspring rats. These ndings provide empirical support towards the implications of MIA in inducing neuroprogressive changes and schizophrenia-like behaviors. They also offer mechanistic evidence verifying the interactions between the pathways hypothesized as the foundation of neuroprogression [22,38].  Tables   Table-1: Shows the list of the genes, primer sequences and their accession number.

Name of Gene
Forward primer Reverse primer Accession number   Figure 1 Gene expression of TLR and in ammasome pathway-related genes in hippocampal brain tissue in the offspring of poly (I:C) and LPS-treated and control rats.