The innate immune toll-like-receptor-2 modulates the depressogenic and anorexiolytic neuroinflammatory response in obstructive sleep apnoea

The increased awareness of obstructive sleep apnoea’s (OSA) links to Alzheimer’s disease and major psychiatric disorders has recently directed an intensified search for their potential shared mechanisms. We hypothesised that neuroinflammation and the microglial TLR2-system may act as a core process at the intersection of their pathophysiology. Moreover, we postulated that inflammatory-response might underlie development of key behavioural and neurostructural changes in OSA. Henceforth, we set out to investigate effects of 3 weeks’ exposure to chronic intermittent hypoxia in mice with or without functional TRL2 (TLR2+/+, C57BL/6-Tyrc-Brd-Tg(Tlr2-luc/gfp)Kri/Gaj;TLR2−/−,C57BL/6-Tlr2tm1Kir). By utilising multimodal imaging in this established model of OSA, a discernible neuroinflammatory response was demonstrated for the first time. The septal nuclei and forebrain were shown as the initial key seed-sites of the inflammatory cascade that led to wider structural changes in the associated neurocircuitry. Finally, the modulatory role for the functional TLR2-system was suggested in aetiology of depressive, anxious and anorexiolytic symptoms in OSA.


Results
An acute TLR2 response in the region of basal forebrain and the septal nuclei. Previous studies have shown that TLR2 regulates the hypoxic/ischaemic brain damage caused by stroke 4,20,21 . To establish whether IH provokes a similar inflammatory response in brain, we used an established mouse model of OSA 19,22 and transgenic mice that bore the dual reporter system luciferase/green fluorescent protein under transcriptional control of the murine TLR2-promoter. TLR2 induction/microglial activation and its spatial and temporal dynamics were investigated in real time using BLI 20 . As previously reported by us and others 20 , the luciferase immunoreactivity co-localized in more than 95% of cells with Iba1 immunostaining 20 (e.g. microglial marker; Supplement). The signals were analysed over a 3-week period following experimental (IH) and control (CTRL) protocol ( Fig. 1A-D; also see SI Figure S1).
Olfactory bulb microglia in mice receive and translate numerous inputs from the brain and the environment and likely serve as sensors and/or modulators of brain inflammation. The subset of olfactory bulb microglial cells in mice was previously shown to continually express TLR2, enabling them to survey the environment in a 'primed' or alert state 20 . Similarly, in our study, a baseline activation over the area of the olfactory bulb and anterior olfactory nucleus was recorded (TLR2 +/+ CTRL: 7.655 × 10 6 ps −1 ; n = 6; TLR2 +/+ IH: 7.821 × 10 6 ps −1 ; n = 7; TLR2 modulates the effects of chronic IH on structural brain changes. Neuroimaging of patients with OSA demonstrates distinct neuroanatomical changes 2 . In order to investigate whether the observed neuroinflammatory response under our experimental conditions leads to similar structural changes, we utilised high resolution ex vivo magnetic resonance imaging (MRI). To fully verify involvement of the TLR2-system, mice with (TLR2 +/+ ) and without (TLR2 −/− ) functional TLR2 gene were imaged after 3 weeks of IH or CTRL protocol ( Fig. 2; SI Figs. S2-S4).
As shown in Fig. 2, comparison of structural brain grey and white matter changes with MRI in TLR2 +/+ IH vs TLR2 +/+ CTRL mice demonstrated coexistent hyper-(green) and hypotrophic (red) cortical, subcortical and white matter changes. Most important enlargements were visible bilaterally in the hippocampi and presubiculi regions while the reduction of volume was most evident bilaterally in the reticular nuclei of the thalamus, dorsal striatum, parahippocampal and piriform cortex and dorsolateral pons, with involvement of distinct parts of periaqueductal grey (PAG), including the dorsal raphe nuclei (DRN) (Fig. 2, also see Supplement). Taken together, the spatio-temporal nature of the demonstrated neuroinflammatory process over the 3 weeks suggested that majority of later structural changes developed in neuroanatomical regions with monosynaptic connections (see Fig. 3) to initial frontal and basal forebrain cortical sites of microglial TLR2 response (Fig. 2).
In addition to structural changes observed by MRI, further immunochemistry analyses examined the effect of TLR2 genotype on the cellular level. The TLR2 genotype appeared protective against demyelinating effects of IH. For example, a widespread IH-induced demyelination of the hippocampus was only demonstrable in TLR2 −/− deficient mice (SI Fig. S7 Table S2). Of those, notably, we demonstrated a prominent up-regulation of the cell adhesion molecule neuroplastin in mice with a functional TLR2-system. This was shown by its strong immunoreactivity signal pattern in all major hippocampal sublayers that contain neurons [e.g. granular layer in dentate gyrus (DG), and pyramidal layers in Cornu Ammonis (CA)] ( Fig. 4; SI Fig. S9). However, under our experimental conditions, the TLR2 deficiency appeared permissive for a more prominent modulation of IHinduced inflammatory stress response via neuroplastin in distinct hippocampal DG stratum granulare (t test for independent samples, t = − 2.72; df = 9; P = 0.023) and moleculare (t = − 3.09; df = 9; P = 0.013), and in CA2 region stratum pyramidale (t = − 2.71; df = 9; P = 0.024) and stratum oriens (t = − 2.61; df = 9; P = 0.028) (SI Fig. S9; also see SI Table S3). Plot of the data is depicted, which is obtained by measuring the luciferase activity (in photon per second, p/s) following intermittent hypoxia (IH; TLR2 +/+ IH; n = 7) and control (C; TLR2 +/+ CTRL; n = 6) conditions in TLR2 +/+ mice. Statistically significant increase of neuroinflammatory response is recorded after 3 days of IH, which remained elevated throughout the protocol (mean ± SD; Wilcoxon signed ranks test, P < 0.05 *compared with baseline values). X axis shows BLI signal in photons/sec/cm2/sr, Y axis depicts day of IH protocol. In B, a representative image of ex vivo TLR2 signal is shown following 1 day of C (control) or IH (intermittent hypoxia) protocol in TLR2 +/+ mouse. The signal is localized to the olfactory bulbs and anterior olfactory nucleus (OB) of both treatment groups at baseline. However, please note its stronger and more widespread posterior spread in IH group (TLR2 +/+ IH). In C, representative three-dimensional reconstruction images of diffuse light imaging tomography are shown following 3 days of IH and C protocol. A second locus of intense TLR2 expression is present in mice exposed to IH only (TLR2 +/+ IH), and it co-localises to the region of brain's septal nuclei (SN). In D, photographs of TLR2 induction in the brain of a single TLR2 +/+ IH mouse are shown, taken at the same time each day, every day during the 3 weeks of investigations (e.g. day one, two three etc.). The colour calibrations at the right are photon counts. Note the occurrence of different scales in various ranges. C control, TLR2 Toll like receptor 2, IH intermittent hypoxia, SN septal nuclei, SD standard deviation, OB olfactory bulb. www.nature.com/scientificreports/ TLR2-induced neuroinflammatory response is localised to brain derived neurotrophic factor, neuroplastin and fibronectin-1-rich neurocircuitry. Taken together, we demonstrated the role for TLR2 in modulation of the neuroinflammatory response in a distinct brain circuitry. We then further considered the molecular origins that might underlie mechanisms behind observed changes. To this end, an investigation of the observed network's modulation in mice with (TLR2 +/+ ) and without functional TLR2 (TLR2 −/− ) was undertaken by linking it with the microregional brain plasticity gene expression profiles ( Fig. 5; see SI Table S5). For this purpose the Allen Brain Mouse Atlas 23 mRNA gene profile expression database and MR parametric t-statistic maps were utilised via a novel mapping method, recently described 24 . Several major neuroplasticity genes were initially found to show a strong association, but only in mice with a functional TLR2-system (SI Table S4). The genes that were linked with TLR2-enabled structural network reorganisations were the brain derived neurotrophic factor (BDNF), neuroplastin, Calcium/calmodulin dependent protein kinase II alpha (CAMK2A), Rho Guanine Nucleotide Exchange Factor 6 (ARHGEF6), Cholecystokinin (CCK), Fibronectin 1 (FN1) and RAS guanyl nucleotide-releasing protein 1 (RASGRP1) (SI Table S4). Further multiple regression analysis suggested the strongest, statistically significant permissive role for BDNF, FN1 and RASGRP1. A significant correlation of longitudinal relaxation time (T1) with BDNF expression was shown (r = 0.819; P = 0.002) (Fig. 6). FN1gene expression was predictive of brain volume increases (r = 0.627; P = 0.039). Conversely, RASGRP1 gene had an inverse relationship to transverse relaxation time (T2, r = − 0.646; P = 0.033) (Fig. 6), suggestive of its protective Neuroinflammatory response: from effects on mood, cognition to effects on weight gain. After describing distinct structural, cellular and molecular basis of the observed inflammatory response, we set out to explore its impact on behavioural changes. Specifically, we wanted to assess if mice in our OSA model shared similar behavioural and cognitive changes to those frequently reported in patients with OSA, including their well-documented struggles with obesity, somnolence, fragile mood and memory, and attention deficits 2,25 . We also wanted to see if those changes were linked to neuroanatomical ROIs that were initially highlighted by our BL and MR imaging: frontal cortex 14 , septal nuclei, ventral hippocampi 26 and PAG 27,28 .
Weight . Firstly, the potential role for the TLR2 system in weight gain was investigated. In our study we demonstrated changes in two brain regions, which were recently proposed as anorexigenic in the neural circuitry    (Fig. 7). Although exposure to IH resulted in significant weight loss in both TLR2 +/+ IH and TLR2 −/− IH mice, compared to their respective controls (Fig. 7, Supplement), TLR2 −/− deficient mice (e.g. both TLR2 TLR2 −/− IH and TLR2 −/− CTRL) overall struggled to gain weight over the period of 3 weeks (Fig. 7). Thus, while mice with functional TLR2 (TLR2 +/+ ) showed a steady state increase in weight, even following the initial loss under IH, TLR2 −/− mice were not able to regain initially lost weight (Fig. 7). This finding is highly suggestive of a link between TLR2-system, neuroinflammation and the weight gain in OSA. Table S6) known to assess and target psychomotor changes, affective and cognitive symptoms were used to investigate the role of TLR2 in neurocognitive changes. We observed two primary findings, which were consistently replicated across several behavioural tests and their parameters (SI Table S6). Firstly, as expected, we demonstrated increased psychomotor agitation and anxiety in all mice exposed to the IH protocol. This behaviour appeared independent of their TLR2-functionality. For example, during the tail suspension test (TST), the mice that were exposed to the experimental IH protocol spent significantly more time trying to escape the uncomfortable setting, and less time being immobile (immobility period:  www.nature.com/scientificreports/ The second finding was unexpected. It was noted that functional TLR2 genotype in mice exposed to IH protocol was linked to a specific "proactive" behavioural endophenotype. This was, for example, demonstrated by shorter latencies to trying to first escape during the TST (TST: TLR2 +/+ IH27.65 ± 7.11 s; TLR2 +/+ CTRL 27.37 ± 12.79 s vs TLR2 −/− IH 45.04 ± 22.08 s; TLR2 −/− CTRL 30.14 ± 12.38 s). Here functional TLR2 genotype appeared to rescue depression-like behaviour noted under experimental IH conditions of repeated stress (Z = − 2.12,P (TLR2 +/+ IH vs TLR2 −/− IH) = 0.027). We then investigated which ROIs might have functionally contributed to this behavioural endophenotype in TLR2 +/+ IH mice. Correlation analyses suggested significant divergent link to (left) frontal cortical (P = 0.02, r = 0.635) and periaqueductal grey region (P = 0.083, r = − 0.499; also see SI Table S1). A significant aberrant connectivity between the two regions was also noted (P = 0.011; r = − 0.66). www.nature.com/scientificreports/ In regard to other behavioural findings, only a statistical trend for more effective spatial navigation and cognitive processing was seen in mice with functional TLR2 (TLR2 +/+ ). For example, in the Y-maze test, the presence of the TLR2 system appeared to partially rescue deficits in spatial acquisition following exposure to IH, otherwise recorded in TLR2 −/− mice, as measured by the path efficiency (path efficiency%: TLR2 +/+ IH1.05 ± 0.16; TLR2 +/+ CTRL1.09 ± 0.20; TLR2 −/− IH0.63 ± 0.14; TLR2 −/− CTRL1.04 ± 0.20; Z = − 1.82, P (TLR2 +/+ IH vs TLR2 −/− IH) = 0.06) (see SI Table S6 for further details).

Discussion
Whilst it has been accepted that OSA promotes a low-grade chronic systemic inflammation 29 , it has been a matter of some significant speculation whether it may also cause neuroinflammation 2 . We here advance that OSA may indeed promote a significant inflammatory response in the brain, that can result in specific structural and behavioural changes. To the best of our knowledge, this is the first direct demonstration of neuroinflammatory response under OSA conditions. We demonstrate that inflammatory spread occurs in the associated distinct neurocircuitry, and notably we report subsequent structural changes that closely correspond those previously recorded in clinical studies of patients with OSA 2,17,30 . Moreover, a clear link with structural changes and further functional and metabolic alterations is suggested by our findings, with possible significant translational clinical implications. More specifically, TLR2 system-driven changes in fronto-brainstem and hippocampal-septal circuitry are demonstrated, with links to agitated behaviour under episodes of stress, and an increased ability to gain weight.
Patients with OSA are known to be prone to obesity 31 , they have well documented cognitive and neuropsychiatric deficits 25 , excessive daytime somnolence, and are more likely to develop depression and anxiety 3,25,30 . They are also known to be particularly prone to traffic and general work accidents, in part through their welldocumented deficits in attention 25 , but also presumably through the erroneous encoding of spatial information in the context of navigation 2,32 . Somewhat surprisingly, our data suggests an early antidepressogenic effect of the neuroinflammatory response in OSA that is TLR2-dependent, and which is functionally linked to a distinct fronto-brainstem subcircuitry. The activation of a similar network in mice has been reported to favour effortful behavioural responses to challenging situations 33 . More specifically, it was shown that a selective activation of a subclass of prefrontal cortex cells, which project to the brainstem, has been able to induce a profound, rapid and reversible effect on selection of the active behavioural states 33 . According to a description of traditional learned-helplessness response 34 , depressed patients do not favour effortful behavioural responses to challenging situations. To the contrary they are, through their own overvalued negative evaluations of challenges, significantly more likely to earlier retire from trying to find solutions out of their predicament. Conversely, in our study, mice demonstrate a clear, at least initially adaptive, 'hyperactive' behavioural response when placed in the challenging situation. They, unlike their TLR2-deficient counterparts, continued to try to find solutions out of their predicament. It would be of paramount interest to see if this initial adaptive behavioural response with time develops into a potentially more dangerous, futile, and energy wasting 'agitated' depressive profile 34,35 , with heightened anxiety component. A similar mixed anxiety and depression endophenotype has indeed been previously described in some patients with OSA 36 , and it has been traditionally linked with higher suicide risks in depressed patients 37 . Strikingly, only recently, in post-mortem brains of depressed patients who committed suicide, TLR2-protein and its mRNA gene expression have been shown significantly increased, especially in their frontal cortical regions 11 . Hence, an early induction of the neuroinflammatory process via TLR2-system in frontal regions and basal forebrain, such as was demonstrated in our mice, may be of particular importance in Figure 7. Significant body weight loss induced by intermittent hypoxia. Weight changes in four investigated groups are shown. IH protocol was associated with significant weight loss after 3 days in mice (e.g. TLR2 +/+ IH and TLR2 −/− IH, F = 11.6, P < 0.001), irrespective of their TLR2 status. However, mice with functional TLR2 gene (TLR2 +/+ IH) were able to regain most of their lost weight by the end of the experimental period. Overall, having a functional TLR2 gene enabled weight gain, while deficient (TLR2 −/− ) mice struggled to regain weight. Y axis denotes time points (days) during the experimental protocol. Illustrative error bars shown (see SI Table S7  www.nature.com/scientificreports/ understanding the neural circuitry underlying pathological behavioural patterns of action selection and motivation in behaviour of patients with OSA 3 . Our data suggests a potential modulatory role to the neuroinflammatory response in regards to feeding 35 and or ability to gain weight. OSA has been associated with significant metabolic changes, diabetes and weight gain, thought to be at least partly modulated through its systemic inflammatory effects 4 . It has been previously proposed that a significant interplay between central nervous inflammation and systemic inflammation might occur in affected patients, which then further modulates bidirectional links between metabolism, weight gain and neurological disorders 12 . More specifically, our results could be taken to propose that activated TLR2system enables continued feeding drive under conditions of repeated stress (Fig. 7). Presumably, similar drive could later cause maladaptive inability to control one's food intake and to lead to an increased link between our emotional states and eating, causing an excessive weight gain in patients with OSA. The feeding circuitry that controls emotional or cognitive aspects of food intake is still largely unknown. However, recently, Sweeney and Yang 26 demonstrated an anorexigenic neural circuit originating from ventral hippocampus to lateral septal nuclei in the brain, revealing a potential therapeutic target for the treatment of anorexia or other appetite disorders. Interestingly, we were able to differentiate this network solely based on the functionality of the TLR2-system in our mice, and severity of changes appeared further exacerbated by the IH stress (also see SI Table S1 for further details). Whilst preliminary, these findings might go some way to suggest an aberrant feeding circuit that controls emotional and cognitive aspects of food intake in patients with OSA.
In conclusion, utilising the rodent model of OSA that verifiably replicates arousals and hypoxaemia in patients with OSA 18 and visualising in vivo TLR2-activation, we were able to demonstrate early activation of microglia in regions of basal forebrain, with later widespread frontal projections, suggesting a pivotal role for TLR2 in brain's response to OSA injury. Our results provide the first in vivo evidence of an OSA-induced inflammatory response, with septal nuclei, the major source of cholinergic input to the hippocampus, being highlighted as a region of particular vulnerability early on in the neuroinflammatory process (Fig. 1C). Moreover, we were also able to link the initial neuroinflammatory response to later hypotrophic and hypertrophic neuroanatomical changes, with potential primary molecular drivers, such are neuroplastin and BDNF, also highlighted by our results. Through these findings, a fingerprint of a distinct OSA-effected neurocircuitry has emerged, with frontal regions and septal nuclei being suggested as an initial TLR2-dependant seed sites.
Our neuroimaging results are in broad agreement with previous findings in patients with OSA where compensatory mechanisms, activation of various homeostatic gene programmes and astroglial neurogenesis have all been proposed to underlie some of the initial functional and later maladaptive changes 17,38 . In further agreement, our data also suggests that differential plastic response to OSA in the brain may depend on regional genes expressional profiles 39 For example our 'MR-gene' mapping findings support permissive and cohesive role for the TLR2-system in the interplay with BDNF, RSGRP1, fibronectin and neuroplastin-driven significant discrete and transformative neurophysiologic and behavioural changes.
Of the listed genes, the enhanced BDNF-levels have long been associated with increased plasticity and promotion of a growth-permissive environment, and its depletion with many neurological and psychiatric disorders 40 . The concept of the interplay between BDNF and TLR2-system in our study might also explain (preconditioning) protective effect on visuo-spatial memory in TLR2 +/+ IH mice, which, however, showed only significant trend. BDNF has been shown to enhance synaptic plasticity and neuron function in response to physical activity, learning and memory, and its baseline expression and activity-dependent upregulation in the hippocampus is believed to be under control of the medial septal nuclei, and important ROI in our study, with an important regulatory role in REM sleep (Fig. 1, SI Fig. S8). Patients with OSA show a rapid decrease in serum and plasma BDNF levels during initiation of the treatment [with positive airway pressure (PAP)-device], likely reflecting enhanced neuronal demand for BDNF in this condition 41 . Similarly, OSA patients had increased TLR2-expressions on blood immune cells, which could be reversed with PAP treatment 42 . TLR2-deficiency has been shown to impair neurogenesis previously 43 and more recently, the TLR2-receptor has been shown to enhance adult neurogenesis in the hippocampal DG after cerebral ischaemia 44 . Of particular note to our findings, the loss of TLR2 has been recently shown to abolish repeated social defeat stress-induced social avoidance and anxiety in mice, and its deficiency mitigated stress-induced neuronal response attenuation, dendritic atrophy, and microglial activation in the medial prefrontal cortex 14 . TLR2 has also been shown to modulate inflammatory response caused by cerebral ischemia and reperfusion via linking to endogenous ligands, such as fibronectin 45 . Fibronectin, on the other hand, has been shown to act as a reparative molecule that promotes cellular growth and its levels are enhanced after brain injury in variety of disorders, including the AD 40,46 . Finally, we have been able to show significantly higher levels of the cell adhesion molecule neuroplastin in mice with functional TLR2-system. This is of note as neuroplastin is known to play a role in synaptic plasticity (e.g. long-term potentiation), formation and a balance of the excitatory/inhibitory synapses 47 , in shaping of brain's cortical thickness 48 . Moreover, its involvement in early tissue response in hippocampi of AD patients has also been recently shown 49 . Interestingly, in TLR2-deficient mice, IH resulted in more prominent upregulation of this molecule (SI Fig. S9B), perhaps suggesting that in the absence or insufficient TLR2 response, neuroplastin might play a more prominent regulatory role during the inflammatory response. It is tempting to postulate that in patients with comorbid AD and OSA, and or in those with impaired TLR2 microglial response, any such compensatory effect could lead to imbalance in excitatory/ inhibitory synapses at the hippocampal level, with serious consequences.
Taken together, the implicated gene programmes also indirectly suggest that modulations in neurites, cytoskeletal and receptor signalling, cell adhesion, axonal sprouting and other extracellular and perineuronal nets likely underlie the observed structural and functional changes. Whilst our findings are striking and theory-forming, our study leaves many questions still unanswered. There are several limitations to our findings behind OSA-injury, notwithstanding that the majority of experiments were done in cross-sectional manner, preventing us from deduction of causality and or direction of noted changes. Ideally, multimodal longitudinal Scientific RepoRtS | (2020) 10:11475 | https://doi.org/10.1038/s41598-020-68299-2 www.nature.com/scientificreports/ in vivo manipulation of the microglia TLR2 in the highlighted ROIs, along with in vivo functional MR and further behavioural, genetic and electroencephalographic studies, should help shed much needed insight into here proposed novel neural mechanisms. Nonetheless, we believe that the distinct regional association between highlighted gene profiles and structural and functional changes, as demonstrated by our data, arguably further indicates a true circuitry-specific, rather than wider systemic, nature of TLR2-modulated neuroinflammatory injury and as such provide a plethora of potential investigational targets for future studies.
MRI and statistical analyses. The MR images were processed using a combination of FSL 56 , ANTs 57 and the QUIT toolbox 58 , as previously described by our group 59 . A group analysis was carried out on Jacobian determinant images with permutation tests and threshold-free cluster enhancement (TFCE) using FSL randomize 60,61 . Data were displayed on the mouse template image, using the dual coding approach 62 : differences were mapped to color hue, and associated t-statistics were mapped to color transparency. Contours were family wise error (FWE) corrected statistically (P < 0.05) significant differences. The Kolmogorov-Smirnov test was used to test the normality of distributions. In addition, for all variables that were normally distributed one-way analysis of variance and Bonferroni corrections were used, as previously described 38 . Pearson correlation coefficients were calculated between all investigated variables and used to determine heatmaps. Wilcoxon test was used for comparison BLI (photon emissions) between each two measures, and for the analysis of the behavioral tests. T test for independent samples was used to analyze differences in neuroplastin immunoreactivity. All statistical analyses had a two-tailed α level of < 0.05 for defining significance and were performed by an experienced biostatistician (M.M.) on the statistical software IBM SPSS Statistics version 23 (www.spss.com).
Neuroplastin immunoreactivity 47 , perfusion and histology for MRI 21 were all performed as previously described in detail by our group. Further methodological description of mice lines, experimental and study procedures, including the undertaken statistical and MR analyses, is additionally available in the Supplement.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.