Mice lacking Casp1, Ifngr and Nos2 genes exhibit altered depressive- and anxiety-like behaviour, and gut microbiome composition

Converging evidence supports the involvement of pro-inflammatory pathways and the gut microbiome in major depressive disorder (MDD). Pre-clinical and clinical studies suggest that decreasing pro-inflammatory signaling may provide clinical benefit in MDD. In this study, we used the chronic unpredictable stress (CUS) paradigm to assess whether mice lacking the pro-inflammatory caspase 1, interferon gamma-receptor, and nitric oxide synthase (Casp1, Ifngr, Nos2)−/− present altered depressive- and anxiety-like behaviour at baseline and in response to CUS. In comparison to wild-type (wt) mice, (Casp1, Ifngr, Nos2)−/− mice displayed decreased depressive- and anxiety-like behaviour, and increased hedonic-like behaviour and locomotor activity at baseline, and resistance to developing anhedonic-like behaviour and a heightened emotional state following stress. Plasma levels of ACTH and CORT did not differ between the triple knockout and wt mice following stress. The faecal microbiome of (Casp1, Ifngr, Nos2)−/− mice differed from that of wt mice at baseline and displayed reduced changes in response to chronic stress. Our results demonstrate that simultaneous deficit in multiple pro-inflammatory pathways has antidepressant-like effects at baseline, and confers resilience to stress-induced anhedonic-like behaviour. Moreover, accompanying changes in the gut microbiome composition suggest that CASP1, IFNGR and NOS2 play a role in maintaining microbiome homeostasis.


Supplementary methods
All procedures were approved by the South Australian Health and Medical Research Institute (SAHMRI) Ethics committee and are in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes (8 th edition, 2013). All efforts were made to minimize animal suffering. Male C57BL/6J mice (wild-type, wt, n=26) aged 60 days were obtained from the SAHMR1 Bioresources Facility (Adelaide, Australia). Age-matched (Casp1, Ifngr, Nos2) -/mice (n=30) with C57BL/6J background were generated by back-crossing mice with individual gene deletions. [1][2][3] After a seven-day acclimatization period, CUS mice were singly housed and control mice were group housed in transparent Plexiglas cages (Green Line IVC Sealsafe PLUS mouse, Tecniplast, Varese, Italy) in a temperature (21°C ±1°C), humidity (50%) and light (12 hour cycles, lights on at 7:00 am) controlled room, with water and food ad libitum. Behavioural testing was performed in the light phase of the light cycle as recommended by Castagne and colleagues. 4 Fresh faecal pellets were collected between 10-11 am with sterile toothpicks on experimental day 41 during weighing procedures. At the endpoint of the experiment, mice were euthanized by cervical dislocation and blood was collected by cardiac puncture in EDTA coated tubes. A timeline of experimental procedures is shown in Supplementary figure 1.

Chronic unpredictable stress
The CUS procedure used in this study is a variation of the procedure previously described as a naturalistic model of depression in rodents. 5 The CUS protocol consisted of chronic exposure (28 days) to various randomly scheduled, low and mild intensity social and environmental stressors, applied each day during the light phase of the light cycle (except for light cycle reversal stress, which was applied during the weekend). A detailed calendar of the CUS protocol is provided in Supplementary table 2. Depending on the duration of the stressor, one (if it lasted more than two hours) or two (if it lasted two hours or less) stressors were applied each day. The schedule was randomized weekly to maximise the degree of unpredictability and to avoid habituation, which is one of the drawbacks in modelling depression in rodents. 6 Briefly, the stressors were: a) two hours restraint in polypropylene restrainers on an open bench, b) eight hours removal of bedding and nesting material, c) eight hours of soiled bedding, obtained by adding 200 mL of autoclaved water to 100 g of bedding, d) eight hours of 45º cage tilting obtained by introducing a Plexiglas "tilter" inside the cage to allow the cage to be returned to the individually ventilated cage rack, e) two hours of predator stress, obtained by introducing in the cage a 5 ml test tube modified with ten 2 mm holes containing two fresh rat faecal pellets, f) five minutes forced swim test, performed once at the beginning and once at the end of the stress period (representing both a stressor and a behavioural test), g) sixteen hours of overnight fasting in clean cages, h) two hours of social stress, consisting in pair housing two mice from different litters in a neutral cage, i) two hours of light cycle disruption during the light phase, and j) forty-eight hours of light cycle reversal over the weekends.
Sucrose preference tests, considered an index of anhedonic-like behaviour, 7 were performed weekly to assess the effectiveness of the CUS procedure.

Behavioural testing
Mice were submitted to open field, elevated plus maze, forced swim, and sucrose preference tests. All tests were videorecorded by a camera coupled to Ethovision XT 10 computer software (Noldus, Wageningen, Holland) for behaviour recognition and scoring.

Open field test
Animals were placed in the centre of a brightly lit novel arena (50cm x 50cm x 50cm) and their activity was recorded for 30 minutes by a camera mounted above the arena. Total distance (locomotor activity) and average velocity (locomotor speed) were used to quantify locomotor activity. The number of visits (centre visits) and the total time spent in the center of the arena (centre time) were used to assess anxiety-like behaviour. The number of defecations was recorded and used as an index of emotionality, 8,9 The arena was carefully cleaned with F10 after each test session.

Forced swim test
To assess depressive-like behaviour, mice were individually placed in a clear Plexiglas cylindrical container (50cm tall, 30cm diameter) containing 30cm of water at 21°C. The amount of time spent floating, swimming and climbing (respectively <12%, 12%< x <18% and >18% activity) was automatically recorded during a 300 second test period by a camera mounted on the side of the cylinders. The percentage of activity corresponding to each behaviour was set by observing pre-recorded videos.

Sucrose preference test
To assess anhedonic-like behaviour, mice were individually housed and given 2 drinking bottles containing a 1% sucrose solution in drinking water for 24h to familiarize them with the novel drink. The following two days, one bottle was replaced with a standard drinking water bottle and mice were given the choice to drink from either bottle for 48 hours (training). On the fourth day (test day), the amount of liquid drunk from either bottle was recorded and the sucrose preference was determined by calculating the percentage of the volume of sucrose drunk over the total volume of fluid drunk. 5,11 Adrenocorticotropic hormone (ACTH) and corticosterone (CORT) measurement ACTH was measured in plasma by using a competitive inhibition ELISA kit following manufacturer's instruction (Cloud-Clone Corp., Wuhan, Hubei, China). Circulating CORT was measured by using a competitive immunoassay ELISA kit following manufacturer's directions (Enzo Life Sciences, Farmingdale, New York, USA).

Statistical analysis
Power analysis was performed based on the effect size seen in a previous pilot study Effect size was reported as partial eta-squared (η 2 p ). If the stress-genotype interaction was significant, it was further assessed as described previously 12 . Statistical analyses of ELISA results were performed by two-tailed unpaired t-test.