Piras et al R1 20-2-20

Patients suffering from autoimmune diseases are more susceptible to mental disorders yet, the existence of specific cellular and molecular mechanisms behind the co-morbidity of these pathologies is far from being fully elucidated. By generating transgenic mice overexpressing Annexin-A1 exclusively in T cells to study its impact in models of autoimmune diseases, we made the unpredicted observation of an increased level of anxiety. Gene microarray of Annexin-A1 CD4+ T cells identified a novel anxiogenic factor, a small protein of approximately 21kDa encoded by the gene 2610019F03Rik which we named Immuno-moodulin. Neutralizing antibodies against Immuno-moodulin reverted the behavioral phenotype of Annexin-A1 transgenic mice and lowered the basal levels of anxiety in wild type mice; moreover, we also found that patients suffering from obsessive compulsive disorders show high levels of Imood in their peripheral mononuclear cells. We thus identify this protein as a novel peripheral determinant that modulates anxiety behavior. Therapies targeting Immuno-moodulin may lead to a new type of treatment for mental disorders through regulation of the functions of the immune system, rather than directly acting on the nervous system. Piras et al Page ! of ! 3 31 The challenging life of patients diagnosed with autoimmune diseases is often further impoverished by the emergence of mental disorders as a major co-morbidity (Coutinho and Vincent, 2016; Khandaker et al., 2017). For instance, if we only consider multiple sclerosis, ~40% of patients suffering from this disease have attempted suicide (Feinstein and Pavisian, 2017; Pompili et al., 2012). The incidence of as major depression are significantly higher and is correlated with the progression of the disease (Lorefice et al., 2015). Finally, a large study conducted on more than 4000 subjects enrolled on the UK Multiple Sclerosis Register showed that anxiety and depression were notably high, with 54.1% of patients scoring > 8 for anxiety and 46.9% scoring >8 for depression on the Hospital Anxiety and Depression Scale (HADS)(Jones et al., 2012). Similar observations have been made for other autoimmune conditions. More than 30% of patients affected by autoimmune hepatitis suffer from schizophrenia (Butt et al., 2006; Cournos et al., 2005). Meta-analysis studies on the prevalence of depression and anxiety in rheumatoid arthritis (Matcham et al., 2013) or systemic lupus erythematosus (Moustafa et al., 2020) patients have revealed a high variability in the prevalence of anxiety or major depression but in any case ranges of incidence from about 10 to 80% depending on the scale that was used. Most strikingly, immunomodulatory therapies for the treatment of autoimmune conditions might aggravate the emergence of these problems (Bosche et al., 2015) thus presenting both clinicians and patients with a paradoxical dilemma: the physical symptoms of autoimmunity might be effectively improved at the expenses of a worsening of the emotional state and wellbeing. This is for instance the case of interferon beta (IFN-β) that is currently used as an effective treatment for multiple sclerosis but its use is limited by the increased incidence of suicidal thoughts in a significant proportion of patients (Goeb et al., 2006). Although some studies have investigated the functional crosstalk between the brain and the immune system (Dantzer, 2018; Marin and Kipnis, 2017) it is still not clear how one system influences the other and if there is a common root or determinant for the emergence of mental disorders in autoimmune conditions. Annexin-A1 (AnxA1) is an endogenous modulator of a variety of physiological and pathological processes ranging from inflammation (Leoni and Nusrat, 2016; Perretti and D'Acquisto, 2009) and cancer (Boudhraa et al., 2016; Guo et al., 2013; Moraes et al., 2018). As with many other multifunctional mediators, AnxA1 plays a homeostatic role in the immune system as it can exert both positive and negative functions depending on the contexts. In the context of T cells, studies have indeed provided contrasting and opposite results showing that it can act as both a positive (D'Acquisto et al., 2007a; D'Acquisto et al., 2007b; D'Acquisto et al., 2013; Huang et al., 2016; Huggins et al., 2009; Mihaylova et al., 2017; Paschalidis et al., 2010; Paschalidis et al., 2009; Sena et al., 2016; Tzelepis et al., 2015) and a negative modulator of T cell activation (Odobasic et al., 2018; Sena et al., 2016; Yang et al., 2013; Yazid et al., 2015). All these studies have been done using either exogenously administered recombinant AnxA1 (and its mimetic) or AnxA1-deficient mice where the protein is absent in every immune cell. Aiming to address these controversies, we have generated a T cell specific AnxA1 transgenic mouse colony (AnxA1tg) to address the biology of this mediator. In this paper, analysis of AnxA1tg response in experimental model of autoimmunity showed exacerbated signs of inflammation. However, besides their autoimmune-prone Piras et al Page ! of ! 4 31 phenotype, AnxA1tg mice displayed an unexpected high level of compulsive digging that was noticeable even in their home cage and at basal physiological settings e.g. in mice that were not subjected to any inflammatory condition. Further exploration on the molecular mechanisms behind this phenotype of anxiety led us to the discovery of a new homeostatic modulator that we named Immuno-moodulin (Imood) because of its discovery in T cells. Immuno-moodulin levels in mice affect their intrinsic basal level of anxiety. In addition, we found that patients suffering from OCD present high levels of Imood in their peripheral blood mononuclear cells. The results of this study expand our knowledge of the complex interactions and intersection between the immune and the central nervous system. Materials and Methods Reagents Unless otherwise stated, all the chemicals were purchased from Sigma. For the convenience of the readers all the antibodies used in the study have been reported in Table 4 as well as in the figure legends. Animals and husbandry Wild type C57BL/6J (B6-CD45.2) and B6.SJL-Ptprca Pepcb/BoyJ (B6-CD45.1) were purchased from Charles River. AnxA1tg mice were generated in the in-house transgenic mouse facility of Queen Mary University of London by pronuclear injection. Mice were housed in groups of 6 per cage under specific-pathogen-free conditions and with free access to food and water. Mice were housed for at least 7 days prior to testing. All experiments were performed during the light phase of the light-dark cycle and no more than 2 tests per day were performed. The results presented in this manuscript were obtained using male mice (6-8 weeks old) because unlike in humans both the intensity and incidence of inflammatory/autoimmune disease are more pronounced in male mice. However, the difference we observed in terms of both immune response and behavioral changes were confirmed and reproducible in female mice (data not shown). All tests were conducted under license from the Home Office and according to the UK Animals (Scientific Procedures) Act, 1986. All experiments were approved and performed under the guidelines of the Ethical Committee for the Use of Animals, Bart’s and The London School of Medicine and Home Office Regulations (57) (PPL 80/8714). To monitory the transgenic colony, genomic DNA were extracted from ear clips by using REDExtract N-AMP –XNAT kit (Sigma, UK) and analysed by PCR with the following s p e c i f i c p r i m e r s f o r A n x A 1 t g : f o r w a r d p r i m e r 5 ’ G TAT G G A AT C T C T C T T T G C C A A G C 3 ’ ; r e v e r s e p r i m e r i s 5 ’ ACHGATATGCACATCAGGAGGG-3’ (Thermo Scientific, UK). The parameters of the PCR reactions are: initial denaturation at 94oC for 3min followed by 30 cycles of denaturation at 94oC for 45sec, annealing at 60oC for45sec and extension time at 72oC for 15sec, and afterwards a final extension step at 72oC for 7 min. All the behavioral tests were video-recorded and analyzed double-blind during the light phase of the light-dark cycle, as previously described(McIlwain et al., 2001). The scoring and analysis of the data was hand-made by at least 2 blinded independent experimenters. All the efforts were made to minimize mouse discomfort in these behavioral experiments. Piras et al Page ! of ! 5 31 Mice were brought to the testing room at least 30 minutes before the start of the test session to allow habituation to the testing environment. All behavioral equipment was thoroughly cleaned and sanitized at the end of the day and in between tests. Unless otherwise specified, standard lighting (about 50 lux) and quiet conditions were maintained throughout each experiment. Generation of T cell-specific AnxA1tg mice To generate the VACD2 AnxA1 FLAG transgenic mice, murine AnxA1 gene was extracted, amplified and tagged with the FLAG epitope. The gene was cloned in TOPO pcDNA3.1 vector for verification of its expression in vitro and finally subcloned in the VACD2 vector (Zhumabekov et al., 1995) for T cell specific expression in the mouse. Finally, the VACD2 AnxA1 FLAG construct was modified and purified for the pronuclear microinjection into the mouse genome. Flow cytometric analysis Single cell suspension of thymocytes or lymphocytes from spleen and lymph nodes were obtained as previously described (Paschalidis et al., 2010; Rattazzi et al., 2013). Briefly, tissues were gently desegregated in RPMI medium supplemented with 100U/ml of penicillin and streptomycin (PAA laboratories, Buckinghamshire, UK) using a 70μm mesh cell strainer (Falcon, UK) and the piston of a 5ml syringe. Cell suspensions from spleen and lymph nodes were layered onto Histopaque-1077 (Sigma-Aldrich, Dorset, England) in a ratio 3:1 and centrifuged at 400g for 10 mins to isolate mononuclear cells. The resulting buffy coat was collected and washed twice with RPMI supplemented with 100U/ml of penicillin and streptomycin