To our knowledge, this is the first study to evaluate the distribution of peripheral monocyte subsets and to analyze markers of monocyte activation and functionality in early-onset OCD. Our results revealed a proinflammatory predisposition of monocytes from OCD patients based on the imbalance of monocyte subpopulations and their over-reactivity to immune stimulation with LPS. Moreover, OCD patients receiving psychoactive medications were found to have an intermediate inflammatory profile compared to untreated patients and controls.

OCD patients showed an increase in the percentage of total monocytes as well as in the CD16+ subset, especially in intermediate monocytes but also in non-classical monocytes, compared with healthy controls. Although there is no previous evidence of an association between altered monocyte frequencies and OCD, some authors have identified high levels of monocytes in related disorders like Tourette’s syndrome (TS) [38] or autism spectrum disorder [39, 40]. However, no differences in the monocyte subset distribution have been found [41].

Together with these alterations in monocyte subsets, we found that isolated peripheral blood monocytes from children with OCD behaved abnormally upon stimulation with LPS, displaying excessive IL-1β, IL-6, GM-CSF, TNF-α, and IL-8 production compared to monocytes from healthy controls. Interestingly, we did not detect any abnormal activation of monocytes from OCD patients in basal conditions, but exposure to an immune challenge was required for the over-reactivity of these cells. Whether this over-reactivity occurs upon other types of immune stimulus such as psychological stress remains to be elucidated. These findings are at odds with results from previous studies with similar methodologies, which have reported either no differences or lower production of several cytokines after LPS stimulation in OCD individuals [42,43,44,45]. However, we should stress that in these studies, the assessments involved different types of samples including other immune cells and all of them were conducted in adult patients, in whom the pathological mechanism may be different [4, 5]. In addition, cytokine production in children has been reported to be different from that in adults [46, 47]. Apart from the studies mentioned above, most authors have investigated cytokine levels in serum or cerebrospinal fluid in OCD [16,17,18, 47, 48] but the results were inconsistent due to methodological and sample differences such as age, age at onset, disease duration, and pharmacological treatment. Interestingly, in our population, monocytes from pediatric OCD patients were able to respond to glucocorticoid exposure in the same way as those from healthy individuals. However, due to the over-reactivity of monocytes from OCD patients after LPS stimulation, cytokine levels remained higher in patients than in controls when treated with dexamethasone. The significant correlation identified between cytokine levels and the percentage of total monocytes indicates that those individuals with higher production of inflammatory cytokines, both in basal conditions and in response to stimulus, were also those with the higher levels of monocytes and thus with a greater global inflammatory state. However, as all the samples were cultured using the same amount of monocytes, we can rule out that the amount of cytokines released was due to the number of monocytes. In addition, as cytokine levels were mainly not correlated with the different monocyte subsets, the higher production of cytokines must be due to a higher activation of monocytes rather than to monocyte subset distribution.

This study also found that OCD patients receiving pharmacological treatment seem to present an intermediate inflammatory profile (including monocyte subset distribution and cytokine production upon stimulation with LPS) that is lower than non-medicated OCD individuals and higher than healthy controls. These results are in accordance with previous evidence suggesting that treatment with antidepressant medications may have anti-inflammatory properties [49, 50]. However, post hoc analysis was not able to detect significant differences between treated and untreated patients, maybe due to the small size of the non-medicated group. If the trend identified here is confirmed in subsequent studies, the combined treatment with antidepressants and anti-inflammatory drugs like glucocorticoids may be a promising therapy for improving obsessive-compulsive symptoms, as observed in patients with major depressive disorder or schizophrenia [51, 52]. In addition, other immune-modulating medications, such as anticytokine agents, may be useful as adjunct therapy in OCD individuals, as observed in a patient with tumor necrosis factor receptor-associated autoinflammatory syndrome (TRAPS), in which psychiatric symptoms, including tics and OCD, showed a remarkable improvement after treatment with the IL-1 blocking agent anakinra [53].

Monocytes, which were found to be increased in OCD patients in the present study, are the main cells implicated in the first immune response upon infection. Among the different monocyte subsets, CD16+ and specifically the intermediate monocytes, which were increased in our OCD population, are considered the most inflammatory subpopulation due to their expression of inflammatory cytokines and other activation markers and due to their expansion in infectious and inflammatory diseases [29,30,31]. Circulating monocytes can be recruited to the central nervous system (CNS) upon exposure to psychosocial stress [25] or when chronic or intense injury occurs in the brain, where they contribute to the inflammatory response with their phagocytic activity and the release of immune mediators such as cytokines [28, 54]. Additionally, peripherally produced cytokines secreted by monocytes, such as the IL-1β, IL-6, GM-CSF, TNF-α, and IL-8 evaluated in this study, not only act peripherally but also enter the CNS where they increase neuroinflammatory responses and affect neurotransmitter availability [55, 56], brain function, and neurodevelopment [57]. These effects occur mainly in the basal ganglia and dorsal anterior cingulate cortex [58], which have been implicated in the pathophysiology of OCD [59]. Hence, monocyte activation upon immune challenges and other potential stressors in genetically susceptible individuals may be involved in the onset, progression, and exacerbation of obsessive-compulsive symptoms, as proposed for other psychiatric diseases such as schizophrenia [23, 60], depression [58] or anxiety [21]. This peripheral activation of monocytes may also suggest microglial activation, since they are the innate immune cells of the CNS. Although microglia originate from myeloid precursors deriving from the yolk sac during embryonic development, with minimal contribution of bone marrow-derived progenitors in adulthood [54, 61], monocytes and microglial cells may exhibit similar responses to systemic stimuli [54, 62]. Activated microglia with a proinflammatory phenotype are unable to remove debris and promote regeneration of the inflamed tissue leading to a failure in immune resolution and neuroprotection [28]. In addition, microglial abnormalities may lead to alterations of synaptic pruning as well as higher release of microglial-derived glutamate, which in turn might have neurotoxic effects on dendrites and synapses [63]. Indeed, it has been suggested that microglial dysregulation may have a role in the pathophysiology of OCD and related diseases like TS or autism. Postmortem studies evaluating gene expression in basal ganglia from TS subjects identified an increase in the expression of monocyte chemotactic factor-1 (MCP-1) [64] and an upregulation of several microglia-related genes [65], pointing to microglia proliferation and activation. In addition, animal models such as Hoxb8 knockout mice or L-histidine decarboxylase (Hdc) knockout mice, exhibiting repetitive behaviors, mostly grooming, have also suggested the participation of microglia in the development of OCD and TS [63, 66, 67].

Taken together, our results indicate an enhanced proinflammatory state in monocytes in early-onset OCD characterized by alterations in monocyte subset distribution as well as higher production of inflammatory cytokines after monocyte stimulation. It should be borne in mind that our sample comprised OCD patients with a variety of comorbidities and that most patients were receiving pharmacological treatment. However, the higher activation state of monocytes from OCD patients identified here was not due to the presence of these conditions. We should also stress the homogeneity of our OCD sample in terms of age, age at onset, or duration of disease, as all participants were children and adolescents; this avoids the possible influence of these confounding factors and allows the study of the specific etiopathogenic mechanism underlying childhood-onset OCD, which may differ from the pathophysiology of the adult disorder [5]. In addition, a larger size of the control sample, at least similar to that of the OCD group, would be preferred. However, due to the frequent difficulties found in the recruitment of healthy children and adolescents, we were not able to include a higher number of controls. Moreover, despite the imbalance in gender between OCD patients and healthy controls and the age difference between the groups, the results of the univariate analysis adjusted for these two factors revealed that the association between OCD and immune parameters that was identified here was independent of their potential confounding effect.

As we used a primary model of isolated monocytes, the enhanced proinflammatory state in OCD patients shown in this study was not influenced by the immune response mediated by other immune cells. This means that we can specifically determine the potential contribution of monocyte lineage cells to the inflammatory response in early-onset OCD. Nevertheless, although the over-activation of monocytes found in the present study was demonstrated by a number of parameters, only peripheral activation markers were measured. Hence, even though peripheral monocytes and circulating cytokines are able to enter the brain [55] and although these peripheral changes may also reflect microglial activation [23], analysis of more inflammatory markers, including central immune changes, is needed in order to understand the complex inflammatory profile underlying OCD. In addition to these limitations, it should also be borne in mind that no adjustments for multiple comparisons were applied due to the exploratory nature of the study and the high correlation of the monocyte-derived products. Hence, the results should be interpreted with caution.