Frederico Azevedo Costa-Pinto

Reversal of axonal loss and disability in a mouse model of progressive multiple sclerosis

Axonal degeneration is an important determinant of progressive neurological disability in multiple sclerosis (MS). Thus, therapeutic approaches promoting neuroprotection could aid the treatment of progressive MS. Here, we used what we believe is a novel water-soluble fullerene derivative (ABS-75) attached to an NMDA receptor antagonist, which combines antioxidant and anti-excitotoxic properties, to block axonal damage and reduce disease progression in a chronic progressive EAE model. Fullerene ABS-75 treatment initiated after disease onset reduced the clinical progression of chronic EAE in NOD mice immunized with myelin-oligodendrocyte glycoprotein (MOG). Reduced disease progression in ABS-75-treated mice was associated with reduced axonal loss and demyelination in the spinal cord. Fullerene ABS-75 halted oxidative injury, CD11b+ infiltration, and CCL2 expression in the spinal cord of mice without interfering with antigen-specific T cell responses. In vitro, fullerene ABS-75 protected neurons from oxidative and glutamate-induced injury and restored glutamine synthetase and glutamate transporter expression in astrocytes under inflammatory insult. Glutamine synthetase expression was also increased in the white matter of fullerene ABS-75-treated animals. Our data demonstrate the neuroprotective effect of treatment with a fullerene compound combined with a NMDA receptor antagonist, which may be useful in the treatment of progressive MS and other neurodegenerative diseases.

Mutual expression of the transcription factors Runx3 and ThPOK regulates intestinal CD4+ T cell immunity

The gut mucosa hosts large numbers of activated lymphocytes that are exposed to stimuli from the diet, microbiota and pathogens. Although CD4+ T cells are crucial for defense, intestinal homeostasis precludes exaggerated responses to luminal contents, whether they are harmful or not. We investigated mechanisms used by CD4+ T cells to avoid excessive activation in the intestine. Using genetic tools to label and interfere with T cell–development transcription factors, we found that CD4+ T cells acquired the CD8-lineage transcription factor Runx3 and lost the CD4-lineage transcription factor ThPOK and their differentiation into the TH17 subset of helper T cells and colitogenic potential, in a manner dependent on transforming growth factor-β (TGF-β) and retinoic acid. Our results demonstrate considerable plasticity in the CD4+ T cell lineage that allows chronic exposure to luminal antigens without pathological inflammation.

Neuroimmune Interactions in Stress

Stress has long been recognized as a putative modulator of immunity. Several clinical and experimental reports point to a role of physical and psychological stressors on progression or resistance to disease. Nonetheless, literature in this field is sometimes controversial due to the wide variety of stressors employed and parameters of immunity analyzed. This variation should not be considered a consequence of methodological inaccuracy. The stress response, although theoretically stereotyped in nature, may lead to slightly different outcomes according to several modifiers. Our group has compared the effects of several stressors over different parameters of brain activity, behavior, immunity and glucocorticoid levels. These data show altogether that while increased turnover of noradrenaline in the hypothalamus, along with anxiety-like behaviors and increase in serum corticosterone are present very often, the magnitude of changes in immunity may vary considerably. Thus, we review data from our group generated over the past decade to support that effects of stressors on immunity and behavior highly depend on their specifics, animal model, frequency, duration, intensity, perception, and coping by the stressed animal.

Avoidance behavior and neural correlates of allergen exposure in a murine model of asthma

Allergic asthma is characterized by intermittent airway obstruction, inflammation, airway hyperreactivity, and increased production of IgE. The pathophysiology of asthma is well understood but little is known about its influences on brain activity and behavior. We recently described the neural correlates of food allergy and its associated modulation of behavior using an experimental model that also generates a T helper type 2 (Th2)-skewed response, with high levels of IgE. Here we show that mice allergic to ovalbumin (OVA) have an increase in the activity of the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA) following a single nasal OVA challenge. Moreover, we adapted a classical passive avoidance test using an OVA aerosol as the aversive stimulus. We found that allergic mice avoid entering the dark compartment of the apparatus that had been previously associated with nebulization of the allergen, while their non-immunized controls still move into the dark side of the test box. Thus, allergic mice have increased activity in areas of the CNS commonly associated with emotionality-related behavioral responses, such as the avoidance of a context previously associated with an unpleasant or harmful situation. Moreover, our findings on the avoidance test illustrate that previous experience with an airborne allergen can modify behavior.

Neural Correlates of IgE‐Mediated Allergy

Abstract:  Although many authors have considered a direct interaction between allergic reactions and behavioral changes, supporting evidence has been elusive. In this series of studies we show that after oral or nasal ovalbumin (OVA) challenge, allergic mice present increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and in the central nucleus of the amygdala (CeA). Mice with food allergy display higher levels of anxiety and increased serum corticosterone levels, and allergy‐activated neurons express corticotropin‐releasing factor (CRF) in the PVN and CeA. OVA‐allergic mice develop aversion to an antigen‐containing solution, and also avoid a dark compartment previously associated with nebulized OVA. Results on brain Fos expression and behavioral data seem compatible with adaptive responses. Removal of IgE by either antibody depletion or the development of oral tolerance precluded all responses analyzed here. C‐sensitive fiber destruction by neonatal capsaicin inhibited the activation in the PVN, but not in the CeA, and decreased the magnitude of food aversion. Cromolyn, a mast cell stabilizer, completely blocked Fos expression in the PVN and CeA, and precluded the development of aversion to the dark compartment associated with nebulized OVA. Employing mice that do not develop an important inflammatory infiltrate following nasal OVA challenge, we found that inflammatory cells are not required at the site of challenge in order to trigger neural or behavioral correlates of murine experimental asthma. Altogether, we have built a solid foundation for understanding neuroimmune interactions during allergic responses that may contribute to the comprehension of psychological disorders associated with allergy.

Role of mast cell degranulation in the neural correlates of the immediate allergic reaction in a murine model of asthma

Experimental airway allergy in mice leads to increased activity in specific hypothalamic and amygdaloid nuclei, and behavioral changes. The experiments described here were designed to determine the role of anaphylactic antibodies, mast cell degranulation, and lung inflammation in the neural and behavioral correlates of an experimental murine asthma-like response. Animals were sensitized intraperitoneally with ovalbumin adsorbed to alum, and challenged by intranasal ovalbumin instillation or aerosol. To induce immunological tolerance, animals were fed ovalbumin in the drinking water for 5 consecutive days, along with primary sensitization. Depletion of IgE was also accomplished with a non-anaphylactic anti-IgE antibody. Mast cell degranulation was inhibited by cromolyn. In addition to BALB/c animals, C3H/HeJ mice were used for their relative resistance to lung allergic inflammation. We confirmed that ovalbumin challenge in allergic mice leads to increased activity in the paraventricular nucleus of the hypothalamus and central nucleus of the amygdala, and avoidance behavior towards an allergen-associated compartment. Moreover, these responses were precluded by oral tolerance or anti-IgE treatment, even in the presence of IgG1. Cromolyn abrogates both responses in the presence of anaphylactic antibodies. Finally, although sensitized C3H/HeJ mice did not develop airway inflammation, they exhibited brain and behavioral changes similar to BALB/c animals. The repercussions of murine allergic asthma on brain and behavior are IgE-dependent, mediated by mast cell degranulation, and do not require a pulmonary inflammatory infiltrate, suggesting that the early phase of this immediate allergic response suffices for the brain activation associated with avoidance behavior towards exposure to the allergen.

Neural pathways involved in food allergy signaling in the mouse brain: role of capsaicin-sensitive afferents.

There is increasing evidence supporting the notion that brain-gut communication is crucial for the manifestation of functional gastrointestinal (GI) disorders. Employing denervation by neonatal capsaicin treatment, we investigated here the role of unmyelinated C-fibers in food allergy signaling in the brain. We found that 90 min after oral ovalbumin (OVA) challenge, allergic mice present increased c-fos expression in emotionality-related brain areas such as the paraventricular nucleus of the hypothalamus (PVN) and the central nucleus of the amygdala (CeA). Food allergy also induced enhanced Fos immunoreactivity in the nucleus of tractus solitarii (NTS) of OVA-immunized animals. We also show that while the degree of Fos staining in the NTS of allergic mice was only diminished by neonatal capsaicin, it was completely blocked in the PVN. However, capsaicin did not modify food allergy-induced c-fos expression in the CeA. In conclusion, this study provides evidence showing that unmyelinated C-fibers are part of the neural pathways involved in food allergy-induced activation of specific brain areas, particularly the PVN and to a lesser extent the NTS.

Anandamide prior to sensitization increases cell-mediated immunity in mice

The endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal organisms and capacity to play a modulatory role on several physiological systems, including modulation of immunity. Many studies have shown that administration of cannabinoids causes inhibitory effects on immune cells, including decreased proliferation and antigen-presenting cell (APC) co-stimulatory activity. In contrast, other groups have shown that some cannabinoids might present stimulatory actions on macrophage activity and T cell activation. Therefore, we aimed to investigate whether a treatment in vivo with a low dose of anandamide (0.1mg/kg) immediately prior to sensitization would have an immunosuppressive or immunostimulatory effect on cell-mediated immunity (Th1 response) in mice. We report here that anandamide, prior to sensitization, was able to increase the Th1 response to ovalbumin in vivo and ex vivo. Anandamide increased delayed type hypersensitivity (DTH), splenocyte proliferation, and IFN-gamma production in a co-culture of adherent and non-adherent splenocytes. Moreover, anandamide prior to sensitization increased both the expression of DC co-stimulatory molecules (CD80/CD86) and IL-12/IL23 (p40) production ex vivo. We have also assessed direct effects of anandamide in the IFN-gamma/IL-4 balance of ConA-stimulated splenocytes in vitro. Anandamide at nanomolar concentrations increased the production of IFN-gamma, while such production decreased at micromolar range. Thus, anandamide induced both the increment of DC activation and IFN-gamma production, which are likely the mechanisms involved in the increase of Th1 response reported here.

Behavior: A Relevant Tool for Brain-immune System Interaction Studies

Neuroimmunomodulation describes the field focused on understanding the mechanisms by which the central nervous system interacts with the immune system, potentially leading to changes in animal behavior. Nonetheless, not many articles dealing with neuroimmunomodulation employ behavior as an analytical endpoint. Even fewer papers deal with social status as a possible modifier of neuroimmune phenomena. In the described sets of experiments, we tackle both, using a paradigm of social dominance and subordination. We first review data on the effects of different ranks within a stable hierarchical relationship. Submissive mice in this condition display more anxiety‐like behaviors, have decreased innate immunity, and show a decreased resistance to implantation and development of melanoma metastases in their lungs. This suggests that even in a stable, social, hierarchical rank, submissive animals may be subjected to higher levels of stress, with putative biological relevance to host susceptibility to disease. Second, we review data on how dominant and submissive mice respond differentially to lipopolysaccharide (LPS), employing a motivational perspective to sickness behavior. Dominant animals display decreased number and frequency in several aspects of behavior, particularly agonistic social interaction, that is, directed toward the submissive cage mate. This was not observed in submissive mice that maintained the required behavior expected by its dominant mate. Expression of sickness behavior relies on motivational reorganization of priorities, which are different along different social ranks, leading to diverse outcomes. We suggest that in vitro assessment of neuroimmune phenomena can only be understood based on the behavioral context in which they occur.

Behavioral effects of LPS in adult, middle-aged and aged mice

Acute infections lead to alterations in behavior, collectively known as sickness behavior, which includes reduction in locomotion, food ingestion, sexual and social behavior, environmental exploration, and sleep profile. Although generally seen as undesired, sickness behavior represents a conserved strategy for animals to overcome disease. Aging process is associated with a variety of changes in immunity, which are referred to as immunosenescence, and include higher mortality by infectious diseases. Few works studied sickness behavior display in old animals. Thus, we sought to investigate the display of sickness related behaviors on aged mice. Adult (3-6 months old), middle-aged (12-15 m) and aged mice (18-22 m) were treated with i.p. LPS (200 microg/kg) and their behaviors were assessed in the open field and in the elevated plus-maze. Exploratory activity was similar in aged mice treated or not with LPS in both apparati. In the open field, locomotion remained at baseline levels; in the elevated plus-maze, there was a time-dependent decrease in motor activity.