Abnormal Whisker-Dependent Behaviors and Altered Cortico-Hippocampal Connectivity in Shank3b-/- Mice.

Abnormal tactile response is an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Patients with Phelan-McDermid syndrome (PMS), caused by mutations in the SHANK3 gene, show ASD-like symptoms associated with aberrant tactile responses. The neural underpinnings of these abnormalities are still poorly understood. Here we investigated, in Shank3b-/- adult mice, the neural substrates of whisker-guided behaviors, a key component of rodents' interaction with the surrounding environment. We assessed whisker-dependent behaviors in Shank3b-/- adult mice and age-matched controls, using the textured novel object recognition (tNORT) and whisker nuisance (WN) test. Shank3b-/- mice showed deficits in whisker-dependent texture discrimination in tNORT and behavioral hypo-responsiveness to repetitive whisker stimulation in WN. Sensory hypo-responsiveness was accompanied by a significantly reduced activation of the primary somatosensory cortex (S1) and hippocampus, as measured by c-fos mRNA induction, a proxy of neuronal activity following whisker stimulation. Moreover, resting-state fMRI showed a significantly reduced S1-hippocampal connectivity in Shank3b mutants, in the absence of altered connectivity between S1 and other somatosensory areas. Impaired crosstalk between hippocampus and S1 might underlie Shank3b-/- hypo-reactivity to whisker-dependent cues, highlighting a potentially generalizable somatosensory dysfunction in ASD.

Somatosensory cortex hyperconnectivity and impaired whisker-dependent responses in Cntnap2-/- mice.

Sensory abnormalities are a common feature in autism spectrum disorders (ASDs). Tactile responsiveness is altered in autistic individuals, with hypo-responsiveness being associated with the severity of ASD core symptoms. Similarly, sensory abnormalities have been described in mice lacking ASD-associated genes. Loss-of-function mutations in CNTNAP2 result in cortical dysplasia-focal epilepsy syndrome (CDFE) and autism. Likewise, Cntnap2-/- mice show epilepsy and deficits relevant with core symptoms of human ASDs, and are considered a reliable model to study ASDs. Altered synaptic transmission and synchronicity found in the cerebral cortex of Cntnap2-/- mice would suggest a network dysfunction. Here, we investigated the neural substrates of whisker-dependent responses in Cntnap2+/+ and Cntnap2-/- adult mice. When compared to controls, Cntnap2-/- mice showed focal hyper-connectivity within the primary somatosensory cortex (S1), in the absence of altered connectivity between S1 and other somatosensory areas. This data suggests the presence of impaired somatosensory processing in these mutants. Accordingly, Cntnap2-/- mice displayed impaired whisker-dependent discrimination in the textured novel object recognition test (tNORT) and increased c-fos mRNA induction within S1 following whisker stimulation. S1 functional hyperconnectivity might underlie the aberrant whisker-dependent responses observed in Cntnap2-/- mice, indicating that Cntnap2 mice are a reliable model to investigate sensory abnormalities that characterize ASDs.

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ T cells.

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8+ T cells. Although, during their differentiation, activated CD8+ T cells may first lose CD28, and CD28- cells may eventually express CD57 as a subsequent step, a population of CD28+ CD57+ (DP) CD8+ T cells can be identified in the peripheral blood. How this population is distinct from CD28- CD57- (DN) CD8+ T cells, and from the better characterized non-activated/early-activated CD28+ CD57- and senescent-like CD28- CD57+ CD8+ T cell subsets is currently unknown. Here, RNA expression of the four CD8+ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to "early" highly differentiated cells, with decreased TNF and IFN-γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co-inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8+ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8+ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8+ T cell subsets displaying defined properties.

The impact of body mass index on adaptive immune cells in the human bone marrow.

BACKGROUND: Obesity has been associated with chronic inflammation and oxidative stress. Both conditions play a determinant role in the pathogenesis of age-related diseases, such as immunosenescence. Adipose tissue can modulate the function of the immune system with the secretion of molecules influencing the phenotype of immune cells. The importance of the bone marrow (BM) in the maintenance of antigen-experienced adaptive immune cells has been documented in mice. Recently, some groups have investigated the survival of effector/memory T cells in the human BM. Despite this, whether high body mass index (BMI) may affect immune cells in the BM and the production of molecules supporting the maintenance of these cells it is unknown. METHODS: Using flow cytometry, the frequency and the phenotype of immune cell populations were measured in paired BM and PB samples obtained from persons with different BMI. Furthermore, the expression of BM cytokines was assessed. The influence of cytomegalovirus (CMV) on T cell subsets was additionally considered, dividing the donors into the CMV- and CMV+ groups. RESULTS: Our study suggests that increased BMI may affect both the maintenance and the phenotype of adaptive immune cells in the BM. While the BM levels of IL-15 and IL-6, supporting the survival of highly differentiated T cells, and oxygen radicals increased in overweight persons, the production of IFNγ and TNF by CD8+ T cells was reduced. In addition, the frequency of B cells and CD4+ T cells positively correlated with BMI in the BM of CMV- persons. Finally, the frequency of several T cell subsets, and the expression of senescence/exhaustion markers within these subpopulations, were affected by BMI. In particular, the levels of bona fide memory T cells may be reduced in overweight persons. CONCLUSION: Our work suggests that, in addition to aging and CMV, obesity may represent an additional risk factor for immunosenescence in adaptive immune cells. Metabolic interventions may help in improving the fitness of the immune system in the elderly.

Oxidative Stress and Immune System Dysfunction in Autism Spectrum Disorders.

Autism Spectrum Disorders (ASDs) represent a group of neurodevelopmental disorders associated with social and behavioral impairments. Although dysfunctions in several signaling pathways have been associated with ASDs, very few molecules have been identified as potentially effective drug targets in the clinic. Classically, research in the ASD field has focused on the characterization of pathways involved in neural development and synaptic plasticity, which support the pathogenesis of this group of diseases. More recently, immune system dysfunctions have been observed in ASD. In addition, high levels of reactive oxygen species (ROS), which cause oxidative stress, are present in ASD patients. In this review, we will describe the major alterations in the expression of genes coding for enzymes involved in the ROS scavenging system, in both ASD patients and ASD mouse models. In addition, we will discuss, in the context of the most recent literature, the possibility that oxidative stress, inflammation and immune system dysfunction may be connected to, and altogether support, the pathogenesis and/or severity of ASD. Finally, we will discuss the possibility of novel treatments aimed at counteracting the interplay between ROS and inflammation in people with ASD.

Peripheral antibody concentrations are associated with highly differentiated T cells and inflammatory processes in the human bone marrow.

BACKGROUND: Antigen-experienced immune cells migrate back to the bone marrow (BM), where they are maintained in BM survival niches for an extended period. The composition of T cell subpopulations in the BM changes with age, leading to an accumulation of highly differentiated T cells and a loss of naïve T cells. While innate immune cells are also affected by age, little is known about interactions between different adaptive immune cell populations maintained in the BM. In this study, the phenotype and function of innate and adaptive immune cells isolated from human BM and peripheral blood (PB) was analysed in detail using flow cytometry, to determine if the accumulation of highly differentiated T and B cells, supported by the BM niches, limits the maintenance of other immune cells, or affects their functions such as providing protective antibody concentrations. RESULTS: Total T cells increase in the BM with age, as do highly differentiated CD8+ T cells which no longer express the co-stimulatory molecule CD28, while natural killer T (NKT) cells, monocytes, B cells, and naïve CD8+ T cells all decrease in the BM with age. A negative correlation of total T cells with B cells was observed in the BM. The percentage of B cells in the BM negatively correlated with highly differentiated CD8+CD28- T cells, replicative-senescent CD8+CD57+ T cells, as well as the CD8+CD28-CD57+ population. Similar correlations were seen between B cells and the frequency of highly differentiated T cells producing pro-inflammatory molecules in the BM. Interestingly, plasma concentrations of diphtheria-specific antibodies negatively correlated with highly differentiated CD8+CD57+ T cells as well as with exhausted central memory CD8+ and CD4+ T cells in the BM. A negative impact on diphtheria-specific antibodies was also observed for CD8+ T cells expressing senescence associated genes such as the cell cycle regulator p21 (CDKN1A), KLRG-1, and elevated levels of reactive oxygen species (ROS). CONCLUSION: Our data suggest that the accumulation and maintenance of highly differentiated, senescent, and exhausted T cells in the BM, particularly in old age, may interfere with the survival of other cell populations resident in the BM such as monocytes and B cells, leading to reduced peripheral diphtheria antibody concentrations as a result. These findings further highlight the importance of the BM in the long-term maintenance of immunological memory.

"Inflamm-aging" influences immune cell survival factors in human bone marrow.

The bone marrow (BM) plays a key role in the long-term maintenance of immunological memory. However, the impact of aging on the production of survival factors for effector/memory T cells and plasma cells in the human BM has not been studied. We now show that the expression of molecules involved in the maintenance of immunological memory in the human BM changes with age. While IL-15, which protects potentially harmful CD8+ CD28- senescent T cells, increases, IL-7 decreases. IL-6, which may synergize with IL-15, is also overexpressed. In contrast, a proliferation-inducing ligand, a plasma cell survival factor, is reduced. IFN-y, TNF, and ROS accumulate in the BM in old age. IL-15 and IL-6 expression are stimulated by IFN-y and correlate with ROS levels in BM mononuclear cells. Both cytokines are reduced by incubation with the ROS scavengers N-acetylcysteine and vitamin C. IL-15 and IL-6 are also overexpressed in the BM of superoxide dismutase 1 knockout mice compared to their WT counterparts. In summary, our results demonstrate the role of inflammation and oxidative stress in age-related changes of immune cell survival factors in the BM, suggesting that antioxidants may be beneficial in counteracting immunosenescence by improving immunological memory in old age.

Hippocampal dysregulation of neurofibromin-dependent pathways is associated with impaired spatial learning in engrailed 2 knock-out mice.

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2(-/-)) mice show anatomical and behavioral "ASD-like" features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2(-/-) mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2(-/-) mice before and after spatial learning testing. When compared with WT littermates, En2(-/-) mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2(-/-) mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2(-/-) mice after MWM. Treatment of En2(-/-) mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.

Legend or Truth: Mature CD4+CD8+ Double-Positive T Cells in the Periphery in Health and Disease.

The expression of CD4 and CD8 co-receptors defines two distinct T cell populations with specialized functions. While CD4+ T cells support and modulate immune responses through different T-helper (Th) and regulatory subtypes, CD8+ T cells eliminate cells that might threaten the organism, for example, virus-infected or tumor cells. However, a paradoxical population of CD4+CD8+ double-positive (DP) T cells challenging this paradigm has been found in the peripheral blood. This subset has been observed in healthy as well as pathological conditions, suggesting unique and well-defined functions. Furthermore, DP T cells express activation markers and exhibit memory-like features, displaying an effector memory (EM) and central memory (CM) phenotype. A subset expressing high CD4 (CD4bright+) and intermediate CD8 (CD8dim+) levels and a population of CD8bright+CD4dim+ T cells have been identified within DP T cells, suggesting that this small subpopulation may be heterogeneous. This review summarizes the current literature on DP T cells in humans in health and diseases. In addition, we point out that strategies to better characterize this minor T cell subset's role in regulating immune responses are necessary.

At the Crossroad Between Resiliency and Fragility: A Neurodevelopmental Perspective on Early-Life Experiences.

Postnatal development of the brain is characterized by sensitive windows during which, local circuitry are drastically reshaped by life experiences. These critical periods (CPs) occur at different time points for different brain functions, presenting redundant physiological changes in the underlying brain regions. Although circuits malleability during CPs provides a valuable window of opportunity for adaptive fine-tuning to the living environment, this aspect of neurodevelopment also represents a phase of increased vulnerability for the development of a variety of disorders. Consistently, accumulating epidemiological studies point to adverse childhood experience as a major risk factor for many medical conditions, especially stress- and anxiety-related conditions. Thanks to creative approaches to manipulate rodents' rearing environment, neurobiologist have uncovered a pivotal interaction between CPs and early-life experiences, offering an interesting landscape to improve our understanding of brain disorders. In this short review, we discuss how early-life experience impacts cellular and molecular players involved in CPs of development, translating into long-lasting behavioral consequences in rodents. Bringing together findings from multiple laboratories, we delineate a unifying theory in which systemic factors dynamically target the maturation of brain functions based on adaptive needs, shifting the balance between resilience and vulnerability in response to the quality of the rearing environment.

Immune dysfunction in the cerebellum of mice lacking the autism candidate gene Engrailed 2.

Immune system dysfunction has been described in autism spectrum disorder. Here we tested the hypothesis that cerebellar defects are accompanied by immune dysfunction in adult mice lacking the autism-candidate gene Engrailed 2 (En2). Gene ontology analyses revealed that biological processes related to immune function were over-represented in the cerebellar transcriptome of En2-/- mice. Pro-inflammatory molecules and chemokines were reduced in the En2-/- cerebellum compared to controls. Conversely, pro-inflammatory molecules were increased in the peripheral blood of mutant mice. Our results suggest a link between immune dysfunction and cerebellar defects detected in En2-/- mice.

T cells, aging and senescence.

The T cell compartment undergoes characteristic changes with age, which contribute to increased incidence and severity of infections and reduced immunogenicity and efficacy of many vaccines in the older population. Production of naïve T cells is severely impaired due to a decreased output of lymphoid cells from the bone marrow and the involution of the thymus. At the same time, antigen-experienced, highly differentiated T cells accumulate resulting in a diminished T cell receptor repertoire. These cells show some similarities with senescent cells, such as shorter telomers, accumulated DNA damage and metabolic changes. Latent infection with Cytomegalovirus also impacts the T cell compartment and aggravates several of its age-associated changes. Loss of CD28 expression is one hallmark of T cells after repeated antigenic stimulation, but CD28- T cells cannot be considered truly senescent as e.g. they are still able to proliferate upon adequate stimulation. Several additional markers have been suggested in order to define a potential fully senescent T cell population, but no consensus definition has been reached so far. It has been postulated that highly differentiated senescent-like T cells are unable to eliminate other senescent cell types. Removal of senescent non-immune cells has been shown to be beneficial for the organism and a reliable definition of senescent T cells is essential for an extension of this concept to T cells.

Natural Antioxidants: A Novel Therapeutic Approach to Autism Spectrum Disorders?

Autism spectrum disorders (ASD) are a group of neurodevelopmental syndromes with both genetic and environmental origins. Several recent studies have shown that inflammation and oxidative stress may play a key role in supporting the pathogenesis and the severity of ASD. Thus, the administration of anti-inflammatory and antioxidant molecules may represent a promising strategy to counteract pathological behaviors in ASD patients. In the current review, results from recent literature showing how natural antioxidants may be beneficial in the context of ASD will be discussed. Interestingly, many antioxidant molecules available in nature show anti-inflammatory activity. Thus, after introducing ASD and the role of the vitamin E/vitamin C/glutathione network in scavenging intracellular reactive oxygen species (ROS) and the impairments observed with ASD, we discuss the concept of functional food and nutraceutical compounds. Furthermore, the effects of well-known nutraceutical compounds on ASD individuals and animal models of ASD are summarized. Finally, the importance of nutraceutical compounds as support therapy useful in reducing the symptoms in autistic people is discussed.

Antioxidants N-Acetylcysteine and Vitamin C Improve T Cell Commitment to Memory and Long-Term Maintenance of Immunological Memory in Old Mice.

Aging is characterized by reduced immune responses, a process known as immunosenescence. Shortly after their generation, antigen-experienced adaptive immune cells, such as CD8+ and CD4+ T cells, migrate into the bone marrow (BM), in which they can be maintained for long periods of time within survival niches. Interestingly, we recently observed how oxidative stress may negatively support the maintenance of immunological memory in the BM in old age. To assess whether the generation and maintenance of immunological memory could be improved by scavenging oxygen radicals, we vaccinated 18-months (old) and 3-weeks (young) mice with alum-OVA, in the presence/absence of antioxidants vitamin C (Vc) and/or N-acetylcysteine (NAC). To monitor the phenotype of the immune cell population, blood was withdrawn at several time-points, and BM and spleen were harvested 91 days after the first alum-OVA dose. Only in old mice, memory T cell commitment was boosted with some antioxidant treatments. In addition, oxidative stress and the expression of pro-inflammatory molecules decreased in old mice. Finally, changes in the phenotype of dendritic cells, important regulators of T cell activation, were additionally observed. Taken together, our data show that the generation and maintenance of memory T cells in old age may be improved by targeting oxidative stress.

The impact of oxidative stress, inflammation, and senescence on the maintenance of immunological memory in the bone marrow in old age.

The bone marrow (BM) provides a preferential survival environment for the long-term maintenance of antigen-experienced adaptive immune cells. After the contact with antigens, effector/memory T cells and plasma cell precursors migrate to the BM, in which they can survive within survival niches in an antigen-independent manner. Despite this, the phenotype of adaptive immune cells changes with aging, and BM niches themselves are affected, leading to impaired long-term maintenance of immunological memory in the elderly as a result. Oxidative stress, age-related inflammation (inflammaging), and cellular senescence appear to play a major role in this process. This review will summarize the age-related changes in T and B cell phenotype, and in the BM niches, discussing the possibility that the accumulation of highly differentiated, senescent-like T cells in the BM during aging may cause inflammation in the BM and promote oxidative stress and senescence. In addition, senescent-like T cells may compete for space with other immune cells within the marrow, partially excluding effector/memory T cells and long-lived plasma cells from the niches.

Human bone marrow adipocytes display distinct immune regulatory properties.

BACKGROUND: The bone marrow (BM) is a major reservoir of resting memory T cells and long-lived plasma cells, capable of providing protection against recurrent infections. Whether the age-related accumulation of adipose tissue in the BM affects the functionality and maintenance of memory cells is not well understood. METHODS: For the first time, we compare human femur marrow adipose tissue (fMAT) and subcutaneous white adipose tissue of the thigh (tsWAT) obtained from the same donors. Therefore, we used microarrays for comparative global gene expression analysis, and employed assays to analyse parameters of adipocyte biology, inflammation and oxidative stress. FINDINGS: We show that fMAT adipocytes differ significantly from tsWAT adipocytes regarding specific gene expression profiles including inflammatory responses and adipogenesis/adipocyte phenotype. Concomitant with considerably lower levels of CD36, a membrane-associated protein important for long-chain fatty acid uptake that is used as maturation marker, fMAT adipocytes are smaller and contain less triglycerides. fMAT adipocytes secrete similar levels of adiponectin and leptin as tsWAT adipocytes, and express increased levels of pro-inflammatory molecules concomitant with an elevated generation of reactive oxygen species (ROS) and impaired function of plasma cells in the BM. INTERPRETATION: Our findings suggest that fMAT is a unique type of adipose tissue containing small adipocytes with lower CD36 protein and triglyceride levels than tsWAT but high adipokine secretion. Moreover, fMAT adipocytes secrete high levels of pro-inflammatory cytokines, contributing to inflammation and impairment of plasma cell function in the BM, suggesting that fMAT has more immune regulatory functions than tsWAT.

Fcµ receptor as a Costimulatory Molecule for T Cells.

Fc receptor for IgM (FcµR)-deficient mice display dysregulated function of neutrophils, dendritic cells, and B cells. The relevance of FcµR to human T cells is still unknown. We show that FcµR is mostly stored inside the cell and that surface expression is tightly regulated. Decreased surface expression on T cells from elderly individuals is associated with alterations in the methylation pattern of the FCMR gene. Binding and internalization of IgM stimulate transport of FcµR to the cell surface to ensure sustained IgM uptake. Concurrently, IgM accumulates within the cell, and the surface expression of other receptors increases, among them the T cell receptor (TCR) and costimulatory molecules. This leads to enhanced TCR signaling, proliferation, and cytokine release, in response to low, but not high, doses of antigen. Our findings indicate that FcµR is an important regulator of T cell function and reveal an additional mode of interaction between B and T cells.

CD8+HLADR+ Regulatory T Cells Change With Aging: They Increase in Number, but Lose Checkpoint Inhibitory Molecules and Suppressive Function.

CD4+ regulatory T cells have been intensively studied during aging, but little is still known about age-related changes of other regulatory T cell subsets. It was, therefore, the goal of the present study to analyze CD8+human leukocyte antigen-antigen D related (HLADR)+ T cells in old age, a cell population reported to have suppressive activity and to be connected to specific genetic variants. We demonstrate a strong increase in the number of CD8+HLADR+ T cells with age in a cohort of female Sardinians as well as in elderly male and female persons from Austria. We also show that CD8+HLADR+ T cells lack classical activation molecules, such as CD69 and CD25, but contain increased numbers of checkpoint inhibitory molecules, such as cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin and mucin protein-3, LAG-3, and PD-1, when compared with their HLADR- counterparts. They also have the capacity to inhibit the proliferation of autologous peripheral blood mononuclear cells. This suppressive activity is, however, decreased when CD8+HLADR+ T cells from elderly persons are analyzed. In accordance with this finding, CD8+HLADR+ T cells from persons of old age contain lower percentages of checkpoint inhibitory molecules than young controls. We conclude that in spite of high abundance of a CD8+ regulatory T cell subset in old age its expression of checkpoint inhibitory molecules and its suppressive function on a per cell basis are reduced. Reduction of suppressive capacity may support uncontrolled subclinical inflammatory processes referred to as "inflamm-aging."

Increased IL-15 Production and Accumulation of Highly Differentiated CD8+ Effector/Memory T Cells in the Bone Marrow of Persons with Cytomegalovirus.

Cytomegalovirus (CMV) has been described as a contributor to immunosenescence, thus exacerbating age-related diseases. In persons with latent CMV infection, the CD8+ T cell compartment is irreversibly changed, leading to the accumulation of highly differentiated virus-specific CD8+ T cells in the peripheral blood. The bone marrow (BM) has been shown to play a major role in the long-term survival of antigen-experienced T cells. Effector CD8+ T cells are preferentially maintained by the cytokine IL-15, the expression of which increases in old age. However, the impact of CMV on the phenotype of effector CD8+ T cells and on the production of T cell survival molecules in the BM is not yet known. We now show, using BM samples obtained from persons who underwent hip replacement surgery because of osteoarthrosis, that senescent CD8+ TEMRA cells with a bright expression of CD45RA and a high responsiveness to IL-15 accumulate in the BM of CMV-infected persons. A negative correlation was found between CMV antibody (Ab) titers in the serum and the expression of CD28 and IL-7Rα in CD8+ [Formula: see text] cells. Increased IL-15 mRNA levels were observed in the BM of CMV+ compared to CMV- persons, being particularly high in old seropositive individuals. In summary, our results indicate that a BM environment rich in IL-15 may play an important role in the maintenance of highly differentiated CD8+ T cells generated after CMV infection.