Aberrant IL-17 Levels in Rodent Models of Autism Spectrum Disorder: A Systematic Review.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder characterised by stereotyped behaviours, specific interests, and impaired communication skills. Elevated levels of pro-inflammatory cytokines, such as interleukin-17A (IL-17A or IL-17), have been implicated as part of immune alterations that may contribute to this outcome. In this context, rodent models have helped elucidate the role of T-cell activation and IL-17 secretion in the pathogenesis of ASD. Regarding the preclinical findings, the data available is contradictory in offspring but not in the pregnant dams, pointing to IL-17 as one of the main drivers of altered behaviour in some models ASD, whilst there are no alterations described in IL-17 levels in others. To address this gap in the literature, a systematic review of altered IL-17 levels in rodent models of ASD was conducted. In total, 28 studies that explored IL-17 levels were included and observed that this cytokine was generally increased among the different models of ASD. The data compiled in this review can help the choice of animal models to study the role of cytokines in the development of ASD, seeking a parallel with immune alterations observed in individuals with this condition. Systematic Review Registration: PROSPERO, identifier CRD42022306558.

Laminin Triggers Neutrophil Extracellular Traps (NETs) and Modulates NET Release Induced by Leishmania amazonensis.

Neutrophils are recruited from the blood and transmigrate through the endothelium to reach tissues, where they are prone to respond through different mechanisms, including the release of neutrophil extracellular traps (NETs). These responses occur in close contact with proteins from the basement membrane and extracellular matrix, where laminins are abundant. Thus, we investigated the interactions between neutrophils and different laminin (LM) isoforms and analyzed the induction of NETs. We showed that neutrophils stimulated with LM isoforms 111, 211, 332, 411, 421, and 511 released NETs. The same occurred when neutrophils interacted with polymerized LMs 111, 411, and 511. LM-induced NETs were partially inhibited by pretreatment of neutrophils with an anti-α6 integrin antibody. Furthermore, NETs triggered by laminins were dependent on elastase and peptidylarginine deiminase (PAD)-4, enzymes that participate in chromatin decondensation. We also found that LMs 411 and LM 511 potentiated the NET release promoted by promastigotes of the protozoan parasite Leishmania, and that NETs stimulated by LMs alone display leishmanicidal activity. The ability of LM to induce NET release may have potential implications for the course of inflammation or infection.

Zika virus disrupts gene expression in human myoblasts and myotubes: Relationship with susceptibility to infection.

The tropism of Zika virus (ZIKV) has been described in the nervous system, blood, placenta, thymus, and skeletal muscle. We investigated the mechanisms of skeletal muscle susceptibility to ZIKV using an in vitro model of human skeletal muscle myogenesis, in which myoblasts differentiate into myotubes. Myoblasts were permissive to ZIKV infection, generating productive viral particles, while myotubes controlled ZIKV replication. To investigate the underlying mechanisms, we used gene expression profiling. First, we assessed gene changes in myotubes compared with myoblasts in the model without infection. As expected, we observed an increase in genes and pathways related to the contractile muscle system in the myotubes, a reduction in processes linked to proliferation, migration and cytokine production, among others, confirming the myogenic capacity of our system in vitro. A comparison between non-infected and infected myoblasts revealed more than 500 differentially expressed genes (DEGs). In contrast, infected myotubes showed almost 2,000 DEGs, among which we detected genes and pathways highly or exclusively expressed in myotubes, including those related to antiviral and innate immune responses. Such gene modulation could explain our findings showing that ZIKV also invades myotubes but does not replicate in these differentiated cells. In conclusion, we showed that ZIKV largely (but differentially) disrupts gene expression in human myoblasts and myotubes. Identifying genes involved in myotube resistance can shed light on potential antiviral mechanisms against ZIKV infection.

Crosstalk Between Innate and T Cell Adaptive Immunity With(in) the Muscle.

Growing evidence demonstrates a continuous interaction between the immune system and the skeletal muscle in inflammatory diseases of different pathogenetic origins, in dystrophic conditions such as Duchenne Muscular Dystrophy as well as during normal muscle regeneration. Although one component of the innate immunity, the macrophage, has been extensively studied both in disease conditions and during cell or gene therapy strategies aiming at restoring muscular functions, much less is known about dendritic cells and their primary immunological targets, the T lymphocytes. This review will focus on the dendritic cells and T lymphocytes (including effector and regulatory T-cells), emphasizing the potential cross talk between these cell types and their influence on the structure and function of skeletal muscle.

Differentiation-dependent susceptibility of human muscle cells to Zika virus infection.

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.

Zika virus targets the human thymic epithelium.

Previous work showed that the thymus can be infected by RNA viruses as HIV and HTLV-1. We thus hypothesized that the thymus might also be infected by the Zika virus (ZIKV). Herein we provide compelling evidence that ZIKV targets human thymic epithelial cells (TEC) in vivo and in vitro. ZIKV-infection enhances keratinization of TEC, with a decrease in proliferation and increase in cell death. Moreover, ZIKV modulates a high amount of coding RNAs with upregulation of genes related to cell adhesion and migration, as well as non-coding genes including miRNAs, circRNAs and lncRNAs. Moreover, we observed enhanced attachment of lymphoblastic T-cells to infected TEC, as well as virus transfer to those cells. Lastly, alterations in thymuses from babies congenitally infected were seen, with the presence of viral envelope protein in TEC. Taken together, our data reveals that the thymus, particularly the thymic epithelium, is a target for the ZIKV with changes in the expression of molecules that are relevant for interactions with developing thymocytes.

Combined methods to evaluate human cells in muscle xenografts.

Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell- and gene- based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space. These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.

Neuroendocrine Control of Macrophage Development and Function.

Macrophages carry out numerous physiological activities that are essential for both systemic and local homeostasis, as well as innate and adaptive immune responses. Their biology is intricately regulated by hormones, neuropeptides, and neurotransmitters, establishing distinct neuroendocrine axes. The control is pleiotropic, including maturation of bone marrow-derived myeloid precursors, cell differentiation into functional subpopulations, cytotoxic activity, phagocytosis, production of inflammatory mediators, antigen presentation, and activation of effector lymphocytes. Additionally, neuroendocrine components modulate macrophage ability to influence tumor growth and to prevent the spreading of infective agents. Interestingly, macrophage-derived factors enhance glucocorticoid production through the stimulation of the hypothalamic-pituitary-adrenal axis. These bidirectional effects highlight a tightly controlled balance between neuroendocrine stimuli and macrophage function in the development of innate and adaptive immune responses. Herein, we discuss how components of neuroendocrine axes impact on macrophage development and function and may ultimately influence inflammation, tissue repair, infection, or cancer progression. The knowledge of the crosstalk between macrophages and endocrine or brain-derived components may contribute to improve and create new approaches with clinical relevance in homeostatic or pathological conditions.

Activated dendritic cells modulate proliferation and differentiation of human myoblasts.

Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

BACKGROUND: The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. METHODS: We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. RESULTS: We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. CONCLUSIONS: We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

CD49d is a disease progression biomarker and a potential target for immunotherapy in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. The immune inflammatory response also contributes to disease progression in DMD patients. In a previous study, we demonstrated higher levels of circulating CD49dhi and CD49ehi T cells in DMD patients compared to healthy control. DMD patients are clinically heterogeneous and the functional defect cannot be correlated with genotype. Therefore, it is important to be able to define reliable noninvasive biomarkers to better define the disease progression at the beginning of clinical trials. RESULTS: We studied 75 DMD patients at different stages of their disease and observed that increased percentages of circulating CD4(+)CD49d(hi) and CD8(+)CD49d(hi) T lymphocytes were correlated with both severity and a more rapid progression of the disease. Moreover, T(+)CD49d(+) cells were also found in muscular inflammatory infiltrates. Functionally, T cells from severely affected patients exhibited higher transendothelial and fibronectin-driven migratory responses and increased adhesion to myotubes, when compared to control individuals. These responses could be blocked with an anti-CD49d monoclonal antibody. CONCLUSION: CD49d can be used as a novel biomarker to stratify DMD patients by predicting disease progression for clinical trials. Moreover, anti-CD49d peptides or antibodies can be used as a therapeutic approach to decrease inflammation-mediated tissue damage in DMD.

Laminin-Mediated Interactions in Thymocyte Migration and Development.

Intrathymic T-cell differentiation is a key process for the development and maintenance of cell-mediated immunity, and occurs concomitantly to highly regulated migratory events. We have proposed a multivectorial model for describing intrathymic thymocyte migration. One of the individual vectors comprises interactions mediated by laminins (LMs), a heterotrimeric protein family of the extracellular matrix. Several LMs are expressed in the thymus, being produced by microenvironmental cells, particularly thymic epithelial cells (TECs). Also, thymocytes and epithelial cells express integrin-type LM receptors. Functionally, it has been reported that the dy/dy mutant mouse (lacking the LM isoform 211) exhibits defective thymocyte differentiation. Several data show haptotactic effects of LMs upon thymocytes, as well as their adhesion on TECs; both effects being prevented by anti-LM or anti-LM receptor antibodies. Interestingly, LM synergizes with chemokines to enhance thymocyte migration, whereas classe-3 semaphorins and B ephrins, which exhibit chemorepulsive effects in the thymus, downregulate LM-mediated migratory responses of thymocytes. More recently, we showed that knocking down the ITGA6 gene (which encodes the α6 integrin chain of LM receptors) in human TECs modulates a large number of cell migration-related genes and results in changes of adhesion pattern of thymocytes onto the thymic epithelium. Overall, LM-mediated interactions can be placed at the cross-road of the multivectorial process of thymocyte migration, with a direct influence per se, as well as by modulating other molecular interactions associated with the intrathymic-trafficking events.

Laminin therapy for the promotion of muscle regeneration.

Muscle regeneration is essentially due to activation of satellite cells, which can be isolated and amplified ex vivo, thus representing good candidates for cell therapy. Accumulating data show that the local microenvironment plays a major role during muscle regeneration. In the satellite cell niche, a major extracellular matrix protein is laminin. Human myoblasts transplanted into immunodeficient mice are preferentially located in laminin-enriched areas. Additionally, laminin-111 enhances myoblast proliferation in vitro and increases expression of the α7ß1 integrin-type laminin receptor. Intramuscular injection of laminin-111 ameliorates muscular pathology in mdx mice, protecting muscle fibers from damage. Moreover, transplantation of human myoblasts with laminin-111 into Rag/mdx immunodeficient recipients improved efficacy of myoblast transplantation, increasing the number of human dystrophin-positive myofibres. Taken together, these data strongly indicate that exogenous laminin can ameliorate the regeneration process in different models of muscular dystrophies and can be instrumental for improving cell therapy aiming at repairing the degeneration/regeneration process in skeletal muscle.

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection.

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.

Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by modifying their kinetics of proliferation and differentiation.

Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

Slowing down differentiation of engrafted human myoblasts into immunodeficient mice correlates with increased proliferation and migration.

We have used a model of xenotransplantation in which human myoblasts were transplanted intramuscularly into immunodeficient Rag2(-/-)γC(-/-) mice, in order to investigate the kinetics of proliferation and differentiation of the transplanted cells. After injection, most of the human myoblasts had already differentiated by day 5. This differentiation correlated with reduction in proliferation and limited migration of the donor cells within the regenerating muscle. These results suggest that the precocious differentiation, already detected at 3 days postinjection, is a limiting factor for both the migration from the injection site and the participation of the donor cells to muscle regeneration. When we stimulated in vivo proliferation of human myoblasts, transplanting them in a serum-containing medium, we observed 5 days post-transplantation a delay of myogenic differentiation and an increase in cell numbers, which colonized a much larger area within the recipient's muscle. Importantly, these myoblasts maintained their ability to differentiate, since we found higher numbers of myofibers seen 1 month postengraftment, as compared to controls. Conceptually, these data suggest that in experimental myoblast transplantation, any intervention upon the donor cells and/or the recipient's microenvironment aimed at enhancing proliferation and migration should be done before differentiation of the implanted cells, e.g., day 3 postengraftment.

In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study.

In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle-derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2(-/-) gammaC(-/-) C5(-/-) mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle-derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.

Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways.

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.