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Hashimoto’s thyroiditis (HT) is an autoimmune and inflammatory disease in which antibodies are directed against the thyroid gland leading to chronic inflammation and hypothyroidism. The autoimmunity against thyroid antigens can be associated to genetic background and environmental factors. Thyroid peroxidase (TPO) and thyroglobulin (TG) are the major autoantigens for characterizing the disease. HT is related to the activation of autoreactive CD4+ T cells, CD8+ cytotoxic T cells and antithyroid antibody producing-B cells. Among several cytokines related to the pathogenesis of HT, a proliferation-inducing ligand (APRIL) has been studied in the context of the establishment and/or maintenance of autoimmune diseases. The role of APRIL in the pathogenesis of HT is still poorly understood. Therefore, the present study aimed to compare APRIL serum concentration in HT patients and healthy donors by ELISA. We observed a significant decrease in APRIL concentration in HT patients when compared to the control group, and a positive correlation between APRIL level and age. Our results suggest that the APRIL molecule can compose the cytokine profile along the inflammatory response in HT, however, other investigations should be proposed to understand its molecular mechanisms via specific receptors and other regulatory loops. A Tireoidite de Hashimoto (TH) é uma doença autoimune e inflamatória na qual os anticorpos são direcionados contra a glândula tireoide, levando à inflamação crônica e ao hipotireoidismo. A tireóide peroxidase (TPO) e a tireoglobulina (TG) são os principais autoantígenos e esta autoimunidade pode estar relacionada a fatores genéticos e ambientais. A TH é caracterizada pela ativação de células T CD4+ autoreativas, células T citotóxicas CD8+ e células B produtoras de anticorpos antitireoidianos. Entre várias citocinas relacionadas à patogênese da TH, um ligante indutor de proliferação (APRIL) tem sido estudado no contexto do estabelecimento e/ou manutenção de doenças autoimunes. O papel da citocina APRIL na patogênese da TH ainda é pouco compreendido, portanto, o presente estudo teve como objetivo dosar a concentração sérica de APRIL em pacientes com TH e indivíduos saudáveis pela técnica de ELISA. Observamos uma diminuição significativa na concentração de APRIL em pacientes com TH quando comparados ao grupo controle, além de uma correlação positiva entre concentração de APRIL e a idade dos pacientes. Nossos resultados sugerem que a molécula de APRIL pode compor o perfil de citocinas ao longo da resposta inflamatória na TH, no entanto, outras investigações devem ser propostas para entender seus mecanismos moleculares por meio de receptores específicos e outras alças regulatórias.

Introduction A proliferation-inducing ligand (APRIL) and B cell activation factor (BAFF) are known to play a significant role in the pathogenesis of several diseases, including BAFF in malaria. The aim of this study was to investigate whether APRIL and BAFF plasma concentrations could be part of inflammatory responses associated with P. vivax and P. falciparum malaria in patients from the Brazilian Amazon. Methods Blood samples were obtained from P. vivax and P. falciparum malaria patients (n = 52) resident in Porto Velho before and 15 days after the beginning of treatment and from uninfected individuals (n = 12). We investigated APRIL and BAFF circulating levels and their association with parasitaemia, WBC counts, and cytokine/chemokine plasma levels. The expression levels of transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) on PBMC from a subset of 5 P. vivax-infected patients were analyzed by flow cytometry. Results APRIL plasma levels were transiently increased during acute P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, in P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL-1, IL-2, IL-4, IL-6, and IL-13 levels. We did not find any association between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL levels. The percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase P. vivax malaria. Conclusion These findings suggest that the APRIL and BAFF inductions reflect different host strategies for controlling infection with each malaria species.

Spinal cord injury (SCI) is a traumatic event that results in motor, sensitive or autonomic function disturbances, which have direct impact on the life quality of the affected individual. Recent studies have shown that attenuation of the inflammatory response after SCI plays a key role in the reestablishment of motor function. Galectin-3 is a pleiotropic molecule belonging to the carbohydrate-ligand lectin family, which is expressed by different cells in different tissues. Studies have shown that galectin-3 induces the recruitment and activation of neutrophils, monocytes/macrophages, lymphocytes and microglia. Thus, the aim of this study was to evaluate the effects of the lack of galectin-3 on the functional outcome, cellular recruitment and morphological changes in tissue, after SCI. C57BL/6 wild-type and galectin-3 knockout mice were used in this study. A vascular clip was used for 1 min to generate a compressive SCI. By BMS we detected that the Gal-3(-/-) presented a better functional outcome during the studied period. This finding is related to a decrease in the injury length and a higher volume of spared white matter at 7 and 42 days post injury (dpi). Moreover, Gal-3(-/-) mice showed a higher number of spared fibers at 28 dpi. Because of the importance of the inflammatory response after SCI and the role that galectin-3 plays in it, we investigated possible differences in the inflammatory response between the analyzed groups. No differences in neutrophils were observed 24h after injury. However, at 3 dpi, the Gal-3(-/-) mice showed more neutrophils infiltrated into the spinal tissue when compared with the WT mice. At this same time point, no differences in the percentage of the CD11b/Arginase1 positive cells were observed. Remarkably, Gal-3(-/-) mice displayed a decrease in CD11b staining at 7 dpi, compared with the WT mice. At the same time, Gal-3(-/-) mice presented a more prominent Arginase1 stained area, suggesting an anti-inflammatory cell phenotype. Taken together, these results demonstrated that the lack of galectin-3 plays a key role in the inflammatory process triggered by SCI, leading to better and early recovery of locomotor function.

Oral transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of T. cruzi Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. In vivo bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. Ex vivo analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP T. cruzi were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant T. cruzi detection and expansion in mouse nasal cavity. Moreover, T. cruzi DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral T. cruzi infection leading to parasite multiplication at the nasal cavity., Author summary Oral transmission of Trypanosoma cruzi associated with food/beverage consumption is presently an important route of infection in Brazil and Venezuela. Colombia, Bolivia, Argentina and Ecuador have also reported to have acute cases of Chagas disease transmission through the oral route. Significant studies about this form of T. cruzi infection are largely lacking. In addition to the classic cardiac involvement, orally-infected patient progress to a highly symptomatic disease and increased mortality rate (8–35%), surpassing the calculated mortality produced by the disease resulting from the biting of infected insect vectors (5–10%). Here, we explored by in vivo bioluminescent images, qPCR and fluorescence microscopy the primary site of parasite entry and multiplication in oral infection (OI). Our results clearly demonstrated that the oral cavity is the main T. cruzi target region in OI, leading to parasite multiplication at the nasal cavity and parasite dissemination to the brain and peripheral tissues. Interestingly, facial edema, paraesthesia of the tongue, gingivitis and dry cough were already described in affected patients. These findings might be associated to our present data, which describe for the first time the nasomaxillary region as the main target tissue following oral T. 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.

Wallerian degeneration (WD) comprises a series of events that includes activation of non-neuronal cells and recruitment of immune cells, creating an inflammatory milieu that leads to extensive nerve fragmentation and subsequent clearance of the myelin debris, both of which are necessary prerequisites for effective nerve regeneration. Previously, we documented accelerated axon regeneration in animals lacking galectin-3 (Gal-3), a molecule associated with myelin clearance. To clarify the mechanisms underlying this enhanced regeneration, we focus here on the early steps of WD following sciatic nerve crush in Gal-3(-/-) mice. Using an in vivo model of nerve degeneration, we observed that removal of myelin debris is more efficient in Gal-3(-/-) than in wild-type (WT) mice; we next used an in vitro phagocytosis assay to document that the phagocytic potential of macrophages and Schwann cells was enhanced in the Gal-3(-/-) mice. Moreover, both RNA and protein levels for the pro-inflammatory cytokines IL-1β and TNF-α, as well as for Toll-like receptor (TLR)-2 and -4, show robust increases in injured nerves from Gal-3(-/-) mice compared to those from WT mice. Collectively, these data indicate that the lack of Gal-3 results in an augmented inflammatory profile that involves the TLR-cytokine pathway, and increases the phagocytic capacity of Schwann cells and macrophages, which ultimately contributes to speeding the course of WD.

Thymus atrophy and thymocyte apoptosis during T. cruzi infection is mediated by both the extrinsic and intrinsic death pathway, and involves caspase activation. Trypanosoma cruzi acute infection leads to thymic atrophy, largely as a result of death of immature DP T cells. In a second vein, the glucocorticoid hormone imbalance promotes DP T cell apoptosis in infected mice. Herein, we assessed the involvement of caspase signaling in thymocyte death during T. cruzi acute infection. BALB/c mice were infected i.p. with 102 trypomastigote forms of T. cruzi and analyzed from 7 to 19 dpi. Thymocyte apoptosis was observed in early stages of infection, increasing along with time postinfection. Immature DN and DP as well as CD4+ and CD8+ thymocytes from infected mice showed increased activation of caspase-8, -9, and -3. In vitro treatment of thymocytes from infected mice with a general caspase inhibitor or the combination of caspase-8- and caspase-9-specific inhibitors increased the number of living thymocytes. Intrathymic injection of the general caspase inhibitor, but not caspase-8 or -9 inhibitors individually, prevented thymic atrophy and thymocyte depletion in infected mice. Moreover, blockade of glucocorticoid receptor activity with RU486 prevented DP thymocyte apoptosis, together with caspase-8 and -9 activation. These findings indicate that DP T cell apoptosis following experimental T. cruzi acute infection is dependent on glucocorticoid stimulation, promoting caspase-8 and -9 activation.

Malaria and Cutaneous Leishmaniasis (CL) are co-endemic throughout large regions in tropical countries and co-infection may impact the evolution of host-parasite interactions. In the present study, we evaluate Malaria/Leishmaniasis disease outcome, Th1/Th2 cytokine levels and the CD4 and CD8 T-cell profiles in a co-infection murine model (BALB/c) of Plasmodium yoelii 17XNL (Py) and Leishmania amazonensis (La) or L. braziliensis (Lb). Malaria parasitaemia was assessed through blood strains stained with Giemsa. Leishmania lesions were monitored with a digital caliper and parasite loads determined by limiting-dilution assay. Serum levels of IFN-γ, TNF, IL-2, IL-4, IL-6, IL-10, and IL-17 were determined using multiplexed bead assay and expression of CD3, CD4, and CD8 T-cells markers were determined by Flow Cytometry in the thymus, spleens and lymph nodes. Parasitaemia in Lb+Py co-infected group was lower than in Py single-infected group, suggesting a protective effect of Lb co-infection in Malaria progression. In contrast, La+Py co-infection increased parasitaemia, patent infection and induced mortality in non-lethal Malaria infection. Regarding Leishmaniasis, Lb+Py co-infected group presented smaller lesions and less ulceration than Lb single-infected animals. In contrast, La+Py co-infected group presented only a transitory delay on the development of lesions when compared to La single-infected mice. Decreased levels of IFN-γ, TNF, IL-6, and IL-10 were observed in the serum of co-infected groups, demonstrating a modulation of Malaria immune response by Leishmania co-infections. We observed an intense thymic atrophy in Py single-infected and co-infected groups, which recovered earlier in co-infected animals. The CD4 and CD8 T cell profiles in thymus, spleens and lymph nodes did not differ between Py single and co-infected groups, except for a decrease in CD4(+)CD8(+) T cells which also increased faster in co-infected mice. Our results suggest that Py and Leishmania co-infection may change disease outcome. Interestingly Malaria outcome can be altered according to the Leishmania specie involved. Alternatively Malaria infection reduced the severity or delayed the onset of leishmanial lesions. These alterations in Malaria and CL development seem to be closely related with changes in the immune response as demonstrated by alteration in serum cytokine levels and thymus/spleens T cell phenotypes dynamics during infection.

Thymocyte differentiation occurs within the thymic microenvironment, consisting of distinct cell types and extracellular matrix (ECM) elements. One of these ECM proteins is laminin. Previous experiments showed that laminin mediates interactions between thymocytes and thymic epithelial cells (TEC) in mice. Since, laminin comprises a family of related isoforms, we searched for laminin isoform expression in the human thymus. We found constitutive gene expression of various laminin chains in TEC preparations, comprising laminin-111 and laminin-211 isoforms. Immunocytochemistry revealed a selective laminin-211 distribution in the thymic lobules. In vitro functional assays revealed that laminin-211 enhances TEC/thymocyte adhesion and thymocyte release from thymic nurse cells, as well as the reconstitution of these complexes. Conversely, these interactions are blocked by monoclonal antibodies specific for laminin-211 and the laminin receptor VLA-6. Our results reinforce the notion that distinct laminin isoforms in the human thymus are relevant for lymphoepithelial interactions.

It is largely established that molecules first discovered in the nervous system are also found in the immune system. Neuropilin-1 (NP-1) was initially identified to mediate semaphorin-induced chemorepulsion during brain development and is also involved in peripheral T cell/dendritic cell interactions. Herein, we studied NP-1 during T cell development in the human thymus. NP-1 is expressed in both cortex and medulla of thymic lobules, being found in distinct CD4/CD8-defined thymocyte subsets. NP-1 is also found in thymic epithelial cells (TEC) in situ and in vitro , and is recruited at the site of TEC–thymocyte contact. Moreover, NP-1 was rapidly up-regulated during thymocyte stimulation by T cell receptor (TCR) and IL-7 or after adhesion to TEC. Semaphorin-3A (Sema-3A), a natural ligand of NP-1, is also present in human thymus, both in TEC and thymocytes, being up-regulated in thymocytes after TCR engagement. Functionally, Sema-3A decreases the adhesion capacity of NP-1 + thymocytes and induces their migration by a repulsive effect. In conclusion, we show here that NP-1/Sema-3A-mediated interactions participate in the control of human thymocyte development.

Pathophysiology of Chagas' disease is not completely defined, although innate and adaptative immune responses are crucial. In acute infection some parasite antigens can activate macrophages, and this may result in pro-inflammatory cytokine production, nitric oxide synthesis, and consequent control of parasitemia and mortality. Cell-mediated immunity in Trypanosoma cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Finally, leukocyte influx towards target tissues is regulated by cytokines, chemokines, and extracellular matrix components which may represent potential therapeutic targets in infected patients. Here we will discuss recent findings on the role of cytokines, chemokines and extracellular matrix components in the regulation of innate and adaptive immunity during T. cruzi infection.

International audience; Diabetes mellitus is a chronic disease that affects over 382 million people worldwide. Type-1 Diabetes (T1D) is classified as an autoimmune disease that results from pancreatic beta-cell destruction and insulin deficiency. Type-2 Diabetes (T2D) is characterized principally by insulin resistance in target tissues followed by decreased insulin production due to beta-cell failure. It is challenging to identify immunological markers such as inflammatory molecules that are triggered in response to changes during the pathogenesis of diabetes. APRIL is an important member of the TNF family and has been linked to chronic inflammatory processes of various diseases since its discovery in 1998. Therefore, this study aimed to evaluate APRIL serum levels in T1D and T2D. For this, we used the ELISA assay to measure serum APRIL levels of 33 T1D and 30 T2D patients, and non-diabetic subjects as control group. Our data showed a decrease in serum APRIL levels in T1D patients when compared with healthy individuals. The same pattern was observed in the group of T2D patients when compared with the control. The decrease of serum APRIL levels in diabetic patients suggests that this cytokine has a role in T1D and T2D. Diabetes is already considered as an inflammatory condition with different cytokines being implicated in its physiopathology. Our data suggest that APRIL can be considered as a potential modulating cytokine in the inflammatory process of diabetes.

We previously showed intrathymic alterations in non-obese diabetic (NOD) mice, including the appearance of giant perivascular spaces, filled with mature thymocytes, intermingled with an extracellular matrix network. This raised the hypothesis of a defect in thymocyte migration with partial arrest of exiting thymocytes in the perivascular spaces. Herein, we investigated the expression of receptors for fibronectin [very late antigen (VLA)-4 and VLA-5] and laminin (VLA-6), known to play a role in thymocyte migration. When compared with two normal and one other autoimmune mouse strains, a decrease of VLA-5 expression in NOD thymocytes was noticed, being firstly observed in late CD4/CD8 double-negative cells, and more pronounced in mature CD4(+) and CD8(+) thymocytes. Functionally, thymocyte exit from the lymphoepithelial complexes, the thymic nurse cells, was reduced. Moreover, NOD thymocyte adhesion to thymic epithelial cells as well as to fibronectin was diminished, and so was the migration of NOD thymocytes through fibronectin-containing transwell chambers. In situ, intra-perivascular space thymocytes were VLA-5-negative, suggesting a correlation between the thymocyte arrest within these structures and loss of VLA-5 expression. Overall, our data reveal impairment in NOD thymocyte migration, and correspond to the first demonstration of a functional fibronectin receptor defect in the immune system.

We previously showed that, in the context of thymic epithelial cells, thymocyte migration is partially controlled by extracellular matrix (ECM)-mediated interactions. Herein we evaluated whether these interactions could be involved in cell migration related events in the context of non-epithelial cells of the thymic microenvironment, the phagocytic cells of the thymic reticulum (PTR). We first showed, by immunocytochemistry, cytofluorometry, and RT-PCR, that PTR produce ECM components, including fibronectin and laminin, and express the corresponding integrin-type receptors, VLA-4, VLA-5, and VLA-6. Thymocytes adhere onto PTR monolayers, with immature CD4(+)CD8(+) cells being predominant. Importantly, such an adhesion is partially mediated by ECM ligands and receptors, since it was impaired by anti-ECM or anti-ECM receptor antibodies. Conjointly, our data reveal that the ECM-dependence for thymocyte adhesion onto the thymic microenvironment is not restricted to the epithelial cells, being also seen when they encounter non-epithelial phagocytic cells.

Cell migration is crucial for thymocyte differentiation, and the cellular interactions involved now begin to be unraveled, with chemokines, extracellular matrix (ECM) proteins, and their corresponding receptors being relevant in such oriented movement of thymocytes. This notion derives from in vitro, ex vivo, and in vivo experimental data, including those obtained in genetically engineered and spontaneous mutant mice. Thymic microenvironmental cells produce both groups of molecules, whereas developing thymocytes express chemokine and ECM receptors. It is important that although chemokines and ECM proteins can drive thymocyte migration per se, a combined role of these molecules likely concurs for the resulting migration patterns of thymocytes in their various differentiation stages. In this respect, among ECM moieties, there are proteins with opposing functions, such as laminin or fibronectin versus galectin-3, which promote, respectively, adhesion and de-adhesion of thymocytes to the thymic microenvironment. How chemokines and ECM are produced and degraded remains to be more clearly defined. Nevertheless, matrix metalloproteinases (MMPs) likely play a role in the intrathymic ECM breakdown. It is interesting that these molecules also degrade chemokines. Thus, the physiological migration of thymocytes should be conceived as a resulting vector of multiple, simultaneous, or sequential stimuli, involving chemokines, adhesive, and de-adhesive ECM proteins. Moreover, these interactions may be physiologically regulated in situ by matrix MMPs and are influenced by hormones. Accordingly, one can predict that pathological changes in any of these loops may result in abnormal thymocyte migration. This actually occurs in the murine infection by the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. In this model, the abnormal release of immature thymocytes to peripheral lymphoid organs is correlated with the higher migratory response to ECM and chemokines. Lastly, the fine dissection of the mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells. The most important function of the thymus is to generate T lymphocytes, which once leaving the organ, are able to colonize specific regions of peripheral lymphoid organs, the T cell zones, where they can mount and regulate cell-mediated, immune responses. This intrathymic T cell differentiation is a complex sequence of biological events, comprising cell proliferation, differential membrane protein expression, gene rearrangements, massive programmed cell death, and cell migration. In this review, we will focus on the mechanisms involved in controlling the migration of thymocytes, from the entrance of cell precursors into the organ to the exit of mature T cells toward peripheral lymphoid organs. Nevertheless, to better comprehend this issue, it appeared worthwhile to briefly comment on some key aspects of thymocyte differentiation and the tissue context in which it takes place, the thymic microenvironment.

We characterized herein the microarchitecture of the equine thymus along with post-natal development (6 months→18 years). Thymuses showed an involutional process, beginning before the puberty and defined by five histological grades, which consider the progressive cortical thymocyte depletion, shrinkage and rearrangement of the epithelial network and increase in extracellular matrix (ECM). A second feature of the equine thymus was the presence of eosinopoiesis, erythropoiesis, mastocytopoiesis and plasmacytogenesis. Additionally, lymphatic vessels, full of lymphocytes, were particularly prominent. Distribution of ECM proteins was heterogeneous, being denser in the medulla, as well as basement membranes of capsule, septa and perivascular spaces, thus similar to the patterns seen in other mammals. In vitro, horse thymic nurse cells produce ECM proteins, which are relevant in thymocyte/epithelial cell interactions. In conclusion, the equine thymus presents morphological and involutional characteristics similar to other mammals, exhibiting particular features, as prominent non-lymphoid hematopoiesis and lymphatic vessels.

Cell migration is a crucial event in the general process of thymocyte differentiation. The cellular interactions involved in the control of this migration are beginning to be defined. At least chemokines and extracellular matrix proteins appear to be part of the game. Cells of the thymic microenvironment produce these two groups of molecules, whereas developing thymocytes express the corresponding receptors. Moreover, although chemokines and extracellular matrix can drive thymocyte migration per se, a combined role for these molecules appears to contribute to the resulting migration patterns of thymocytes in their various stages of differentiation. The dynamics of chemokine and extracellular matrix production and degradation is not yet well understood. However, matrix metalloproteinases are likely to play a role in the breakdown of intrathymic extracellular matrix contents. Thus, the physiological migration of thymocytes should be envisioned as a resulting vector of multiple, simultaneous and/or sequential stimuli involving chemokines, adhesive and de-adhesive extracellular matrix proteins, as well as matrix metalloproteinases. Accordingly, it is conceivable that any pathological change in any of these loops may result in the alteration of normal thymocyte migration. This seems to be the case in murine infection by the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease. A better knowledge of the physiological mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells.

Objectives: Thyroid hormones exert immunomodulatory activities and the thymus is one of their target organs. We previously showed that triiodothyronine (T3) modulates thymic hormone production and extracellular matrix (ECM) expression by mouse thymic epithelial cells (TEC). This concept is enlarged herein by studying the effects of T3 in human TEC preparations including primary cultures derived from thymic nurse cell complexes, as well as human and murine TEC lines. Methods and Results: We observed that in all cases, ECM ligands and receptors (such as fibronectin, laminin, VLA-5 and VLA-6) are enhanced in vitro, as ascertained by immunocytochemistry, ELISA and cytofluorometry. Moreover, thymocyte adhesion to these TEC preparations is augmented by T3. Interestingly, TEC-thymocyte adhesion is also upregulated when thymocytes from T3-treated mice adhere to untreated TEC cultures. Such an enhancing effect of T3 upon TEC-thymocyte interactions is likely due to the increase in the expression of ECM ligands and receptors, since it is prevented when T3-treated TEC cultures are incubated with anti-ECM antibodies prior to the adhesion assay. We then tested whether T3 could modulate interactions between thymocytes and nonepithelial microenvironmental cells, exemplified herein by the phagocytic cells of the mouse thymic reticulum. In fact, in vitro treatment of these cells with T3 increases ECM ligands and receptors and augments their ability to adhere to thymocytes. Lastly, using immunochemistry-based assays, we showed the presence of the nuclear T3 receptor in all thymic microenvironmental cell preparations. Conclusion: Our data show that T3 upregulates ECM-mediated heterocellular interactions of thymocytes with distinct thymic microenvironmental cells, in both humans and mice.

Interactions between thymocytes and thymic epithelial cell (TEC) can be modulated by growth hormone via insulin-like growth factor-1 (IGF-1). In this study, we showed IGF-1 and IGF-1 receptor mRNA expression by human and murine TEC and thymocytes. Functionally, IGF-1 stimulates extracellular matrix production by human TEC. Moreover, pretreatment of murine TEC with IGF-1 increases their adhesion to thymocytes. Interestingly, we observed an increase in the frequency of CD4–CD8–CD90+ T cells which adhered to pretreated TEC, supporting the concept that IGF-1 may also act indirectly on intrathymic T cell differentiation and migration through the thymic epithelium.

GH3B6 cells, a rat pituitary tumor cell line, synthesize and secrete large amounts of prolactin (PRL) in vitro. In the present work, we evaluated the capacity of these cells to express extracellular matrix (ECM) components and receptors in vitro. The expression of laminin (LN), fibronectin (FN) and type IV collagen (CIV) was investigated by immunofluorescence assays. In comparison to PRL distribution, where around 50-70% of the cells contained PRL concentrated in the Golgi region, a variable immunolabeling for the three ECM components could be observed in the majority of GH3B6 cells. Importantly, this pattern was not modified when cells were cultured in the presence of 30 nM thyroliberin (TRH). The expression of the ECM receptors: alpha5beta1 (FN receptor), alpha6beta1 (LN receptor) and CD44 (hyaluronic acid receptor) could be demonstrated by cytofluorometric analysis. Using biochemical procedures, we analyzed the synthesis and secretion of glycosaminoglycans (GAGs). The cells synthesized and secreted mainly heparan sulfate (75%) with a minor amount of chondroitin sulfate/dermatan sulfate. In an attempt to evaluate the individual contribution of the ECM components to influence cell morphology and PRL distribution in vitro, GH3B6 cells were cultivated separately on LN, FN and CIV substrates. Under all conditions, it was possible to observe an increase of cell adherence to the substrate, accompanied with changes of cellular morphology, characterized by the appearance of cytoplasmatic processes. However, no changes on PRL distribution could be observed. Our results suggest that endocrine tumor cell lines are involved in synthesis of ECM components and receptors.

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus. J. Cell. Physiol. 185:68–79, 2000. © 2000 Wiley-Liss, Inc.

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S-Na2SO4 and 3H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S-sulfate/3H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions. J Cell Physiol 178:51–62, 1999. © 1999 Wiley-Liss, Inc.

Thymocytes undergo a complex process of differentiation, largely dependent on interactions with the thymic microenvironment, a tridimensional cellular network formed by epithelial cells, macrophages, dendritic cells, and fibroblasts. One key cellular interaction involves the TCR-CD3 complex expressed by thymocytes with MHC-peptide complexes present on microenvironmental cells. Additionally, thymic epithelial cells (TEC) interact with thymocytes via soluble polypeptides such as thymic hormones and interleukins, as well as through extracellular matrix (ECM) ligands and receptors. Such types of heterotypic interactions are under neuroendocrine control. For example, thymic endocrine function, represented by thymulin production, is up-regulated, both in vivo and in vitro, by thyroid and pituitary hormones, including prolactin and growth hormone. We also showed that these peptides enhance the expression of ECM ligands and receptors, as well as the degree of TEC-thymocyte adhesion. In addition, we studied the thymic nurse cell complex, used herein as an in vitro model for ECM-mediated intrathymic T-cell migration. We observed that T-cell migration is also hormonally regulated as ascertained by the thymocyte entrance into and exit from these lymphoepithelial complexes. Taken together these data clearly illustrate the concept that neuroendocrine circuits exert a pleiotropic control on thymus physiology. Lastly, the intrathymic production of classic hormones such as prolactin and growth hormone suggests that, in addition to endocrine circuits, paracrine and autocrine interactions mediated by these peptides and their respective receptors may exist in the thymus, thus influencing both lymphoid and microenvironmental compartments of the organ.

The thymus is a primary lymphoid organ in which bone marrow-derived T-cell precursors undergo differentiation, leading to migration of positively selected thymocytes to the T-cell-dependent areas of secondary lymphoid organs. This organ can undergo atrophy, caused by several endogenous and exogenous factors such as ageing, hormone fluctuations, and infectious agents. This paper will focus on emerging data on the thymic atrophy caused by infectious agents. We present data on the dynamics of thymus lymphocytes during acuteTrypanosoma cruziinfection, showing that the resulting thymus atrophy comprises the abnormal release of thymic-derived T cells and may have an impact on host immune response.

It is well demonstrated that the thymus gland is under neuroendocrine control. Thymic endocrine function can be modulated by a variety of hormones including those secreted by the thyroid gland. This prompted us to investigate putative influences of T3 in further aspects of thymus physiology. We showed that T3-treated animals exhibited an increase in thymus weight, cellularity and cycling cells. Moreover, Thy1+ thymocytes as well as CD4-CD8 defined subsets were augmented in absolute numbers, whereas PgP.1+ cells increased in both absolute and percentage values. In parallel, the total numbers of thymic nurse cells were also increased. Regarding the expression of extracellular matrix components (ECM) by microenvironmental cells, we observed an enhancement in the intrathymic ECM upon T3 in vivo treatment. Similar effects were found in vitro by treating a thymic epithelial cell line or thymic nurse cell-derived epithelial cultures with T3. This treatment also increased the expression of ECM receptors by thymic epithelial cultures. Interestingly, an enhancement in thymocyte/thymic epithelial cell adhesion ratio was observed after T3 treatment of epithelial cells. Our data suggest that T3 exerts a pleiotropic effect upon thymus physiology, stimulating thymocyte differentiation, not only by modulating epithelial cell hormonal secretion but also their production of ECM proteins and respective receptors.

We describe herein the expression of the VLA6 complex by murine thymic epithelial cells (TEC). The immunohistochemical distribution revealed that VLA6 is found in both thymic medullary and subcapsullary areas. Moreover, studies by immunoelectron microscopy revealed a membrane labeling of the VLA6 molecule, including at desmosomal sites. By means of immunoblotting, immunoprecipitation, and affinity chromatography of extracts from a mouse TEC line, we further demonstrated that VLA6 is a laminin (LN) receptor in these cells. In keeping with this finding, we showed that TEC adhesion, spreading, and proliferation were enhanced in vitro by LN. The fact that VLA6 is also expressed by the large majority of thymocytes raised the hypothesis that it might be involved in LN-mediated TEC-thymocyte interactions. Interestingly, in vitro experiments showed that there is an increase in the TEC-thymocyte adhesion upon glucocorticoid hormone treatment, a situation in which the expression of VLA6 as well as LN is enhanced. Most importantly, this adhesion can be reversed by pre-treating TEC with an anti-alpha 6 integrin mAb. Additionally, spontaneous in vitro thymocyte release by thymic nurse cell complexes was enhanced by LN and partially blocked by anti-alpha 6 or anti-beta 1 antibodies. Our results suggest that VLA6 is involved in LN-mediated TEC-thymocyte interactions that can be relevant for thymic microenvironmental cell physiology and intrathymic T cell differentiation events.

Little is known about the immunologic consequences from endocrine changes observed in diabetes. Since a preserved thymic microenvironment is of critical importance for the T cell development and maturation, we have examined the thymus from alloxan-diabetic mice. An intense thymic atrophy accompanied by changes in histological pattern and in thymocyte subpopulations were observed in diabetic mice. Laminin and fibronectin, which are closely associated with thymocytes maturation, were evaluated, but, only laminin presented an altered distribution and density in thymuses from diabetes group. the expression of fibronectin and laminin receptors was found to be decreased in diabetic mice. There was also intense decrease in the expression of two important chemokines for thymus, CCL25 and CXCL12, and in the CCR9 (CCL25 receptor), but the expression of CXCR4 (CXCL12 receptor) did not drop on cells. However, no significant difference was observed in the in vitro thymocytes migratory capacity from diabetic mice. The results show significant alterations in thymus microenvironment in diabetes and offer insights for studies involving endocrine influences on lymphatic organs and T cell maturation.

Following infection, lymphocytes expand exponentially and differentiate into effector cells to control infection and coordinate the multiple effector arms of the immune response. Soon after this expansion, the majority of antigen-specific lymphocytes die, thus keeping homeostasis, and a small pool of memory cells develops, providing long-term immunity to subsequent reinfection. The extent of infection and rate of pathogen clearance are thought to determine both the magnitude of cell expansion and the homeostatic contraction to a stable number of memory cells. This straight correlation between the kinetics of T cell response and the dynamics of lymphoid tissue cell numbers is a constant feature in acute infections yielded by pathogens that are cleared during the course of response. However, the regional dynamics of the immune response mounted against pathogens that are able to establish a persistent infection remain poorly understood. Herein we discuss the differential lymphocyte dynamics in distinct central and peripheral lymphoid organs following acute infection by Trypanosoma cruzi, the causative agent of Chagas disease. While the thymus and mesenteric lymph nodes undergo a severe atrophy with massive lymphocyte depletion, the spleen and subcutaneous lymph nodes expand due to T and B cell activation/proliferation. These events are regulated by cytokines, as well as parasite-derived moieties. In this regard, identifying the molecular mechanisms underlying regional lymphocyte dynamics secondary to T. cruzi infection may hopefully contribute to the design of novel immune intervention strategies to control pathology in this infection.

In the context of the cross-talk between the neuroendocrine and immune systems, it is well known that growth hormone (GH) exerts physiological effects in central as well as peripheral compartments of the immune system. GH modulates a variety of thymic functions. For example, GH upregulates proliferation of thymocytes and thymic epithelial cells. Accordingly, GH-transgenic mice, as well as animals and humans treated with exogenous GH, exhibit an enhanced cellularity in the thymus organ. GH also stimulates the secretion of thymic hormones, cytokines, and chemokines by the thymic microenvironment as well as the production of extracellular matrix proteins. These effects lead to an increase in thymocyte migratory responses and intrathymic traffic of developing T cells, including the export of thymocytes from the thymus organ, as ascertained by experimental studies with intrathymic injection of GH in normal mice and with GH-transgenic animals. Because GH promotes a replenishment of the thymus and an increase of thymocyte export, it has been applied as a potential adjuvant therapeutic agent in the treatment of immunodeficiencies associated with thymic atrophy.

During acute infection with Trypanosoma cruzi, the causative agent of Chagas´ disease, the thymus undergoes intense atrophy followed by a premature escape of CD4+CD8+ immature cortical thymocytes. Here we report a pivotal role for the endogenous lectin galectin-3 in accelerating death of thymocytes and migration of these cells away from the thymus after T. cruzi infection. We observed a pronounced increase in galectin-3 expression that paralleled the extensive depletion of CD4+CD8+ immature thymocytes after infection. In vitro, recombinant galectin-3 induced increased levels of death in cortical immature thymocytes. Consistent with the role of galectin-3 in promoting cell death, thymuses from gal-3-/- mice did not show cortical thymocyte depletion after parasite infection in vivo. In addition, galectin-3 accelerated laminin-driven CD4+CD8+ thymocyte migration in vitro and in vivo induced exportation of CD4+CD8+ cells from the thymus to the peripheral compartment. Our findings provide evidence of a novel role for galectin-3 in the regulation of thymus physiology and identify a potential mechanism based on protein-glycan interactions in thymic atrophy associated with acute T. cruzi infection Fil: Silva Monteiro, Elizangela. Instituto Oswaldo Cruz; Brasil Fil: Reis Lorenzato, Luciana. Instituto Oswaldo Cruz; Brasil Fil: Kenji Nihei, Oscar. Instituto Oswaldo Cruz; Brasil Fil: Junqueira, Mara. Universidade de Sao Paulo; Brasil Fil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Kaiyuan Hsu, Daniel. University of California; Estados Unidos Fil: Liu, Fu Tong. University of California; Estados Unidos Fil: Savino, Wilson. Instituto Oswaldo Cruz; Brasil Fil: Chammas, Roger. Universidade de Sao Paulo; Brasil Fil: Serra Villaverde, Dea Maria. Instituto Oswaldo Cruz; Brasil

Previous evidence indicates that GH modulates thymic cell migration. In this study we approached this issue in vivo, studying thymocyte migration in GH transgenic animals and in normal mice treated intrathymically with GH. Extracellular matrix and chemokines are involved in thymocyte migration. In this respect, thymocyte adhesion to laminin was higher in GH-treated animals than controls, and the numbers of migrating cells in laminin-coated Transwells was higher in GH-transgenic and GH-injected mice. Additionally, CXC chemokine ligand 12 (CXCL12)-driven migration was higher in GH-Tg and GH-treated animals compared with controls. Interestingly, although CXCR4 expression on thymocytes did not change in GH-Tg mice, the CXCL12 intrathymic contents were higher. We found that CXCL12, in conjunction with laminin, would additionally enhance the migration of thymocytes previously exposed to high concentrations of GH in vivo. Lastly, there was an augmentation of recent thymic emigrants in lymph nodes from GH-Tg and GH-injected animals. In conclusion, enhanced thymocyte migration in GH transgenic mice as well as GH-injected mice results at least partially from a combined action of laminin and CXCL12. Considering that GH is presently being used as an adjuvant therapeutic agent in immunodeficiencies, including AIDS, the concepts defined herein provide important background knowledge for future GH-based immune interventions.

Several T cell abnormalities have been described in the course of acute Trypanosoma cruzi infection in mice, including severe effects on the thymus. In the present study, looking at the expression of extracellular matrix ligands in the thymus, we observed that deposits of fibronectin and laminin increased progressively during the course of infection, reaching a maximum at the peak of parasitemia and thymic atrophy. Concomitantly, membrane expression of fibronectin and laminin receptors (VLA-4, VLA-5 and VLA-6) was also enhanced on thymocyte subsets of infected mice. These results correlated with changes in intrathymic thymocyte migration ability during the acute phase of infection, when a higher fibronectin-dependent transmigratory activity of CD4(+)CD8(+) thymocytes was observed. Strikingly, we detected higher frequency of immature and high VLA-expressing CD4(+)CD8(+) T cells in the peripheral lymphoid organs of infected mice at the peak of parasitemia. These cells seemed to be thymus dependent, since significantly lower amounts of them were found in thymectomized mice, and some of them carry "prohibited" Vbeta segments of the TCR. Our data suggest an imbalance in the intrathymic cell trafficking following acute T. cruzi infection, likely due to dysregulated extracellular matrix-dependent interactions.

The process of thymocyte differentiation occurs within the context of the thymic microenvironment, in which T cell precursors interact with thymic microenvironmental cells and extracellular matrix. Here we studied the expression of galectin-3, a beta-galactoside binding lectin, in the thymus of young adult mice. Galectin-3 was found mainly in the medulla and to a lesser extent in the cortex. We further showed that distinct microenvironmental elements, such as thymic epithelial cells, the epithelial component of thymic nurse complexes and phagocytic cells of the thymic reticulum produce, secrete and accumulate galectin-3 on the cell surface. Functionally, galectin-3-enriched medium inhibited in vitro thymocyte interactions with thymic microenvironmental cells, accelerated the release of thymocytes from thymic nurse cells and inhibited the reconstitution of these lymphoepithelial complexes. These effects were blocked by exogenous lactose (Galbeta1-4Glc), but not melibiose (Galalpha1-6Glc), and by a monospecific anti-galectin-3 antibody. Recombinant galectin-3 also inhibited thymocyte/thymic epithelial cell interactions. Our data indicate that intrathymically produced galectin-3 disrupts thymocyte/microenvironmental cell interactions, thus acting as a de-adhesion molecule.

The thymic microenvironment plays a key role in the intrathymic T-cell differentiation. It is composed of a tridimensional network of epithelial cells whose physiology is controlled by extrinsic circuits such as neuroendocrine axes. Herein we show that the expression of extracellular matrix ligands and receptor by cultured thymic epithelial cells is upregulated by prolactin (PRL) and growth hormone (GH), the latter apparently occurring via insulin-like growth factor I (IGF-I). Thymocyte release from the lymphoepithelial complexes, thymic nurse cells, as well as the reconstitution of these complexes are enhanced by PRL, GH or IGF-I. Treatment of a mouse thymic epithelial cell line with these hormones induced an increase in thymocyte adhesion, an effect significantly prevented in the presence of antibodies to fibronectin, laminin or respective receptors VLA-5 and VLA-6. Our data suggest that the in vitro changes in thymocyte/thymic epithelial cell interactions induced by pituitary hormones are partially mediated by the enhancement of extracellular matrix ligands and receptors.

Acute Trypanosoma cruzi infection induces alterations in both lymphoid and microenvironmental compartments of the thymus. This prompted us to investigate whether the lymphoepithelial complex thymic nurse cell (TNC) was comprised in the thymic pathology occurring in experimental Chagas' disease. The isolation of TNCs from acutely T. cruzi-infected mice revealed a reduction in TNC numbers that paralleled thymic atrophy. This decrease does not seem to be stress-related since it was not seen following glucocorticoid hormone injection. Moreover, an increased intra-TNC cell death in complexes from infected animals was noticed. In addition, acute T. cruzi infection induced a decrease in size and granularity of TNC complexes, as well as several ultrastructural alterations indicating cell damage. The epithelial component of TNCs, independent of being infected in vitro or derived from infected animals, showed an enhancement of extracellular matrix proteins that is likely related to the enhanced thymocyte release observed in these complexes. Conjointly, these data show that TNCs are importantly affected in acute experimental T. cruzi infection, possibly contributing to the previously observed alterations in thymocyte differentiation.

Extracellular matrix (ECM) proteins influence cell migration and differentiation in a variety of cell systems. Within the thymus, the ECM distribution pattern is conserved among various mammalian species, but its physiological role is not completely understood. Interferon-gamma (IFN-gamma), a cytokine produced by thymocytes, is able to in vitro modulate ECM production by thymic epithelial cells (TEC) in a dose-dependent biphasic pattern. In the same model, we determined herein that the expression of VLA-5 and VLA-6 (fibronectin and laminin receptors, respectively) were upregulated by low doses of IFN-gamma, whereas high doses of the cytokine induced an opposite effect. In a second in vitro system, we evidenced that thymocyte adhesion to a TEC line was also modulated by IFN-gamma. Importantly, such effects were due to the biphasic modulation of ECM ligands and receptors since they could be specifically prevented by preincubating the TEC cultures with anti-ECM or anti-ECM receptor antibodies. Additionally, spontaneous thymocyte release from thymic nurse cells was similarly biphasically modulated by IFN-gamma. Our data provide support for the notion that thymocyte-derived products can play a role in the dialogue that exists in the thymus, involving differentiating thymocytes and microenvironmental cells. Moreover, we bring arguments indicating that one of the implicated mechanisms involved is the modulation of ECM ligands and receptors by the thymic epithelium.

Extracellular matrix (ECM) proteins can influence cell migration and differentiation in a variety of cell systems. Within the thymus, these molecules are heterogeneously distributed, and their physiological role is poorly understood. This prompted us to carry out in vitro studies using the thymic nurse cell (TNC) model. We observed that fibronectin and laminin accelerate spontaneous in vitro release of thymocytes from TNC, whereas anti-ECM antibodies exhibited a blocking effect. Similar results were obtained with anti-ECM receptor reagents. Moreover, these antibodies abrogated in vitro reconstitution of TNC complexes and thymocyte adhesion to TNC-derived epithelial cultures. Our results indicate that lymphocyte traffic in TNC (comprising both entrance into and exit from the epithelial structure) is affected by interactions involving extracellular matrix ligands and receptors. In this respect, the dynamic analysis of thymic nurse cell complexes should be regarded as a relevant in vitro tool for functional studies of distinct adhesion molecules in intrathymic lymphocyte traffic.

Intrathymic T-cell migration and differentiation is not completely understood. Here, Wilson Savino and colleagues argue that certain interactions between differentiating thymocytes and thymic epithelial cells are mediated by extracellular matrix proteins and that these interactions influence intrathymic migration events and thymocyte differentiation.

The thymic stroma plays a critical role in the generation of T lymphocytes by direct cell-to-cell contacts as well as by secreting growth factors or hormones. The thymic epithelial cells, responsible for thymic hormone secretion, include morphologically and antigenically distinct subpopulations that may exert different roles in thymocyte maturation. The recent development of thymic epithelial cell lines provided an interesting model for studying thymic epithelial influences on T cell differentiation. Treating mouse thymocytes by supernatants from one of TEC line (IT-76M1), we observed an induction of thymocyte proliferation and an increase in the percentages of CD4-/CD8- thymocytes. This proliferation was largely inhibited when thymocytes were incubated with IT-76M1 supernatants together with an anti-thymulin monoclonal antibody, but could be enhanced by pretreating growing epithelial cells by triiodothyronine. We suggest that among the target cells for thymulin within the thymus, some putative precursors of early phenotype might be included.

Os microRNAs são pequenos RNAs, não-codificantes que funcionam como reguladores a nível pós-transcricional da expressão gênica. Nos últimos anos, novas evidências demonstram o papel importante dos microRNAs na regulação e desenvolvimento do sistema imune. Apesar da função de poucos microRNAs ser conhecida, a sua expressão alterada vêm sendo associada a patogênese de diversas doenças autoimunes, incluindo a artrite reumatóide (AR). Recentemente a expressão desregulada de uma série de microRNAs está sendo descrita em pacientes com AR, e o papel patogênico de apenas uma parte deles foi investigada em modelos animais. A artrite reumatóide é uma doença autoimune sistêmica caracterizada por um intenso processo inflamatório na sinóvia, podendo causar destruição óssea e articular. Os linfócitos T apresentam papel importante na indução, manutenção e progressão da doença. A artrite induzida por colágeno é um modelo animal amplamente utilizado por suas características fisiopatológicas muito similares à doença em humanos. A linhagem de camundongos DBA-1/J desenvolve a doença após imunização e booster com colágeno do tipo II, enquanto que a linhagem DBA-2/J se mostra refratária. Isso confere um sistema modelo de susceptibilidade/resistência à artrite, que pode ser estudado em diferentes abordagens. O objetivo do nosso estudo foi identificar o perfil transcricional e as redes de interação entre um grupo de microRNAs e seus respectivos alvos nos timócitos e linfócitos T CD3+ periféricos nos camundongos da linhagem DBA-1/J e DBA-2/J. Para a avaliação da expressão gênica, utilizou-se a tecnologia de microarrays. O uso de programas de análise e para a construção das redes foi imprescindível. Os resultados encontrados evidenciam uma expressão diferenciada de mRNAs e microRNAs em timócitos e linfócitos T CD3+ periféricos entre as duas linhagens utilizadas. Novos microRNAs foram encontrados nos diferentes estágios de desenvolvimento do linfócito T. Nas redes de interação microRNA-RNAm obtidas, genes importantes associados aos processos de sistema imune, adesão e diferenciação celular, apoptose, recombinação, ativação de linfócitos T e resposta inflamatória, foram encontrados como potenciais alvos. Além disso, em uma perspectiva clínica, baseados nos resultados obtidos em camundongo, nos encontramos a expressão do miR-505 nos linfócitos T de pacientes com AR. Nossos resultados contribuem para a melhor compreensão dos mecanismos molecular envolvidos na resistência/susceptibilidade a CIA. MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate the expression of multiple protein-encoding genes at the post-transcriptional level. During the last several years, evidence has emerged to show their critical role for the regulation and development of immune system. Although the function of most mammalian miRNAs has yet to be determined, their aberrant expression has been associated with several autoimmune conditions, including rheumatoid arthritis (RA). Recently, the deregulated expression of a dozen miRNAs has been reported in patients with RA, and the pathogenic role of only a few of these has been investigated in experimental mouse models. RA is a systemic autoimmune disorder mainly characterized by the inflammation of synovial tissue that can lead to destruction of bone and cartilage. The role of effectors T cells in induction, maintenance and progression of the disease is now becoming better understood. Collagen-induced arthritis is an animal model widely studied due to its similarities to human disease. The DBA-1/J mouse strain develops arthritis after immunization process and booster with Type II collagen, and the DBA-2/J strain is refractory to the disease induction. This offers an useful susceptibility/resistance model-system to study RA. The aim of this study was to identify the expression profiles and interaction networks between a set of microRNAs and their mRNA targets in thymocytes and peripheral CD3+ T lymphocytes in DBA-1/J and DBA-2/J mice strain. For this purpose we used the microarray technology to evaluate the expression of the miRNAs and mRNAs as possible targets involved in this process. The use of bioinformatics software to reconstruct the networks was essential. The results show differential expression of mRNAs and miRNAs in thymocytes and peripheral CD3+ T lymphocytes between both strains. New miRNAs were found during all the stages of T cells development. The microRNA-mRNA interaction networks obtained in this study showed that important genes related to apoptose, immune system, recombination, cell adhesion and differentiation, inflammatory process and T cell activation were found as potential targets. In addition, in a clinical prospects, based on the results obtained in mice, we found the expression of the miRNA miR-505 in T cells of RA patients. Our results contribute to a better understand of the molecular mechanisms evolved in the resistance/susceptibility to CIA.

A tolerância imunológica é a propriedade essencial do sistema imune que controla as reações patológicas contra antígenos do próprio. O timo é visto como o principal órgão envolvido com a indução de tolerância aos antígenos próprios que são expressos pelas células tímicas (tolerância central), enquanto que a indução de tolerância aos antígenos relacionados a outros tecidos (TRAs) tem sido atribuída aos mecanismos de tolerância extratímica (tolerância periférica). Entretanto, a evidência da expressão de TRAs de órgãos e tecidos parenquimais no timo pelas células medulares epiteliais (mTECs) de camundongos e de humanos a qual foi referida como expressão gênica promíscua (PGE) reforçou a concepção de tolerância central de TRAs. O controle molecular dessa expressão tem sido atribuído ao gene Aire (Auto immune regulator) que é um regulador de transcrição. No presente estudo, procurou-se retratar a expressão gênica promíscua no timo de camundongos NOD (Non Obese Diabetic) por meio da análise da expressão gênica em grande escala, ou seja, descrevendo seu transcriptoma usando a tecnologia de cDNA microarrays. Para a análise dos dados utilizamos programas de bioinformática dedicados a microarrays bem como dados de bancos para a caracterização da PGE e susceptibilidade genética ao diabetes melitus do tipo 1 (DM-1). Três conjuntos de resultados puderam ser evidenciados. No primeiro conjunto observou-se a ocorrência da PGE de antígenos tecidos/órgãos parenquimatosos (TRAs) em timos recém removidos e em in vitro em cultura ATOC de camundogos NOD pré-autoimunes e autoimunes (diabéticos). O segundo conjunto de resultados consistiu na análise do efeito da inativação do transcrito de gene Aire na expressão gênica do timo de camundongos NOD in vitro em cultura ATOC. Finalmente, no último conjunto de dados, demonstrou-se que certos genes de TRAs com expressão promíscua, se encontram em regiões cromossômicas de susceptibilidade ao DM - 1 (idds). Como três deles (Il-4, Cd4 e Cdk4) são diretamente relacionados com a patogenia do DM-1 em camundongos foi possível estabelecer um paralelo entre PGE e susceptibilidade genética. Immunologic tolerance is an essential property of the immune system, which controls immune reactions directed against the body self components. The thymus is seen as the main organ involved with the tolerance induction to self antigens, which are expressed by the thymic cells (central tolerance), while the tolerance induction to the diverse other peripheral tissues and organs is attributed to extra thymic mechanisms (peripheral tolerance). Nevertheless, the evidence for the expression of peripheral tissue related antigens (TRAs) in the thymus by the medullary thymic epithelial cells (mTECs) of mice and humans, which have been termed to as promiscuous gene expression (PGE), has contributed to the concept of central tolerance to TRAs. The molecular control of such gene expression has been attributed to the Aire (autoimmune regulator) gene, which plays a role as a transcriptional regulator. In the present study, we searched to picture PGE in the thymus of NOD (non obese diabetic) mice by means of high throughput gene expression, analyzing the transcriptome by the cDNA microarray method. To analyzing data we used bioinformatics programs dedicated to microarrays and specialized data banks to characterize PGE and genetic susceptibility to type 1 diabetes mellitus (DM-1). Studying pre and autoimmune NOD mice, we evidentiate three sets of results. In the first set, it was observed the occurrence of PGE of parenchymal tissue/organs antigens (TRAs) in fresh thymuses and in thymuses cultured in vitro in adult thymus organ cultures (ATOC). The second set of results consisted in the analysis of the effect of in vitro (ATOC) Aire gene silencing on PGE. Finally, in the third data set, we demonstrated that certain promiscuously expressed genes are positioned in DM-1 genetic susceptibility chromosomal regions (idds). As three of such genes (IL4, Cd4 and Cdk4) are directly associated to the DM-1 pathogenesis in mice, it was possible to establishing a parallel between PGE in the thymus and genetic susceptibility to this autoimmune disease.