Gerardo A. Mirkin

Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.

In recent years it has become clear that the therapeutic properties of bone marrow-derived mesenchymal stromal cells (MSC) are related not only to their ability to differentiate into different lineages but also to their capacity to suppress the immune response. We here studied the influence of MSC on macrophage function. Using mouse thioglycolate-elicited peritoneal macrophages (M) stimulated with LPS, we found that MSC markedly suppressed the production of the inflammatory cytokines TNF-α, IL-6, IL-12p70 and interferon-γ while increased the production of IL-10 and IL-12p40. Similar results were observed using supernatants from MSC suggesting that factor(s) constitutively released by MSC are involved. Supporting a role for PGE2 we observed that acetylsalicylic acid impaired the ability of MSC to inhibit the production of inflammatory cytokines and to stimulate the production of IL-10 by LPS-stimulated M. Moreover, we found that MSC constitutively produce PGE2 at levels able to inhibit the production of TNF-α and IL-6 by activated M. MSC also inhibited the up-regulation of CD86 and MHC class II in LPS-stimulated M impairing their ability to activate antigen-specific T CD4+ cells. On the other hand, they stimulated the uptake of apoptotic thymocytes by M. Of note, MSC turned M into cells highly susceptible to infection with the parasite Trypanosoma cruzi increasing more than 5-fold the rate of M infection. Using a model of inflammation triggered by s.c. implantation of glass cylinders, we found that MSC stimulated the recruitment of macrophages which showed a low expression of CD86 and the MHC class II molecule Iab and a high ability to produce IL-10 and IL-12p40, but not IL-12 p70. In summary, our results suggest that MSC switch M into a regulatory profile characterized by a low ability to produce inflammatory cytokines, a high ability to phagocyte apoptotic cells, and a marked increase in their susceptibility to infection by intracellular pathogens.

IL-10 inhibits the NF-κB and ERK/MAPK-mediated production of pro-inflammatory mediators by up-regulation of SOCS-3 in Trypanosoma cruzi-infected cardiomyocytes.

Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease.

Redirection of the Immune Response to the Functional Catalytic Domain of the Cystein Proteinase Cruzipain Improves Protective Immunity against Trypanosoma cruzi Infection

Despite the strong immune responses elicited after natural infection with Trypanosoma cruzi or vaccination against it, parasite survival suggests that these responses are insufficient or inherently inadequate. T. cruzi contains a major cystein proteinase, cruzipain, which has a catalytic N-terminal domain and a C-terminal extension. Immunizations that employed recombinant cruzipain or its N- and C-terminal domains allowed evaluation of the ability of cruzipain to circumvent responses against the catalytic domain. This phenomenon is not a property of the parasite but of cruzipain itself, because recombinant cruzipain triggers a response similar to that of cruzipain during natural or experimental infection. Cruzipain is not the only antigen with a highly immunogenic region of unknown function that somehow protects an essential domain for parasite survival. However, our studies show that this can be reverted by using the N-terminal domain as a tailored immunogen able to redirect host responses to provide enhanced protection.

Different Trypanosoma cruzi strains promote neuromyopathic damage mediated by distinct T lymphocyte subsets.

The proliferative response of CD4 and CD8 T lymphocytes obtained from C3H/HeN mice chronically infected with Trypanosoma cruzi strains that differ in virulence, tropism and immunogenicity, was assayed against skeletal muscle, sciatic nerve and spinal cord homogenates. Although both CD4 and CD8 T lymphocytes from mice infected with the RA strain strongly proliferated against the nervous system, no response against skeletal muscle antigens was detected. CD4 and CD8 T lymphocytes from mice infected with the K‐98 clone (from CA‐I strain) showed low proliferative response against all the antigens assayed. To determine whether the proliferation patterns showed correlation with T cell‐mediated neuromuscular damage, passive cell transfer studies were performed. Fifteen days after transfer of CD4 T cells from RA‐infected donors (CD4‐RA), normal syngeneic recipients displayed exclusively nervous tissue damage, such as perineural, endoneural and/or meningeal inflammatory infiltrates, with predominance of CD4 T cells. Fifteen days after transfer of CD4 T lymphocytes from mice infected with K‐98 (CD4‐K98), recipients showed inflammatory infiltrates only in skeletal muscle, where CD4 T lymphocytes and macrophages were predominant cells. Recipients of CD8 T cells from RA‐infected mice (CD8‐RA) showed lesions in both spinal cord and sciatic nerves. Higher percentages of CD8 T cells were observed in comparison with the recipients of CD4‐RA or CD4‐K98. In contrast, CD8 T cells from K‐98‐infected donors (CD8‐K98) did not induce tissue damage. These results provide evidence that mice infected with T. cruzi populations that differ in their biological characteristics show diverse immune mechanisms that may be involved in the pathogenesis of peripheral nervous system damage.

Chagas' disease: reactivity against homologous tissues induced by different strains of Trypanosoma cruzi.

We have previously reported that the mechanisms of neuromyopathic damage induced by Trypanosoma cruzi are mediated by T cells and are parasite-strain dependent. In the present study our aim was to determine whether the humoral response against muscle and nervous system is also parasite-strain dependent. Of the sera from mice infected with RA and CA-I. T. cruzi strains, 93% reacted against antigens of the nervous system (sciatic nerve, spinal cord and brain). No differences in the ability to recognize heart antigens were found between RA (48%) and CA-I (63%) antisera. Reactivity against skeletal muscle was only relevant in anti-CA-I sera at 270 days post-infection. Each of the antisera assayed in Western blots developed a particular antigenic pattern, but 3 antigens in the nervous system of molecular weight 48, 60 and 70 kDa were detected by 42, 28 and 23% of the sera, respectively. On the other hand, deposits of IgG were observed at the interstitial matrix in sciatic nerve and as endomisial and sarcolemmal patterns in skeletal muscle by IFAT for both RA and CA-I antisera. Absorption of sera with parasite antigens did not abolish the autoreactivity. Our results suggest that major serum autoreactivity from T. cruzi-infected mice is not parasite-strain dependent and does not arise from molecular mimicry.

Role of PPARs in Trypanosoma cruzi Infection: Implications for Chagas Disease Therapy

Chagas disease, which is caused by Trypanosoma cruzi (T. cruzi), remains a substantial public health concern and an important cause of morbidity and mortality in Latin America. T. cruzi infection causes an intense inflammatory response in diverse tissues by triggering local expression of inflammatory mediators, which results in the upregulation of the levels of cytokines and chemokines, and important cardiac alterations in the host, being one of the most characteristic damages of Chagas disease. Therefore, controlling the inflammatory reaction becomes critical for the control of the proliferation of the parasite and of the evolution of Chagas disease. The nuclear receptors known as peroxisome proliferator-activated receptors (PPARs) have emerged as key regulators of lipid metabolism and inflammation. The precise role of PPAR ligands in T. cruzi infection or in Chagas disease is poorly understood. This review summarizes our knowledge about T. cruzi infection as well as about the activation of PPARs and the potential role of their ligands in the resolution of inflammation, with the aim to address a new pharmacological approach to improve the host health.

Impaired Trypanosoma cruzi-specific IFN-γ secretion by T cells bearing the BV9 T-cell receptor is associated with local IL-10 production in non-lymphoid tissues of chronically infected mice

The role of non-lymphoid tissue T cells expressing the BV9 family T-cell receptor (TCRBV9) was studied in mice chronically infected with the Trypanosoma cruzi. Heart and skeletal muscles had higher frequencies and ratios of CD8+ TCRBV9+ to CD4+ TCRBV9+ T cells than lymph nodes. Also, homing experiments of CFSE-labeled T cells showed preferential homing of TCRBV9+ T cells to heart tissue. In vitro proliferation assays showed higher [3H]thymidine uptake by non-lymphoid tissue TCRBV9+ T cells than lymph node TCRBV9+ T cells co-cultured with antigen-presenting cells (APC), in response to T. cruzi amastigote antigens (TcAg). Lymph node TCRBV9+ T cells secreted IFN-gamma and IL-10, but not IL-4, upon stimulation with TcAg in the presence of APC. Moreover, non-lymphoid tissue-derived TCRBV9+ T cells showed impairment of IFN-gamma, no IL-4 production, and higher levels of IL-10 secretion under the same conditions. Our results show that T. cruzi-specific IFN-gamma- and IL-10-producing TCR BV9+ T cells develop in the mouse lymph nodes during chronic infection with T. cruzi. Upon homing to non-lymphoid parasitized tissues, IFN-gamma secretion might subside due to the overt secretion of IL-10, of which TCRBV9+ T cells represent a significant source.

Treatment with a New Peroxisome Proliferator-Activated Receptor Gamma Agonist, Pyridinecarboxylic Acid Derivative, Increases Angiogenesis and Reduces Inflammatory Mediators in the Heart of Trypanosoma cruzi-Infected Mice

Trypanosoma cruzi infection induces an intense inflammatory response in diverse host tissues. The immune response and the microvascular abnormalities associated with infection are crucial aspects in the generation of heart damage in Chagas disease. Upon parasite uptake, macrophages, which are involved in the clearance of infection, increase inflammatory mediators, leading to parasite killing. The exacerbation of the inflammatory response may lead to tissue damage. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear transcription factor that exerts important anti-inflammatory effects and is involved in improving endothelial functions and proangiogenic capacities. In this study, we evaluated the intermolecular interaction between PPARγ and a new synthetic PPARγ ligand, HP24, using virtual docking. Also, we showed that early treatment with HP24, decreases the expression of NOS2, a pro-inflammatory mediator, and stimulates proangiogenic mediators (vascular endothelial growth factor A, CD31, and Arginase I) both in macrophages and in the heart of T. cruzi-infected mice. Moreover, HP24 reduces the inflammatory response, cardiac fibrosis and the levels of inflammatory cytokines (TNF-α, interleukin 6) released by macrophages of T. cruzi-infected mice. We consider that PPARγ agonists might be useful as coadjuvants of the antiparasitic treatment of Chagas disease, to delay, reverse, or preclude the onset of heart damage.

Redirection of the immune response to the functional catalytic domain of cruzipain improves protective immunity against Trypanosoma cruzi infection

Despite the strong immune responses elicited after natural infection with Trypanosoma cruzi or vaccination against it, parasite survival suggests that these responses are insufficient or inherently inadequate. T. cruzi contains a major cystein proteinase, cruzipain, which has a catalytic N-terminal domain and a C-terminal extension. Immunizations that employed recombinant cruzipain or its N- and C-terminal domains allowed evaluation of the ability of cruzipain to circumvent responses against the catalytic domain. This phenomenon is not a property of the parasite but of cruzipain itself, because recombinant cruzipain triggers a response similar to that of cruzipain during natural or experimental infection. Cruzipain is not the only antigen with a highly immunogenic region of unknown function that somehow protects an essential domain for parasite survival. However, our studies show that this can be reverted by using the N-terminal domain as a tailored immunogen able to redirect host responses to provide enhanced protection.

Treatment with Fenofibrate plus a low dose of Benznidazole attenuates cardiac dysfunction in experimental Chagas disease

Trypanosoma cruzi induces serious cardiac alterations during the chronic infection. Intense inflammatory response observed from the beginning of infection, is critical for the control of parasite proliferation and evolution of Chagas disease. Peroxisome proliferator-activated receptors (PPAR)-α, are known to modulate inflammation. In this study we investigated whether a PPAR-α agonist, Fenofibrate, improves cardiac function and inflammatory parameters in a murine model of T. cruzi infection. BALB/c mice were sequentially infected with two T. cruzi strains of different genetic background. Benznidazole, commonly used as trypanocidal drug, cleared parasites but did not preclude cardiac pathology, resembling what is found in human chronic chagasic cardiomyopathy. Fenofibrate treatment restored to normal values the ejection and shortening fractions, left ventricular end-diastolic, left ventricular end-systolic diameter, and isovolumic relaxation time. Moreover, it reduced cardiac inflammation and fibrosis, decreased the expression of pro-inflammatory (IL-6, TNF-α and NOS2) and heart remodeling mediators (MMP-9 and CTGF), and reduced serum creatine kinase activity. The fact that Fenofibrate partially inhibited NOS2 expression and NO release in the presence of a PPAR-α non-competitive inhibitor, suggested it also acted through PPAR-α-independent pathways. Since IκBα cytosolic degradation was inhibited by Fenofibrate, it can be concluded that the NFκB pathway has a role in its effects. Thus, we demonstrate that Fenofibrate acts through PPAR-α-dependent and -independent pathways. Our study shows that combined treatment with Fenofibrate plus Benznidazole is able both to reverse the cardiac dysfunction associated with the ongoing inflammatory response and fibrosis and to attain parasite clearance in an experimental model of Chagas disease.