Annexin A1 improves immune responses and control of tissue parasitism during Leishmania amazonensis infection in BALB/c mice.

Leishmaniases, a group of diseases caused by the species of the protozoan parasite Leishmania, remains a significant public health concern worldwide. Host immune responses play a crucial role in the outcome of Leishmania infections, and several mediators that regulate inflammatory responses are potential targets for therapeutic approaches. Annexin A1 (AnxA1), an endogenous protein endowed with anti-inflammatory and pro-resolving properties, has emerged as a potential player. We have shown that during L. braziliensis infection, deficiency of AnxA1 exacerbates inflammatory responses but does not affect parasite burden. Here, we have investigated the role of AnxA1 in L. amazonensis infection, given the non-healing and progressive lesions characteristic of this infectious model. Infection of AnxA1 KO BALB/c mice resulted in increased lesion size and tissue damage associated with higher parasite burdens and enhanced inflammatory response. Notably, therapeutic application of the AnxA1 peptidomimetic Ac2-26 improves control of parasite replication and increases IL-10 production in vivo and in vitro, in both WT and AnxA1 KO mice. Conversely, administration of WRW4, an inhibitor of FPR2/3, resulted in larger lesions and decreased production of IL-10, suggesting that the effects of AnxA1 during L. amazonensis infection are associated with the engagement of these receptors. Our study illuminates the role of AnxA1 in L. amazonensis infection, demonstrating its impact on the susceptibility phenotype of BALB/c mice. Furthermore, our results indicate that targeting the AnxA1 pathway by using the Ac2-26 peptide could represent a promising alternative for new treatments for leishmaniasis.

Temporary Shutdown of ERK1/2 Phosphorylation Is Associated With Activation of Adaptive Immune Cell Responses and Disease Progression During Leishmania amazonensis Infection in BALB/c Mice.

Leishmania spp. infection outcomes are dependent on both host and parasite factors. Manipulation of host signaling pathways involved in the generation of immune responses is thought to be one of the most common mechanisms used by parasites for persistence within the host. Considering the diversity of pathologies caused by different Leishmania spp., it is plausible that significant differences may exist in the mechanisms of host cell manipulation by each parasite species, which may have implications when developing new vaccine or treatment strategies. Here we show that in L. braziliensis-infection in BALB/c mice, a model of resistance, activation of ERK1/2 coincides with the peak of inflammatory responses and resolution of tissue parasitism. In contrast, in the susceptibility model of L. amazonensis-infection, an early silent phase of infection is observed, detected solely by quantification of parasite loads. At this early stage, only basal levels of P-ERK1/2 are observed. Later, after a brief shutdown of ERK1/2 phosphorylation, disease progression is observed and is associated with increased inflammation, lesion size and tissue parasitism. Moreover, the short-term down-regulation of ERK1/2 activation affected significantly downstream inflammatory pathways and adaptive T cell responses. Administration of U0126, a MEK/ERK inhibitor, confirmed this phenomenon, since bigger lesions and higher parasite loads were seen in infected mice that received U0126. To investigate how kinetics of ERK1/2 activation could affect the disease progression, U0126 was administered to L. amazonensis-infected animals earlier than the P-ERK1/2 switch off time-point. This intervention resulted in anticipation of the same effects on inflammatory responses and susceptibility phenotype seen in the natural course of infection. Additionally, in vitro inhibition of ERK1/2 affected the phagocytosis of L. amazonensis by BMDMs. Collectively, our findings reveal distinct temporal patterns of activation of inflammatory responses in L. braziliensis and L. amazonensis in the same animal background and a pivotal role for a brief and specific shutdown of ERK1/2 activation at late stages of L. amazonensis infection. Since activation of inflammatory responses is a crucial aspect for the control of infectious processes, these findings may be important for the search of new and specific strategies of vaccines and treatment for tegumentary leishmaniasis.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

Intestinal microbiota regulates tryptophan metabolism following oral infection with Toxoplasma gondii.

INTRODUCTION: The intestinal microbiota plays an important role in modulating host immune responses. Oral Toxoplasma gondii infection can promote intestinal inflammation in certain mice strains. The IDO-AhR axis may control tryptophan (Trp) metabolism constituting an important immune regulatory mechanism in inflammatory settings. AIMS: In the present study, we investigated the role of the intestinal microbiota on Trp metabolism during oral infection with T gondii. METHODS AND RESULTS: Mice were treated with antibiotics for four weeks and then infected with T gondii by gavage. Histopathology and immune responses were evaluated 8 days after infection. We found that depletion of intestinal microbiota by antibiotics contributed to resistance against T gondii infection and led to reduced expression of AhR on dendritic and Treg cells. Mice depleted of Gram-negative bacteria presented higher levels of systemic Trp, downregulation of AhR expression and increased resistance to infection whereas depletion of Gram-positive bacteria did not affect susceptibility or expression of AhR on immune cells. CONCLUSION: Our findings indicate that the intestinal microbiota can control Trp availability and provide a link between the AhR pathway and host-microbiota interaction in acute infection with T gondii.

Frequency of CDH1 germline variants and contribution of dietary habits in early age onset gastric cancer patients in Brazil.

INTRODUCTION: The contribution of CDH1 germline variants to gastric cancer burden among young adults is unknown in Brazil. We aimed to evaluate the frequency of CDH1 germline variants and the diet/lifestyle habits in early age onset gastric cancer (EOGC, ≤ 55 years old) patients. METHODOLOGY: From 2013 to 2015, a total of 88 unrelated and consecutive patients diagnosed with EOGC were enrolled. All CDH1 exons and intronic boundaries were sequenced, and large genomic rearrangements were screened by MLPA. CDH1 transcription analysis was performed for variants that could potentially induce an effect on splicing. The diet and lifestyle habits of EOGC patients were compared to Brazilian population diet and lifestyle, obtained from governmental databases. RESULTS: Of 88 patients, the mean age at EOGC diagnosis was 39 years and 55% fulfilled the criteria for hereditary diffuse gastric cancer. The majority of the tumors were diffuse (74%) and poorly differentiated (80%). In total, 4 novel missense variants of uncertain significance (VUS) were identified: c.313T>A, c.387G>T, c.1676G>A, and c.1806C>A. The MLPA results revealed no rearrangements and CDH1 transcription analysis for variants of interest were inconclusive. EOGC patients had a higher red (OR:2.6, 95%CI:1.4-4.9) and processed (OR:3.1, 95%CI:1.6-6.0) meat intake and higher fruit consumption (OR:0.4, 95%IC:0.3-0.7) compared to eating habits of the Brazilian population. CONCLUSIONS: No unequivocal pathogenic germline CDH1 variants were identified in Brazilian EOGC patients. Dietary habits may be associated with the EOGC development.

Sulfate-reducing bacteria stimulate gut immune responses and contribute to inflammation in experimental colitis.

The intestinal microbiota is critical for mammalian immune system development and homeostasis. Sulfate-reducing bacteria (SRB) are part of the normal gut microbiota, but their increased levels may contribute to colitis development, likely in association with hydrogen sulfide (H2S) production. Here, we investigated the effects of SRB in the gut immune response in germ-free mice, and in experimental colitis. After 7days of colonization with Desulfovibrio indonesiensis or with a human SRB consortium (from patients with colitis), germ-free mice exhibited alterations in the colonic architecture, with increased cell infiltration in the lamina propria. SRB colonization upregulated the Th17 and Treg profiles of cytokine production/cell activation, in T cells from mesenteric lymph nodes. These alterations were more pronounced in mice colonized with the human SRB consortium, although D. indonesiensis colonization produced higher levels of H2S. Importantly, the colon of C57BL/6 mice with colitis induced by TNBS or oxazolone had increased SRB colonization, and the administration of D. indonesiensis to mice with TNBS-induced colitis clearly exacerbated the alterations in colonic architecture observed in the established disease, and also increased mouse weight loss. We conclude that SRB contribute to immune response activation in the gut and play an important role in colitis development.

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease.

Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.

Prognostic Value of ADAMTS Proteases and Their Substrates in Epithelial Ovarian Cancer.

BACKGROUND: ADAMTS are metalloproteases with disintegrin and thrombospondin motifs. They are secreted proteases playing a role in biological processes such as inflammation, angiogenesis, and urogenital development. ADAMTS have specific substrates, such as the proteoglycans (PG) versican, aggrecan, and brevican. Despite data indicating a role of ADAMTS in tumor invasion and metastases, effects played by these molecules in cancer progression are still controversial. In ovarian cancer, the importance of ADAMTS gene mutations was recently described and related to chemotherapy outcome. OBJECTIVE: To analyze protein levels of ADAMTS-1, -4, and -5, and TIMP-3 in human ovarian cancer classified as benign, borderline, or malignant. We also assessed the expression of the ADAMTS substrates aggrecan, brevican, and versican in these neoplasms. Correlations between overall survival and protein expression were performed. METHODS: Tumors were classified according to the WHO Classification of Tumors of Female Reproductive Organs. Protein and PG expression was studied by immunohistochemistry. Differences in labeling were analyzed by percent measurements of stained areas. RESULTS: ADAMTS-1, ADAMTS-5, and its tissue inhibitor TIMP-3 are increased in borderline and malignant tumors compared to benign neoplasms. Aggrecan and versican levels were increased in malignant subtypes compared to benign ovarian cancer. Higher ADAMTS-1, TIMP-3, and versican expression was associated with a shorter overall survival. CONCLUSIONS: Comparison of protease, TIMP-3, and substrate expression showed that in malignant tumors all ADAMTS and TIMP-3 expression levels were significantly raised compared to the substrates studied.

IFN-γ-Dependent Recruitment of CD4(+) T Cells and Macrophages Contributes to Pathogenesis During Leishmania amazonensis Infection.

Interferon gamma (IFN-γ) is a key factor in the protection of hosts against intracellular parasites. This cytokine induces parasite killing through nitric oxide and reactive oxygen species production by phagocytes. Surprisingly, during Leishmania amazonensis infection, IFN-γ plays controversial roles. During in vitro infections, IFN-γ induces the proliferation of the amastigote forms of L. amazonensis. However, this cytokine is not essential at the beginning of an in vivo infection. It is not clear why IFN-γ does not mediate protection during the early stages of infection. Thus, the aim of our study was to investigate the role of IFN-γ during L. amazonensis infection. We infected IFN-γ(-/-) mice in the footpad and followed the development of leishmaniasis in these mice compared with that in WT mice. CD4(+) T lymphocytes and macrophages migrated earlier to the site of infection in the WT mice, and the earlier migration of these 2 cell types was associated with lesion development and parasite growth, respectively. These differences in the infiltrate populations were explained by the increased expression of chemokines in the lesions of the WT mice. Thus, we propose that IFN-γ plays a dual role during L. amazonensis infection; it is an important inducer of effector mechanisms, particularly through inducible nitric oxide synthase expression, and conversely, it is a mediator of inflammation and pathogenesis through the induction of the expression of chemokines. Our data provided evidence for a pathogenic effect of IFN-γ production during leishmaniasis that was previously unknown.

IL-18 contributes to susceptibility to Leishmania amazonensis infection by macrophage-independent mechanisms.

We evaluated the role of IL-18 during Leishmania amazonensis infection in C57BL/6 mice, using IL-18KO mice. We showed that IL-18 is involved in susceptibility to L. amazonensis, since IL-18KO mice presented reduced lesions and parasite loads. Because macrophages are the host cells of the parasite, we investigated if macrophages were involved in IL-18-mediated susceptibility to L. amazonensis. We showed that macrophages obtained from WT or IL-18KO responded similarly to L. amazonensis infection. Moreover, we showed that C57BL/6 macrophages do not respond to IL-18, since they do not express IL-18R. Therefore, macrophages are not involved in IL-18-mediated susceptibility to L. amazonensis.

The multifaceted role of commensal microbiota in homeostasis and gastrointestinal diseases.

The gastrointestinal tract houses a complex and diverse community of microbes. In recent years, an increased understanding of the importance of intestinal microbiota for human physiology has been gained. In the steady state, commensal microorganisms have a symbiotic relationship with the host and possess critical and distinct functions, including directly influencing immunity. This means that recognition of commensal antigens is necessary for the development of complete immune responses. Therefore, the immune system must face the challenge of maintaining mucosal homeostasis while dealing with undue passage of commensal or pathogenic microbes, as well as the host nutritional status or drug use. Disruption of this fine balance has been associated with the development of several intestinal inflammatory diseases. In this review, we discuss the mechanisms involved in the modulation of host-microbe interactions and how the breakdown of this homeostatic association can lead to intestinal inflammation and pathology.

Short-term protection conferred by Leishvacin® against experimental Leishmania amazonensis infection in C57BL/6 mice.

To date, there is no vaccine available against human leishmaniasis. Although some vaccination protocols can induce immunity in murine models, they fail to induce protection in humans. The reasons for that remain unclear. The aim of the present study was to characterize the changes in the pattern of the immune response during subcutaneous vaccination with Leishvacin® in mice. We also investigated whether IFN-γ and nitric oxide synthase are indispensable for the protection elicited by the vaccine. C57BL/6 WT vaccinated mice showed smaller lesions and fewer numbers of parasites in footpads until 8 weeks post-infection. Up to this time, they produced higher levels of IFN-γ, IL-2, IL-4, IL-17A and IL-10 and higher specific antibody response than control non-vaccinated mice. Moreover, we showed that IFN-γ, most likely by induction of iNOS expression, is essential for immunity. However, after 12 weeks of infection, we observed loss of difference in lesion size and parasite burden between the groups. Loss of resistance was associated with the disappearance of differences in cytokine patterns between vaccinated and control mice, but not of antibody response, which remained different until a later time of infection. The reversal of resistance to L. amazonensis could not be explained by upregulation of regulatory cytokines. Our data point to a subversion of the host immune response by L. amazonensis even when a protective response was previously induced.

The transcription factors Thpok and LRF are necessary and partly redundant for T helper cell differentiation.

T helper (Th) cells are critical for defenses against infection and recognize peptides bound to class II major histocompatibility complex (MHC II) molecules. Although transcription factors have been identified that direct Th cells into specific effector fates, whether a "master" regulator controls the developmental program common to all Th cells remains unclear. Here, we showed that the two transcription factors Thpok and LRF share this function. Although disruption of both factors did not prevent the generation of MHC II-specific T cells, these cells failed to express Th cell genes or undergo Th cell differentiation in vivo. In contrast, T cells lacking Thpok, which only displayed LRF-dependent functions, contributed to multiple effector responses, both in vitro and in vivo, with the notable exception of Th2 cell responses that control extracellular parasites. These findings identify the Thpok-LRF pair as a core node of Th cell differentiation and function.

Acute gastrointestinal infection induces long-lived microbiota-specific T cell responses.

The mammalian gastrointestinal tract contains a large and diverse population of commensal bacteria and is also one of the primary sites of exposure to pathogens. How the immune system perceives commensals in the context of mucosal infection is unclear. Here, we show that during a gastrointestinal infection, tolerance to commensals is lost, and microbiota-specific T cells are activated and differentiate to inflammatory effector cells. Furthermore, these T cells go on to form memory cells that are phenotypically and functionally consistent with pathogen-specific T cells. Our results suggest that during a gastrointestinal infection, the immune response to commensals parallels the immune response against pathogenic microbes and that adaptive responses against commensals are an integral component of mucosal immunity.

Characterization of chronic cutaneous lesions from TNF-receptor-1-deficient mice infected by Leishmania major.

Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis.

Essential role for retinoic acid in the promotion of CD4(+) T cell effector responses via retinoic acid receptor alpha.

Vitamin A and its metabolite, retinoic acid (RA) are implicated in the regulation of immune homeostasis via the peripheral induction of regulatory T cells. Here we showed RA was also required to elicit proinflammatory CD4(+) helper T cell responses to infection and mucosal vaccination. Retinoic acid receptor alpha (RARα) was the critical mediator of these effects. Antagonism of RAR signaling and deficiency in RARα (Rara(-/-)) resulted in a cell-autonomous CD4(+) T cell activation defect, which impaired intermediate signaling events, including calcium mobilization. Altogether, these findings reveal a fundamental role for the RA-RARα axis in the development of both regulatory and inflammatory arms of adaptive immunity and establish nutritional status as a broad regulator of adaptive T cell responses.

Monoassociation with probiotic Lactobacillus delbrueckii UFV-H2b20 stimulates the immune system and protects germfree mice against Listeria monocytogenes infection.

In the present study, we investigated the protective effects of Lactobacillus delbrueckii UFV-H2b20 on the resistance to Listeria monocytogenes infection in gnotobiotic mice. Germfree mice or monoassociated mice were infected with L. monocytogenes, and the microbiological and immunological responses were evaluated after 1, 3, and 5 days of infection. Monoassociation with L. delbrueckii was capable of protecting mice against death caused by L. monocytogenes and induced a faster clearance of the bacteria in the liver, spleen, and peritoneal cavity at days 1, 3, and 5 post-infection. Also, monoassociated mice displayed less liver injury than germfree mice. The production of TNF-α in the serum, peritoneal cavity, and gut was augmented in monoassociated mice. Likewise, the levels of IFN-γ found on supernatants of spleen cells cultures were higher after the monoassociation. In addition, increased production of nitric oxide in peritoneal cell cultures supernatants and in serum was observed in mice that received L. delbrueckii. The monoassociation with L. delbrueckii induced higher production of IL-10 in the mucosal immune system. We conclude that monoassociation with L. delbrueckii UFV-H2b20 protects mice from death caused by L. monocytogenes infection by favoring effector responses while preventing their immunopathological consequences.

Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection.

Using a model of lethal oral infection with Toxoplasma gondii, we examined the fate of both induced and natural regulatory T (Treg) cells in the face of strong inflammatory responses occurring in a tolerogenic-prone environment. We found that during highly T helper 1 (Th1) cell-polarized mucosal immune responses, Treg cell numbers collapsed via multiple pathways, including blockade of Treg cell induction and disruption of endogenous Treg cell homeostasis. In particular, shutdown of interleukin 2 (IL-2) in the highly Th1 cell-polarized environment triggered by infection directly contributes to Treg cell incapacity to suppress effector responses and eventually leads to immunopathogenesis. Furthermore, we found that environmental cues provided by both local dendritic cells and effector T cells can induce the expression of T-bet transcription factor and IFN-gamma by Treg cells. These data reveal a mechanism for Th1 cell pathogenicity that extends beyond their proinflammatory program to limit Treg cell survival.

Probiotics protect mice against experimental infections.

OBJECTIVES: Our group has concerned itself with the study of the effect of probiotics on the resistance to infections using experimental models. Here, we will focus on evidence that the UFV-H2b20 strain of Lactobacillus delbrueckii var. bulgaricus may be considered a probiotic and has protective effects on mice against a variety of bacterial infections. METHODS: Germ-free, monoassociated, and conventional mice were used. Mice were treated with probiotics and challenged with Escherichia coli, Salmonella enterica serovar Typhimurium, or Listeria monocytogenes, and the outcome of infection was measured as mortality, quantification of bacteria in target organs, and systemic of local cytokine production. RESULTS: L. delbrueckii increased clearance of E. coli and production of systemic inflammatory cytokines. This strain also protected monoassociated and conventional mice against infection with S. enterica serovar Typhimurium. Finally, monoassociated mice were more resistant to L. monocytogenes as measured by mortality and the number of bacteria in spleen and liver. In addition, monoassociated mice challenged with L. monocytogenes showed increased production of inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) and nitric oxide. Interestingly, interleukin-10 levels were not altered by monoassociation or infection. CONCLUSIONS: L. delbrueckii UFV-H2b20 protects mice against infection, apparently by eliciting the up-regulation of production of inflammatory cytokines.