Tuft-Cell-Derived Leukotrienes Drive Rapid Anti-helminth Immunity in the Small Intestine but Are Dispensable for Anti-protist Immunity.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

The pathology of the spleen in lethal canine babesiosis caused by Babesia rossi.

To provide useful information based on the macropathology, histopathology and immunohistochemical investigation in the spleens of dogs with Babesia rossi infection. Control spleens were collected from four healthy dogs euthanized for welfare reasons. Nine dogs that died naturally because of a mono-infection with Babesia rossi were selected for the diseased group. One haematoxylin-and-eosin-stained section of splenic tissue from each of the infected and control dogs was examined under the light microscope. Immunohistochemical markers were applied to characterize different immunocyte populations. The application of analytic software enabled semi-quantitative comparison of leucocyte subpopulations. Routine splenic histopathology revealed diffuse intermingling of white and red pulp from infected dogs with a clear loss of distinction between these zones. Immunohistochemistry revealed an increase in the proportion of tissue resident and bone marrow origin macrophages in the infected spleens. Apart from a few remnant lymphocytes within the peri-arteriolar lymphatic sheaths and follicles, the majority of the immunocytes redistributed to the red pulp, supporting the observation of white and red pulp intermingling. The majority of our findings are in agreement with histomorphological descriptions of the spleen in a variety of noncanid mammalian hosts with lethal malaria or babesiosis.

Injection of T3SS effectors not resulting in invasion is the main targeting mechanism of Shigella toward human lymphocytes.

The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4+ T, and CD8+ T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.

Type 2 innate lymphoid cells control eosinophil homeostasis.

Eosinophils are specialized myeloid cells associated with allergy and helminth infections. Blood eosinophils demonstrate circadian cycling, as described over 80 years ago, and are abundant in the healthy gastrointestinal tract. Although a cytokine, interleukin (IL)-5, and chemokines such as eotaxins mediate eosinophil development and survival, and tissue recruitment, respectively, the processes underlying the basal regulation of these signals remain unknown. Here we show that serum IL-5 levels are maintained by long-lived type 2 innate lymphoid cells (ILC2) resident in peripheral tissues. ILC2 cells secrete IL-5 constitutively and are induced to co-express IL-13 during type 2 inflammation, resulting in localized eotaxin production and eosinophil accumulation. In the small intestine where eosinophils and eotaxin are constitutive, ILC2 cells co-express IL-5 and IL-13; this co-expression is enhanced after caloric intake. The circadian synchronizer vasoactive intestinal peptide also stimulates ILC2 cells through the VPAC2 receptor to release IL-5, linking eosinophil levels with metabolic cycling. Tissue ILC2 cells regulate basal eosinophilopoiesis and tissue eosinophil accumulation through constitutive and stimulated cytokine expression, and this dissociated regulation can be tuned by nutrient intake and central circadian rhythms.

Host-pathogen interaction in Toxoplasma gondii-infected mixed chicken blood cell cultures.

Toxoplasma gondii has the ability to infect various nucleated cell types in different hosts. The aim of the present study was to investigate which chicken blood cells were targeted by T. gondii in a mixed blood cell culture similar to in vivo conditions and to evaluate parasite-host cell interactions. The study consisted of two subsequent experiments. In experiment 1, we applied T. gondii tachyzoites (ME49) at a multiplicity of infection of 1 tachyzoite per blood cell and examined parasite replication, cytokine, and inducible nitric oxide synthase (iNOS) mRNA expression between 1 h and 48 h post-infection (p.i.) by quantitative PCR. By using T. gondii RH-GFP tachyzoites expressing the green fluorescent protein (GFP) in experiment 2, we aimed for visualizing infected cells by confocal laser scanning microscopy (CLSM) and flow cytometric analysis at 24 h p.i. The parasite replication curve showed a massive decrease of parasite stages until 24 h p.i. followed by an approximately plateau phase. We observed mainly significantly increased iNOS mRNA expression levels in T. gondii-infected culture compared to uninfected cells. Flow cytometry and CLSM data confirmed monocytes/macrophages as main target cells for T. gondii. Moreover, different lymphocytes like B cells and cytotoxic T cells seem to be targeted to a low extent. Our findings indicate that monocytes/macrophages play a key role during T. gondii infection in chicken as host cells and triggering of immune response. To the best of our knowledge, this is the first report of a mixed chicken blood cell culture experimentally infected with T. gondii.

5-HT3A serotonin receptor in the gastrointestinal tract: the link between immune system and enteric nervous system in the digestive form of Chagas disease.

Chagas disease is caused by Trypanosoma cruzi and remains one of the most neglected diseases in Latin America. One of its clinical forms is Chagas megacolon. Despite being known for more than half a century, detailed causes are still obscure. Recent evidence indicates a close relationship between the immune system and the enteric nervous system in the etiology of chagasic megacolon pathology. It is believed that low expression of the 5-HT3A serotonin receptor on lymphocytes could be linked to megacolon development. To test this hypothesis, this work investigated the distribution of CD4, CD8, and CD20 lymphocytes and their 5-HT3A receptor expression. The results demonstrated that Chagas patients without megacolon present a higher expression of the 5-HT3A receptor in all analyzed lymphocytes compared with Chagas patients with megacolon. These data suggest that the high expression of this receptor may lead to immunomodulation and prevent the development of Chagas megacolon.

Increased Innate Lymphoid Cell 3 and IL-17 Production in Mouse Lamina Propria Stimulated with Giardia lamblia.

Innate lymphoid cells (ILCs) are key players during an immune response at the mucosal surfaces, such as lung, skin, and gastrointestinal tract. Giardia lamblia is an extracellular protozoan pathogen that inhabits the human small intestine. In this study, ILCs prepared from the lamina propria of mouse small intestine were incubated with G. lamblia trophozoites. Transcriptional changes in G. lamblia-exposed ILCs resulted in identification of activation of several immune pathways. Secretion of interleukin (IL)-17A, IL-17F, IL-1ß, and interferon-γ was increased, whereas levels of IL-13, IL-5, and IL22, was maintained or reduced upon exposure to G. lamblia. Goup 3 ILC (ILC3) was found to be dominant amongst the ILCs, and increased significantly upon co-cultivation with G. lamblia trophozoites. Oral inoculation of G. lamblia trophozoites into mice resulted in their presence in the small intestine, of which, the highest number of parasites was detected at the 5 days-post infection. Increased ILC3 was observed amongst the ILC population at the 5 days-post infection. These findings indicate that ILC3 from the lamina propria secretes IL-17 in response to G. lamblia, leading to the intestinal pathology observed in giardiasis.

Tumor suppressor p53 induces apoptosis of host lymphocytes experimentally infected by Leishmania major, by activation of Bax and caspase-3: a possible survival mechanism for the parasite.

Apoptosis of infected host macrophages by Leishmania spp. is mainly addressed as one of the survival mechanisms of the parasite. However, there is no eligible data about whether tumor suppressor p53 could induce the apoptosis of host lymphocytes-treated Leishmania major via the mitochondrial intrinsic pathway. In this study, the amastigotes of L. major obtained from ten cutaneous leishmaniases (CL) patients were separately isolated and cultured in N.N.N and RPMI 1640 media. L. major was definitely confirmed by targeting Cyt b gene following sequencing. Subsequently, 2-3 × 106 lymphocytes obtained from ten healthy individuals were isolated and co-cultured with 1-2 × 106 L. major promastigotes. Following 6 h of exposure time, the enzymatic activity of caspase-3 was determined by fluorometric assay in each L. major-treated lymphocytes and cell control (only lymphocyte). The mRNA expressions of Bax, Bcl-2, p53, and caspase-3 genes were assessed by quantitative real-time-PCR analysis following 6 to 9 h of exposure times. The Bcl-2 mRNA expression in L. major-treated lymphocytes was 100-fold down-regulated relative to cell control. The mRNA expressions of p53 and caspase-3 were over-expressed 1.8- and 3.2-fold up-regulated relative to control lymphocytes, respectively. The Bax/Bcl-2 ratio and caspase-3 activity were higher than the control group (Pv <0.05). The current new findings indicate that the apoptotic effects of L. major-treated host lymphocytes dependent on p53 tumor suppressor via mitochondrial pathway may probably address as an auxiliary survival mechanism of L. major in CL patients. However, here is much work ahead to figure out the multiple functions played by apoptosis in the evasion of L. major.

Intranasal immunization with recombinant Toxoplasma gondii actin depolymerizing factor confers protective efficacy against toxoplasmosis in mice.

BACKGROUND: Toxoplasma gondii is an opportunistic protozoan closely associated with AIDS and vertical transmission. T. gondii actin depolymerizing factor (TgADF) plays an important role in actin cytoskeleton remodeling, and it is required to invade host cells. TgADF was a promising vaccine candidate. To observe the immunological changes and protective efficacy of recombinant TgADF protein (rTgADF) against T. gondii infection, we optimized the intranasal immunization dose of rTgADF and analyzed the survival rate and tachyzoite loads in mouse tissues after oral challenge with T. gondii tachyzoites. RESULTS: rTgADF was prepared, purified, and combined with mouse anti-His antibody and rabbit anti-T. gondii serum. After intranasal immunization with 10 µg, 20 µg, 30 µg, or 40 µg of rTgADF, the 30-µg group elicited high levels of secretory IgA (sIgA) in nasal, intestinal, and vesical washes, raised IgG titres in the sera, strong proliferation of splenocytes, and increased secretion of IL-2 and IFN-γ when compared with the control group. When the mice were orally challenged with T. gondii, an increase in the survival rate (36.36 %) and a decrease in the tachyzoite loads in the liver (67.77 %) and brain (51.01 %) were observed. CONCLUSIONS: Our findings demonstrate that intranasal immunization with rTgADF can simultaneously trigger mucosal and systemic immune responses and protect the mice against T. gondii infection.

γδ Intraepithelial Lymphocyte Migration Limits Transepithelial Pathogen Invasion and Systemic Disease in Mice.

BACKGROUND & AIMS: Intraepithelial lymphocytes that express the γδ T-cell receptor (γδ IELs) limit pathogen translocation across the intestinal epithelium by unknown mechanisms. We investigated whether γδ IEL migration and interaction with epithelial cells promote mucosal barrier maintenance during enteric infection. METHODS: Salmonella typhimurium or Toxoplasma gondii were administered to knockout (KO) mice lacking either the T cell receptor δ chain (Tcrd) or CD103, or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of green fluorescent protein (GFP)-tagged γδ T cells within the small intestinal mucosa of mice infected with DsRed-labeled S typhimurium. Mixed bone marrow chimeras were generated to assess the effects of γδ IEL migration on early pathogen invasion and chronic systemic infection. RESULTS: Morphometric analyses of intravital video microscopy data showed that γδ IELs rapidly localized to and remained near epithelial cells in direct contact with bacteria. Within 1 hour, greater numbers of T gondii or S typhimurium were present within mucosae of mice with migration-defective occludin KO γδ T cells, compared with controls. Pathogen invasion in Tcrd KO mice was quantitatively similar to that in mice with occludin-deficient γδ T cells, whereas invasion in CD103 KO mice, which have increased migration of γδ T cells into the lateral intercellular space, was reduced by 63%. Consistent with a role of γδ T-cell migration in early host defense, systemic salmonellosis developed more rapidly and with greater severity in mice with occludin-deficient γδ IELs, relative to those with wild-type or CD103 KO γδ IELs. CONCLUSIONS: In mice, intraepithelial migration to epithelial cells in contact with pathogens is essential to γδ IEL surveillance and immediate host defense. γδ IEL occludin is required for early surveillance that limits systemic disease.

Comparing the immune responses of two genetically B-complex disparate Fayoumi chicken lines to Eimeria tenella.

The present study was conducted to compare the susceptibility of congenic Fayoumi lines to Eimeria tenella infection and to assess genetic differences in Eimeria egression. Chickens were orally inoculated with 5 × 10(4) sporulated E. tenella oocysts and challenged with 5 × 10(6) oocysts on the 10th day after the primary infection. The Fayoumi M5.1 line exhibited higher levels of body weight gain, less oocyst shedding and higher percentages of B and CD4(+)/CD8(+) T cells than the M15.2 chickens. These results demonstrate that M5.1 line is more resistant to E. tenella infection than M15.2 line. Furthermore, the percentage of sporozoite egress from peripheral blood mononuclear cells (PBMCs) was higher in the M5.1 line. The results of this study suggest that enhanced resistance of Fayoumi M5.1 to E. tenella infection may involve heightened cell-mediated and adaptive immunity, resulting in reduced intracellular development of Eimeria parasites.

Role of the polymorphic immunodominant molecule in entry of Theileria parva sporozoites into bovine lymphocytes.

Theileria parva is a tick-transmitted apicomplexan parasite that infects cattle and African buffalo. In cattle, it causes a fatal lymphoproliferative disease called East Coast fever. The polymorphic immunodominant molecule (PIM) is expressed by two stages of the parasite: the sporozoite, which is inoculated by the tick to infect mammalian lymphocytes, and the schizont, the established intralymphocytic stage. Here, we demonstrate that monoclonal antibodies (MAb) to PIM can reduce the ability of sporozoites to infect bovine lymphocytes in vitro. This reduction appears to be due to blocking of sporozoite attachment by binding of the MAb to several regions of PIM. Interestingly, one MAb, which recognizes an epitope in the central variable region of PIM, did not inhibit sporozoite infectivity. We also demonstrate that PIM antigen, as a recombinant molecule, can also reduce sporozoite infectivity in vitro by blocking both attachment and internalization of sporozoites. Electron microscopic studies showed that PIM is present in microspheres below the sporozoite surface and is transported to the parasite surface soon after contact with bovine lymphocytes. The results suggest that at least two sporozoite molecules, PIM and the previously described p67, are involved in the entry of T. parva into mammalian lymphocytes.

Salivary gland homogenates from wild-caught sand flies Lutzomyia flaviscutellata and Lutzomyia (Psychodopygus) complexus showed inhibitory effects on Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis infection in BALB/c mice.

During the natural transmission of Leishmania parasites, the infected sand fly female regurgitates promastigotes into the host's skin together with its saliva. It has been reported that vector saliva contains immunomodulatory molecules that facilitate the establishment of infection. Thus, the main objective of this study was to evaluate the specificity of Lutzomyia (Lu.) flaviscutellata and Lu. (Psychodopygus) complexus salivas on the infectivity of Leishmania (L.) (Leishmania) amazonensis and L. (Viannia) braziliensis, respectively. BALB/c mice were inoculated into the skin of hind footpad with L. (L.) amazonensis and L. (V.) braziliensis promastigotes in the absence or presence of Lu. flaviscutellata and Lu. (P.) complexus salivary gland homogenates (SGHs). The evolution of the infection was evaluated by lesion size, histopathological analysis and determination of the parasite load in the skin biopsies collected from the site of infection at 4 and 8 weeks PI. The lesion size and the parasite load of both groups of mice infected in the presence of SGHs were smaller than the control groups. The histopathological features showed that the inflammatory reaction was less prominent in the groups of mice infected in the presence of both SGHs when compared to the control group. The results showed that the presence of SGHs of Lu. flaviscutellata and Lu. (P.) complexus led to induction of processes that were disadvantageous to parasite establishment during infection by L. (L.) amazonensis and L. (V.) braziliensis. An inhibitory effect on Leishmania infection could be observed in both groups inoculated with SGHs, especially when the SGH from Lu. (P.) complexus was used.

Target evaluation of deoxyhypusine synthase from Theileria parva the neglected animal parasite and its relationship to Plasmodium.

East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy.

Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease.

Necrotic enteritis (NE) is a re-emerging disease as a result of increased restriction on the use of antibiotics in poultry. However, the molecular mechanisms underlying the pathogenesis of NE are unclear. Small RNA transcriptome analysis was performed using spleen and intestinal intraepithelial lymphocytes (IEL) from 2 inbred chicken lines selected for resistance or susceptibility to Marek's disease (MD) in an experimentally induced model of avian NE to investigate whether microRNA (miRNA) control the expression of genes associated with host response to pathogen challenge. Unique miRNA represented only 0.02 to 0.04% of the total number of sequences obtained, of which 544 were unambiguously identified. Hierarchical clustering revealed that most of miRNA in IEL were highly expressed in the MD-susceptible line 7.2 compared with MD-resistant line 6.3. Reduced CXCL14 gene expression was correlated with differential expression of several unique miRNA in MD-resistant chickens, whereas TGFßR2 gene expression was correlated with altered gga-miR-216 miRNA levels in MD-susceptible animals. In conclusion, miRNA profiling and deep sequencing of small RNA in experimental models of infectious diseases may be useful for further understanding of host-pathogen interactions, and for providing insights into genetic markers of disease resistance.

[Evaluation of the genotoxic effect of malaria in a cohort of patients in Medellín and Quibdó]. / Evaluación del efecto genotóxico en pacientes con malaria de una región de Colombia.

INTRODUCTION: During malaria infection, both parasite and host are under the effects of oxidative stress due to the increased production of reactive oxygen species, which can induce DNA damage by its genotoxic effects. OBJECTIVE: To evaluate genotoxic effects in human lymphocytes in a cohort of patients with malaria from Medellin and Quibdó. METHODS: We performed an observational cross sectional study in 100 individuals with malaria and 100 healthy controls. Patients infected with Plasmodium consulting the Institute Colombiano of Medicina Tropical of Medellin and the Hospital Ismael Roldán Valencia of Quibdó were included. Genotoxic effects (genetic damage) was analysed by electrophoresis using alkaline single cell gel (Commet assay). RESULTS: The average of tail length of malaria samples (26.9±9.8) was significantly higher than of controls (14.8±3.2) (p<0.01). CONCLUSION: In our study population, malaria infection was associated with increased genotoxicity, while other variables such as smoking, antimalarial treatment, and occupation were not.

Innate lymphoid cells in immunity and disease.

The family of innate lymphoid cells (ILCs) comprises of natural killer (NK) cells, Rorγt-dependent ILCs (lymphoid tissue inducer (LTi) cells, ILC22, and ILC17), and type 2 ILCs. Apart from a common requirement for inhibitor of DNA binding 2 (Id2) expression and common γ-chain (γc) signaling, the differentiation of ILC populations is regulated by distinct transcription factors. ILCs play fundamental roles in processes such as cytotoxicity, lymphoid organogenesis, intestinal homeostasis, immunity against infections, and wound healing. However, the dysregulation of ILCs has been implicated in autoimmune and inflammatory diseases. Here, we will review the recent advances in ILC development and their roles in immunity and disease, with a primary focus on type 2 ILCs.

Influence of Toxoplasma gondii acute infection on cholinesterase activities of Wistar rats.

Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.