Dysbiosis in Inflammatory Bowel Disease: Pathogenic Role and Potential Therapeutic Targets

Microbe–host communication is essential to maintain vital functions of a healthy host, and its disruption has been associated with several diseases, including Crohn’s disease and ulcerative colitis, the two major forms of inflammatory bowel disease (IBD). Although individual members of the intestinal microbiota have been associated with experimental IBD, identifying microorganisms that affect disease susceptibility and phenotypes in humans remains a considerable challenge. Currently, the lack of a definition between what is healthy and what is a dysbiotic gut microbiome limits research. Nevertheless, although clear proof-of-concept of causality is still lacking, there is an increasingly evident need to understand the microbial basis of IBD at the microbial strain, genomic, epigenomic, and functional levels and in specific clinical contexts. Recent information on the role of diet and novel environmental risk factors affecting the gut microbiome has direct implications for the immune response that impacts the development of IBD. The complexity of IBD pathogenesis, involving multiple distinct elements, suggests the need for an integrative approach, likely utilizing computational modeling of molecular datasets to identify more specific therapeutic targets

Dysbiosis in Inflammatory Bowel Disease: Pathogenic Role and Potential Therapeutic Targets

Microbe–host communication is essential to maintain vital functions of a healthy host, and its disruption has been associated with several diseases, including Crohn’s disease and ulcerative colitis, the two major forms of inflammatory bowel disease (IBD). Although individual members of the intestinal microbiota have been associated with experimental IBD, identifying microorganisms that affect disease susceptibility and phenotypes in humans remains a considerable challenge. Currently, the lack of a definition between what is healthy and what is a dysbiotic gut microbiome limits research. Nevertheless, although clear proof-of-concept of causality is still lacking, there is an increasingly evident need to understand the microbial basis of IBD at the microbial strain, genomic, epigenomic, and functional levels and in specific clinical contexts. Recent information on the role of diet and novel environmental risk factors affecting the gut microbiome has direct implications for the immune response that impacts the development of IBD. The complexity of IBD pathogenesis, involving multiple distinct elements, suggests the need for an integrative approach, likely utilizing computational modeling of molecular datasets to identify more specific therapeutic targets

Pyrimidinergic Receptor Activation Controls <i>Toxoplasma gondii</i> Infection in Macrophages

<div><p>Infection by the protozoan parasite <i>Toxoplasma gondii</i> is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. <i>T</i>. <i>gondii</i> is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with <i>T</i>. <i>gondii</i> tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca²⁺-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y₂ and P2Y₄ and P2Y₆ showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls <i>T</i>. <i>gondii</i> infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against <i>T</i>. <i>gondii</i> infection.</p></div

<i>T</i>. <i>gondii</i> tachyzoites that egress prematurely from nucleotide-treated macrophages have reduced infectivity.

<p>Mouse peritoneal macrophages infected with tachyzoites at a 5:1 ratio were treated with 100 μM UTP or UDP for 30 min. Immediately after nucleotide treatment, prematurely egressed parasites were recovered from culture supernatants and allowed to interact with cultures of freshly harvested peritoneal macrophages for 24 h. Cells were then processed for light microscopy analysis of the % of infected cells (A) and the infection index (B). Untreated control parasites represented those that had not invaded the untreated cells 2 h post-interaction. Cytoplasmic vacuole acidification was analyzed by fluorescence microscopy using the acidic compartment probe Lysotracker red (C). Arrows indicate parasites inside host cells. Phagolysosomal fusion inhibition (with lack of Lysotracker red staining) was observed in cells infected with control parasites obtained from infected mice (Fresh). In contrast, in cultures infected with parasites rescued from nucleotide-treated cells (UTP or UDP), tachyzoites were found inside acidic (Lysotracker red-positive) parasitophorous vacuoles, indicating that phagolysosomal fusion occurred during infection. (A, B) Data represent mean and SEM of three independent experiments. * p < 0.05; ** p < 0.001; *** p < 0.0001, relative to untreated controls.</p

<i>Toxoplasma gondii</i> tachyzoites that egressed prematurely from nucleotide-treated cells could not inhibit phagolysosomal fusion in subsequent infections.

<p>Parasites that egressed prematurely from infected and nucleotide treated cells were recovered from culture supernatants and allowed to interact with freshly harvested macrophages for 2 h, and then processed for immunofluorescence to detect the tachyzoite surface protein SAG-1 (in green) and the lysosomal membrane protein LAMP-1 (in red). Phagolysosome fusion occurred in cells infected with prematurely egressed parasites (UTP and UDP samples), as evidenced by SAG-1 and LAMP-1 co-localization (yellow in the overlay). Phagolysosomal fusion also occurred in cells containing fixed tachyzoites (Fixed; positive control for fusion), but did not occur in cells infected with parasites freshly harvested from infected mice (Fresh; negative control for fusion).</p

Treatment with the P2Y agonist nucleotides reduces <i>T</i>. <i>gondii</i> infection in peritoneal macrophages.

<p>Mouse peritoneal macrophages were infected with <i>T</i>. <i>gondii</i> tachyzoites for 2h and then treated with nucleotides for 30 minutes. (A) Infected cells stained with panotic, showing that the parasite load was reduced after 18h of infection. Black arrows indicate parasitophorus vacuoles containing <i>T</i>. <i>gondii</i> tachyzoites. (B-C) Treatment with UTP reduced the percentage of infected cells (B) and the number of parasites per host cell (infection index; (C), in a dose-dependent manner. Data represent standard error of mean (SEM) of five independent experiments (D) Nucleotide treatment reduced the % of infection, and this effect was reversed by pre-treatment with 100 μM of the P2 antagonist suramin (for 30 minutes before (100 μM) nucleotide treatment). Data represent mean and standard error of mean (SEM) of three independent experiments; * significantly different relative to untreated; #, significantly different relative to the corresponding nucleotide-treated group not pre-incubated with suramin. *,^# p < 0.05; * *, ^{# #} p < 0.001; * * *, ^{# # #} p < 0.0001.</p

The activation of different P2Y receptor subtypes reduce <i>T</i>. <i>gondii</i> infection in peritoneal macrophages.

<p>Mouse peritoneal macrophages were infected with <i>T</i>. <i>gondii</i> tachyzoites at a ratio of 5:1 for 2 h and then treated with specific P2Y receptor agonists and antagonists for 30 min, prior to infection index determination. (A) Treatment 2 Thio-UTP at concentrations of 0.05 or 0.1 μM (to activate P2Y₄) and 0.5 or 1 μM of 2 Thio-UTP (to activate P2Y₂) led to similar reductions in the infection index relative to untreated controls. (B) The infection index was also reduced after treatment with MRS 2693, a specific P2Y₆ agonist, and MRS 2693 effect was totally blocked by pre-treatment with the specific P2Y₆ antagonist MRS 2578. The 20, 50, 100 represent on the x-axis concentration of the agonist/antagonist. * statistically significant relative to untreated. # statistically significant relative to reduction of infection index. Data represent mean and SEM of three independent experiments. * p < 0,05; **,^## p < 0.001; ***, ^### p < 0.0001.</p

Effect of treatment with nucleotide agonists of P2Y receptors in the production of nitric oxide (NO), reactive oxygen species (ROS) and cell death by <i>Toxoplasma gondii</i>-infected macrophages.

<p>Mouse peritoneal macrophages were kept uninfected or were infected with <i>T</i>. <i>gondii</i> tachyzoites (at a 5:1 ratio of tachyzoites to host cells) for 2 h, and then treated with 100 μM of ATP, UTP or UDP during 30–90 min. Then, cell supernatants were analyzed for the levels of the inflammatory mediator NO 30 min later (A), and cells were analyzed for ROS production (B), using the Griess reagent method (indirect NO quantification via nitrite level measurements) and dihydroethidium (DHE) fluorescence, respectively. Neither nucleotide treatment nor infection resulted in statistically significant changes in NO levels (A). As expected, 1mM ATP increased ROS production by macrophages, although this effect was significantly less pronounced in infected cells (B). In contrast, other nucleotide treatments did not alter ROS production by uninfected or <i>T</i>. <i>gondii</i>-infected macrophages (B). Necrotic cell death was analyzed by measuring lactate dehydrogenase (LDH) activity in culture supernatants harvested 4h after nucleotide treatment (C). Treatment with 0.1% Triton X-100 was used as a positive control for necrosis. Nucleotide treatment did no induce necrosis in uninfected or <i>T</i>. <i>gondii</i>-infected cells. Apoptotic cell death was analyzed by flow cytometry using ethidium bromide, to identify cells with < 2C DNA content (D). As a positive control for apoptosis induction, cells were treated with 5 μM staurosporine (STP) for 24 h before analyze. Treatment with nucleotides for 12 h did not induced apoptosis in uninfected or <i>T</i>. <i>gondii</i>-infected macrophages. Data represent mean and SEM of three independent experiments.</p

Scanning Electron Microscopy (SEM) of <i>Toxoplasma gondii</i>-infected macrophages treated with UTP or UDP.

<p>Mouse peritoneal infected-macrophages treated with UTP or UTP for 15 minutes were incubated with 0.1% triton X-100 for 2 min before fixation for SEM (A-C), to remove the host cell plasma membrane, or dry-cleaved with adhesive tape (D-F), to expose the cytoplasm containing parasites. (A) the micrograph shows no visible parasite out of parasitophorus vacuoles. Figs (B) and (C) show parasites interacting with cytoplasmic structures. (D) the micrograph shows a parasite inside a parasitophorous vacuole, and interacting with the intravacuolar network (arrow), as expected during normal infection. Figs (E) and (F) shows egressing parasite from UTP- and UDP-treated cells, respectively, with extruded conoid structure, typical of parasites in active egress.</p

<i>Toxoplasma gondii</i> tachyzoites that egressed prematurely from nucleotide-treated cells are captured passively by host cells.

<p> Mouse peritoneal macrophages infected with tachyzoites at a 5:1 ratio were treated with 100 μM UTP (for 30 min) or 5 μM of the Ca²⁺ ionophore ionomycin (for 15 minutes). After nucleotide or ionophore treatment, prematurely egressed parasites were recovered from culture supernatants and allowed to interact with freshly harvested peritoneal macrophages, for 2 hours. Samples were examined either immediately (A and B) or 24 hours after treatment with cytochalasin D (C). The % of infected cells (A and C) and the infection index (B) were then estimated by light microscopy analysis. (D) Egressed parasites were allowed to interact with fibroblast cultures for 2 hours, and then the infection index was estimated by light microscopy analysis. * p < 0.05; ** p < 0.001; *** p < 0.0001, relative to samples “Fresh”. ## p < 0.001; ### p < 0.0001, relative to samples not treated with cytochalasin D. @ p<0.05; @@ p<0.001, for UTP vs Ca²⁺ ionophore treatments.</p