Comprehensive Kinetic Survey of Intestinal, Extra-Intestinal and Systemic Sequelae of Murine Ileitis Following Peroral Low-Dose Toxoplasma gondii Infection

We have recently shown that following peroral low-dose Toxoplasma gondii infection susceptible mice develop subacute ileitis within 10 days. Data regarding long-term intestinal and extra-intestinal sequelae of infection are scarce, however. We therefore challenged conventional C57BL/6 mice with one cyst of T. gondii ME49 strain by gavage and performed a comprehensive immunopathological survey 10, 36, and 57 days later. As early as 10 days post-infection, mice were suffering from subacute ileitis as indicated by mild-to-moderate histopathological changes of the ileal mucosa. Furthermore, numbers of apoptotic and proliferating/regenerating epithelial cells as well as of T and B lymphocytes in the mucosa and lamina propria of the ileum were highest at day 10 post-infection, but declined thereafter, and were accompanied by enhanced pro-inflammatory mediator secretion in ileum, colon and mesenteric lymph nodes that was most pronounced during the early phase of infection. In addition, subacute ileitis was accompanied by distinct shifts in the commensal gut microbiota composition in the small intestines. Remarkably, immunopathological sequelae of T. gondii infection were not restricted to the intestines, but could also be observed in extra-intestinal tissues including the liver, kidneys, lungs, heart and strikingly, in systemic compartments that were most prominent at day 10 post-infection. We conclude that the here provided long-term kinetic survey of immunopathological sequalae following peroral low-dose T. gondii infection provides valuable corner stones for a better understanding of the complex interactions within the triangle relationship of (parasitic) pathogens, the host immunity and the commensal gutmicrobiota during intestinal inflammation. The low-dose T. gondii infection model may be applied as valuable gut inflammation model in future pre-clinical studies in order to test potential treatment options for intestinal inflammatory conditions in humans

Toxoplasma co-infection prevents Th2 differentiation and leads to a helminth- specific Th1 response

Nematode infections, in particular gastrointestinal nematodes, are widespread and co-infections with other parasites and pathogens are frequently encountered in humans and animals. To decipher the immunological effects of a widespread protozoan infection on the anti-helminth immune response we studied a co-infection with the enteric nematode Heligmosomoides polygyrus in mice previously infected with Toxoplasma gondii. Protective immune responses against nematodes are dependent on parasite-specific Th2 responses associated with IL-4, IL-5, IL-13, IgE, and IgG1 antibodies. In contrast, Toxoplasma gondii infection elicits a strong and protective Th1 immune response characterized by IFN-γ, IL-12, and IgG2a antibodies. Co-infected animals displayed significantly higher worm fecundity although worm burden remained unchanged. In line with this, the Th2 response to H. polygyrus in co-infected animals showed a profound reduction of IL-4, IL-5, IL-13, and GATA-3 expressing T cells. Co-infection also resulted in the lack of eosinophilia and reduced expression of the Th2 effector molecule RELM-β in intestinal tissue. In contrast, the Th1 response to the protozoan parasite was not diminished and parasitemia of T. gondii was unaffected by concurrent helminth infection. Importantly, H. polygyrus specific restimulation of splenocytes revealed H. polygyrus-reactive CD4+ T cells that produce a significant amount of IFN-γ in co-infected animals. This was not observed in animals infected with the nematode alone. Increased levels of H. polygyrus-specific IgG2a antibodies in co-infected mice mirrored this finding. This study suggests that polarization rather than priming of naive CD4+ T cells is disturbed in mice previously infected with T. gondii. In conclusion, a previous T. gondii infection limits a helminth-specific Th2 immune response while promoting a shift toward a Th1-type immune response

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis. Methodology/Principal Findings Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti- inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro- inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner. Conclusion/Significance Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases

Ly6Chi Monocytes Provide a Link between Antibiotic-Induced Changes in Gut Microbiota and Adult Hippocampal Neurogenesis

Antibiotics, though remarkably useful, can also cause certain adverse effects. We detected that treatment of adult mice with antibiotics decreases hippocampal neurogenesis and memory retention. Reconstitution with normal gut flora (SPF) did not completely reverse the deficits in neurogenesis unless the mice also had access to a running wheel or received probiotics. In parallel to an increase in neurogenesis and memory retention, both SPF-reconstituted mice that ran and mice supplemented with probiotics exhibited higher numbers of Ly6Chi monocytes in the brain than antibiotic-treated mice. Elimination of Ly6Chi monocytes by antibody depletion or the use of knockout mice resulted in decreased neurogenesis, whereas adoptive transfer of Ly6Chi monocytes rescued neurogenesis after antibiotic treatment. We propose that the rescue of neurogenesis and behavior deficits in antibiotic-treated mice by exercise and probiotics is partially mediated by Ly6Chi monocytes

The Central Nervous System Contains ILC1s That Differ From NK Cells in the Response to Inflammation

Innate lymphoid cells (ILCs) are tissue resident cells with organ-specific properties. Here, we show that the central nervous system (CNS) encompasses ILCs. In particular, CD3-NK1.1+ cells present in the murine CNS comprise natural killer (NK) cells, ILC1s, intermediate ILC1s (intILC1s) and ex-ILC3s. We investigated the properties of CNS-ILC1s in comparison with CNS-NK cells during steady state and experimental autoimmune encephalomyelitis (EAE). ILC1s characteristically express CXCR3, CXCR6, DNAM-1, TRAIL, and CD200R and display heightened TNF-α production upon stimulation. In addition, ILC1s express perforin and are able to degranulate, although in a lesser extent than NK cells. Within the CNS compartments, ILC1s are enriched in the choroid plexus where very few NK cells are present, and also reside in the brain parenchyma and meninges. During EAE, ILC1s maintain stable IFN-γ and TNF-α levels while in NK cells the production of these cytokines increases as EAE progresses. Moreover, the amount of ILC1s and intILC1s increase in the parenchyma during EAE, but in contrast to NK cells, they show no signs of local proliferation. The upregulation in the inflamed brain of chemokines involved in ILC1 migration, such as CXCL9, CXCL10, and CXCL16 may lead to a recruitment of ILC1s from meninges or choroid plexus into the brain parenchyma. In sum, CNS-ILC1 phenotype, distribution and moderate inflammatory response during EAE suggest that they may act as gatekeepers involved in the control of neuroinflammation

Pituitary Adenylate Cyclase-Activating Polypeptide—A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD)

Innate Lymphoid Cells in Protection, Pathology, and Adaptive Immunity During Apicomplexan Infection

Apicomplexans are a diverse and complex group of protozoan pathogens including Toxoplasma gondii, Plasmodium spp., Cryptosporidium spp., Eimeria spp., and Babesia spp. They infect a wide variety of hosts and are a major health threat to humans and other animals. Innate immunity provides early control and also regulates the development of adaptive immune responses important for controlling these pathogens. Innate immune responses also contribute to immunopathology associated with these infections. Natural killer (NK) cells have been for a long time known to be potent first line effector cells in helping control protozoan infection. They provide control by producing IL-12 dependent IFNγ and killing infected cells and parasites via their cytotoxic response. Results from more recent studies indicate that NK cells could provide additional effector functions such as IL-10 and IL-17 and might have diverse roles in immunity to these pathogens. These early studies based their conclusions on the identification of NK cells to be CD3–, CD49b+, NK1.1+, and/or NKp46+ and the common accepted paradigm at that time that NK cells were one of the only lymphoid derived innate immune cells present. New discoveries have lead to major advances in understanding that NK cells are only one of several populations of innate immune cells of lymphoid origin. Common lymphoid progenitor derived innate immune cells are now known as innate lymphoid cells (ILC) and comprise three different groups, group 1, group 2, and group 3 ILC. They are a functionally heterogeneous and plastic cell population and are important effector cells in disease and tissue homeostasis. Very little is known about each of these different types of ILCs in parasitic infection. Therefore, we will review what is known about NK cells in innate immune responses during different protozoan infections. We will discuss what immune responses attributed to NK cells might be reconsidered as ILC1, 2, or 3 population responses. We will then discuss how different ILCs may impact immunopathology and adaptive immune responses to these parasites

Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

Background Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far. Methodology/Principal Findings Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra- intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals. Conclusion/Significance NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner