Microbiome Composition in Both Wild-Type and Disease Model Mice Is Heavily Influenced by Mouse Facility

Murine models have become essential tools for understanding the complex interactions between gut microbes, their hosts, and disease. While many intra-facility factors are known to influence the structure of mouse microbiomes, the contribution of inter-facility variation to mouse microbiome composition, especially in the context of disease, remains under-investigated. We replicated microbiome experiments using identical mouse lines housed in two separate animal facilities and report drastic differences in composition of microbiomes based upon animal facility of origin. We observed facility-specific microbiome signatures in the context of a disease model [the Ednrb (endothelin receptor type B) Hirschsprung disease mouse] and in normal C57BL/6J mice. Importantly, these facility differences were independent of cage, sex, or sequencing-related influence. In addition, we investigated the reproducibility of microbiome dysbiosis previously associated with Ednrb-/- (knock-out; KO) mice. While we observed genotype-based differences in composition between wild-type (WT) and KO mice, these differences were inconsistent with the previously reported conclusions. Furthermore, the genotype-based differences were not identical across animal facilities. Despite this, through differential abundance testing, we identified several conserved candidate taxa and candidate operational taxonomic units that may play a role in disease promotion or protection. Overall, our findings raise the possibility that previously reported microbiome-disease associations from murine studies conducted in a single facility may be heavily influenced by facility-specific effects. More generally, these results provide a strong rationale for replication of mouse microbiome studies at multiple facilities, and for the meticulous collection of metadata that will allow the confounding effects of facility to be more specifically identified

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

Plasmacytoid DC from Aged Mice Down-Regulate CD8 T Cell Responses by Inhibiting cDC Maturation after Encephalitozoon cuniculi Infection

Age associated impairment of immune function results in inefficient vaccination, tumor surveillance and increased severity of infections. Several alterations in adaptive immunity have been observed and recent studies report age related declines in innate immune responses to opportunistic pathogens including Encephalitozoon cuniculi. We previously demonstrated that conventional dendritic cells (cDC) from 9-month-old animals exhibit sub-optimal response to E. cuniculi infection, suggesting that age associated immune senescence begins earlier than expected. We focused this study on how age affects plasmacytoid DC (pDC) function. More specifically how aged pDC affect cDC function as we observed that the latter are the predominant activators of CD8 T cells during this infection. Our present study demonstrates that pDC from middle-aged mice (12 months) suppress young (8 week old) cDC driven CD8 T cell priming against E. cuniculi infection. The suppressive effect of pDC from older mice decreased maturation of young cDC via cell contact. Aged mouse pDC exhibited higher expression of PD-L1 and blockade of their interaction with cDC via this molecule restored cDC maturation and T cell priming. Furthermore, the PD-L1 dependent suppression of cDC T cell priming was restricted to effector function of antigen-specific CD8 T cells not their expansion. To the best of our knowledge, the data presented here is the first report highlighting a cell contact dependent, PD-L1 regulated, age associated defect in a DC subpopulation that results in a sub-optimal immune response against E. cuniculi infection. These results have broad implications for design of immunotherapeutic approaches to enhance immunity for aging populations

Possible activating receptor and NK cell subpopulation involvement in recognition of <i>T</i>. <i>gondii</i>-infected cells.

<p>The subpopulation of NK cells important for IFNγ-dependent protection, defined by specific activating (immunoreceptor tyrosine-based activating motif [ITAM]) receptors, is unknown. <b>A.</b> Infection of a target cell by <i>T</i>. <i>gondii</i> could induce stress, resulting in expression of ULBPs (Rae1, Mult1, or others), or alter self (MICA/B), Ly49-specific ligands, and/or NCR1 ligands (NCR1L, possible molecules vimentin or heparan sulfate glycoproteins [HSGP]). <b>B.</b> ULBPs or altered self-molecules would be recognized by NKG2D. Parasite-produced Ly49 ligands would be recognized by Ly49H or Ly49D. Host-derived NCR1 ligands would be recognized by NKp46 (NCR1). MHC Class I (MHCI) could be recognized by immunoreceptor tyrosine-based inhibitory motif (ITIM) receptors and SHP1/SHIP1 could impact signaling. <b>C.</b> NK cell-activating ligands that are recognized would activate the NK cell to produce cytokines (IFNγ), be cytotoxic, proliferate, and promote survival via signaling from either NKG2D-associated DAP10 or DAP12-dependent activation of Vav2/3/Sos1/PI3K or Syk/ZAP70/PI3K-dependent pathways, respectively, Ly49-associated DAP12-dependent activation of Syk/ZAP70/PI3K or NKp46-associated FcγR, and CD3ζ chain-dependent activation of Syk/ZAP70/PI3K signaling. Additional receptors not shown in figure include CD94/NKG2C, 2B4, FcRγIII, TRAIL, and IL-12R.</p

Multiple roles for NK cells during <i>T</i>. <i>gondii</i> infection.

<p>Natural killer (NK) cells function in different phases of immunity in response to parasite infection. <b>Step 1: Innate</b>. During the innate response, <i>T</i>. <i>gondii</i> infection stimulated production of inflammatory cytokines IL-1β, IFNα/β, IL-6, IL-12, IL-15, and IL-18, driving NK cell production of IFNγ. This results in early control of parasite infection by targeting intracellular parasites. IL-6 can stimulate NK cell IL-17 production. The importance of NK cell IL-17 is not well understood. Cytotoxic (CTL) response by NK cells is also induced; however, the importance of this function for control of acute parasite infection is not well known. Other factors important for NK cell responses include CD28, STAT4, Tbet, and NfκB family members (cRel, p50). Eomesodermin (Eomes) role is unclear. <b>Step 2: Regulation</b>. NK cells produce IL-10 and regulate innate responses by down-regulating IL-12 and possibly other cytokines. This is aryl hydrocarbon receptor (AHR)-dependent. Whether NK cell IL-10 can impact CD4 and CD8 T cell responses is not known. <b>Step 3: Adaptive</b>. NK cells can participate in adaptive immunity as memory-like cells. NK cells may be important for (2°) secondary <i>T</i>. <i>gondii</i> infections. Whether NK cells that experience <i>T</i>. <i>gondii</i> infection early live long-term or develop memory-like features and the mechanisms behind these cell-intrinsic fates are unknown.</p

Long-Term Immunity to Lethal Acute or Chronic Type II Toxoplasma gondii Infection Is Effectively Induced in Genetically Susceptible C57BL/6 Mice by Immunization with an Attenuated Type I Vaccine Strain▿

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice, regardless of the route of challenge infection. Remarkably, cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts, cps1-1 strain-elicited CD8+ immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition, we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively, these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain