Of Mice and Men: Studying Innate and Adaptive Immunity Against the Epstein-Barr Virus

The Epstein-Barr virus (EBV) is a lymphotropic -herpes virus infecting over 90% of the human adult population. A striking feature that the virus shares with other -herpes viruses is its oncogenic potential. This transforming property can be observed as B cell transformation in vitro and lymphomas as well as epithelial cancers in vivo, but most immunocompetent individuals control EBV infection successfully without the occurrence of disease. Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56brightCD16- NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. In fact, 100-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection against EBV-mediated B cell transformation in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The lack of an animal model of EBV infection prevents assignment of a protective value to immune subsets in vivo. We generated a small animal model that can be infected with EBV by reconstituting NOD-scid c -/- mice with CD34+ hematopoietic stem cells. We demonstrated that primary T cell responses in these humanized mice control infection with EBV. These T cell responses were HLA restricted and partially specific for EBV derived peptides. In HLA-A2 transgenic animals T cell responses against lytic EBV antigens dominated over recognition of latent EBV antigens during early phases of infection similarly to human EBV carriers. This mouse model recapitulates features of symptomatic primary EBV infection, and generates T cell mediated immune control that resists oncogenic transformation. We were also able to demonstrate that humanized mice develop functional human NK cells, this will allow us now to study the contributions of NK cells to innate immune control of EBV in vivo in the future

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-γ

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56brightCD16− NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen

PrsA2 (CD630_35000) of Clostridioides difficile Is an Active Parvulin-Type PPIase and a Virulence Modulator

Clostridioides difficile is the main cause for nosocomial antibiotic associated diarrhea and has become a major burden for the health care systems of industrial countries. Its main virulence factors, the small GTPase glycosylating toxins TcdA and TcdB, are extensively studied. In contrast, the contribution of other factors to development and progression of C. difficile infection (CDI) are only insufficiently understood. Many bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) have been described in the context of virulence. Among them are the parvulin-type PrsA-like PPIases of Gram-positive bacteria. On this basis, we identified CD630_35000 as the PrsA2 homolog in C. difficile and conducted its enzymatic and phenotypic characterization in order to assess its involvement during C. difficile infection. For this purpose, wild type CdPrsA2 and mutant variants carrying amino acid exchanges mainly in the PPIase domain were recombinantly produced. Recombinant CdPrsA2 showed PPIase activity toward the substrate peptide Ala-Xaa-Pro-Phe with a preference for positively charged amino acids preceding the proline residue. Mutation of conserved residues in its active site pocket impaired the enzymatic activity. A PrsA2 deficient mutant was generated in the C. difficile 630Δerm background using the ClosTron technology. Inactivation of prsA2 resulted in a reduced germination rate in response to taurocholic acid, and in a slight increase in resistance to the secondary bile acids LCA and DCA. Interestingly, in the absence of PrsA2 colonization of mice by C. difficile 630 was significantly reduced. We concluded that CdPrsA2 is an active PPIase that acts as a virulence modulator by influencing crucial processes like sporulation, germination and bile acid resistance resulting in attenuated mice colonization

Characterization of serum biomarkers and antibody responses against Prevotella spp. in preclinical and new-onset phase of rheumatic diseases

IntroductionThe characterization of the influence of the microbiota on the development and drug responses during rheumatic diseases has intensified in recent years. The role of specific bacteria during disease development has become a central research question. Notably, several lines of evidence point to distinct microbes, e.g., Prevotella copri (P. copri) being targeted by antibodies in clinical phases of rheumatic diseases.MethodsIn the present study, we compiled a broad collection of human serum samples from individuals at risk of developing RA, chronic RA patients as well as patients with new-onset of rheumatic diseases. We evaluated the presence of inflammatory biomarkers in our serum collection as well as serum antibody responses against novel, genetically distinct isolates of P. copri and several oral pathobionts.ResultsOur analysis revealed the presence of increased levels of inflammatory markers already in pre-clinical and new onset rheumatoid arthritis. However, antibody reactivity against the microbes did not differ between patient groups. Yet, we observed high variability between the different P. copri strains. We found total serum IgG levels to slightly correlate with IgG antibody responses against P. copri, but no relation between the latter and presence or prevalence of P. copri in the intestine.DiscussionIn conclusion, our work underlined the importance of strain-level characterization and its consideration during further investigations of host-microbiota interactions and the development of microbiome-based therapeutic approaches for treating rheumatic diseases

Caspase-11 Activation in Response to Bacterial Secretion Systems That Access the Host Cytosol

Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense

Transgenic expression of human signal regulatory protein alpha in Rag2−/−γc −/− mice improves engraftment of human hematopoietic cells in humanized mice

Transplantation of human hematopoietic stem cells into severely immunocompromised newborn mice allows the development of a human hematopoietic and immune system in vivo. NOD/scid/γc−/− (NSG) and BALB/c Rag2−/ −γc−/− mice are the most commonly used mouse strains for this purpose and a number of studies have demonstrated the high value of these model systems in areas spanning from basic to translational research. However, limited cross-reactivity of many murine cytokines on human cells and residual host immune function against the xenogeneic grafts results in defective development and maintenance of human cells in vivo. Whereas NSG mice have higher levels of absolute human engraftment than similar mice on a BALB/c background, they have a shorter lifespan and NOD ES cells are unsuitable for the complex genetic engineering that is required to improve human hematopoiesis and immune responses by transgenesis or knockin of human genes. We have generated mice that faithfully express a transgene of human signal regulatory protein alpha (SIRPa), a receptor that negatively regulates phagocytosis, in Rag2−/−γc−/− mice on a mixed 129/BALB/c background, which can easily be genetically engineered. These mice allow significantly increased engraftment and maintenance of human hematopoietic cells reaching levels comparable to NSG mice. Furthermore, we found improved functionality of the human immune system in these mice. In summary, hSIRPa-transgenic Rag2−/−γc−/− mice represent a unique mouse strain supporting high levels of human cell engraftment, which can easily be genetically manipulated

The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure

Extension of the Segatella copri complex to 13 species with distinct large extrachromosomal elements and associations with host conditions

The Segatella copri (formerly Prevotella copri) complex (ScC) comprises taxa that are key members of the human gut microbiome. It was previously described to contain four distinct phylogenetic clades. Combining targeted isolation with large-scale metagenomic analysis, we defined 13 distinct Segatella copri-related species, expanding the ScC complex beyond four clades. Complete genome reconstruction of thirteen strains from seven species unveiled the presence of genetically diverse large circular extrachromosomal elements. These elements are consistently present in most ScC species, contributing to intra- and inter-species diversities. The nine species-level clades present in humans display striking differences in prevalence and intraspecies genetic makeup across human populations. Based on a meta-analysis, we found reproducible associations between members of ScC and the male sex and positive correlations with lower visceral fat and favorable markers of cardiometabolic health. Our work uncovers genomic diversity within ScC, facilitating a better characterization of the human microbiome

Imbalanced gut microbiota fuels hepatocellular carcinoma development by shaping the hepatic inflammatory microenvironment

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide, and therapeutic options for advanced HCC are limited. Here, we observe that intestinal dysbiosis affects antitumor immune surveillance and drives liver disease progression towards cancer. Dysbiotic microbiota, as seen in Nlrp6(-/-) mice, induces a Toll-like receptor 4 dependent expansion of hepatic monocytic myeloid-derived suppressor cells (mMDSC) and suppression of T-cell abundance. This phenotype is transmissible via fecal microbiota transfer and reversible upon antibiotic treatment, pointing to the high plasticity of the tumor microenvironment. While loss of Akkermansia muciniphila correlates with mMDSC abundance, its reintroduction restores intestinal barrier function and strongly reduces liver inflammation and fibrosis. Cirrhosis patients display increased bacterial abundance in hepatic tissue, which induces pronounced transcriptional changes, including activation of fibro-inflammatory pathways as well as circuits mediating cancer immunosuppression. This study demonstrates that gut microbiota closely shapes the hepatic inflammatory microenvironment opening approaches for cancer prevention and therapy. Steatohepatitis is a chronic hepatic inflammation associated with increased risk of hepatocellular carcinoma progression. Here the authors show that intestinal dysbiosis in mice lacking the inflammasome sensor molecule NLRP6 aggravates steatohepatitis and accelerates liver cancer progression, a process that can be delayed by antibiotic treatment.Peer reviewe

A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called 'Pig intestinal bacterial collection' (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota