A Dual-Pathogen Mitral Valve Endocarditis Caused by Coxiella burnetii and Streptococcus gordonii-Which Came First?

Infective endocarditis (IE) is still a life-threatening disease with high morbidity and mortality. While usually caused by a single bacterium, poly-microbial infective endocarditis (IE) is rare. Here, we report a (blood-culture-negative) dual pathogen mitral valve IE caused by Coxiella burnetii and Streptococcus gordonii: A 53-year-old woman was presented to an internal medicine department with abdominal pain for further evaluation. Within the diagnostic work up, transthoracic echocardiography (TTE) revealed an irregularly shaped echogenic mass (5 × 13 mm) adherent to the edge of the posterior mitral valve leaflet and protruding into the left atrium. As infected endocarditis was suspected, blood cultures were initially obtained, but they remained negative. Chronic Q fever infection was diagnosed using serologic testing. After the occurrence of cerebral thromboembolic events, the patient was admitted for mitral valve surgery. Intraoperatively, a massively destructed mitral valve with adhering vegetations was noted. Examination of the mitral valve by broad-range bacterial polymerase chain reaction (PCR) and amplicon sequencing confirmed Coxiella burnetii infection and yielded Streptococcus gordonii as the second pathogen. Based on the detailed diagnosis, appropriate antibiotic therapy of both pathogens was initiated, and the patient could be discharged uneventfully on the 11th postoperative day after a successful minimal-invasive mitral valve replacement.

CD83 expressed by macrophages is an important immune checkpoint molecule for the resolution of inflammation.

Excessive macrophage (Mφ) activation results in chronic inflammatory responses or autoimmune diseases. Therefore, identification of novel immune checkpoints on Mφ, which contribute to resolution of inflammation, is crucial for the development of new therapeutic agents. Herein, we identify CD83 as a marker for IL-4 stimulated pro-resolving alternatively activated Mφ (AAM). Using a conditional KO mouse (cKO), we show that CD83 is important for the phenotype and function of pro-resolving Mφ. CD83-deletion in IL-4 stimulated Mφ results in decreased levels of inhibitory receptors, such as CD200R and MSR-1, which correlates with a reduced phagocytic capacity. In addition, CD83-deficient Mφ upon IL-4 stimulation, show an altered STAT-6 phosphorylation pattern, which is characterized by reduced pSTAT-6 levels and expression of the target gene Gata3. Concomitantly, functional studies in IL-4 stimulated CD83 KO Mφ reveal an increased production of pro-inflammatory mediators, such as TNF-α, IL-6, CXCL1 and G-CSF. Furthermore, we show that CD83-deficient Mφ have enhanced capacities to stimulate the proliferation of allo-reactive T cells, which was accompanied by reduced frequencies of Tregs. In addition, we show that CD83 expressed by Mφ is important to limit the inflammatory phase using a full-thickness excision wound healing model, since inflammatory transcripts (e.g. Cxcl1, Il6) were increased, whilst resolving transcripts (e.g. Ym1, Cd200r, Msr-1) were decreased in wounds at day 3 after wound infliction, which reflects the CD83 resolving function on Mφ also in vivo. Consequently, this enhanced inflammatory milieu led to an altered tissue reconstitution after wound infliction. Thus, our data provide evidence that CD83 acts as a gatekeeper for the phenotype and function of pro-resolving Mφ.

STAT1 coordinates intestinal epithelial cell death during gastrointestinal infection upstream of Caspase-8.

Intestinal homeostasis and the maintenance of the intestinal epithelial barrier are essential components of host defense during gastrointestinal Salmonella Typhimurium infection. Both require a strict regulation of cell death. However, the molecular pathways regulating epithelial cell death have not been completely understood. Here, we elucidated the contribution of central mechanisms of regulated cell death and upstream regulatory components during gastrointestinal infection. Mice lacking Caspase-8 in the intestinal epithelium are highly sensitive towards bacterial induced enteritis and intestinal inflammation, resulting in an enhanced lethality of these mice. This phenotype was associated with an increased STAT1 activation during Salmonella infection. Cell death, barrier breakdown and systemic infection were abrogated by an additional deletion of STAT1 in Casp8ΔIEC mice. In the absence of epithelial STAT1, loss of epithelial cells was abolished which was accompanied by a reduced Caspase-8 activation. Mechanistically, we demonstrate that epithelial STAT1 acts upstream of Caspase-8-dependent as well as -independent cell death and thus might play a major role at the crossroad of several central cell death pathways in the intestinal epithelium. In summary, we uncovered that transcriptional control of STAT1 is an essential host response mechanism that is required for the maintenance of intestinal barrier function and host survival.

Bile Acids and Microbiota: Multifaceted and Versatile Regulators of the Liver-Gut Axis.

After their synthesis from cholesterol in hepatic tissues, bile acids (BAs) are secreted into the intestinal lumen. Most BAs are subsequently re-absorbed in the terminal ileum and are transported back for recycling to the liver. Some of them, however, reach the colon and change their physicochemical properties upon modification by gut bacteria, and vice versa, BAs also shape the composition and function of the intestinal microbiota. This mutual interplay of both BAs and gut microbiota regulates many physiological processes, including the lipid, carbohydrate and energy metabolism of the host. Emerging evidence also implies an important role of this enterohepatic BA circuit in shaping mucosal colonization resistance as well as local and distant immune responses, tissue physiology and carcinogenesis. Subsequently, disrupted interactions of gut bacteria and BAs are associated with many disorders as diverse as Clostridioides difficile or Salmonella Typhimurium infection, inflammatory bowel disease, type 1 diabetes, asthma, metabolic syndrome, obesity, Parkinson's disease, schizophrenia and epilepsy. As we cannot address all of these interesting underlying pathophysiologic mechanisms here, we summarize the current knowledge about the physiologic and pathogenic interplay of local site microbiota and the enterohepatic BA metabolism using a few selected examples of liver and gut diseases.

Arginase impedes the resolution of colitis by altering the microbiome and metabolome.

Arginase 1 (Arg1), which converts l-arginine into ornithine and urea, exerts pleiotropic immunoregulatory effects. However, the function of Arg1 in inflammatory bowel disease (IBD) remains poorly characterized. Here, we found that Arg1 expression correlated with the degree of inflammation in intestinal tissues from IBD patients. In mice, Arg1 was upregulated in an IL-4/IL-13- and intestinal microbiota-dependent manner. Tie2-Cre Arg1fl/fl mice lacking Arg1 in hematopoietic and endothelial cells recovered faster from colitis than Arg1-expressing (Arg1fl/fl) littermates. This correlated with decreased vessel density, compositional changes in intestinal microbiota, diminished infiltration by myeloid cells, and an accumulation of intraluminal polyamines that promote epithelial healing. The proresolving effect of Arg1 deletion was reduced by an l-arginine-free diet, but rescued by simultaneous deletion of other l-arginine-metabolizing enzymes, such as Arg2 or Nos2, demonstrating that protection from colitis requires l-arginine. Fecal microbiota transfers from Tie2-Cre Arg1fl/fl mice into WT recipients ameliorated intestinal inflammation, while transfers from WT littermates into Arg1-deficient mice prevented an advanced recovery from colitis. Thus, an increased availability of l-arginine as well as altered intestinal microbiota and metabolic products accounts for the accelerated resolution from colitis in the absence of Arg1. Consequently, l-arginine metabolism may serve as a target for clinical intervention in IBD patients.

The Role of Adaptor Proteins in the Biology of Natural Killer T (NKT) Cells.

Adaptor proteins contribute to the selection, differentiation and activation of natural killer T (NKT) cells, an innate(-like) lymphocyte population endowed with powerful immunomodulatory properties. Distinct from conventional T lymphocytes NKT cells preferentially home to the liver, undergo a thymic maturation and differentiation process and recognize glycolipid antigens presented by the MHC class I-like molecule CD1d on antigen presenting cells. NKT cells express a semi-invariant T cell receptor (TCR), which combines the Vα14-Jα18 chain with a Vß2, Vß7, or Vß8 chain in mice and the Vα24 chain with the Vß11 chain in humans. The avidity of interactions between their TCR, the presented glycolipid antigen and CD1d govern the selection and differentiation of NKT cells. Compared to TCR ligation on conventional T cells engagement of the NKT cell TCR delivers substantially stronger signals, which trigger the unique NKT cell developmental program. Furthermore, NKT cells express a panoply of primarily inhibitory NK cell receptors (NKRs) that control their self-reactivity and avoid autoimmune activation. Adaptor proteins influence NKT cell biology through the integration of TCR, NKR and/or SLAM (signaling lymphocyte-activation molecule) receptor signals or the variation of CD1d-restricted antigen presentation. TCR and NKR ligation engage the SH2 domain-containing leukocyte protein of 76kDa slp-76 whereas the SLAM associated protein SAP serves as adaptor for the SLAM receptor family. Indeed, the selection and differentiation of NKT cells selectively requires co-stimulation via SLAM receptors. Furthermore, SAP deficiency causes X-linked lymphoproliferative disease with multiple immune defects including a lack of circulating NKT cells. While a deletion of slp-76 leads to a complete loss of all peripheral T cell populations, mutations in the SH2 domain of slp-76 selectively affect NKT cell biology. Furthermore, adaptor proteins influence the expression and trafficking of CD1d in antigen presenting cells and subsequently selection and activation of NKT cells. Adaptor protein complex 3 (AP-3), for example, is required for the efficient presentation of glycolipid antigens which require internalization and processing. Thus, our review will focus on the complex contribution of adaptor proteins to the delivery of TCR, NKR and SLAM receptor signals in the unique biology of NKT cells and CD1d-restricted antigen presentation.

Friend or Foe? The Ambiguous Role of Innate Lymphoid Cells in Cancer Development.

The development of immunotherapies represents a major advance towards the effective eradication of malignant tumors. So far, therapeutic approaches have largely focused on T lymphocytes, but the innate arm of the immune system might be similarly important. Innate lymphoid cells (ILCs) are rapidly-responding cells that are functionally analogous to diverse T cell subsets. In recent years these cells have attracted enormous attention owing to their pleiotropic effects in early host defense to infection and organ pathologies. ILCs might also represent promising targets in the context of cancer therapy because they are an innate immune cell population endowed with potent immunomodulatory properties. In this review we discuss the impact of the three ILC subsets and the signature cytokines they release on cancer development and tumor growth.

Synthetic Lipoteichoic Acid Glycans Are Potential Vaccine Candidates to Protect from Clostridium difficile Infections.

Infections with Clostridium difficile increasingly cause morbidity and mortality worldwide. Bacterial surface glycans including lipoteichoic acid (LTA) were identified as auspicious vaccine antigens to prevent colonization. Here, we report on the potential of synthetic LTA glycans as vaccine candidates. We identified LTA-specific antibodies in the blood of C. difficile patients. Therefore, we evaluated the immunogenicity of a semi-synthetic LTA-CRM197 glycoconjugate. The conjugate elicited LTA-specific antibodies in mice that recognized natural LTA epitopes on the surface of C. difficile bacteria and inhibited intestinal colonization of C. difficile in mice in vivo. Our findings underscore the promise of synthetic LTA glycans as C. difficile vaccine candidates.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of ß2-microglobulin (ß2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-ß2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice.

Involvement of interleukin-18 in the pathogenesis of human eosinophilic esophagitis.

IL-18 is induced in food allergy and EoE is food allergen-induced disease. Therefore, we tested the hypothesis whether IL-18 is involved in food allergen-induced EoE pathogenesis. Accordingly, we examined normal SPT+ and SPT- EoE patient blood and biopsy samples for IL-18, IL-18Rα, ICAM and VCAM expression. Herein, we show increased IL-18 level is highly significant in food allergen SPT+ compared to SPT- EoE patients. We also report that IL-18Rα+ cells and mRNA levels are induced in the esophageal biopsies of EoE patients and blood IL-18 levels correlate with esophageal eosinophilia (P<0.01). Additionally, we report that the levels of esophageal eosinophil and mast cells correlate with ICAM expression in human EoE. Mechanistically, we show that IL-18 in vitro stimulates iNKT cells and endothelial cells and induce eosinophil active cytokines IL-5 and IL-13. We provide the evidence that IL-18 is critical cytokine involved in activation of iNKT cells and ICAM in promoting human EoE.

Cytokines, signaling pathways, and effector molecules required for the control of Leishmania (Viannia) braziliensis in mice.

Cutaneous leishmaniasis is caused by protozoan parasites of the genus Leishmania. The mechanisms of pathogen control have been established primarily in the mouse model of Leishmania major infection, but they might not hold true for other Leishmania species associated with cutaneous disease. Here, we analyzed the role of cytokines, signaling components, and effector molecules in the control of New World cutaneous leishmaniasis due to L. braziliensis. Unlike L. major, L. braziliensis caused small, nonulcerative, and self-healing skin swelling in C57BL/6 mice, as well as BALB/c mice. In contrast to the results obtained for L. mexicana, mice deficient for interleukin-12 or its key signaling molecule, signal transducer and activator of transcription 4, rapidly succumbed to severe visceral leishmaniasis. Infection of tumor necrosis factor knockout mice with L. braziliensis led to progressive, nonhealing skin lesions with erosions and hemorrhagic ulcerations, but in contrast to the results with L. major, only 20 to 30% of the mice developed fatal visceral disease. As seen with L. major, mice with a deleted inducible nitric oxide synthase gene (iNOS(-/-)) were unable to contain L. braziliensis in the skin, whereas the control of the parasite in the spleen remained unimpaired. Unlike what happens in L. major infections, NADPH oxidase had no impact on the course of disease in L. braziliensis-infected mice. These results not only define essential components of a protective immune response to L. braziliensis but also illustrate that the requirements for the control of cutaneous leishmaniasis vary between different parasite species.

A modified alpha-galactosyl ceramide for staining and stimulating natural killer T cells.

CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.

Lysosomal glycosphingolipid recognition by NKT cells.

NKT cells represent a distinct lineage of T cells that coexpress a conserved alphabeta T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking beta-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity.

The role of type I interferons in non-viral infections.

For a long time, the family of type I interferons (IFN-alpha/beta) has received little attention outside the fields of virology and tumor immunology. In recent years, IFN-alpha/beta regained the interest of immunologists, due to the phenotypic and functional characterization of IFN-alpha/beta-producing cells, the definition of novel immunomodulatory functions and signaling pathways of IFN-alpha/beta, and the observation that IFN-alpha/beta not only exerts antiviral effects but is also relevant for the pathogenesis or control of certain bacterial and protozoan infections. This review summarizes the current knowledge on the production and function of IFN-alpha/beta during non-viral infections in vitro and in vivo.

Protection against progressive leishmaniasis by IFN-beta.

Type I IFNs (IFN-alphabeta) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-beta protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (10(6)) or low (10(4)) number of Leishmania major parasites, respectively. IFN-beta treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-gamma and IL-12 in the draining lymph node. Low, but not high doses of IFN-beta caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-beta-induced increase of IFN-gamma production was dependent on STAT4. Protection by IFN-beta strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-beta led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-beta as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.

The control of Leishmania (Leishmania) major by TNF in vivo is dependent on the parasite strain.

Previous studies provided evidence that the role of TNF in the control of Leishmania (Leishmania) major might vary with the parasite strain. Here, we analyzed the development and outcome of cutaneous leishmaniasis in C57BL/6 wild-type (B6.WT) and TNF-deficient (B6. TNF(-/-)) mice infected with two different isolates of L. (L.) major (FRIEDLIN vs. BNI). L. (L.) major BNI caused progressive, fatal disease in B6.TNF(-/-) mice. In contrast, B6.TNF(-/-) mice infected with the L. (L.) major FRIEDLIN strain exhibited partial resistance characterized by chronic, non-healing skin lesions without lethality. Analysis of the tissue parasite numbers showed that the numbers of L. (L.) major FRIEDLIN and BNI parasites were comparable in footpads and lymph nodes of B6.TNF(-/-) mice, whereas in the spleen the parasite numbers were strikingly lower in the case of L. (L.) major FRIEDLIN. In vitro, cytokine-activated inflammatory macrophages killed L. (L.) major FRIEDLIN more efficiently than L. (L.) major BNI. These results suggest that in the absence of TNF, the course of leishmaniasis depends on the biology of the inoculated L. (L.) major strain, which most likely explains the previously published discrepant results on the role of TNF in leishmaniasis.