Thomas Hehlgans

Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Human β-Defensin 2 and 3 and Their Mouse Orthologs Induce Chemotaxis through Interaction with CCR2

β-defensins play a dual role during immune response. Their direct antimicrobial properties contribute to the local innate immune response by combating microbial invasions. Furthermore, previous studies revealed the capacity of certain β-defensin family members to chemoattract immature dendritic cells and CD45RO+ CD4+ T cells through chemokine receptor CCR6. However, because β-defensins also chemoattract macrophages and monocytes, which do not express CCR6, efforts have been made to identify other receptors for these polypeptides. In this study, we demonstrate the capacity of human β-defensin (hBD)2 and 3 and their mouse orthologs, β-defensin 4 and 14, to interact with CCR2, a chemokine receptor expressed on monocytes, macrophages, and neutrophils. These β-defensins, fused to the Fc region of human IgG1, showed binding to CCR2-transfected HEK293 cells, as revealed by flow cytometry. The β-defensin fusion proteins also induced CCR2-specific chemotaxis of transfected HEK293 cells, human peripheral blood monocytes, and mouse peritoneal exudate cells in a dose-dependent manner. Preincubation of human monocytes with CCL2/MCP-1, the chemokine ligand for CCR2, abolished migration induced by β-defensins. Conversely, preincubation with hBD2:Ig or hBD3:Ig inhibited MCP-1 induced migration. Peritoneal exudate cells from CCR2-deficient mice failed to migrate toward these fusion proteins. In conclusion, the β-defensins used in this study contribute to the innate and adaptive immune response in their role as chemoattractants. Our data indicate that hBD2 and hBD3, together with their mouse orthologs (β-defensin 4 and 14), are chemotactic for a broad spectrum of leukocytes in a CCR6- and CCR2-dependent manner.

Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 μmol/L to 11.8 ± 2.8 μmol/L in all post-transplant samples and to 3.5 ± 3 μmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

The Lymphotoxin β Receptor Is Critically Involved in Controlling Infections with the Intracellular Pathogens Mycobacterium tuberculosis and Listeria monocytogenes

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-γ and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LTβR pathway. When mice deficient in LTα or LTβ were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LTαβ heterotrimers in resistance to infection. Indeed, mice deficient in the receptor for LTα1β2 heterotrimers (LTβR-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LTβR-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LTβR-KO mice had similar transcript levels of TNF and IFN-γ and recruited similar numbers of CD3+ T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LTβR is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LTβR-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LTα1β2 heterotrimers via the LTβR is an essential prerequisite for containment of intracellular pathogens.

Endothelial cell–specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation

Endothelial cell ablation of the lymphotoxin-β receptor results in failure to develop peripheral lymph nodes and normal high endothelial venues, which impairs lymphocyte homing.

Identification and Biological Characterization of Mouse beta-defensin 14, the orthologue of human beta-defensin 3.

β-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, it became evident that certain members of the β-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses by interacting with the CC-chemokine receptor CCR6. We have identified mouse β-defensin 14 (mBD14, Defb14) as an orthologue of human β-defensin 3 (hBD3 or DEFB103). Based on primary structural analysis, mBD14 demonstrates greater (68%) homology to its human orthologue, containing three conserved cystein linkages, characteristic for the β-defensin super family. mBD14 is expressed in a wide variety of tissues including spleen, colon, and tissues of the upper and lower respiratory tract. Interestingly, we also detected mBD14 expression in immature CD11c+ bone marrow-derived dendritic cells. The expression of mBD14 can be induced by Toll-like receptor agonists such as lipopolysaccharide and poly(I:C) and by pro-inflammatory stimuli e.g. tumor necrosis factor and interferon-γ. Furthermore, expression of mBD14 seems to be regulated by activation of the intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene analysis. We prepared a recombinant mBD14-Ig fusion protein that retained potent antimicrobial activity against several Escherichia coli strains but not against various Gram-positive Staphylococcus aureus strains. hBD3 and also the newly identified mBD14 were chemotactic for cells expressing the mouse CC-chemokine receptor CCR6. In addition, both hBD3 and mBD14 were chemotactic for freshly isolated mouse resident peritoneal cells. Thus, mBD14, based on structural and functional similarities, appears to be an orthologue of hBD3.

Lymphotoxin-β Receptor Activation by Activated T Cells Induces Cytokine Release from Mouse Bone Marrow-Derived Mast Cells

Lymphotoxin-β receptor (LTβR) signaling is known to play a key role in embryonic lymphoid organ formation as well as maintenance of lymphoid architecture. Activation of the LTβR is induced by either the heterotrimeric lymphotoxin-α1β2 (LTα1β2) or the homotrimeric LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV gpD for herpes virus entry mediator, a receptor expressed by T lymphocyte). Both ligands are expressed on activated lymphocytes. As mast cells reside in close proximity to activated T cells in some inflammatory tissues, we examined the expression of LTβR on bone marrow-derived mast cells and asked whether the LTβR-ligand interaction would allow communication between mast cells and activated T cells. We found that mast cells express LTβR at the mRNA as well as at the protein level. To investigate LTβR-specific mast cell activation, the LTβR on BMMC from either wild-type or LTβR-deficient mice was stimulated with recombinant mouse LIGHT or agonistic mAbs in the presence of ionomycin. LTβR-specific release of the cytokines IL-4, IL-6, TNF, and the chemokines macrophage inflammatory protein 2 and RANTES was detected. Moreover, coculture of mast cells with T cells expressing the LTβR ligands also entailed the release of these cytokines. Interference with a specific LTβR inhibitor resulted in significant suppression of mast cell cytokine release. These data clearly show that LTβR expressed on mast cells can transduce a costimulatory signal in T cell-dependent mast cell activation.

Mouse Aorta Smooth Muscle Cells Differentiate Into Lymphoid Tissue Organizer-Like Cells on Combined Tumor Necrosis Factor Receptor-1/Lymphotoxin β-Receptor NF-κB Signaling

Objective—Mouse aorta smooth muscle cells (SMC) express tumor necrosis factor receptor superfamily member 1A (TNFR-1) and lymphotoxin &bgr;-receptor (LT&bgr;R). Circumstantial evidence has linked the SMC LT&bgr;R to tertiary lymphoid organogenesis in hyperlipidemic mice. Here, we explored TNFR-1 and LT&bgr;R signaling in cultured SMC. Methods and Results—TNFR-1 signaling activated the classical RelA NF-&kgr;B pathway, whereas LT&bgr;R signaling activated the classical RelA and alternative RelB NF-&kgr;B pathways, and both signaling pathways synergized to enhance p100 inhibitor processing to the p52 subunit of NF-&kgr;B. Microarrays showed that simultaneous TNFR-1/LT&bgr;R activation resulted in elevated mRNA encoding leukocyte homeostatic chemokines CCL2, CCL5, CXCL1, and CX3CL1. Importantly, SMC acquired features of lymphoid tissue organizers, which control tertiary lymphoid organogenesis in autoimmune diseases through hyperinduction of CCL7, CCL9, CXCL13, CCL19, CXCL16, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1. TNFR-1/LT&bgr;R cross-talk resulted in augmented secretion of lymphorganogenic chemokine proteins. Supernatants of TNFR-1/LT&bgr;R–activated SMC markedly supported migration of splenic T cells, B cells, and macrophages/dendritic cells. Experiments with ltbr−/− SMC indicated that LT&bgr;R-RelB activation was obligatory to generate the lymphoid tissue organizer phenotype. Conclusion—SMC may participate in the formation of tertiary lymphoid tissue in atherosclerosis by upregulation of lymphorganogenic chemokines involved in T-lymphocyte, B-lymphocyte, and macrophage/dendritic cell attraction.

Tumor necrosis factor induces tumor necrosis via tumor necrosis factor receptor type 1-expressing endothelial cells of the tumor vasculature.

Activation of endothelial cells, fibrin deposition, and coagulation within the tumor vasculature has been shown in vivo to correlate with the occurrence of tumor necrosis factor (TNF)-induced tumor necrosis in mice. In the present study we investigated which target cells mediate the TNF-induced necrosis in fibrosarcomas grown in wild type (wt), TNF receptor type 1-deficient (TNFRp55-/-), and TNF receptor type 2-deficient (TNFRp75-/-) mice. TNF administration resulted in tumor necrosis exclusively in wt and TNFRp75-/-, but not in TNFRp55-/- mice, indicating a dependence of TNF-mediated tumor necrosis on the expression of TNF receptor type 1. However, using wt and TNFRp55-/- fibrosarcomas in wt mice, we found that TNF-mediated tumor necrosis was completely independent of TNF receptor type 1 expression in tumor cells. Thus we could exclude any direct tumoricidal effect of TNF in this model. Soluble TNF induced leukostasis in wt and TNFRp75-/- mice but not in TNFRp55-/- mice. TNF-induced leukostasis in TNFRp55-/- mice was restored by adoptive bone marrow transplantation of wt hematopoietic cells, but TNF failed to induce tumor necrosis in these chimeric mice. Because TNF administration resulted in both activation and focal damage of tumor endothelium, TNF receptor type 1-expressing cells of the tumor vasculature, likely to be endothelial cells, appear to be target cells for mediating TNF-induced tumor necrosis.

Specific Binding and Chemotactic Activity of mBD4 and Its Functional Orthologue hBD2 to CCR6-expressing Cells

β-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, members of the β-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses, which are in part mediated by the CC-chemokine receptor CCR6. Here we report the expression of recombinant mBD4 and its human orthologue hBD2 fused to the constant domain of human IgG1 to obtain correct folding and to increase stability and solubility using the Drosophila S2 expression system. Purified recombinant mBD4:Ig and hBD2:Ig fusion proteins retained potent antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, these β-defensin fusion proteins showed specific binding to CCR6-expressing cells as revealed by flow cytometry. Interestingly, although hBD2:Ig bound to both human and mouse CCR6-expressing cells, mBD4:Ig did only bind to mCCR6-expressing cells but not to hCCR6-expressing cells. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CC-chemokine receptor CCR6. The chemokine ligand CCL20 competed with the β-defensin fusion proteins for specific binding to CCR6 as analyzed by fluorescence-activated cell sorter analysis. Both β-defensin fusion proteins demonstrated chemotactic activity for cells expressing the mouse CCR6 receptor, but mBD4:Ig did not induce chemotactic activity of cells expressing human CCR6. This result supports our finding that mBD4 does not interact with human CCR6-expressing cells. Further evidence for specific interaction of the β-defensin fusion proteins with CCR6-expressing cells is demonstrated by the observation that CCL20 and β-defensin fusion proteins desensitize each other in inducing chemotactic activity. In addition both mBD4:Ig and hBD2:Ig demonstrated CCR6-independent chemotaxis of freshly isolated mouse resident peritoneal cells and human peripheral blood mononuclear cells, indicating the interaction with another chemotaxis-inducing receptor. Thus, the β-defensin fusion proteins used in this study retained their biological activity and are a feasible tool to identify and analyze specific β-defensin receptor interactions.