Pathogenesis of chronic Immunosuppression induced by a defective Differentiation of dendritic cells after Sepsis

Sepsis ist verbunden mit der Entwicklung einer Immunsuppression und Organdysfunktion, die zu Multiorganversagen und dem Tod der Patienten führen kann. Die Komplikationen bei septischen Patienten umfassen eine Persistenz der Primärinfektion und eine erhöhte Anfälligkeit für Sekundärinfektionen. Dendritische Zellen, die äußerst potente Antigen-präsentierende Zellen darstellen, spielen eine Schlüsselrolle bei der Initiierung einer T-Helferzell-Antwort, welche essentiell für die effektive Abwehr von bakteriellen Infektionen ist. Während einer akuten Sepsis entwickeln DC in der Milz eine Dysfunktion, die sich auszeichnet durch eine verminderte Fähigkeit das TH1-polarisierende Zytokin IL-12 zu produzieren und so eine protektive TH1-Zell Antwort zu induzieren, während die Produktion des anti-inflammatorischen Zytokins IL-10 erhöht ist. Im Rahmen dieser Arbeit wurde mittels CLP-Modell, welches über die Freisetzung von Bakterien nach Ligation und Punktion des Darms eine Sepsis in Mäusen induziert, untersucht, ob eine fehlerhafte Differenzierung von DC aus dem Knochenmark die Dysfunktion von DC vermittelt und so die Entwicklung einer Immunsuppression während Sepsis begünstigt. Vier Tage nach Induktion einer subletalen Sepsis, wenn die Mäuse keine Anzeichen einer Erkrankung mehr zeigten, bestand eine anhaltende Immunsuppression der Mäuse, charakterisiert durch eine erhöhte Anfälligkeit für P. aeruginosa in Kombination mit einem veränderten Lungen-Zytokin-Profil. Die DC aus der Milz zeigten eine Dysfunktion, die sich in einer verminderten IL-12 Produktion bei gleichzeitig erhöhter IL-10 Expression äußerte. BMDC, differenziert aus dem Knochenmark von septischen Versuchstieren, sezernierten nach Stimulation mit bakteriellen Komponenten vermehrt IL-10 und hemmten eine Polarisierung von TH1-Zellen in vivo. In Abwesenheit von IL-10 oder NK-Zellen in vivo konnte eine TH1-Hemmung vermindert werden. In vitro zeigten die DC aus dem Knochenmark septischer Tiere eine Suppression der IFN-gamma Produktion von naiven NK-Zellen. Adoptiver Transfer von DC aus septischen Tieren vermittelte weiterhin eine erhöhte Anfälligkeit gegenüber Pseudomonas in naiven Tieren. Eine subletale Sepsis induziert demnach eine abweichende Differenzierung von DC aus dem Knochenmark, eventuell vermittelt durch eine Veränderung der zellulären Zusammensetzung des Knochenmarks. So entstehen dysfunktionale DC, die lymphoide und nicht-lymphoide Organe besiedeln, eine protektive TH1-Antwort hemmen, und eine Immunsuppression vermitteln können. Eine effektive Immunabwehr während und nach einer Sepsis wird verhindert. Strategien, die zur Aufhebung der Dysfunktionen von DC während einer Sepsis führen, könnten daher Patienten nach Überleben einer Sepsis vor tödlichen Sekundärinfektionen schützen.Sepsis is associated with the development of chronic immunosuppression and organ-dysfunction which may lead to multiple organ failure and death of the patient. Persistency of the primary infection and an enhanced susceptibility to secondary infections are complications of septic patients. Dendritic cells, the most potent antigen-presenting cells, play a key role in initiating a protective T helper (Th) cell response, which is essential to fend off bacterial infections. During acute sepsis splenic dendritic cells (DC) develop a dysfunction which is characterized by an impaired capacity to secrete the Th1-polarizing cytokine IL-12 and to induce a protective Th1 cell response, while the production of the anti-inflammatory cytokine IL-10 is enhanced. Within the scope of this thesis the release of bacteria after ligation and puncture of the cecum (CLP model) induced a sub lethal polymicrobial sepsis in mice. It was investigated whether sepsis mediates a defect in the differentiation of DC from progenitors in the bone marrow and thereby favors the development of immunosuppression. Four day after CLP when mice recovered from sepsis a sustained immunosuppression existed. This comprised an enhanced susceptibility to P. aeruginosa and a modified cytokine pattern of the lung of septic animals. DC from the spleen of septic mice displayed a dysfunction which was characterized by a reduced expression of IL-12 and an enhanced production of IL-10. After stimulation with bacterial components bone marrow-derived dendritic cells (BMDC) from septic animals released extended levels of IL-10 and showed a reduced capacity to promote Th1-cell polarization in vivo. This inhibition of Th1 cells was counteracted by the absence of NK cells in vivo. In vitro BMDC from septic mice suppressed the IFN-gamma production of naive NK cells. Adoptive transfer of DC from septic animals mediated an enhanced susceptibility of naive mice to P. aeruginosa. Thus, sepsis induces a reprogramming of DC precursors in the bone marrow, possibly by an altered cellular composition of the bone marrow, towards dysfunctional DC that repopulate lymphoid and non-lymphoid organs, impede a protective Th1 response and mediate immunosuppression. A favorable immune response is thereby avoided. Restoration of the differentiation of competent dendritic cells during or after sepsis might allow an effective therapy to protect post-septic patients from the risks of secondary infections

Combination immunotherapy with anti-PD-L1 antibody and depletion of regulatory T cells during acute viral infections results in improved virus control but lethal immunopathology

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied

Treatment with Helicobacter pylori-derived VacA attenuates allergic airway disease

BACKGROUND: Asthma is an incurable heterogeneous disease with variations in clinical and underlying immunological phenotype. New approaches could help to support existing therapy concepts. Neonatal infection of mice with Helicobacter pylori or administration of H. pylori-derived extracts or molecules after birth have been shown to prevent the development of allergic airway disease later in life. This study evaluated the potential therapeutic efficacy of H. pylori vacuolating cytotoxin A (VacA) in allergic airway inflammation and investigated the underlying immunological mechanisms for its actions. METHODS: Murine models of allergic airway diseases, and murine and human in vitro models were used. RESULTS: In both an acute model and a therapeutic house dust mite model of allergic airway disease, treatment with H. pylori-derived VacA reduced several asthma hallmarks, including airway hyperresponsiveness, inflammation and goblet cell metaplasia. Flow cytometry and ELISA analyses revealed induction of tolerogenic dendritic cells (DC) and FoxP3 positive regulatory T cells (Tregs), and a shift in the composition of allergen-specific immunoglobulins. Depletion of Tregs during treatment with VacA reversed treatment-mediated suppression of allergic airway disease. Human monocyte derived DCs (moDC) that were exposed to VacA induced Tregs in co-cultured naïve autologous T cells, replicating key observations made in vivo. CONCLUSION: H. pylori-derived VacA suppressed allergic airway inflammation via induction of Tregs in both allergic airway disease models. These data suggest that the immunomodulatory activity of VacA could potentially be exploited for the prevention and treatment of allergic airway disease

Depletion of Foxp3(+) regulatory T cells is accompanied by an increase in the relative abundance of Firmicutes in the murine gut microbiome

A reciprocal interaction exists between the gut microbiota and the immune system. Regulatory T (Treg) cells are important for controlling immune responses and for maintaining the intestinal homeostasis but their precise influence on the gut microbiota is unclear. We studied the effects of Treg cell depletion on inflammation of the intestinal mucosa and analysed the gut microbiota before and after depletion of Treg cells using the DEpletion of REGulatory T cells (DEREG) mouse model. DNA was extracted from stool samples of DEREG mice and wild‐type littermates at different time‐points before and after diphtheria toxin application to deplete Treg cells in DEREG mice. The V3/V4 region of the 16S rRNA gene was used for studying the gut microbiota with Illumina MiSeq paired ends sequencing. Multidimensional scaling separated the majority of gut microbiota samples from late time‐points after Treg cell depletion in DEREG mice from samples of early time‐points before Treg cell depletion in these mice and from gut microbiota samples of wild‐type mice. Treg cell depletion in DEREG mice was accompanied by an increase in the relative abundance of the phylum Firmicutes and by intestinal inflammation in DEREG mice 20 days after Treg cell depletion, indicating that Treg cells influence the gut microbiota composition. In addition, the variables cage, breeding and experiment number were associated with differences in the gut microbiota composition and these variables should be respected in murine studies

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells.

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely on SCF in vivo in some microenvironments, with potential implications for graft-versus-host disease and antitumor immunity

Interleukin-33 signaling exacerbates experimental infectious colitis by enhancing gut permeability and inhibiting protective Th17 immunity.

A wide range of microbial pathogens is capable of entering the gastrointestinal tract, causing infectious diarrhea and colitis. A finely tuned balance between different cytokines is necessary to eradicate the microbial threat and to avoid infection complications. The current study identified IL-33 as a critical regulator of the immune response to the enteric pathogen Citrobacter rodentium. We observed that deficiency of the IL-33 signaling pathway attenuates bacterial-induced colitis. Conversely, boosting this pathway strongly aggravates the inflammatory response and makes the mice prone to systemic infection. Mechanistically, IL-33 mediates its detrimental effect by enhancing gut permeability and by limiting the induction of protective T helper 17 cells at the site of infection, thus impairing host defense mechanisms against the enteric pathogen. Importantly, IL-33-treated infected mice supplemented with IL-17A are able to resist the otherwise strong systemic spreading of the pathogen. These findings reveal a novel IL-33/IL-17A crosstalk that controls the pathogenesis of Citrobacter rodentium-driven infectious colitis. Manipulating the dynamics of cytokines may offer new therapeutic strategies to treat specific intestinal infections

Intestinal helminth infection drives carcinogenesis in colitis-associated colon cancer

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract, strongly associated with an increased risk of colorectal cancer development. Parasitic infections caused by helminths have been shown to modulate the host’s immune response by releasing immunomodulatory molecules and inducing regulatory T cells (Tregs). This immunosuppressive state provoked in the host has been considered as a novel and promising approach to treat IBD patients and alleviate acute intestinal inflammation. On the contrary, specific parasite infections are well known to be directly linked to carcinogenesis. Whether a helminth infection interferes with the development of colitis-associated colon cancer (CAC) is not yet known. In the present study, we demonstrate that the treatment of mice with the intestinal helminth Heligmosomoides polygyrus at the onset of tumor progression in a mouse model of CAC does not alter tumor growth and distribution. In contrast, H. polygyrus infection in the early inflammatory phase of CAC strengthens the inflammatory response and significantly boosts tumor development. Here, H. polygyrus infection was accompanied by long- lasting alterations in the colonic immune cell compartment, with reduced frequencies of colonic CD8+ effector T cells. Moreover, H. polygyrus infection in the course of dextran sulfate sodium (DSS) mediated colitis significantly exacerbates intestinal inflammation by amplifying the release of colonic IL-6 and CXCL1. Thus, our findings indicate that the therapeutic application of helminths during CAC might have tumor-promoting effects and therefore should be well-considered