7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Professor

Neutrophils sensitize Klebsiella pneumoniae for antibiotic-mediated bacterial killing

Infection Immunology

Introduction: Multidrug-resistant bacteria such as K. pneumoniae represent important health threats due inefficacy of antibiotics. First line innate immune defense through neutrophils plays a crucial role in the resolution of infections. However, the contribution of the immune system to effective antibiotic therapy remains unappreciated and hard-to-measure.Goals: Here we hypothesized that neutrophils might enhance the effectiveness of antibiotic treatment against antibiotic-resistant K. pneumoniae.Methods: For this study, the Robert-Koch-Institut provided clinical isolates of K. pneumoniae. AST was performed and interpreted according to EUCAST criteria. Isolates were selected based on MICs. Neutrophils were isolated from healthy volunteers (ethics approval #2021-253). Phagocytosis was assessed with gentamycin protection assay. IL-8 was measured by ELISA. Expression of CD62L, CD66b and CD63 was analyzed by flow cytometry. Serum resistance was assessed in 50% normal human serum.Results: Neutrophils failed to kill clinical isolates of K. pneumoniae. However, their presence synergistically increased the effect of meropenem and cefotaxime on bacterial killing. This effect was specific for cell wall-active antibiotics, e.g. 3rd generation cephalosporins and carbapenems and was not obtained with tigecycline. It was further dependent on the strain and on the respective MIC. Of note, the synergy of neutrophils and antibiotics enables killing of bacteria tested as intermediate and resistant. It did not correlate with susceptibility to complement-mediated killing nor expression of activation markers on neutrophils. Moreover, most strains were not phagocytosed, suggesting an extracellular mechanism underlying the observed synergy.Summary: Although K. pneumoniae was resistant to neutrophil killing, our data suggest that these cells can play a role in control of the infection and bacterial killing in the presence of cell wall-targeting antibiotics. Further experiments will elucidate the cellular events supporting K. pneumoniae killing.Funding: European Commission Innovative Training Network (ITN) Training towards Innovative Personalized Antibiotic Therapy (TIPAT).

Oral Presentation

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals, and that single nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs), but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33&quot;s influence on antibacterial defense was dependent on housing conditions of the mice, and mediated by the modulatory effect of IL-33 on the microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We identify a mechanism, dependent on both genetic and environmental factors, that impacts the efficacy of antibacterial immune defense and thus susceptibility to pneumonia.

Deubiquitinating enzyme OTUB1 is essential for intracellular control of Salmonella Typhimurium in macrophages during infection.

Objectives: The bacterium Salmonella Typhimurium (STm) causes non-typhoidal gastrointestinal tract disease upon ingestion of contaminated food or water. STm first invades intestinal epithelial cells and then infects basolaterally located macrophages. Macrophages play an important role in the control of the pathogen but also serve as a niche for intracellular bacterial growth. Invasion and replication within the macrophages trigger activation of pro-inflammatory signaling pathways, which are essential for anti-bacterial responses. These pathways are tightly regulated by posttranslational modifications including ubiquitination/deubiquitination. The deubiquitinating enzyme (DUB) OTUB1 is one such versatile DUB, which preferentially cleaves K48 ubiquitin chains from substrates or can bind to E2 ubiquitin-conjugating enzymes to prevent the ubiquitination of the substrate and thereby regulate signaling. The objective of this project is to study the role of OTUB1 in macrophages during Salmonella infection.Methods: We generated conditional OTUB1-deficient mice (LysM-Cre OTUB1fl/fl) with deletion of OTUB1 in macrophages. LysM-Cre OTUB1fl/fl and control OTUB1fl/fl mice were orally infected with SL1344 wild-type STm. For in vitro infection, OTUB1-deficient and -competent bone marrow-derived macrophages (BMDM) were used.Results: Upon oral infection with STm, LysM-Cre OTUB1fl/fl mice harbored higher bacterial load and showed increased inflammation in the spleen and liver compared to the OTUB1fl/fl mice. In concurrence, OTUB1-deficient BMDMs were unable to control STm in vitro. The impaired control of Salmonella in-OTUB1-deficient BMDM was paralleled by impaired activation of protective pro-inflammatory NF-κB and MAPK signaling and decreased production of anti-bacterial nitric oxide (NO). Mechanistically, OTUB1 removes the K48-like ubiquitin chain, stabilizes the E2-conjugating enzymes UBC13 and UBCH5c, and thereby fosters NF-KB and MAPK-mediated production of antibacterial NO in macrophages.Conclusions: These data identify OTUB1 as a critical regulator of UBC13 and UBCH5c mediated anti-STm response in macrophages.

Prof.Dr.

Temporal single-cell RNA sequencing captures macrophage reprogramming by Salmonella

While macrophages are well-equipped to track and kill foreign invaders, they paradoxically constitute a replicative niche for several intracellular pathogens. For instance, the intracellular Gram-negative bacteria Salmonella enterica serovar Typhimurium can turn infected macrophages into a favourable intracellular niche by secreting bacterial proteins effectors within the host cytoplasm. Typically, macrophages induce a pro-inflammatory program (coined M1) upon bacterial invasion to control and kill the bacteria. However, Salmonella can reverse macrophage polarization towards an anti-inflammatory state (so-called M2) permissive to bacterial survival and replication. The understanding of the transcriptional cascade leading to this reprogramming on the macrophage side remains unknown. Here, we leveraged temporal single-cell RNA-sequencing and RNA metabolic labelling to resolve the transcriptomic response of mouse bone marrow-derived macrophages during the first hours following Salmonella challenge. The initial and stereotypical response driving infected macrophages to M1 polarization was characterized by the pathogen detection and the strong induction of NF-κB signaling. Heterogeneity emerged in the macrophage population at 6 hours following infection, with some infected cells switching to the M2 phenotype. This critical point determined the final polarization state of macrophages. The M2 macrophage population was marked by a clear dampening of the initial inflammatory response and the induction of specific gene modules, concurrently to the activation of the Salmonella pathogenicity island 2 (SPI2). We identified transcription factors marking the initiation of the reprogramming trajectory and correlated to the M2 polarization even at later timepoints. Overall, our work highlights a weak point in macrophage wiring exploited by a pathogen to overcome cellular immunity and induce host cell reprogramming.

PD. Dr.

Prof. Dr. med.

Dietary L-arginine supplementation cures chronic L. mexicana infection

Control of Leishmania (L.) requires IFNγ-dependent type 2 nitric oxide (NO) synthase (NOS2), an enzyme that converts L-arginine into citrulline and NO. NOS2 activity is counteracted by arginase (ARG) 1 and 2, which are induced by Th2 cytokines and cleave L-arginine into urea and ornithine, a precursor of polyamines necessary for cell proliferation. Recently, we observed that the expression of ARG1 steadily increased in L. mexicana-infected BALB/c and C57BL/6 mice during disease manifestation. Here, we studied the functional role of host cell arginases during L. mexicana induced chronic cutaneous leishmaniasis (CL).L.mexicana infected C57BL/6 WT mice developed non-healing chronic CL, whereas mice with deletion of ARG1 in monocytes and macrophages (Arg1 ΔCx3cr1) exhibited a strongly reduced pathology and ultimately resolved their skin lesions despite parasite persistence. Mice lacking IL-10 in CD4+ T cells (Il10ΔCd4) revealed IL-10 as a factor inducing ARG1 during infection. Flow cytometry and scRNAseq analyses of skin lesions showed an enrichment of dermal myeloid subpopulations in WT mice, including a dominant cluster of ARG1+NOS2+ inflammatory macrophages that developed from recruited monocytes in an ARG1- and IFNγ-dependent manner. These macrophages expressed high levels of CXCL9/10, thereby entertaining a self-perpetuating cycle of inflammation, and served as a replicative niche for the parasites, because ARG1 restricted NOS2-mediated killing due to substrate competition. Additionally, the accumulation of ARG1+NOS2+ macrophages led to a long-lasting depletion of L-arginine in the skin as measured by metabolomics, which was not observed in ARG1-deficient healer mice. Based on this finding we treated infected mice with dietary L-arginine via the drinking water. Oral supplementation with L-arginine was able to prevent chronic CL in a prophylactic setting. Most strikingly, infected sick WT mice could be cured by the L-arginine treatment in a therapeutic setting.Thus, metabolic reprogramming by L-arginine can resolve chronic skin inflammation in L. mexicana infection, which is induced and maintained by an ARG1-dependent pathogenic differentiation of monocytes.

Dendritic cell specific deletion of OTUD7b protects mice from experimental cerebral malaria

Cerebral malaria (CM) is a severe life-threatening neurological complication of human malaria. Studies in murine experimental cerebral malaria (ECM) have shown that dendritic cells (DCs) prime CD8+ T cells, which migrate to the brain and cause disturbance of the blood-brain-barrier (BBB). The intrinsic mechanisms leading to DC survival permitting the development of CD8+ T cell-mediated immunopathology are yet incompletely understood. The pleiotropic cytokine TNF, which is highly expressed in both CM and ECM, regulates both cell survival and cell death depending on the ubiquitination status of signaling molecules. However, our molecular understanding of the highly dynamic processes of ubiquitination and deubiquitination is still fragmentary. Here, we newly identified that the ovarian tumor deubiquitinating enzyme 7b (OTUD7b) prevents TNF-induced apoptosis of DCs, thereby enabling efficient priming of pathogen-specific CD8+ T cells and development of ECM. Mechanistically, OTUD7b stabilizes TRAF2 in both human and murine DCs by preventing its K48-ubiquitination and proteasomal degradation. TRAF2 in turn facilitates K63-linked polyubiquitination of RIPK1, which mediates (i) activation of NF-kB and MAP kinases, (ii) production of IL-12, and (iii) expression of anti-apoptotic molecules cFLIP and Bcl-xL. Consequently, mice with DC-specific OTUD7b-deficiency were protected from ECM due to DC apoptosis and a failure to induce CD8+ T cell-mediated brain pathology, Taken together our study identifies OTUD7b as a molecular switch deciding on DC survival and death in both human and murine DC and qualifies it as a potential therapeutic target to manipulate DC responses.

Oral Session
Infection Immunology

Appointment

Chair

Topic

Programme

Oral SessionInfection Immunology

Appointment

Chair

Topic

Programme

Oral Session
Infection Immunology