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Mack, M.

Publications and source records attributed to Mack, M..

32 records · Page 2Linked to original sources

RAG suppresses group 2 innate lymphoid cells

Antigen specificity is the central trait distinguishing adaptive from innate immune function. Assembly of antigen-specific T cell and B cell receptors occurs through V(D)J recombination mediated by the Recombinase Activating Gene endonucleases RAG1 and RAG2 (collectively called RAG). In the absence of RAG, mature T and B cells do not develop and thus RAG is critically associated with adaptive immune function. In addition to adaptive T helper 2 (Th2) cells, group 2 innate lymphoid cells (ILC2s) contribute to type 2 immune responses by producing cytokines like Interleukin-5 (IL-5) and IL-13. Although it has been reported that RAG expression modulates the function of innate natural killer (NK) cells, whether other innate immune cells such as ILC2s are affected by RAG remains unclear. We find that in RAG-deficient mice, ILC2 populations expand and produce increased IL-5 and IL-13 at steady state and contribute to increased inflammation in atopic dermatitis (AD)-like disease. Further, we show that RAG modulates ILC2 function in a cell-intrinsic manner independent of the absence or presence of adaptive T and B lymphocytes. Lastly, employing multiomic single cell analyses of RAG1 lineage-traced cells, we identify key transcriptional and epigenomic ILC2 functional programs that are suppressed by a history of RAG expression. Collectively, our data reveal a novel role for RAG in modulating innate type 2 immunity through suppression of ILC2s.

immunology↗

Mutation of the P. falciparum flavokinase confers resistance to roseoflavin and 8-aminoriboflavin

We previously found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilisation. To determine the mechanism of action of roseoflavin in P. falciparum, we generated roseoflavin-resistant parasites by in vitro evolution over 27 weeks. The roseoflavin-resistant parasites were found to be four times more resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Resistant parasites were subjected to whole genome sequencing and a missense mutation (T2015A), leading to an amino acid exchange (L672H), was detected in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin (vitamin B2) into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the observed phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene via a single-crossover recombination. The IC50 values for roseoflavin (RoF) and 8-aminoriboflavin against the RoF-resistant parasites created through in vitro evolution were indistinguishable from the IC50 values for parasites in which the missense mutation was specifically introduced into the native PfFK. To investigate this mutation, we generated two parasite lines episomally-expressing GFP-tagged versions of either the wild type or mutant forms of flavokinase. We found that PfFK-GFP localises to the parasite cytosol and that immunopurified PfFK-GFP was active and phosphorylated riboflavin into flavin mononucleotide. The L672H mutation caused a reduction of the binding affinity, especially for the substrate RoF, which explains the resistance phenotype. The mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonised by increasing the extracellular concentration of riboflavin, consistent with the compound also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite growth by inhibiting FMN production, in addition to the generation of toxic flavin cofactor analogues.

microbiology↗

Expansion of Disease Specific Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis

BackgroundImmune checkpoint inhibitors (ICIs), antibodies targeting PD-1/PD-L1 or CTLA4 have revolutionized cancer management but are associated with devastating immune-related adverse events (irAEs) including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI-myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. While much has been learned regarding the role of T-cells in ICI-myocarditis, little is understood regarding the identity, transcriptional diversity, and functions of infiltrating macrophages. MethodsWe employed an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization and molecular imaging and antibody neutralization studies. ResultsWe observed marked increases in CCR2+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9, Cxcl10, Gbp2b, and Fcgr4 that originated from CCR2+ monocytes. Importantly, a similar macrophage population expressing CXCL9, CXCL10, and CD16 (human homologue of mouse FcgR4) was found selectively expanded in patients with ICI myocarditis compared to other forms of heart failure and myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-{gamma} and CXCR3 signaling pathways. Depleting CD8+T-cells, macrophages, and blockade of IFN-{gamma} signaling blunted the expansion of Cxcl9+Cxcl10+macrophages in the heart and attenuated myocarditis suggesting that this interaction was necessary for disease pathogenesis. ConclusionThese data demonstrate that ICI-myocarditis is associated with the expansion of a specific population of IFN-{gamma} induced inflammatory macrophages and suggest the possibility that IFN-{gamma} blockade may be considered as a treatment option for this devastating condition.

immunology↗

IgG4 serum levels are not elevated in cases of Post-COVID syndrome

Recently, unexpectedly high virus-specific IgG4 levels were reported after more than two mRNA vaccinations. Class switch towards IgG4 occurs after long-term antigen exposure, downregulates immune responses and is associated with several autoimmune diseases. Here, we examined differences in antigen-specific IgG subtypes in serum samples from 64 Post-COVID patients and an equally sized cohort of convalescent controls. In both cohorts, the relative amounts of spike protein-specific IgG subtypes were comparable. IgG1 was the most frequent, followed by IgG3, IgG2, and IgG4. A difference between cohorts was observed only for IgG2, which was significantly lower in the Post-COVID cohort. Further analysis of the reactive IgG4 revealed a small but significant difference for the spike protein receptor-binding domain but not for the spike ectodomain. Since the total IgG4 levels are very low, we do not expect a biologically relevant role in Post-COVID syndrome. However, reduced virus-specific IgG2 levels could contribute to the persistence of SARS-CoV-2, causing chronic inflammation in the setting of Post-COVID syndrome.

immunology↗

Synthetic STING agonists elicit powerful vaccine adjuvancy providing robust central memory and anti-tumour effects

Drugs that target the innate immune sensor STING are known to be effective in modulating the immune infiltrate of the tumour microenvironment. STING agonists have potential to enhance responses to checkpoint inhibitor therapy, however, their ability to influence and shape adaptive immune responses is poorly understood. Here, we investigated the impact of a range of synthetic STING agonists on antigen specific CD8+ T-cell responses to soluble antigen using the murine OT-1 adoptive transfer model with Ovalbumin as the antigen to monitor T cell responses. Our data demonstrate that synthetic STING agonists are able to stimulate antigen specific T-cell expansion in response to challenge in mice. This effect required expression of STING, an intact myeloid compartment and Type-I IFN and TNF signalling. Expanded T-cells post treatment differed from those induced by the established immune adjuvant, anti-CD40 antibody through lower induction of the immune checkpoint receptor PD-1. Furthermore, our data revealed a marked increase in the induction and persistence of CD8+ central memory cells after STING agonist and antigen challenge. Finally, we demonstrate that following rechallenge, STING agonism produced larger secondary responses that could be translated into enhanced tumour protection and survival. Therefore, synthetic STING agonists are capable of acting as potent immune adjuvants and can induce robust memory formation leading to better recall and tumour control. Critically, these benefits along with the lower expression of PD-1, have implications for their use as adjuvants for multiple immunotherapy and vaccine applications.

immunology↗

Monocyte-derived peritoneal macrophages protect C57BL/6 mice against surgery-induced adhesions

Peritoneal adhesions commonly occur after abdominal or pelvic surgery. These scars join internal organs to each other or to the cavity wall and can present with abdominal or pelvic pain, and bowel obstruction or female infertility. The mechanisms underlying adhesion formation remain unclear and thus, effective treatments are not forthcoming. Peritoneal macrophages accumulate after surgery and previous studies have attributed either pro- or anti-scarring properties to these cells. We propose that there are complex and nuanced responses after surgery with respect to both resident and also monocyte-derived peritoneal macrophage subpopulations. Moreover, we contend that differences in responses of specific macrophage subpopulations in part explain the risk of developing peritoneal scars. We characterised alterations in peritoneal macrophage subpopulations after surgery-induced injury using two strains of mice, BALB/c and C57BL/6, with known differences in macrophage response post-infection. At 14 days post-surgery, BALB/c mice displayed more adhesions compared with C57BL/6 mice. This increase in scarring correlated with a lower influx of monocyte-derived macrophages at day 3 post-surgery. Moreover, BALB/c mice showed distinct macrophage repopulation dynamics after surgery. To confirm a role for monocyte-derived macrophages, we used Ccr2-deficient mice as well as antibody-mediated depletion of CCR2 expressing cells during initial stages of adhesion formation. Both Ccr2-deficient and CCR2-depleted mice showed a significant increase in adhesion formation associated with the loss of peritoneal monocyte influx. These findings revealed an important protective role for monocyte-derived cells in reducing adhesion formation after surgery.

immunology↗

Roseoflavin, a natural riboflavin analogue, possesses in vitro and in vivo antiplasmodial activity

The ability of the human malaria parasite Plasmodium falciparum to access and utilise vital nutrients is critical to its growth and proliferation. Molecules that interfere with these process could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilisation of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focussing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4-5 days. Our data are consistent with riboflavin metabolism and/or the utilisation of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.

pharmacology and toxicology↗

Targeting riboswitches with synthetic small RNAs for metabolic engineering

Our growing knowledge of the diversity of non-coding RNAs in natural systems and our deepening knowledge of RNA folding and function have fomented the rational design of RNA regulators. Based on that knowledge, we designed and implemented a small RNA (sRNA) tool to target bacterial riboswitches and activate gene expression. The synthetic sRNA is suitable for regulation of gene expression both in cell-free and in cellular systems. It targets riboswitches to promote the antitermination folding regardless the cognate metabolite concentration. Therefore, it prevents transcription termination increasing gene expression up to 103-fold. We successfully used sRNA arrays for multiplex targeting of riboswitches. Finally, we used the synthetic sRNA to engineer an improved riboflavin producer strain. The easiness to design and construct, and the fact that the riboswitch-targeting sRNA works as a single genome copy, make it an attractive tool for engineering industrial metabolite-producing strains. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/449321v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@11437b3org.highwire.dtl.DTLVardef@54e712org.highwire.dtl.DTLVardef@e657aorg.highwire.dtl.DTLVardef@1286b30_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Live imaging of SARS-CoV-2 infection in mice reveals neutralizing antibodies require Fc function for optimal efficacy

Neutralizing antibodies (NAbs) are effective in treating COVID-19 but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment in prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. We could visualize virus spread sequentially from the nasal cavity to the lungs and thereafter systemically to various organs including the brain, which culminated in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct Fab-mediated neutralization, Fc effector interactions of NAbs with monocytes, neutrophils and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2.

immunology↗

Global analysis of protein arginine methylation

Quantitative information about the levels and dynamics of post-translational modifications (PTMs) is critical for an understanding of cellular functions. Protein arginine methylation (ArgMet) is an important subclass of PTMs and is involved in a plethora of (patho)physiological processes. However, due to the lack of methods for global analysis of ArgMet, the link between ArgMet levels, dynamics and (patho)physiology remains largely unknown. We utilized the high sensitivity and robustness of Nuclear Magnetic Resonance (NMR) spectroscopy to develop a general method for the quantification of global protein ArgMet. Our NMR-based approach enables the detection of protein ArgMet in purified proteins, cells, organoids, and mouse tissues. We demonstrate that the process of ArgMet is a highly prevalent PTM and can be modulated by small-molecule inhibitors and metabolites and changes in cancer and during ageing. Thus, our approach enables to address a wide range of biological questions related to ArgMet in health and disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/428036v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@37a0f1org.highwire.dtl.DTLVardef@273ebdorg.highwire.dtl.DTLVardef@87fcd6org.highwire.dtl.DTLVardef@1e84c07_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease.

immunology↗

Cold Exposure Protects from Neuroinflammation Through Immunologic Reprogramming

Autoimmunity is energetically costly, but the impact of a metabolically active state on immunity and immune-mediated diseases is unclear. Ly6Chi monocytes are key effectors in CNS autoimmunity with elusive role in priming naive autoreactive T cells. Here we provide unbiased analysis of the immune changes in various compartments during cold exposure, and show that this energetically costly stimulus markedly ameliorates active experimental autoimmune encephalomyelitis (EAE). Cold exposure decreases MHCII on monocytes at steady-state and in various inflammatory mouse models, and suppresses T cell priming and pathogenicity through the modulation of monocytes. Genetic, or antibody-mediated monocyte depletion, or adoptive transfer of Th1- or Th17-polarized cells for EAE abolish the cold-induced effects on T cells or EAE, respectively. These findings provide a mechanistic link between environmental temperature and neuroinflammation, and suggest competition between cold-induced metabolic adaptations and autoimmunity as energetic trade-off beneficial for the immune-mediated diseases.

immunology↗

Gradual centriole maturation associates with the mitotic surveillance pathway in mouse development

Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4-/- embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4-/- embryos. The data indicates that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.

developmental biology↗

Macrophages can either inhibit or enhance endometriosis depending on their origin

Macrophages are intimately involved in the pathophysiology of endometriosis, a chronic inflammatory disorder characterized by the growth of endometrial-like tissue (lesions) outside the uterus. By combining genetic and pharmacological monocyte and macrophage depletion strategies we determined the ontogeny and function of macrophages in a mouse model of induced endometriosis. We demonstrate that lesion-resident macrophages are derived from eutopic endometrial tissue, infiltrating large peritoneal macrophages (LpM) and monocytes. Furthermore, we found endometriosis to trigger continuous recruitment of monocytes and expansion of CCR2+ LpM. Depletion of eutopic endometrial macrophages results in smaller endometriosis lesions, whereas constitutive inhibition of monocyte recruitment significantly reduces peritoneal macrophage populations and increased the number of lesions. We propose a putative model whereby endometrial macrophages are pro-endometriosis whilst newly-recruited monocyte-derived macrophages, possibly in LpM form, are anti-endometriosis. These observations highlight the importance of monocyte-derived macrophages in limiting disease progression.

immunology↗