Molecular and Functional Dynamics of Hematopoietic Progenitor Cell Fate During Ontogeny

Hematopoietic stem and progenitor cells (HSPCs) in the fetus and the adult possess distinct molecular signatures that regulate cell fate and change their susceptibility to initiation and progression of hematological malignancies, such as leukemia. It is well established that fetal and adult hematopoiesis are functionally distinct processes; however, the underlying molecular mechanisms that govern ontogenic differences in normal and malignant hematopoiesis have thus far been poorly explored on the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in HSPCs during ontogeny controls both the outcome of normal hematopoiesis and the susceptibility to initiation of leukemia, and that the proteomic make–up of the leukemia-initiating cell (LIC) has an instructive role in determining the pathogenic outcome of the resulting cancer. Here, we have addressed our hypothesis by implementing mass spectrometry (MS)-based quantitative proteomics in combination with advanced functional assays to comprehensively characterize factors that are responsible for the differences in fetal and adult hematopoiesis in both a normal and a malignant setting.Our initial work resulted in the identification of numerous proteins and biological processes that distinguish fetal and adult Lin- Sca-1+ cKit+ (LSK) HSPCs, with particularly strong differences between the two cell types in processes related to immune response, protection against reactive oxygen species (ROS) and proteolysis. We showed that fetal and adult HSPCs exhibit distinct responses to modulation of Type I interferon (IFN) signaling and neutrophil serine protease (NSP) activity. We subsequently investigated redox homeostasis in fetal and adult HSPCs, and showed that in line with the lower expression of proteins involved in antioxidant defense, the fetal HSPC proteome has a significantly higher oxidation level than the adult, and undergoes even further oxidation upon leukemia initiation.Our subsequent work focused on characterization of ontogenic proteomic and functional changes that occur in lineage-biased hematopoietic progenitor cells (HPCs). We revealed an intrinsically programmed difference in the lineage bias of fetal and adult lymphomyeloid multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte monocyte progenitors (GMPs), with the adult cells showing a considerably stronger myeloid potential compared to the fetal. The difference in myeloid potential was in part due to a significantly lower expression of Irf8 and a concomitant defect in production of mature monocytes in fetal relative to adult GMPs, which could be partially rescued by increasing Irf8 expression.Finally, we focused our investigations on leukemia driven by the MLL-ENL fusion oncogene, a translocation which represents a highly prevalent mutational event in in utero-derived infant acute lymphoblastic leukemia (ALL). We showed that fetal MLL-ENL-expressing LMPPs possess an intrinsic bias towards generation of immature lymphoid cells ex vivo. In a transplantation model, however, expression of MLL-ENL in fetal as well as adult LMPPs drives acute myeloid leukemia (AML) in adult recipients, suggesting that an interplay between intra- and extracellular factors determines disease progression and outcome in MLLr leukemia. Molecularly, we found that MLL-ENL-mediated leukemia initiation is hallmarked by a differentiation arrest and metabolic reprogramming in fetal as well as adult cells. Remarkably, while expression of proteins associated with inflammation is increased in adult leukemic relative to wild-type (WT) LMPPs, such signatures instead appear to be suppressed in fetal-origin leukemia. Thus, we identified shared as well as ontogeny-specific features of MLL-ENL-mediated leukemogenesis which could potentially be exploited in the development of age-tailored anti-cancer therapies. Collectively, this thesis work has uncovered numerous novel ontogeny-specific molecular and functional features of fetal and adult HSPCs, both in normal development and in leukemia. The results from the included studies will aid in guiding future efforts aimed at developing novel strategies for prevention and treatment of pediatric and adult acute leukemia

Characterization of the CD177 interaction with the ANCA antigen proteinase 3

Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti- neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. In most individuals, a subpopulation of neutrophils also produce CD177, proposed to present additional PR3 on the surface, resulting in CD177neg/mPR3low and CD177pos/mPR3high neutrophil subsets. A positive correlation has been shown between mPR3 abundance, disease incidence, and clinical outcome. We present here a detailed investigation of the PR3:CD177 complex, verifying the interaction, demonstrating the effect of binding on PR3 proteolytic activity and explaining the accessibility of major PR3-ANCA epitopes. We observed high affinity PR3:CD177 complex formation by surface plasmon resonance. Using flow cytometry and a PR3-specific FRET assay, we found that CD177 binding reduced the proteolytic activity of PR3 in vitro using purified proteins, in neutrophil degranulation supernatants containing wtPR3 and directly on mPR3high neutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3high neutrophils showed no migration advantage in vitro or in vivo when migrating from the blood into the oral cavity. We illuminate details of the PR3:CD177 interaction explaining mPR3 membrane orientation and proteolytic activity with relevance to ANCA activation of the distinct mPR3 neutrophil populations

Consequences of cathepsin C inactivation for membrane exposure of proteinase 3, the target antigen in autoimmune vasculitis

Membrane-bound proteinase 3 (PR3(m)) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3(m) triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3(m) and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefevre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3(m) expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3(m) on PLS neutrophils, whereas the total amount of PR3(m) on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34(+) hematopoietic stem cell model. Human CD34(+) hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3(m), cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised

Pulmonary Tuberculosis in Children: A Forgotten Disease?

Even today, tuberculosis in childhood is a disease that is often undiagnosed and undertreated. In the absence of therapy with antituberculosis drugs, children in the first years of life have a high degree of severe forms and mortality. In these children, symptoms are often not very specific and can easily be confused with other diseases of bacterial, viral or fungal etiology, making diagnosis more difficult. Nevertheless, the introduction of new diagnostic techniques has allowed a more rapid identification of the infection. Indeed, Interferon gamma release assay (IGRA) is preferred to the Mantoux, albeit with obvious limitations in children aged <2 years. While the Xpert Mtb/RIF Ultra test is recommended as an initial diagnostic investigation of the gastric aspirate and/or stools in children with signs and symptoms of pulmonary tuberculosis. The drugs used in the treatment of susceptible and resistant TB are the same as those used in adults but doses and combinations are different in the pediatric age. In children, brief therapy is preferable in both the latent infection and the active disease, as a significant reduction in side effects is obtained

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.Peer reviewe

Antimicrobial peptides as potential anti-tubercular leads: A concise review

Despite being considered a public health emergency for the last 25 years, tuberculosis (TB) is still one of the deadliest infectious diseases, responsible for over a million deaths every year. The length and toxicity of available treatments and the increasing emergence of multidrugresistant strains of Mycobacterium tuberculosis renders standard regimens increasingly inefficient and emphasizes the urgency to develop new approaches that are not only cost-and time-effective but also less toxic. Antimicrobial peptides (AMP) are small cationic and amphipathic molecules that play a vital role in the host immune system by acting as a first barrier against invading pathogens. The broad spectrum of properties that peptides possess make them one of the best possible alternatives for a new “post-antibiotic” era. In this context, research into AMP as potential anti-tubercular agents has been driven by the increasing danger revolving around the emergence of extremely-resistant strains, the innate resistance that mycobacteria possess and the low compliance of patients towards the toxic anti-TB treatments. In this review, we will focus on AMP from various sources, such as animal, non-animal and synthetic, with reported inhibitory activity towards Mycobacterium tuberculosis.This research was funded by Fundação para a Ciência e Tecnologia (FCT), Portugal, through projects UIDB/50006/2020, and PTDC/BTM-SAL/29786/2017

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections - Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.Peer reviewe

The role of neutrophil extracellular traps in the pathogenesis of periodontal diseases

This thesis investigated neutrophil extracellular traps (NETs) in the pathogenesis of periodontal diseases, including chronic periodontitis, experimental gingivitis and Papillon Lefèvre syndrome (PLS). $In$ $vitro$ assays investigated the interactions between periodontal bacteria and peripheral neutrophils isolated by discontinuous Percoll gradients, and demonstrated differential NET release in response to bacteria. Interestingly, NETs entrapped all periodontal bacteria assayed to some extent; however bacterial growth and survival were not impeded. A longitudinal intervention clinical study of chronic periodontitis patients and matched healthy controls revealed no differences in peripheral NET production; however NET production by patients decreased following non-surgical treatment. Furthermore, a subset of patients displayed impeded NET degradation by plasma that was restored following disease treatment; this may be the result of increased circulating immunoglobulins and free light chains (FLCs) pre-treatment. Peripheral NET production did not change throughout the experimental gingivitis model study; however NET release was impeded in PLS patients relative to healthy controls. Additional $in$ $vitro$ studies demonstrated that cigarette smoking had an inhibitory effect on NET release. Collectively, this thesis indicates that NETs contribute to innate immunity, however, given that periodontitis pathogenesis is characterised by aberrant neutrophil responses, NETs may also be involved in the progression of the disease

Three Wavelength Substrate System of Neutrophil Serine Proteinases

Neutrophil serine proteases, including elastase, proteinase 3, and cathepsin G, are closely related enzymes stored in similar amounts in azurophil granules and released at the same time from triggered neutrophils at inflammatory sites. We have synthesized new fluorescence resonance energy transfer (FRET) substrates with different fluorescence donor–acceptor pairs that allow all three proteases to be quantified at the same time and in the same reaction mixture. This was made possible because the fluorescence emission spectra of the fluorescence donors do not overlap and because the values of the specificity constants were in the same range. Thus, similar activities of proteases can be measured with the same sensitivity. In addition, these substrates contain an N-terminal 2-(2-(2-aminoethoxy)­ethoxy)­acetic acid (PEG) moiety that makes them cell permeable. Using the mixture of these selected substrates, we were able to detect the neutrophil serine protease (NSP) activity on the activated neutrophil membrane and in the neutrophil lysate in a single measurement. Also, using the substrate mixture, we were in a position to efficiently determine NSP activity in human serum of healthy individuals and patients with diagnosed Wegener disease or <i>microscopic polyangiitis</i>