Persisting IL-18 levels after COVID-19 correlate with markers of cardiovascular inflammation reflecting potential risk of CVDs development.

COVID-19 manifestation is associated with a strong immune system activation leading to inflammation and subsequently affecting the cardiovascular system. The objective of the study was to reveal possible interconnection between prolongated inflammation and the development or exacerbation of long-term cardiovascular complications after COVID-19. We investigated correlations between humoral and cellular immune system markers together with markers of cardiovascular inflammation/dysfunction during COVID-19 onset and subsequent recovery. We analyzed 22 hospitalized patients with severe COVID-19 within three timepoints (acute, 1 and 6 months after COVID-19) in order to track the impact of COVID-19 on the long-term decline of the cardiovascular system fitness and eventual development of CVDs. Among the cytokines dysregulated during COVID-19 changes, we showed significant correlations of IL-18 as a key driver of several pathophysiological changes with markers of cardiovascular inflammation/dysfunction. Our findings established novel immune-related markers, which can be used for the stratification of patients at high risk of CVDs for further therapy.

Influence of protein corona on the interaction of glycogen-siRNA constructs with ex vivo human blood immune cells.

Glycogen-nucleic acid constructs i.e., glycoplexes are emerging promising platforms for the alteration of gene expression and transcription. Understanding the interaction of glycoplexes with human blood components, such as serum proteins and peripheral blood mononuclear cells (PBMCs), is important to overcome immune cell activation and control biodistribution upon administration of the glycoplexes in vivo. Herein, we investigated the interactions of polyethylene glycol (PEG)ylated and non-PEGylated glycoplexes carrying siRNA molecules with PBMCs isolated from the blood of healthy donors. We found that both types of glycoplexes were non-toxic and were primarily phagocytosed by monocytes without triggering a pro-inflammatory interleukin 6 cytokine production. Furthermore, we investigated the role of the protein corona on controlling the internalization efficiency in immune cells - we found that the adsorption of serum proteins, in particular haptoglobin, alpha-1-antitrypsin and apolipoprotein A-II, onto the non-PEGylated glycoplexes, significantly reduced the uptake of the glycoplexes by PBMCs. Moreover, the non-PEGylated glycoplexes were efficient in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) knockdown in monocytic THP-1 cell line. This study provides an insight into the rational design of glycogen-based nanocarriers for the safe delivery of siRNA without eliciting unwanted immune cell activation and efficient siRNA activity upon its delivery.

Polymorphonuclear Cells Show Features of Dysfunctional Activation During Fatal Sepsis.

Sepsis and septic shock remain leading causes of morbidity and mortality for patients in the intensive care unit. During the early phase, immune cells produce various cytokines leading to prompt activation of the immune system. Polymorphonuclear leukocytes (PMNs) respond to different signals producing inflammatory factors and executing their antimicrobial mechanisms, resulting in the engulfment and elimination of invading pathogens. However, excessive activation caused by various inflammatory signals produced during sepsis progression can lead to the alteration of PMN signaling and subsequent defects in their functionality. Here, we analyzed samples from 34 patients in septic shock, focusing on PMNs gene expression and proteome changes associated with septic shock. We revealed that, compared to those patients who survived longer than five days, PMNs from patients who had fulminant sepsis were characterized by a dysfunctional hyper-activation, show altered metabolism, and recent exit from the cell cycle and signs of cellular lifespan. We believe that this multi-omics approach, although limited, pinpoints the alterations in PMNs' functionality, which may be rescued by targeted treatments.

Immunosuppression Affects Neutrophil Functions: Does Calcineurin-NFAT Signaling Matter?

Neutrophils are innate immune cells with important roles in antimicrobial defense. However, impaired or dysregulated neutrophil function can result in host tissue damage, loss of homeostasis, hyperinflammation or pathological immunosuppression. A central link between neutrophil activation and immune outcomes is emerging to be the calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway, which is activated by neutrophil detection of a microbial threat via pattern recognition receptors and results in inflammatory cytokine production. This potent pro-inflammatory pathway is also the target of several immunosuppressive drugs used for the treatment of autoimmune disorders, during solid organ and hematopoietic cell transplantations, and as a part of anti-cancer therapy: but what effects these drugs have on neutrophil function, and their broader consequences for immune homeostasis and microbial defense are not yet known. Here, we bring together the emerging literature describing pathology- and drug- induced neutrophil impairment, with particular focus on their effects on calcineurin-NFAT signaling in the innate immune compartment.

Targeted volatolomics of human monocytes: Comparison of 2D-GC/TOF-MS and 1D-GC/Orbitrap-MS methods.

Blood is a complex biological matrix providing valuable information on nutritional, metabolic, and immune status. The detection of blood biomarkers requires sensitive analytical methods because analytes are at very low concentrations. Peripheral blood monocytes play a crucial role in inflammatory processes, and the metabolites released by monocytes during these processes might serve as important signalling molecules and biomarkers of particular physiological states. Headspace solid-phase microextraction (HS-SPME) combined with two different mass spectrometric platforms, two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (2D-GC/TOF-MS) and one-dimensional gas chromatography coupled to Orbitrap mass spectrometry (GC/Orbitrap-MS), were applied for the investigation of volatile organic compounds (VOCs) produced by human peripheral blood monocytes. An optimized method was subsequently applied for the characterization of changes in VOCs induced by lipopolysaccharides (LPS) and zymosan (ZYM) stimulation. Overall, the 2D-GC/TOF-MS and the 1D-GC/Orbitrap-MS analyses each yielded about 4000 and 400 peaks per sample, respectively. In total, 91 VOCs belonging to eight different chemical classes were identified. The samples were collected in two fractions, conditioned media for monitoring extracellularly secreted molecules and cell pellet samples to determine the intracellular composition of VOCs. Alcohols, ketones, and hydrocarbons were the main chemical classes of the metabolic profile identified in cell fractions. Aldehydes, acids and cyclic compounds were characteristic of the conditioned media fraction. Here we demonstrate that HS-SPME-2D-GC/TOF-MS is more suitable for the identification of specific VOC profiles produced by human monocytes than 1D-GC/Orbitrap-MS. We define the signature of VOCs occurring early after monocyte activation and characterise the signalling compounds released by immune cells into media.

NFAT signaling in human mesenchymal stromal cells affects extracellular matrix remodeling and antifungal immune responses.

Mesenchymal stromal cells (MSCs) combined with calcineurin-nuclear factor of activated T cell (CN-NFAT) inhibitors are being tested as a treatment for graft-versus-host disease (GvHD). The immunosuppressive properties of MSCs seem beneficial; however, their response during fungal infection, which is an important cause of mortality in patients with GvHD , is unknown. We report that MSCs phagocytose the fungal component zymosan, resulting in phosphorylation of spleen tyrosine kinase (Syk), increase in cytosolic calcium levels, and ultimately, increase in NFAT1 nuclear translocation. RNA sequencing analysis of zymosan-treated MSCs showed that CN-NFAT inhibition affects extracellular matrix (ECM) genes but not cytokine expression that is under the control of the NF-κB pathway. When coculturing MSCs or decellularized MSC-ECM with human peripheral blood mononuclear cells (PBMCs), selective NFAT inhibition in MSCs decreased cytokine expression by PBMCs. These findings reveal a dual mechanism underlying the MSC response to zymosan: while NF-κB directly controls inflammatory cytokine expression, NFAT impacts immune-cell functions by regulating ECM remodeling.

Immunosenescence in Childhood Cancer Survivors and in Elderly: A Comparison and Implication for Risk Stratification.

The population of childhood cancer survivors (CCS) has grown rapidly in recent decades. Although cured of their original malignancy, these individuals are at increased risk of serious late effects, including age-associated complications. An impaired immune system has been linked to the emergence of these conditions in the elderly and CCS, likely due to senescent immune cell phenotypes accompanied by low-grade inflammation, which in the elderly is known as "inflammaging." Whether these observations in the elderly and CCS are underpinned by similar mechanisms is unclear. If so, existing knowledge on immunosenescent phenotypes and inflammaging might potentially serve to benefit CCS. We summarize recent findings on the immune changes in CCS and the elderly, and highlight the similarities and identify areas for future research. Improving our understanding of the underlying mechanisms and immunosenescent markers of accelerated immune aging might help us to identify individuals at increased risk of serious health complications.

Differences in monocyte subsets are associated with short-term survival in patients with septic shock.

Sepsis is characterized by dynamic changes of the immune system resulting in deregulated inflammation and failure of homoeostasis and can escalate to septic shock. Circulating monocytes and other innate immune cells are among the first ones to recognize and clear pathogens. Monocytes have an important role in sepsis and septic shock and have been studied as potential diagnostic markers. In total, forty-two patients with septic shock were recruited and blood samples obtained within first 12 hours of ICU admission. We showed that frequency of classical and intermediate monocytes assessed at the time of admission to the intensive care unit are significantly distinct in patients with septic shock who survived longer that five days from those who died. These parameters correlate significantly with differences in serum levels of inflammatory cytokines MCP-1, IL-6, IL-8, IL-10, and IL-18, and with the proportion of helper and cytotoxic T cells. The described changes in frequency of monocyte subsets and their activation status may predict short-term septic shock survival and help with fast identification of the group of vulnerable patients, who may profit from tailored therapy.

The Effect of Mindfulness-Based Stress Reduction (MBSR) on Depression, Cognition, and Immunity in Mild Cognitive Impairment: A Pilot Feasibility Study.

BACKGROUND: Mindfulness-based programs have shown a promising effect on several health factors associated with increased risk of dementia and the conversion from mild cognitive impairment (MCI) to dementia such as depression, stress, cognitive decline, immune system and brain structural and functional changes. Studies on mindfulness in MCI subjects are sparse and frequently lack control intervention groups. OBJECTIVE: To determine the feasibility and the effect of mindfulness-based stress reduction (MBSR) practice on depression, cognition and immunity in MCI compared to cognitive training. METHODS: Twenty-eight MCI subjects were randomly assigned to two groups. MBSR group underwent 8-week MBSR program. Control group underwent 8-week cognitive training. Their cognitive and immunological profiles and level of depressive symptoms were examined at baseline, after each 8-week intervention (visit 2, V2) and six months after each intervention (visit 3, V3). MBSR participants completed feasibility questionnaire at V2. RESULTS: Twenty MCI patients completed the study (MBSR group n=12, control group n=8). MBSR group showed significant reduction in depressive symptoms at both V2 (p=0.03) and V3 (p=0.0461) compared to the baseline. There was a minimal effect on cognition - a group comparison analysis showed better psychomotor speed in the MBSR group compared to the control group at V2 (p=0.0493) but not at V3. There was a detectable change in immunological profiles in both groups, more pronounced in the MBSR group. Participants checked only positive/neutral answers concerning the attractivity/length of MBSR intervention. More severe cognitive decline (PVLT≤36) was associated with the lower adherence to home practice. CONCLUSION: MBSR is well-accepted potentially promising intervention with positive effect on cognition, depressive symptoms and immunological profile.

Childhood survivors of high-risk neuroblastoma show signs of immune recovery and not immunosenescence.

Neuroblastoma survivors show signs of immunosenescence early after therapy in CD8+ T cell compartment and elevated plasma TNF-α but in later follow-up immune recovery comes into play. Whether the recovery phenotype is long lasting or transient remains to be elucidated, however, late adverse effects often occur in childhood cancer survivors.

Roles of IL-2 in bridging adaptive and innate immunity, and as a tool for cellular immunotherapy.

IL-2 was initially characterized as a T cell growth factor in the 1970s, and has been studied intensively ever since. Decades of research have revealed multiple and diverse roles for this potent cytokine, indicating a unique linking role between adaptive and innate arms of the immune system. Here, we review the literature showing that IL-2 is expressed in a plethora of cell types across the immune system, where it has indispensable functions in orchestrating cellular interactions and shaping the nature and magnitude of immune responses. Emerging from the basic research that has revealed the molecular mechanisms and the complexity of the biologic actions of IL-2, several immunotherapeutic approaches have now focused on manipulating the levels of this cytokine in patients. These strategies range from inhibition of IL-2 to achieve immunosuppression, to the application of IL-2 as a vaccine adjuvant and in cancer therapies. This review will systematically summarize the major findings in the field and identify key areas requiring further research in order to realize the potential of IL-2 in the treatment of human diseases.

Calcineurin inhibitors reduce NFAT-dependent expression of antifungal pentraxin-3 by human monocytes.

Calcineurin (CN) inhibitors are effective clinical immunosuppressants but leave patients vulnerable to potentially fatal fungal infections. This study tested the hypothesis that CN inhibition interferes with antifungal immune defenses mediated by monocytes. We showed that NFAT is expressed by human monocytes, and is activated by exposure to fungal ligands. We confirmed that NFAT translocation potently activated target gene transcription using a human monocytic reporter cell line. Inhibition of CN-NFAT by cyclosporine A significantly reduced monocyte production of TNF-α, IL-10, and MCP-1 proteins in response to pattern recognition receptor ligands as well as to Aspergillus fumigatus conidia. Moreover, we revealed that human monocytes express the antifungal protein pentraxin-3 under control of NFAT. In conclusion, clinical CN inhibitors have the potential to interfere with the novel NFAT-dependent pentraxin-3 pathway as well as antifungal cytokine production in human monocytes, thereby impeding monocyte-mediated defenses against fungal infection in immune-suppressed patients.

Draft Genome Sequence of the Panton-Valentine Leucocidin-Producing Staphylococcus aureus Sequence Type 154 Strain NRL 08/001, Isolated from a Fatal Case of Necrotizing Pneumonia.

Panton-Valentine leucocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) strains cause life-threatening diseases. We present a draft genome sequence of PVL-positive MRSA sequence type 154 (ST154) strain NRL 08/001, isolated from a fatal case of necrotizing pneumonia. The genome consists of 2.9 Mb over 39 contigs and harbors novel composite island staphylococcal cassette chromosome mec element (SCCmec)-mercury composite type 2B&5.

The Telomerase Complex Directly Controls Hematopoietic Stem Cell Differentiation and Senescence in an Induced Pluripotent Stem Cell Model of Telomeropathy.

Telomeropathies are rare disorders associated with impaired telomere length control mechanisms that frequently result from genetic mutations in the telomerase complex. Dyskeratosis congenita is a congenital progressive telomeropathy in which mutation in the telomerase RNA component (TERC) impairs telomere maintenance leading to accelerated cellular senescence and clinical outcomes resembling premature aging. The most severe clinical feature is perturbed hematopoiesis and bone-marrow failure, but the underlying mechanisms are not fully understood. Here, we developed a model of telomerase function imbalance using shRNA to knockdown TERC expression in human induced pluripotent stem cells (iPSCs). We then promoted in vitro hematopoiesis in these cells to analyze the effects of TERC impairment. Reduced TERC expression impaired hematopoietic stem-cell (HSC) differentiation and increased the expression of cellular senescence markers and production of reactive oxygen species. Interestingly, telomere length was unaffected in shTERC knockdown iPSCs, leading to conclusion that the phenotype is controlled by non-telomeric functions of telomerase. We then assessed the effects of TERC-depletion in THP-1 myeloid cells and again observed reduced hematopoietic and myelopoietic differentiative potential. However, these cells exhibited impaired telomerase activity as verified by accelerated telomere shortening. shTERC-depleted iPSC-derived and THP-1-derived myeloid precursors had lower phagocytic capacity and increased ROS production, indicative of senescence. These findings were confirmed using a BIBR1532 TERT inhibitor, suggesting that these phenotypes are dependent on telomerase function but not directly linked to telomere length. These data provide a better understanding of the molecular processes driving the clinical signs of telomeropathies and identify novel roles of the telomerase complex other than regulating telomere length.

High-Throughput Screening of Senescence Markers in Hematopoietic Stem Cells Derived from Induced Pluripotent Stem Cells.

The successful development and characterization of human induced pluripotent stem cells (iPSCs) provides a powerful tool to study the molecular mechanisms that control cell fate decisions and differentiation toward distinct lineages. Here we focus on the ability of donors derived iPSCs to differentiate toward hematopoietic progenitor cells and on the analysis of their telomere length. The ability to screen telomere length in individual donors is important for defining cellular senescence, which correlates with their differentiation potential toward hematopoietic lineages. We have modified iPSC culture protocol and telomere length analysis to suit for high throughput screening of telomere length in large number of individual donors. This approach can be used to demonstrate the heterogeneity or changes of telomere length and its shortening as an exclusion criterion for selection of suitable donors for future stem cell therapies.

Chronic Inflammation in Immune Aging: Role of Pattern Recognition Receptor Crosstalk with the Telomere Complex?

Age-related decline in immunity is characterized by stem cell exhaustion, telomere shortening, and disruption of cell-to-cell communication, leading to increased patient risk of disease. Recent data have demonstrated that chronic inflammation exerts a strong influence on immune aging and is closely correlated with telomere length in a range of major pathologies. The current review discusses the impact of inflammation on immune aging, the likely molecular mediators of this process, and the various disease states that have been linked with immunosenescence. Emerging findings implicate NF-κB, the major driver of inflammatory signaling, in several processes that regulate telomere maintenance and/or telomerase activity. While prolonged triggering of pattern recognition receptors is now known to promote immunosenescence, it remains unclear how this process is linked with the telomere complex or telomerase activity. Indeed, enzymatic control of telomere length has been studied for many decades, but alternative roles of telomerase and potential influences on inflammatory responses are only now beginning to emerge. Crosstalk between these pathways may prove to be a key molecular mechanism of immunosenescence. Understanding how components of immune aging interact and modify host protection against pathogens and tumors will be essential for the design of new vaccines and therapies for a wide range of clinical scenarios.

Calcineurin-NFAT signalling in myeloid leucocytes: new prospects and pitfalls in immunosuppressive therapy.

Myeloid leucocytes mediate host protection against infection and critically regulate inflammatory responses in body tissues. Pattern recognition receptor signalling is crucial for myeloid cell responses to pathogens, but growing evidence suggests an equally potent role for Calcineurin-NFAT signalling in control of myeloid cell function. All major subsets of myeloid leucocytes employ Calcineurin-NFAT signalling during immune responses to pathogens and/or tissue damage, but the influence this pathway exerts on pathogen clearance and host susceptibility to infection is not fully understood. Recent data from experimental models indicate that Calcineurin-NFAT signalling is essential for infection control, and calcineurin inhibitors used in transplantation medicine (including cyclosporine A and tacrolimus) are now being tested for efficacy in a diverse range of inflammatory conditions and autoimmune pathologies. Efforts to repurpose calcineurin inhibitor drugs for new therapeutic applications may yield rapid improvements in clinical outcomes, but the potential impact of these compounds on myeloid cell function in treated patients is largely unknown. Here we discuss Calcineurin-NFAT control of myeloid leucocyte function in the context of recent therapeutic developments and ongoing clinical studies.