Pharmacometabolomics applied to low-dose interleukin-2 treatment in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The immunosuppressive functions of regulatory T lymphocytes (Tregs) are impaired in ALS, and correlate to disease progression. The phase 2a IMODALS trial reported an increase in Treg number in ALS patients following the administration of low-dose (ld) interleukin-2 (IL-2). We propose a pharmacometabolomics approach to decipher metabolic modifications occurring in patients treated with ld-IL-2 and its relationship with Treg response. Blood metabolomic profiles were determined on days D1, D64, and D85 from patients receiving 2 MIU of IL-2 (n = 12) and patients receiving a placebo (n = 12). We discriminated the three time points for the treatment group (average error rate of 42%). Among the important metabolites, kynurenine increased between D1 and D64, followed by a reduction at D85. The percentage increase of Treg number from D1 to D64, as predicted by the metabolome at D1, was highly correlated with the observed value. This study provided a proof of concept for metabolic characterization of the effect of ld-IL-2 in ALS. These data could present advances toward a personalized medicine approach and present pharmacometabolomics as a key tool to complement genomic and transcriptional data for drug characterization, leading to systems pharmacology.

Post-COVID Trajectory of Pentraxin 3 Plasma Levels Over 6 Months and Their Association with the Risk of Developing Post-Acute Depression and Anxiety.

BACKGROUND AND OBJECTIVES: Clinical manifestations of coronavirus disease 2019 (COVID-19) often persist after acute disease resolution. Underlying molecular mechanisms are unclear. The objective of this original article was to longitudinally measure plasma levels of markers of the innate immune response to investigate whether they associate with and predict post-COVID symptomatology. METHODS: Adult patients with previous severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection during the first pandemic wave who underwent the 6-month multidisciplinary follow-up were included. Plasma levels of pentraxin 3 (PTX3), the complement components C3a and C5a, and chitinase-3 like-protein-1 (CHI3L1) were measured at hospital admission during acute disease (baseline) and at 1 and 6 months after hospital discharge. Associations with post-COVID-19 sequelae at 6 months were investigated using descriptive statistic and multiple regression models. RESULTS: Ninety-four COVID-19 patients were included. Baseline PTX3, C5a, C3a, and CHI3L1 did not predict post-COVID-19 sequelae. The extent of the reduction of PTX3 over time (delta PTX3) was associated with lower depressive and anxiety symptoms at 6 months (both p < 0.05). When entering sex, age, need for intensive care unit or non-invasive ventilation during hospital stay, psychiatric history, and baseline PTX3 as nuisance covariates into a generalized linear model (GLM), the difference between baseline and 6-month PTX3 levels (delta PTX3) significantly predicted depression (χ2 = 4.66, p = 0.031) and anxiety (χ2 = 4.68, p = 0.031) at 6 months. No differences in PTX3 levels or PTX3 delta were found in patients with or without persistent or new-onset other COVID-19 symptoms or signs at 6 months. Plasma levels of C3a, C5a, and CHI3L1 did not correlate with PTX3 levels at either time point and failed to associate with residual or de novo respiratory or systemic clinical manifestations of the disease at 6 months. CONCLUSIONS: A lower reduction of plasma PTX3 after acute COVID-19 associates with the presence of depression and anxiety, suggesting an involvement of inflammation in post-COVID-19 psychopathology and a potential role of PTX3 as a biomarker.

Neutrophils Mediate Protection Against Colitis and Carcinogenesis by Controlling Bacterial Invasion and IL22 Production by γδ T Cells.

Neutrophils are the most abundant leukocytes in human blood and play a primary role in resistance against invading microorganisms and in the acute inflammatory response. However, their role in colitis and colitis-associated colorectal cancer is still under debate. This study aims to dissect the role of neutrophils in these pathologic contexts by using a rigorous genetic approach. Neutrophil-deficient mice (Csf3r-/- mice) were used in classic models of colitis and colitis-associated colorectal cancer and the role of neutrophils was assessed by histologic, cellular, and molecular analyses coupled with adoptive cell transfer. We also performed correlative analyses using human datasets. Csf3r-/- mice showed increased susceptibility to colitis and colitis-associated colorectal cancer compared with control Csf3r+/+ mice and adoptive transfer of neutrophils in Csf3r-/- mice reverted the phenotype. In colitis, Csf3r-/- mice showed increased bacterial invasion and a reduced number of healing ulcers in the colon, indicating a compromised regenerative capacity of epithelial cells. Neutrophils were essential for γδ T-cell polarization and IL22 production. In patients with ulcerative colitis, expression of CSF3R was positively correlated with IL22 and IL23 expression. Moreover, gene signatures associated with epithelial-cell development, proliferation, and antimicrobial response were enriched in CSF3Rhigh patients. Our data support a model where neutrophils mediate protection against intestinal inflammation and colitis-associated colorectal cancer by controlling the intestinal microbiota and driving the activation of an IL22-dependent tissue repair pathway.

Loss of interleukin 1 signaling causes impairment of microglia- mediated synapse elimination and autistic-like behaviour in mice.

In the last years, the hypothesis that elevated levels of proinflammatory cytokines contribute to the pathogenesis of neurodevelopmental diseases has gained popularity. IL-1 is one of the main cytokines found to be elevated in Autism spectrum disorder (ASD), a complex neurodevelopmental condition characterized by defects in social communication and cognitive impairments. In this study, we demonstrate that mice lacking IL-1 signaling display autistic-like defects associated with an excessive number of synapses. We also show that microglia lacking IL-1 signaling at early neurodevelopmental stages are unable to properly perform the process of synapse engulfment and display excessive activation of mammalian target of rapamycin (mTOR) signaling. Notably, even the acute inhibition of IL-1R1 by IL-1Ra is sufficient to enhance mTOR signaling and reduce synaptosome phagocytosis in WT microglia. Finally, we demonstrate that rapamycin treatment rescues the defects in IL-1R deficient mice. These data unveil an exclusive role of microglial IL-1 in synapse refinement via mTOR signaling and indicate a novel mechanism possibly involved in neurodevelopmental disorders associated with defects in the IL-1 pathway.

Complement-pentraxins synergy: Navigating the immune battlefield and beyond.

The complement is a crucial immune defense system that triggers rapid immune responses and offers efficient protection against foreign invaders and unwanted host elements, acting as a sentinel. Activation of the complement system occurs upon the recognition of pathogenic microorganisms or altered self-cells by pattern-recognition molecules (PRMs) such as C1q, collectins, ficolins, and pentraxins. Recent accumulating evidence shows that pentraxins establish a cooperative network with different classes of effector PRMs, resulting in synergistic effects in complement activation. This review describes the complex interaction of pentraxins with the complement system and the implications of this cooperative network for effective host defense during pathogen invasion.

The Yin Yang of Complement and Cancer.

Cancer-related inflammation is a crucial component of the tumor microenvironment (TME). Complement activation occurs in cancer and supports the development of an inflammatory microenvironment. Complement has traditionally been considered a mechanism of immune resistance against cancer, and its activation is known to contribute to the cytolytic effects of antibody-based immunotherapeutic treatments. However, several studies have recently revealed that complement activation may exert protumoral functions by sustaining cancer-related inflammation and immunosuppression through different molecular mechanisms, targeting both the TME and cancer cells. These new discoveries have revealed that complement manipulation can be considered a new strategy for cancer therapies. Here we summarize our current understanding of the mechanisms by which the different elements of the complement system exert antitumor or protumor functions, both in preclinical studies and in human tumorigenesis. Complement components can serve as disease biomarkers for cancer stratification and prognosis and be exploited for tumor treatment.

Soluble interleukin-1 receptor type 2 plasma levels in Parkinson's disease: relationship with cardiac autonomic profile before and after peripheral mechanical somatosensory stimulation.

Introduction: Systemic inflammation promotes neurodegeneration in Parkinson's disease (PD). Interleukin-1 receptor type 2 (sIL-1R2) plasma levels increase during inflammation. Data on sIL-1R2 in PD patients and its relationship with PD cardiac autonomic profile are limited, given the possible anti-inflammatory effect of vagal activation. Previously, automated mechanical peripheral somatosensory stimulation (AMPSS) enhanced cardiac vagal modulation. Objectives were to 1) evaluate sIL-1R2 plasma concentrations in PD patients and healthy controls and 2) investigate the correlations between sIL-1R2 and cardiac autonomic indices obtained by spectrum analysis of heart rate variability before and after AMPSS. Methods: sIL-1R2 plasma levels were assessed in 48 PD patients and 50 healthy controls. Electrocardiogram and beat-by-beat arterial pressure were recorded at baseline and after 5 AMPSS sessions in 16 PD patients. Results: PD patients had higher sIL-1R2 levels than controls. In the PD subgroup, an inverse correlation between sIL-1R2 and HFnu was found. There was a negative correlation between changes induced by AMPSS on HFnu and sIL-1R2. Discussion: Higher sIL-1R2 levels in PD patients reflect the inflammatory dysregulation associated with the disease. In PD patients, higher sIL-1R2 was associated with reduced cardiovagal tone. Increased cardiovagal modulation following AMPSS was associated with lower sIL-1R2 levels in Parkinson's disease patients, suggesting inflammatory state improvement.

A Multilayered Imaging and Microfluidics Approach for Evaluating the Effect of Fibrinolysis in Staphylococcus aureus Biofilm Formation.

The recognition of microbe and extracellular matrix (ECM) is a recurring theme in the humoral innate immune system. Fluid-phase molecules of innate immunity share regulatory roles in ECM. On the other hand, ECM elements have immunological functions. Innate immunity is evolutionary and functionally connected to hemostasis. Staphylococcus aureus (S. aureus) is a major cause of hospital-associated bloodstream infections and the most common cause of several life-threatening conditions such as endocarditis and sepsis through its ability to manipulate hemostasis. Biofilm-related infection and sepsis represent a medical need due to the lack of treatments and the high resistance to antibiotics. We designed a method combining imaging and microfluidics to dissect the role of elements of the ECM and hemostasis in triggering S. aureus biofilm by highlighting an essential role of fibrinogen (FG) in adhesion and formation. Furthermore, we ascertained an important role of the fluid-phase activation of fibrinolysis in inhibiting biofilm of S. aureus and facilitating an antibody-mediated response aimed at pathogen killing. The results define FG as an essential element of hemostasis in the S. aureus biofilm formation and a role of fibrinolysis in its inhibition, while promoting an antibody-mediated response. Understanding host molecular mechanisms influencing biofilm formation and degradation is instrumental for the development of new combined therapeutic approaches to prevent the risk of S. aureus biofilm-associated diseases.

Genetics of autoinflammation instructs selective IL-1 targeting.

Interleukin-1 (IL-1) is a primary pro-inflammatory cytokine requiring tightly controlled negative regulation. In this issue of Immunity, Wang et al.,1 inspired by an IL-1 receptor missense mutation associated with unleashed IL-1-mediated inflammation, design a new drug to selectively inhibit IL-1.

IL-1R8: A molecular brake of anti-tumor and anti-viral activity of NK cells and ILC.

Interleukin-1 receptor family members (ILRs) and Toll-Like Receptors (TLRs) play pivotal role in immunity and inflammation and are expressed by most cell types including cells of both the innate and adaptive immune system. In this context, IL-1 superfamily members are also important players in regulating function and differentiation of adaptive and innate lymphoid cells. This system is tightly regulated in order to avoid uncontrolled activation, which may lead to detrimental inflammation contributing to autoimmune or allergic responses. IL-1R8 (also known as TIR8 or SIGIRR) is a member of the IL-1R family that acts as a negative regulator dampening ILR and TLR signaling and as a co-receptor for human IL-37. Human and mouse NK cells, that are key players in immune surveillance of tumors and infections, express high level of IL-1R8. In this review, we will summarize our current understanding on the structure, expression and function of IL-1R8 and we will also discuss the emerging role of IL-1R8 as an important checkpoint regulating NK cells function in pathological conditions including cancer and viral infections.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

Long pentraxin 3 (PTX3) levels predict death, intubation and thrombotic events among hospitalized patients with COVID-19.

Background: PTX3 is an important mediator of inflammation and innate immunity. We aimed at assessing its prognostic value in a large cohort of patients hospitalized with COVID-19. Methods: Levels of PTX3 were measured in 152 patients hospitalized with COVID-19 at San Gerardo Hospital (Monza, Italy) since March 2020. Cox regression was used to identify predictors of time from admission to in-hospital death or mechanical ventilation. Crude incidences of death were compared between patients with PTX3 levels higher or lower than the best cut-off estimated with the Maximally Selected Rank Statistics Method. Results: Upon admission, 22% of the patients required no oxygen, 46% low-flow oxygen, 30% high-flow nasal cannula or CPAP-helmet and 3% MV. Median level of PTX3 was 21.7 (IQR: 13.5-58.23) ng/ml. In-hospital mortality was 25% (38 deaths); 13 patients (8.6%) underwent MV. PTX3 was associated with risk of death (per 10 ng/ml, HR 1.08; 95%CI 1.04-1.11; P<0.001) and death/MV (HR 1.04; 95%CI 1.01-1.07; P=0.011), independently of other predictors of in-hospital mortality, including age, Charlson Comorbidity Index, D-dimer and C-reactive protein (CRP). Patients with PTX3 levels above the optimal cut-off of 39.32 ng/ml had significantly higher mortality than the others (55% vs 8%, P<0.001). Higher PTX3 plasma levels were found in 14 patients with subsequent thrombotic complications (median [IQR]: 51.4 [24.6-94.4] versus 21 [13.4-55.2]; P=0.049). Conclusions: High PTX3 levels in patients hospitalized with COVID-19 are associated with a worse outcome. The evaluation of this marker could be useful in prognostic stratification and identification of patients who could benefit from immunomodulant therapy.

Macrophages as tools and targets in cancer therapy.

Tumour-associated macrophages are an essential component of the tumour microenvironment and have a role in the orchestration of angiogenesis, extracellular matrix remodelling, cancer cell proliferation, metastasis and immunosuppression, as well as in resistance to chemotherapeutic agents and checkpoint blockade immunotherapy. Conversely, when appropriately activated, macrophages can mediate phagocytosis of cancer cells and cytotoxic tumour killing, and engage in effective bidirectional interactions with components of the innate and adaptive immune system. Therefore, they have emerged as therapeutic targets in cancer therapy. Macrophage-targeting strategies include inhibitors of cytokines and chemokines involved in the recruitment and polarization of tumour-promoting myeloid cells as well as activators of their antitumorigenic and immunostimulating functions. Early clinical trials suggest that targeting negative regulators (checkpoints) of myeloid cell function indeed has antitumor potential. Finally, given the continuous recruitment of myelomonocytic cells into tumour tissues, macrophages are candidates for cell therapy with the development of chimeric antigen receptor effector cells. Macrophage-centred therapeutic strategies have the potential to complement, and synergize with, currently available tools in the oncology armamentarium.

Intrahepatic CD69+Vδ1 T cells re-circulate in the blood of patients with metastatic colorectal cancer and limit tumor progression.

BACKGROUND: More than 50% of all patients with colorectal cancer (CRC) develop liver metastases (CLM), a clinical condition characterized by poor prognosis and lack of reliable prognostic markers. Vδ1 cells are a subset of tissue-resident gamma delta (γδ) T lymphocytes endowed with a broad array of antitumor functions and showing a natural high tropism for the liver. However, little is known about their impact in the clinical outcomes of CLM. METHODS: We isolated human γδ T cells from peripheral blood (PB) and peritumoral (PT) tissue of 93 patients undergone surgical procedures to remove CLM. The phenotype of freshly purified γδ T cells was assessed by multiparametric flow cytometry, the transcriptional profiles by single cell RNA-sequencing, the functional annotations by Gene Ontology enrichment analyses and the clonotype by γδ T cell receptor (TCR)-sequencing. RESULTS: The microenvironment of CLM is characterized by a heterogeneous immune infiltrate comprising different subsets of γδ tumor-infiltrating lymphocytes (TILs) able to egress the liver and re-circulate in PB. Vδ1 T cells represent the largest population of γδ TILs within the PT compartment of CLM that is greatly enriched in Vδ1 T effector (TEF) cells expressing constitutive high levels of CD69. These Vδ1 CD69+ TILs express a distinct phenotype and transcriptional signature, show high antitumor potential and correlate with better patient clinical outcomes in terms of lower numbers of liver metastatic lesions and longer overall survival (OS). Moreover, intrahepatic CD69+ Vδ1 TILs can egress CLM tissue to re-circulate in PB, where they retain a phenotype, transcriptional signature and TCR clonal repertoires resembling their liver origin. Importantly, even the increased frequencies of the CD69+ terminally differentiated (TEMRA) Vδ1 cells in PB of patients with CLM significantly correlate with longer OS. The positive prognostic score of high frequencies of CD69+ TEMRA Vδ1 cells in PB is independent from the neoadjuvant chemotherapy and immunotherapy regimens administered to patients with CLM prior surgery. CONCLUSIONS: The enrichment of tissue-resident CD69+ Vδ1 TEMRA cells re-circulating at high frequencies in PB of patients with CLM limits tumor progression and represents a new important clinical tool to either predict the natural history of CLM or develop alternative therapeutic protocols of cellular therapies.

Complement activation in cancer: Effects on tumor-associated myeloid cells and immunosuppression.

Cancer-related inflammation plays a central role in the establishment of tumor-promoting mechanisms. Tumor-associated myeloid cells, which engage in complex interactions with cancer cells, as well as stromal and tumor immune infiltrating cells, promote cancer cell proliferation and survival, angiogenesis, and the generation of an immunosuppressive microenvironment. The complement system is one of the inflammatory mechanisms activated in the tumor microenvironment. Beside exerting anti-tumor mechanisms such as complement-dependent cytotoxicity and phagocytosis induced by therapeutic monoclonal antibodies, the complement system may promote immunosuppression and tumor growth and invasiveness, in particular, through the anaphylatoxins which target both leukocytes and cancer cells. In this review, we will discuss complement-mediated mechanisms acting on leukocytes, in particular on cells of the myelomonocytic cell lineage (macrophages, neutrophils, myeloid derived suppressor cells), which promote myeloid cell recruitment and functional skewing, leading to immunosuppression and resistance to tumor-specific immunity. Pre-clinical studies, which have elucidated the role of complement in activating pro-tumor mechanisms in myeloid cells, showing the relevance of these mechanisms in human, and therapeutic approaches based on complement targeting support the hypothesis that complement directly and indirectly interferes with many of the effector pathways associated with the cancer-immunity cycle, suggesting the relevance of complement targeting to improve responses to immunotherapeutic approaches.

Pentraxin 3 Inhibits Complement-driven Macrophage Activation to Restrain Granuloma Formation in Sarcoidosis.

Rationale: Sarcoidosis is a multisystemic inflammatory disease characterized by the formation of granulomas in response to persistent stimuli. The long pentraxin PTX3 (pentraxin 3) has emerged as a component of humoral innate immunity with essential functions in the resolution of inflammation, but its role during granuloma formation is unknown. Objectives: To evaluate PTX3 as a modulator of pathogenic signals involved in granuloma formation and inflammation in sarcoidosis. Methods: Peripheral blood mononuclear cells obtained from patients with sarcoidosis harboring loss-of-function genetic variants and gene-deleted mice were used to assess the role of PTX3 in experimental models of granuloma formation in vitro and in vivo. The identified mechanisms of granulomatous inflammation were further evaluated in tissue and BAL samples and correlated with the disease course. Measurements and Main Results: We have identified a molecular link between PTX3 deficiency and the pathogenic amplification of complement activation to promote granuloma formation. Mechanistically, PTX3 deficiency licensed the complement component C5a-mediated activation of the metabolic checkpoint kinase mTORC1 (mammalian target of rapamycin complex 1) and the reprogramming of macrophages toward increased glycolysis to foster their proliferation and aggregation. This process sustained the further recruitment of granuloma-promoting immune cells and the associated proinflammatory microenvironment and influenced the clinical course of the disease. Conclusions: Our results identify PTX3 as a pivotal molecule that regulates complement-mediated signaling cues in macrophages to restrain granulomatous inflammation and highlight the therapeutic potential of this signaling axis in targeting granuloma formation in sarcoidosis.