Neutrophils Driving Unconventional T Cells Mediate Resistance against Murine Sarcomas and Selected Human Tumors.

Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4- CD8- unconventional αß T cells (UTCαß). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαß associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTCαß polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors.

IL-1R8 is a checkpoint in NK cells regulating anti-tumour and anti-viral activity.

Interleukin-1 receptor 8 (IL-1R8, also known as single immunoglobulin IL-1R-related receptor, SIGIRR, or TIR8) is a member of the IL-1 receptor (ILR) family with distinct structural and functional characteristics, acting as a negative regulator of ILR and Toll-like receptor (TLR) downstream signalling pathways and inflammation. Natural killer (NK) cells are innate lymphoid cells which mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses. NK cells mediate resistance against haematopoietic neoplasms but are generally considered to play a minor role in solid tumour carcinogenesis. Here we report that IL-1R8 serves as a checkpoint for NK cell maturation and effector function. Its genetic blockade unleashes NK-cell-mediated resistance to hepatic carcinogenesis, haematogenous liver and lung metastasis, and cytomegalovirus infection.

The Long Pentraxin 3 Contributes to Joint Inflammation in Gout by Facilitating the Phagocytosis of Monosodium Urate Crystals.

The purpose of this study was to investigate the role of pentraxin 3 (PTX3), a pivotal component of the innate immune system, in gout. Levels of PTX3 and IL-1ß in human samples were evaluated by ELISA. Development of murine gout was evaluated through the levels of cytokines (PTX3, CXCL1, and IL-1ß) and neutrophil recruitment into the joint cavity. Phagocytosis of monosodium urate (MSU) crystals and caspase-1 activation were determined by flow cytometer. Acute gout patients showed elevated concentration of PTX3 in plasma and synovial fluid as compared with healthy and osteoarthritic subjects. Moreover, there was a positive correlation between intra-articular PTX3 and IL-1ß levels. PTX3 was induced in the periarticular tissue of mice postinjection of MSU crystals. Importantly, Ptx3-deficient mice showed reduced inflammation in response to MSU crystal injection compared with wild-type mice, including reduction of neutrophil recruitment into the joint cavity and IL-1ß and CXCL1 production. Interestingly, addition of PTX3 in vitro enhanced MSU crystal phagocytosis by monocytes and resulted in higher production of IL-1ß by macrophages. This contribution of PTX3 to the phagocytosis of MSU crystals and consequent production of IL-1ß occurred through a mechanism mainly dependent on FcγRIII. Thus, our results suggest that PTX3 acts as a humoral pattern recognition molecule in gout facilitating MSU crystal phagocytosis and contributing to the pathogenesis of gouty arthritis.

Role of a fluid-phase PRR in fighting an intracellular pathogen: PTX3 in Shigella infection.

Shigella spp. are pathogenic bacteria that cause bacillary dysentery in humans by invading the colonic and rectal mucosa where they induce dramatic inflammation. Here, we have analyzed the role of the soluble PRR Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity. Mice that had been intranasally infected with S. flexneri were rescued from death by treatment with recombinant PTX3. In vitro PTX3 exerts the antibacterial activity against Shigella, impairing epithelial cell invasion and contributing to the bactericidal activity of serum. PTX3 is produced upon LPS-TLR4 stimulation in accordance with the lipid A structure of Shigella. In the plasma of infected patients, the level of PTX3 amount only correlates strongly with symptom severity. These results signal PTX3 as a novel player in Shigella pathogenesis and its potential role in fighting shigellosis. Finally, we suggest that the plasma level of PTX3 in shigellosis patients could act as a biomarker for infection severity.

Fluid phase recognition molecules in neutrophil-dependent immune responses.

The innate immune system comprises both a cellular and a humoral arm. Neutrophils are key effector cells of the immune and inflammatory responses and have emerged as a major source of humoral pattern recognition molecules (PRMs). These molecules, which include collectins, ficolins, and pentraxins, are specialised in the discrimination of self versus non-self and modified-self and share basic multifunctional properties including recognition and opsonisation of pathogens and apoptotic cells, activation and regulation of the complement cascade and tuning of inflammation. Neutrophils act as a reservoir of ready-made soluble PRMs, such as the long pentraxin PTX3, the peptidoglycan recognition protein PGRP-S, properdin and M-ficolin, which are stored in neutrophil granules and are involved in neutrophil effector functions. In addition, other soluble PRMs, such as members of the collectin family, are not expressed in neutrophils but can modulate neutrophil-dependent immune responses. Therefore, soluble PRMs are an essential part of the innate immune response and retain antibody-like effector functions. Here, we will review the expression and general function of soluble PRMs, focusing our attention on molecules involved in neutrophil effector functions.

Occurrence and significance of tumor-associated neutrophils in patients with colorectal cancer.

Inflammatory cells are an essential component of the tumor microenvironment. Neutrophils have emerged as important players in the orchestration and effector phase of innate and adaptive immunity. The significance of tumor-associated neutrophils (TAN) in colorectal cancer (CRC) has been the subject of conflicting reports and the present study was designed to set up a reliable methodology to assess TAN infiltration in CRC and to evaluate their clinical significance. CD66b and myeloperoxidase (MPO) were assessed as candidate neutrophil markers in CRC using immunohistochemistry. CD66b was found to be a reliable marker to identify TAN in CRC tissues, whereas MPO also identified a subset of CD68(+) macrophages. CRC patients (n = 271) (Stages I-IV) were investigated retrospectively by computer-assisted imaging on whole tumor sections. TAN density dramatically decreases in Stage IV patients as compared to Stage I-III. At Cox analysis, higher TAN density was associated with better prognosis. Importantly, multivariate analysis showed that prognostic significance of TAN can be influenced by clinical stage and 5-fluorouracil(5-FU)-based chemotherapy. On separate analysis of Stage III patients (n = 178), TAN density had a dual clinical significance depending on the use of 5-FU-based chemotherapy. Unexpectedly, higher TAN density was associated with better response to 5-FU-based chemotherapy. Thus, TAN are an important component of the immune cell infiltrate in CRC and assessment of TAN infiltration may help identify patients likely to benefit from 5-FU-based chemotherapy. These results call for a reassessment of the role of neutrophils in cancer using rigorous quantitative methodology.

The pentraxins PTX3 and SAP in innate immunity, regulation of inflammation and tissue remodelling.

Pentraxins are a superfamily of fluid phase pattern recognition molecules conserved in evolution and characterized by a cyclic multimeric structure. C-reactive protein (CRP) and serum amyloid P component (SAP) constitute the short pentraxin arm of the superfamily. CRP and SAP are produced in the liver in response to IL-6 and are acute phase reactants in humans and mice respectively. In addition SAP has been shown to affect tissue remodelling and fibrosis by stabilizing all types of amyloid fibrils and by regulating monocyte to fibrocyte differentiation. Pentraxin 3 (PTX3) is the prototype of the long pentraxin arm. Gene targeted mice and genetic and epigenetic studies in humans suggest that PTX3 plays essential non-redundant roles in innate immunity and inflammation as well as in tissue remodelling. Recent studies have revealed the role of PTX3 as extrinsic oncosuppressor, able to tune cancer-related inflammation. In addition, at acidic pH PTX3 can interact with provisional matrix components promoting inflammatory matrix remodelling. Thus acidification during tissue repair sets PTX3 in a tissue remodelling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

Integrating metabolic profiling of pancreatic juice with transcriptomic analysis of pancreatic cancer tissue identifies distinct clinical subgroups.

Introduction: Metabolic reprogramming is a hallmark feature of pancreatic ductal adenocarcinoma (PDAC). A pancreatic juice (PJ) metabolic signature has been reported to be prognostic of oncological outcome for PDAC. Integration of PJ profiling with transcriptomic and spatial characterization of the tumor microenvironment would help in identifying PDACs with peculiar vulnerabilities. Methods: We performed a transcriptomic analysis of 26 PDAC samples grouped into 3 metabolic clusters (M_CL) according to their PJ metabolic profile. We analyzed molecular subtypes and transcriptional differences. Validation was performed by multidimensional imaging on tumor slides. Results: Pancreatic juice metabolic profiling was associated with PDAC transcriptomic molecular subtypes (p=0.004). Tumors identified as M_CL1 exhibited a non-squamous molecular phenotype and demonstrated longer survival. Enrichment analysis revealed the upregulation of immune genes and pathways in M_CL1 samples compared to M_CL2, the group with worse prognosis, a difference confirmed by immunofluorescence on tissue slides. Enrichment analysis of 39 immune signatures by xCell confirmed decreased immune signatures in M_CL2 compared to M_CL1 and allowed a stratification of patients associated with longer survival. Discussion: PJ metabolic fingerprints reflect PDAC molecular subtypes and the immune microenvironment, confirming PJ as a promising source of biomarkers for personalized therapy.

High-Resolution Analysis of Mononuclear Phagocytes Reveals GPNMB as a Prognostic Marker in Human Colorectal Liver Metastasis.

Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell-cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

Oncogenic KRAS-Induced Protein Signature in the Tumor Secretome Identifies Laminin-C2 and Pentraxin-3 as Useful Biomarkers for the Early Diagnosis of Pancreatic Cancer.

KRAS mutations characterize pancreatic cell transformation from the earliest stages of carcinogenesis, and are present in >95% of pancreatic ductal adenocarcinoma (PDAC) cases. In search of novel biomarkers for the early diagnosis of PDAC, we identified the proteins secreted by the normal human pancreatic cell line (HPDE) recently transformed by inducing the overexpression of the KRASG12V oncogene. We report a proteomic signature of KRAS-induced secreted proteins, which was confirmed in surgical tumor samples from resected PDAC patients. The putative diagnostic performance of three candidates, Laminin-C2 (LAMC2), Tenascin-C (TNC) and Pentraxin-3 (PTX3), was investigated by ELISA quantification in two cohorts of PDAC patients (n = 200) eligible for surgery. Circulating levels of LAMC2, TNC and PTX3 were significantly higher in PDAC patients compared to the healthy individuals (p < 0.0001). The Receiver Operating Characteristics (ROC) curve showed good sensitivity (1) and specificity (0.63 and 0.85) for LAMC2 and PTX3, respectively, but not for TNC, and patients with high levels of LAMC2 had significantly shorter overall survival (p = 0.0007). High levels of LAMC2 and PTX3 were detected at early stages (I−IIB) and in CA19-9-low PDAC patients. In conclusion, pancreatic tumors release LAMC2 and PTX3, which can be quantified in the systemic circulation, and may be useful in selecting patients for further diagnostic imaging.

The Long Pentraxin PTX3 Controls Klebsiella Pneumoniae Severe Infection.

Klebsiella pneumoniae is a common pathogen in human sepsis. The emergence of multidrug-resistant K. pneumoniae strains represents a major clinical challenge in nosocomial and community acquired infections. The long pentraxin PTX3, a key component of humoral innate immunity, is involved in resistance to selected pathogens by promoting opsonophagocytosis. We investigated the relevance of PTX3 in innate immunity against K. pneumoniae infections using Ptx3-/- mice and mouse models of severe K. pneumoniae infections. Local and systemic PTX3 expression was induced following K. pneumoniae pulmonary infection, in association with the up-regulation of TNF-α and IL-1ß. PTX3 deficiency in mice was associated with higher bacterial burden and mortality, release of pro-inflammatory cytokines as well as IL-10 in the lung and systemically. The analysis of the mechanisms responsible of PTX3-dependent control of K. pneumoniae infection revealed that PTX3 did not interact with K. pneumoniae, or promote opsonophagocytosis. The comparison of susceptibility of wild-type, Ptx3-/-, C3-/- and Ptx3-/- /C3-/- mice to the infection showed that PTX3 acted in a complement-independent manner. Lung histopathological analysis showed more severe lesions in Ptx3-/- mice with fibrinosuppurative, necrotizing and haemorrhagic bronchopneumonia, associated with increased fibrin deposition in the lung and circulating fibrinogen consumption. These findings indicate that PTX3 contributes to the control of K. pneumoniae infection by modulating inflammatory responses and tissue damage. Thus, this study emphasizes the relevance of the role of PTX3 as regulator of inflammation and orchestrator of tissue repair in innate responses to infections.

Interference of the CadC regulator in the arginine-dependent acid resistance system of Shigella and enteroinvasive E. coli.

A typical pathoadaptive mutation of Shigella and enteroinvasive Escherichia coli (EIEC) is the inactivation of the cad locus which comprises the genes necessary for lysine decarboxylation, an enzyme involved in pH homoeostasis. In E. coli, the cadBA operon, encoding lysine decarboxylase (CadA) and a lysine cadaverine antiporter (CadB), is submitted to the control of CadC, a positive activator whose gene maps upstream the operon, and is transcribed independently from the same strand. CadC is an integral inner membrane protein which acts both, as signal sensor and as transcriptional regulator responding to the low pH and lysine signals. Analysis of the molecular rearrangements responsible for the loss of lysine decarboxylase activity in Shigella and EIEC has revealed that the inactivation of the cadC gene is a common feature. The 3 major adaptive acid resistance (AR) systems - AR1, AR2, and AR3 - are known to be activated at low pH by Shigella and E. coli, allowing them to withstand extremely acid conditions. In this study, evaluating the survival of S. flexneri, S. sonnei, and EIEC strains complemented with a functional cadC gene and challenged at low pH, we present evidence that CadC negatively regulates the expression of the arginine-dependent adaptive acid-resistance system (AR3), encoded by the adi locus while it has no effect on the expression of AR1 and AR2 systems. Moreover, since our results indicate that in enteroinvasive strains the presence of CadC reduces the expression of the arginine decarboxylase encoding gene adiA, it is possible to hypothesize that the loss of functionality of lysine decarboxylase is counterbalanced by a higher expression of the adi system, and that CadC, besides specifically affecting the regulation of the cadBA operon, is also relevant to other systems responding to low pH.

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Macrophage morphology correlates with single-cell diversity and prognosis in colorectal liver metastasis.

It has long been known that in vitro polarized macrophages differ in morphology. Stemming from a conventional immunohistology observation, we set out to test the hypothesis that morphology of tumor-associated macrophages (TAMs) in colorectal liver metastasis (CLM) represents a correlate of functional diversity with prognostic significance. Density and morphological metrics of TAMs were measured and correlated with clinicopathological variables. While density of TAMs did not correlate with survival of CLM patients, the cell area identified small (S-TAM) and large (L-TAM) macrophages that were associated with 5-yr disease-free survival rates of 27.8% and 0.2%, respectively (P < 0.0001). RNA sequencing of morphologically distinct macrophages identified LXR/RXR as the most enriched pathway in large macrophages, with up-regulation of genes involved in cholesterol metabolism, scavenger receptors, MERTK, and complement. In single-cell analysis of mononuclear phagocytes from CLM tissues, S-TAM and L-TAM signatures were differentially enriched in individual clusters. These results suggest that morphometric characterization can serve as a simple readout of TAM diversity with strong prognostic significance.

Metabolome of Pancreatic Juice Delineates Distinct Clinical Profiles of Pancreatic Cancer and Reveals a Link between Glucose Metabolism and PD-1+ Cells.

Better understanding of pancreatic diseases, including pancreatic ductal adenocarcinoma (PDAC), is an urgent medical need, with little advances in preoperative differential diagnosis, preventing rational selection of therapeutic strategies. The clinical management of pancreatic cancer patients would benefit from the identification of variables distinctively associated with the multiplicity of pancreatic disorders. We investigated, by 1H nuclear magnetic resonance, the metabolomic fingerprint of pancreatic juice (the biofluid that collects pancreatic products) in 40 patients with different pancreatic diseases. Metabolic variables discriminated PDAC from other less aggressive pancreatic diseases and identified metabolic clusters of patients with distinct clinical behaviors. PDAC specimens were overtly glycolytic, with significant accumulation of lactate, which was probed as a disease-specific variable in pancreatic juice from a larger cohort of 106 patients. In human PDAC sections, high expression of the glucose transporter GLUT-1 correlated with tumor grade and a higher density of PD-1+ T cells, suggesting their accumulation in glycolytic tumors. In a preclinical model, PD-1+ CD8 tumor-infiltrating lymphocytes differentially infiltrated PDAC tumors obtained from cell lines with different metabolic consumption, and tumors metabolically rewired by knocking down the phosphofructokinase (Pfkm) gene displayed a decrease in PD-1+ cell infiltration. Collectively, we introduced pancreatic juice as a valuable source of metabolic variables that could contribute to differential diagnosis. The correlation of metabolic markers with immune infiltration suggests that upfront evaluation of the metabolic profile of PDAC patients could foster the introduction of immunotherapeutic approaches for pancreatic cancer.

Macrophages in Colorectal Cancer Liver Metastases.

Tumor-associated macrophages (TAMs) provide a nurturing microenvironment for metastasis and are concomitantly key determinants of the efficacy of anticancer strategies. TAM represent an extremely heterogeneous population in terms of cell morphology, functions, and tissue localization. Colorectal liver metastases (CLM) display a high heterogeneity, responsible for a wide array of clinical presentations and responsiveness to treatments. In the era of precision medicine, there is a critical need of reliable prognostic markers to improve patient stratification, and, for their predominance in metastatic tissues, TAMs are emerging as promising candidates.

The two-faced role of cad genes in the virulence of pathogenic Escherichia coli.

In enterobacteria, acid stress induces expression of the cad system which is involved in maintaining intracellular pH at levels compatible with cell survival. Despite its crucial role, the cad operon is silenced in Shigella and in other pathogenic Escherichia coli. In the present review, we will address the question of why and how the cad locus has been sacrificed for the sake of optimal expression of virulence traits.

Glucose availability enhances lipopolysaccharide production and immunogenicity in the opportunistic pathogen Acinetobacter baumannii.

AIM: Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis. MATERIALS & METHODS: We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages. RESULTS: Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium. CONCLUSION: We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

Expression and function of IL-1R8 (TIR8/SIGIRR): a regulatory member of the IL-1 receptor family in platelets.

AIMS: Platelets express functional interleukin-1 receptor-1 (IL-1R1) as well as a repertoire of toll-like receptors (TLRs) involved in platelet activation, platelet-leucocyte reciprocal activation, and immunopathology. IL-1R8, also known as single Ig IL-1-related receptor (SIGIRR) or TIR8, is a member of the IL-1R family that negatively regulates responses to IL-1R family members and TLRs. In the present study, we addressed the expression of IL-1R8 in platelets and megakaryocytes and its role in the control of platelet activation during inflammatory conditions and thromboembolism. METHODS AND RESULTS: Here, we show by flow cytometry analysis, western blot, confocal microscopy, and quantitative real-time polymerase chain reaction that IL-1R8 is expressed on human and mouse platelets at high levels and on megakaryocytes. IL-1R8-deficient mice show normal levels of circulating platelets. Homotypic and heterotypic (platelet-neutrophil) aggregation triggered by Adenosine DiPhosphate (ADP) and IL-1 or lipopolysaccharide (LPS) was increased in IL-1R8-deficient platelets. IL-1R8-deficient mice showed increased soluble P-selectin levels and increased platelet-neutrophil aggregates after systemic LPS administration. Commensal flora depletion and IL-1R1 deficiency abated platelet hyperactivity and the increased platelet/neutrophil aggregation observed in Il1r8(-/-) mice in vitro and in vivo, suggesting a key role of IL-1R8 in regulating platelet TLR and IL-1R1 function. In a mouse model of platelet-dependent pulmonary thromboembolism induced by ADP administration, IL-1R8-deficient mice showed an increased frequency of blood vessel complete obstruction. CONCLUSION: These results show that platelets, which have a large repertoire of TLRs and IL-1 receptors, express high levels of IL-1R8, which plays a non-redundant function as a regulator of thrombocyte activity in vitro and in vivo.