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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.

Supplementary Tables

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.

Supplementary Figures 1-4, Tables S1 and S5

Supplementary Table S4

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.

Supplementary figure legends

Supplementary Figures

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.

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.

Macrophage morphology could be used to stratify metastasis risk for CRC patients., Using macrophage morphology in human colorectal cancer liver metastasis, Donadon et al. in this issue of JEM (https://doi.org/10.1084/jem.20191847) provide a window into lipid metabolism and foamy macrophages, which accrue in numerous pathological states and here are shown to have clinical application.

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.

Summary 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 αβ T cells (UTCαβ). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαβ 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 UTCαβ polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors., Graphical Abstract, Highlights • Neutrophils mediate antitumor response by sustaining an IL-12/IFNγ-dependent pathway • Neutrophils are essential for unconventional αβ T cell (UTCαβ) type 1 polarization • Type 1 UTCαβ possess an innate-like phenotype and display antitumor potential in vivo • Neutrophil infiltration is associated with good prognosis in selected human tumors, Tumor-associated neutrophils (TANs) have mainly been portrayed as tumor-promoters. Here, we describe a novel antitumor pathway in which TANs promote IL-12 production by macrophages, leading to type 1 polarization of a subset of unconventional αβ T cell (UTCαβ). Type 1 UTCαβ possess an innate-like phenotype and antitumor potential in vivo. In selected human tumors, neutrophil infiltration is associated with type 1 immunity and better clinical outcome.

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-1β in human samples were evaluated by ELISA. Development of murine gout was evaluated through the levels of cytokines (PTX3, CXCL1, and IL-1β) 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-1β 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-1β and CXCL1 production. Interestingly, addition of PTX3 in vitro enhanced MSU crystal phagocytosis by monocytes and resulted in higher production of IL-1β by macrophages. This contribution of PTX3 to the phagocytosis of MSU crystals and consequent production of IL-1β 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.

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.

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 .

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) (stage I to stage IV) were investigated retrospectively by computer-assisted imaging on whole tumor sections. TAN density dramatically decreases in stage IV patients 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. This article is protected by copyright. All rights reserved.

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.

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(1). Natural killer (NK) cells are innate lymphoid cells which mediate resistance against pathogens and contribute to the activation and orientation of adaptive immune responses(2-4). NK cells mediate resistance against haematopoietic neoplasms but are generally considered to play a minor role in solid tumour carcinogenesis(5-7). 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.

Background 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, tumor-associated macrophages (TAMs) emerge as promising candidates. The aim of this study was to test the presence of discrete TAM populations in CLM patients on the basis of morphology, and to test the impact of TAMs morphology on recurrence-free survival (RFS). Methods NCT038888638 is a single-center study conducted in a tertiary-referral university hospital that examined a cohort of patients with CLMs that underwent hepatectomy between 2005 and 2017. TAMs cell density, cell area and cell perimeter were systematically quantified in 3 non-contiguous and non-overlapping areas of CLM sections by means of immuno-reactive area of CD163+ macrophages. The association of TAMs metrics and RFS was tested by using receiver operating characteristics (ROC) curves, multivariate Cox regression analysis and survival analysis. Results A cohort of 101 CLM patients resected between 2005 and 2017 was considered. Among density (AUC=0.555; 95%CI=0.410-0.701; P = 0.449), perimeter (AUC=0.526; 95%CI=0.383-0.671; P = 0.708) and area (AUC=0.791; 95%CI=0.572-0.841; P = 0.006) ofCD163+ TAMs, only the latter was significantly associated with differences in survival time. Small and large TAMs, as defined by using the best cutoff value extrapolated from the ROC curve (area: 60.39 μm2; Se = 0.79; Sp = 0.44), were clearly associated with significantly different 5-year RFS rates of 27.8% and 0.2% respectively (P Conclusions Macrophage morphology is critically associated with prognosis of CLM patients. The results reported here support that accurate quantitative morphometric characterization of TAMs can serve as an easily quantifiable correlate of functional diversity with prognostic significance. Clinical trial identification NCT038888638. Legal entity responsible for the study The authors. Funding Associazione Italiana per la Ricerca sul Cancro (AIRC). Disclosure All authors have declared no conflicts of interest.

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.

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 polyamine profile of Shigella, the etiological agent of bacillary dysentery in humans, differs markedly from that of E. coli, its innocuous commensal ancestor. Pathoadaptive mutations such as the loss of cadaverine and the increase of spermidine favour the full expression of the virulent phenotype of Shigella. Spermidine levels affect the expression of the MdtJI complex, a recently identified efflux pump belonging to the small multi-drug resistance family of transporters. In the present study, we have addressed the regulation of the mdtJI operon in Shigella by asking which factors influence its expression as compared to E. coli. In particular, after identifying the mdtJI promoter by primer extension analysis, in vivo transcription assays and gel-retardation experiments were carried out to get insight on the silencing of mdtJI in E. coli. The results indicate that H-NS, a major nucleoid protein, plays a key role in repressing the mdtJI operon by direct binding to the regulatory region. In the Shigella background mdtJI expression is increased by the high levels of spermidine typically found in this microorganism and by VirF, the plasmid-encoded regulator of the Shigella virulence regulatory cascade. We also show that the expression of mdtJI is stimulated by bile components. Functional analyses reveal that MdtJI is able to promote the excretion of putrescine, the spermidine precursor. This leads us to consider the MdtJI complex as a possible safety valve allowing Shigella to maintain spermidine to a level optimally suited to survival within infected macrophages and, at the same time, prevent toxicity due to spermidine over-accumulation.

"Objective: Functional polarization of human monocyte-derived macrophages (MDMs) into M1 cells leads to inhibition of R5 HIV-1 replication and viral DNA synthesis in comparison to control, unpolarized cells together with CD4 downregulation from the cell surface and upregulation of CCR5-binding chemokine secretion. We here investigated whether a postentry restriction of virus replication is also induced by M1 polarization of MDM.. \t . . Design: MDM were first polarized to M1 cells by 18 h stimulation with interferon-[gamma] and tumor necrosis factor-[alpha]; the cytokines were then removed and the cells were infected with vesicular stomatitis virus G-protein pseudotyped enhanced green fluorescence protein HIV-1 (HIV-GFP) generating a single-round infection cycle.. \t . . Methods: HIV-1 expression was monitored in terms of eGFP expression by fluorescence activated cell sorter (FACS) analysis and real-time PCR analysis of total HIV-1 gag DNA, 2-long terminal repeat DNA, proviral DNA, and multiply spliced RNA transcripts. Expression of apolipopoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3G (APOBEC3G), and APOBEC3A was tested by western blotting and FACS analysis.. \t . . Results: Inhibition of HIV-GFP expression was observed in M1-MDM along with impaired viral DNA synthesis, delayed proviral integration, and reduced proviral transcription. Although APOBEC3G levels were similar in M1 and unpolarized MDM, APOBEC 3A was selectively expressed only by M1 cells.. \t . . Conclusion: M1 polarization of in-vitro differentiated primary MDM determines a transient, but profound restriction of HIV-1 replication affecting multiple (entry and postentry) steps in the virus life cycle likely involving the upregulated expression of APOBEC3A."

International audience; The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.

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.

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-1β. 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.