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IntroductionDozens of vaccines have been approved or authorized internationally in response to the ongoing SARS-CoV-2 pandemic, covering a range of modalities and routes of delivery. For example, mucosal delivery of vaccines via the intranasal (i.n.) route has been shown to improve protective mucosal responses in comparison to intramuscular (i.m.) delivery. As we gain knowledge of the limitations of existing vaccines, it is of interest to understand if changes in product presentation or combinations of multiple vaccine modalities can further improve immunological outcomes.MethodsWe investigated a commercial-stage SARS-CoV-2 receptor binding domain (RBD) antigen adjuvanted with a clinical-stage TLR-7/8 agonist (3M-052) formulated on aluminum oxyhydroxide (Alum). In a murine immunogenicity model, we compared i.n. and i.m. dosing of the RBD-3M-052-Alum vaccine. We measured the magnitude of antibody responses in serum and lungs, the antibody-secreting cell populations in bone marrow, and antigen-specific cytokine-secreting splenocyte populations. Similarly, we compared different heterologous and homologous prime-boost regimens using the RBD-3M-052-Alum vaccine and a clinical-stage self-amplifying RNA (saRNA) vaccine formulated on a nanostructured lipid carrier (NLC) using the i.m. route alone. Finally, we developed a lyophilized presentation of the RBD-3M-052-Alum vaccine and compared it to the liquid presentation and a heterologous regimen including a previously characterized lyophilized form of the saRNA-NLC vaccine.Results and discussionWe demonstrate that i.n. dosing of the RBD-3M-052-Alum vaccine increased IgA titers in the lung by more than 1.5 logs, but induced serum IgG titers 0.8 logs lower, in comparison to i.m. dosing of the same vaccine. We also show that the homologous prime-boost RBD-3M-052-Alum regimen led to the highest serum IgG and bronchial IgA titers, whereas the homologous saRNA-NLC regimen led to the highest splenocyte interferon-γ response. We found that priming with the saRNA-NLC vaccine and boosting with the RBD-3M-052-Alum vaccine led to the most desirable immune outcome of all regimens tested. Finally, we show that the lyophilized RBD-3M-052-Alum vaccine retained its immunological characteristics. Our results demonstrate that the route of delivery and the use of heterologous regimens each separately impacts the resulting immune profile, and confirm that multi-product vaccine regimens can be developed with stabilized presentations in mind.

IntroductionThe lymphatic system is a multifaceted regulator of tissue homeostasis and an integral part of immune responses. Previous studies had shown that subsets of lymphatic endothelial cells (LEC) express PTX3, an essential component of humoral innate immunity and tissue homeostasis.MethodsIn the present study using whole-mount imaging and image-based morphometric quantifications, Ptx3-targeted mice and in vivo functional analysis, we investigated the involvement of PTX3 in shaping and function of the lymphatic vasculature.ResultsWe found that PTX3 is localized in the extracellular matrix (ECM) surrounding human and murine lymphatic vessels (LV). In murine tissues, PTX3 was localized in the ECM close to LV terminals and sprouting. Ptx3-deficient mice showed LV abnormalities in the colon submucosa and diaphragm, including a disorganized pattern and hyperplasia of initial LV capillaries associated with altered distribution of tight junction-associated molecules. Mice with LEC-restricted PTX3 gene inactivation showed morphological and organization abnormalities similar to those observed in Ptx3-deficient animals. Ptx3-deficient mice showed defective fluid drainage from footpads and defective dendritic cell (DC) trafficking.DiscussionThus, PTX3 is strategically localized in the ECM of specialized LV, playing an essential role in their structural organization and immunological function.

Soluble pattern recognition molecules (PRMs) are a heterogenous group of proteins that recognize pathogen- and danger-associated molecular patterns (PAMPs and DAMPs, respectively), and cooperate with cell-borne receptors in the orchestration of innate and adaptive immune responses to pathogenic insults and tissue damage. Amongst soluble PRMs, pentraxins are a family of highly conserved proteins with distinctive structural features. Originally identified in the early 1990s as an early inflammatory gene, PTX3 is the prototype of long pentraxins. Unlike the short pentraxin C reactive protein (CRP), whose expression is mostly confined to the liver, PTX3 is made by several immune and non-immune cells at sites of infection and inflammation, where it intercepts fundamental aspects of infection immunity, inflammation, and tissue remodeling. Of note, PTX3 cross talks to components of the complement system to control cancer-related inflammation and disposal of pathogens. Also, it is an essential component of inflammatory extracellular matrices (ECMs) through crosslinking of hyaluronic acid and turn-over of provisional fibrin networks that assemble at sites of tissue injury. This functional diversity is mediated by unique structural characteristics whose fine details have been unveiled only recently. Here, we revisit the structure/function relationships of this long pentraxin in light of the most recent advances in its structural biology, with a focus on the interplay with complement and the emerging roles as a component of the ECM. Differences to and similarities with the short pentraxins are highlighted and discussed.

Introduction: Dozens of vaccines have been approved or authorized internationally in response to the ongoing SARS-CoV-2 pandemic, covering a range of modalities and routes of delivery. For example, mucosal delivery of vaccines via the intranasal (i.n.) route has been shown to improve protective mucosal responses in comparison to intramuscular (i.m.) delivery. As we gain knowledge of the limitations of existing vaccines, it is of interest to understand if changes in product presentation or combinations of multiple vaccine modalities can further improve immunological outcomes. Methods: We investigated a commercial-stage SARS-CoV-2 receptor binding domain (RBD) antigen adjuvanted with a clinical-stage TLR-7/8 agonist (3M-052) formulated on aluminum oxyhydroxide (Alum). In a murine immunogenicity model, we compared i.n. and i.m. dosing of the RBD-3M-052-Alum vaccine. We measured the magnitude of antibody responses in serum and lungs, the antibody-secreting cell populations in bone marrow, and antigen-specific cytokine-secreting splenocyte populations. Similarly, we compared different heterologous and homologous prime-boost regimens using the RBD-3M-052-Alum vaccine and a clinical-stage self-amplifying RNA (saRNA) vaccine formulated on a nanostructured lipid carrier (NLC) using the i.m. route alone. Finally, we developed a lyophilized presentation of the RBD-3M-052-Alum vaccine and compared it to the liquid presentation and a heterologous regimen including a previously characterized lyophilized form of the saRNA-NLC vaccine. Results and discussion: We demonstrate that i.n. dosing of the RBD-3M-052-Alum vaccine increased IgA titers in the lung by more than 1.5 logs, but induced serum IgG titers 0.8 logs lower, in comparison to i.m. dosing of the same vaccine. We also show that the homologous prime-boost RBD-3M-052-Alum regimen led to the highest serum IgG and bronchial IgA titers, whereas the homologous saRNA-NLC regimen led to the highest splenocyte interferon-γ response. We found that priming with the saRNA-NLC vaccine and boosting with the RBD-3M-052-Alum vaccine led to the most desirable immune outcome of all regimens tested. Finally, we show that the lyophilized RBD-3M-052-Alum vaccine retained its immunological characteristics. Our results demonstrate that the route of delivery and the use of heterologous regimens each separately impacts the resulting immune profile, and confirm that multi-product vaccine regimens can be developed with stabilized presentations in mind. [ABSTRACT FROM AUTHOR]

Introduction: The lymphatic system is a multifaceted regulator of tissue homeostasis and an integral part of immune responses. Previous studies had shown that subsets of lymphatic endothelial cells (LEC) express PTX3 , an essential component of humoral innate immunity and tissue homeostasis. Methods: In the present study using whole-mount imaging and image-based morphometric quantifications, Ptx3 -targeted mice and in vivo functional analysis, we investigated the involvement of PTX3 in shaping and function of the lymphatic vasculature. Results: We found that PTX3 is localized in the extracellular matrix (ECM) surrounding human and murine lymphatic vessels (LV). In murine tissues, PTX3 was localized in the ECM close to LV terminals and sprouting. Ptx3 -deficient mice showed LV abnormalities in the colon submucosa and diaphragm, including a disorganized pattern and hyperplasia of initial LV capillaries associated with altered distribution of tight junction-associated molecules. Mice with LEC-restricted PTX3 gene inactivation showed morphological and organization abnormalities similar to those observed in Ptx3 -deficient animals. Ptx3-deficient mice showed defective fluid drainage from footpads and defective dendritic cell (DC) trafficking. Discussion: Thus, PTX3 is strategically localized in the ECM of specialized LV, playing an essential role in their structural organization and immunological function. [ABSTRACT FROM AUTHOR]

Even though cancer patients are generally considered more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the mechanisms driving their predisposition to severe forms of coronavirus disease 2019 (COVID-19) have not yet been deciphered. Since metabolic disorders are associated with homeostatic frailty, which increases the risk of infection and cancer, we asked whether we could identify immunometabolic pathways intersecting with cancer and SARS-CoV-2 infection. Thanks to a combined flow cytometry and multiomics approach, here we show that the immunometabolic traits of COVID-19 cancer patients encompass alterations in the frequency and activation status of circulating myeloid and lymphoid subsets, and that these changes are associated with i) depletion of tryptophan and its related neuromediator tryptamine, ii) accumulation of immunosuppressive tryptophan metabolites (i.e., kynurenines), and iii) low nicotinamide adenine dinucleotide (NAD+) availability. This metabolic imbalance is accompanied by altered expression of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs), with a distinctive downregulation of IL-6 and upregulation of IFNγ mRNA expression levels. Altogether, our findings indicate that cancer not only attenuates the inflammatory state in COVID-19 patients but also contributes to weakening their precarious metabolic state by interfering with NAD+-dependent immune homeostasis.

Osteomyelitis (OM) is an infectious disease of the bone predominantly caused by the opportunistic bacterium Staphylococcus aureus (S. aureus). Typically established upon hematogenous spread of the pathogen to the musculoskeletal system or contamination of the bone after fracture or surgery, osteomyelitis has a complex pathogenesis with a critical involvement of both osteal and immune components. Colonization of the bone by S. aureus is traditionally proposed to induce functional inhibition and/or apoptosis of osteoblasts, alteration of the RANKL/OPG ratio in the bone microenvironment and activation of osteoclasts; all together, these events locally subvert tissue homeostasis causing pathological bone loss. However, this paradigm has been challenged in recent years, in fact osteoblasts are emerging as active players in the induction and orientation of the immune reaction that mounts in the bone during an infection. The interaction with immune cells has been mostly ascribed to osteoblast-derived soluble mediators that add on and synergize with those contributed by professional immune cells. In this respect, several preclinical and clinical observations indicate that osteomyelitis is accompanied by alterations in the local and (sometimes) systemic levels of both pro-inflammatory (e.g., IL-6, IL-1α, TNF-α, IL-1β) and anti-inflammatory (e.g., TGF-β1) cytokines. Here we revisit the role of osteoblasts in bacterial OM, with a focus on their secretome and its crosstalk with cellular and molecular components of the bone microenvironment and immune system.

BackgroundPTX3 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.MethodsLevels 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.ResultsUpon 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

BackgroundEarly prognostic stratification of patients with sepsis is a difficult clinical challenge. Aim of this study was to evaluate novel molecules in association with clinical parameters as predictors of 90-days mortality in patients admitted with sepsis at Humanitas Research Hospital.MethodsPlasma samples were collected from 178 patients, diagnosed based on Sepsis-3 criteria, at admission to the Emergency Department and after 5 days of hospitalization. Levels of pentraxin 3 (PTX3), soluble IL-1 type 2 receptor (sIL-1R2), and of a panel of pro- and anti-inflammatory cytokines were measured by ELISA. Cox proportional-hazard models were used to evaluate predictors of 90-days mortality.ResultsCirculating levels of PTX3, sIL-1R2, IL-1β, IL-6, IL-8, IL-10, IL-18, IL-1ra, TNF-α increased significantly in sepsis patients on admission, with the highest levels measured in shock patients, and correlated with SOFA score (PTX3: r=0.44, p

Trichuriasis is one of the most common neglected tropical diseases of the world’s poorest people. A recombinant vaccine composed of Tm-WAP49, an immunodominant antigen secreted by adult Trichuris stichocytes into the mucosa of the cecum to which the parasite attaches, is under development. The prototype is being evaluated in a mouse model of Trichuris muris infection, with the ultimate goal of producing a mucosal vaccine through intranasal delivery. Intranasal immunization of mice with Tm-WAP49 formulated with the adjuvant OCH, a truncated analog of alpha-GalCer with adjuvanticity to stimulate natural killer T cells (NKT) and mucosal immunity, induced significantly high levels of IgG and its subclasses (IgG1 and IgG2a) in immunized mice. This also resulted in a significant reduction of worm burden after challenge with T. muris-infective eggs. The addition of QS-21 adjuvant to this vaccine formulation further reduced worm counts. The improved protection from the dual-adjuvanted vaccine correlated with higher serum antibody responses (IgG, IgG1, IgG2a, IgA) as well as with the induction of antigen-specific IgA in the nasal mucosa. It was also associated with the robust cellular responses including functional subsets of CD4 T cells producing IL-4, and cytotoxic CD8 T cells expressing granzyme B. The worm reduction achieved by mucosal immunization was higher than that induced by subcutaneous immunization. Intranasal immunization also induced a significantly higher nasal mucosa-secreted antigen-specific IgA response, as well as higher functional cellular responses including CD4+IL4+ (Th1) and CD8+GnzB+ (Th2) T cells, and antigen-specific INFγ-producing T cells in both spleen and MLNs and antibody-producing B cells (CD19+B220+/B220+GL7+). Mucosal immunization further induced long-term T lymphocyte memory with increased central (CD62L+CD44+) and effector (CD62L-CD44+) memory subsets of both CD4 and CD8 T cells at 60 days after the last immunization. In summary, intranasal immunization with recombinant Tm-WAP49 protein induced strong protection versus murine trichuriasis. It represents a promising vaccination approach against intestinal nematodes.

Control of human ascariasis, the most prevalent neglected tropical disease globally affecting 450 million people, mostly relies on mass drug administration of anthelmintics. However, chemotherapy alone is not efficient due to the high re-infection rate for people who live in the endemic area. The development of a vaccine that reduces the intensity of infection and maintains lower morbidity should be the primary target for infection control. Previously, our group demonstrated that immunization with crude Ascaris antigens in mice induced an IgG-mediated protective response with significant worm reduction. Here, we aimed to develop a multipeptide chimera vaccine based on conserved B-cell epitopes predicted from 17 common helminth proteomes using a bioinformatics algorithm. More than 480 B-cell epitopes were identified that are conserved in all 17 helminths. The Ascaris-specific epitopes were selected based on their reactivity to the pooled sera of mice immunized with Ascaris crude antigens or infected three times with A. suum infective eggs. The top 35 peptides with the strongest reactivity to Ascaris immune serum were selected to construct a chimeric antigen connected in sequence based on conformation. This chimera, called ASCVac-1, was produced as a soluble recombinant protein in an Escherichia coli expression system and, formulated with MPLA, was used to immunize mice. Mice immunized with ASCVac-1/MPLA showed around 50% reduced larvae production in the lungs after being challenged with A. suum infective eggs, along with significantly reduced inflammation and lung tissue/function damage. The reduced parasite count and pathology in infected lungs were associated with strong Th2 immune responses characterized by the high titers of antigen-specific IgG and its subclasses (IgG1, IgG2a, and IgG3) in the sera and significantly increased IL-4, IL-5, IL-13 levels in lung tissues. The reduced IL-33 titers and stimulated eosinophils were also observed in lung tissues and may also contribute to the ASCVac-1-induced protection. Taken together, the preclinical trial with ASCVac-1 chimera in a mouse model demonstrated its significant vaccine efficacy associated with strong IgG-based Th2 responses, without IgE induction, thus reducing the risk of an allergic response. All results suggest that the multiepitope-based ASCVac-1 chimera is a promising vaccine candidate against Ascaris sp. infections.

The ubiquitous mold Aspergillus fumigatus is the major etiologic agent of invasive aspergillosis, a life-threatening infection amongst immune compromised individuals. An increasing body of evidence indicates that effective disposal of A. fumigatus requires the coordinate action of both cellular and humoral components of the innate immune system. Early recognition of the fungal pathogen, in particular, is mediated by a set of diverse soluble pattern recognition molecules (PRMs) that act as “ancestral antibodies” inasmuch as they are endowed with opsonic, pro-phagocytic and killing properties. Pivotal is, in this respect, the contribution of the complement system, which functionally cooperates with cell-borne pattern recognition receptors (PRRs) and other soluble PRMs, including pentraxins. Indeed, complement and pentraxins form an integrated system with crosstalk, synergism, and regulation, which stands as a paradigm of the interplay between PRMs in the mounting and orchestration of antifungal immunity. Following upon our past experience with the long pentraxin PTX3, a well-established immune effector in the host response to A. fumigatus, we recently reported that this fungal pathogen is targeted in vitro and in vivo by the short pentraxin Serum Amyloid P component (SAP) too. Similar to PTX3, SAP promotes phagocytosis and disposal of the fungal pathogen via complement-dependent pathways. However, the two proteins exploit different mechanisms of complement activation and receptor-mediated phagocytosis, which further extends complexity and integration of the complement-pentraxin crosstalk in the immune response to A. fumigatus. Here we revisit this crosstalk in light of the emerging roles of SAP as a novel PRM with antifungal activity.

AimsTo determine the relationship between PTX3 systemic and synovial levels and the clinical features of rheumatoid arthritis (RA) in a cohort of early, treatment naïve patients and to explore the relevance of PTX3 expression in predicting response to conventional-synthetic (cs) Disease-Modifying-Anti-Rheumatic-Drugs (DMARDs) treatment.MethodsPTX3 expression was analyzed in 119 baseline serum samples from early naïve RA patients, 95 paired samples obtained 6-months following the initiation of cs-DMARDs treatment and 43 healthy donors. RNA-sequencing analysis and immunohistochemistry for PTX3 were performed on a subpopulation of 79 and 58 synovial samples, respectively, to assess PTX3 gene and protein expression. Immunofluorescence staining was performed to characterize PTX3 expressing cells within the synovium.ResultsCirculating levels of PTX3 were significantly higher in early RA compared to healthy donors and correlated with disease activity at baseline and with the degree of structural damages at 12-months. Six-months after commencing cs-DMARDs, a high level of PTX3, proportional to the baseline value, was still detectable in the serum of patients, regardless of their response status. RNA-seq analysis confirmed that synovial transcript levels of PTX3 correlated with disease activity and the presence of mediators of inflammation, tissue remodeling and bone destruction at baseline. PTX3 expression in the synovium was strongly linked to the degree of immune cell infiltration, the presence of ectopic lymphoid structures and seropositivity for autoantibodies. Accordingly, PTX3 was found to be expressed by numerous synovial cell types such as plasma cells, fibroblasts, vascular and lymphatic endothelial cells, macrophages, and neutrophils. The percentage of PTX3-positive synovial cells, although significantly reduced at 6-months post-treatment as a result of global decreased cellularity, was similar in cs-DMARDs responders and non-responders.ConclusionThis study demonstrates that, early in the disease and prior to treatment modification, the level of circulating PTX3 is a reliable marker of RA activity and predicts a high degree of structural damages at 12-months. In the joint, PTX3 associates with immune cell infiltration and the presence of ectopic lymphoid structures. High synovial and peripheral blood levels of PTX3 are associated with chronic inflammation characteristic of RA. Additional studies to determine the mechanistic link are required.

PTX3 is a soluble pattern recognition molecule (PRM) belonging to the humoral innate immune system, rapidly produced at inflammatory sites by phagocytes and stromal cells in response to infection or tissue injury. PTX3 interacts with microbial moieties and selected pathogens, with molecules of the complement and hemostatic systems, and with extracellular matrix (ECM) components. In wound sites, PTX3 interacts with fibrin and plasminogen and favors a timely removal of fibrin-rich ECM for an efficient tissue repair. Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown origin, associated with excessive ECM deposition affecting tissue architecture, with irreversible loss of lung function and impact on the patient’s life quality. Maccarinelli et al. recently demonstrated a protective role of PTX3 using the bleomycin (BLM)-induced experimental model of lung fibrosis, in line with the reported role of PTX3 in tissue repair. However, the mechanisms and therapeutic potential of PTX3 in IPF remained to be investigated. Herein, we provide new insights on the possible role of PTX3 in the development of IPF and BLM-induced lung fibrosis. In mice, PTX3-deficiency was associated with worsening of the disease and with impaired fibrin removal and subsequently increased collagen deposition. In IPF patients, microarray data indicated a down-regulation of PTX3 expression, thus suggesting a potential rational underlying the development of disease. Therefore, we provide new insights for considering PTX3 as a possible target molecule underlying therapeutic intervention in IPF.

Helminths, including nematodes, cestodes and trematodes, are complex parasitic organisms that infect at least one billion people globally living in extreme poverty. Helminthic infections are associated with severe morbidity particularly in young children who often harbor the highest burden of disease. While each helminth species completes a distinct life cycle within the host, several helminths incite significant lung disease. This impact on the lungs occurs either directly from larval migration and host immune activation or indirectly from a systemic inflammatory immune response. The impact of helminths on the pulmonary immune response involves a sophisticated orchestration and activation of the host innate and adaptive immune cells. The consequences of activating pulmonary host immune responses are variable with several helminthic infections leading to severe, pulmonary compromise while others providing immune tolerance and protection against the development of pulmonary diseases. Further delineation of the convoluted interface between helminth infection and the pulmonary host immune responses is critical to the development of novel therapeutics that are critically needed to prevent the significant global morbidity caused by these parasites.

The innate immune system is equipped with a number of germ-line encoded soluble pattern recognition molecules (PRMs) that collectively mediate the humoral host response to infection and damage in cooperation with cells and tissues of the immune and non-immune compartments. Despite the impressive diversity in structure, source, and regulation across PRMs, these all share remarkably similar functions inasmuch as they recognize microbes and damaged tissues, activate complement, exert opsono-phagocytic activities, and regulate inflammation. The long pentraxin 3 (PTX3) is a prototypic soluble PRM. Long known as a major player in innate immunity, inflammation and matrix remodeling, only recently has PTX3 emerged as a mediator of bone homeostasis in rodents and humans. Ptx3-targeted mice exhibit reduced trabecular volume during bone development, and impaired callus mineralization following experimental fracture. The murine gene is expressed in vivo by non-hematopoietic periosteal cells in the early phases of fracture healing, and in vitro by maturing osteoblasts. Human osteoblasts do express the PTX3 protein, whose levels positively correlate with bone density in vivo and osteoblast proliferation and maturation in vitro, thus pointing to a role in bone deposition. Contrasting evidence, however, suggest osteoclastogenesis-promoting effects of PTX3, where its expression has been associated with periodontitis, arthritis, and bone metastasis, conditions hallmarked by inflammation and bone resorption. Here, we review past and recent literature on the functions exerted by this long pentraxin in bone biology, with major emphasis on physiological skeletal remodeling, fracture healing, and chronic diseases of the bone.

The long pentraxin PTX3 is a member of the pentraxin family produced locally by stromal and myeloid cells in response to proinflammatory signals and microbial moieties. The prototype of the pentraxin family is C reactive protein (CRP), a widely-used biomarker in human pathologies with an inflammatory or infectious origin. Data so far describe PTX3 as a multifunctional protein acting as a functional ancestor of antibodies and playing a regulatory role in inflammation. Cardiovascular disease (CVD) is a leading cause of mortality worldwide, and inflammation is crucial in promoting it. Data from animal models indicate that PTX3 can have cardioprotective and atheroprotective roles regulating inflammation. PTX3 has been investigated in several clinical settings as possible biomarker of CVD. Data collected so far indicate that PTX3 plasma levels rise rapidly in acute myocardial infarction, heart failure and cardiac arrest, reflecting the extent of tissue damage and predicting the risk of mortality.

The innate immune system comprises a cellular and a humoral arm. Humoral pattern recognition molecules include complement components, collectins, ficolins, and pentraxins. These molecules are involved in innate immune responses by recognizing microbial moieties and damaged tissues, activating complement, exerting opsonic activity and facilitating phagocytosis, and regulating inflammation. The long pentraxin PTX3 is a prototypic humoral pattern recognition molecule that, in addition to providing defense against infectious agents, plays several functions in tissue repair and regulation of cancer-related inflammation. Characterization of the PTX3 molecular structure and biochemical properties, and insights into its interactome and multiple roles in tissue damage and remodeling support the view that microbial and matrix recognition are evolutionarily conserved functions of humoral innate immunity molecules.

The first line of defense in innate immunity is provided by cellular and humoral mediators. Pentraxins are a superfamily of phylogenetically conserved humoral mediators of innate immunity. PTX3, the first long pentraxin identified, is a soluble pattern recognition molecule rapidly produced by several cell types in response to primary pro-inflammatory signals and microbial recognition. PTX3 acts as an important mediator of innate immunity against pathogens of fungal, bacterial and viral origin, and as a regulator of inflammation, by modulating complement activation and cell extravasation, and facilitating pathogen recognition by myeloid cells. In sepsis, PTX3 plasma levels are associated with severity of the condition, patient survival, and response to therapy. In combination with other established biomarkers, PTX3 could improve stratification of sepsis patients and thus, complement the system of classification and monitoring of this disease.

Pentraxins and complement defense collagens are soluble recognition proteins that sense pathogens and altered-self elements, and trigger immune responses including complement activation. PTX3 has been shown to interact with the globular recognition domains (gC1q) of the C1q protein of the classical complement pathway, thereby modulating complement activity. The C1q-PTX3 interaction has been characterized previously by site-specific mutagenesis using individual gC1q domains of each of the three C1q chains. The present study is aimed at revisiting this knowledge taking advantage of full-length recombinant C1q. Four mutations targeting exposed amino acid residues in the gC1q domain of each of the C1q chains (LysA200Asp-LysA201Asp, ArgB108Asp-ArgB109Glu, TyrB175Leu, and LysC170Glu) were introduced in recombinant C1q and the interaction properties of the mutants were analyzed using surface plasmon resonance. All C1q mutants retained binding to C1r and C1s proteases and mannose-binding lectin-associated serine proteases, indicating that the mutations did not affect the function of the collagen-like regions of C1q. The effect of these mutations on the interaction of C1q with PTX3 and IgM, and both the PTX3- and IgM-mediated activation of the classical complement pathway were investigated. The LysA200Asp-LysA201Asp and LysC170Glu mutants retained partial interaction with PTX3 and IgM, however they triggered efficient complement activation. In contrast, the ArgB108Asp-ArgB109Glu mutation abolished C1q binding to PTX3 and IgM, and significantly decreased complement activation. The TyrB175Leu mutant exhibited decreased PTX3- and IgM-dependent complement activation. Therefore, we provided evidence that, in the context of the full length C1q protein, a key contribution to the interaction with both PTX3 and IgM is given by the B chain Arg residues that line the side of the gC1q heterotrimer, with a minor participation of a Lys residue located at the apex of gC1q. Furthermore, we generated recombinant forms of the human PTX3 protein bearing either D or A at position 48, a polymorphic site of clinical relevance in a number of infections, and observed that both allelic variants equally recognized C1q.

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity.

Pentraxin 3 (PTX3) is an inflammatory mediator acting as a fluid-phase pattern recognition molecule and playing an essential role in innate immunity and matrix remodeling. Inflammatory mediators also contribute to skeletal homeostasis, operating at multiple levels in physiological and pathological conditions. This study was designed to investigate the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (μCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted female and male mice (ptx3−/−) had lower trabecular bone volume than their wild-type (ptx3+/+) littermates (BV/TV by μCT: 3.50 ± 1.31 vs 6.09 ± 1.17 for females, p

Vaccinology aims to understand what factors drive vaccine-induced immunity and protection. For many vaccines, however, the mechanisms underlying immunity and protection remain incompletely characterized at best, and except for neutralizing antibodies induced by viral vaccines, few correlates of protection exist. Recent omics and systems biology big data platforms have yielded valuable insights in these areas, particularly for viral vaccines, but in the case of more complex vaccines against bacterial infectious diseases, understanding is fragmented and limited. To fill this gap, the EC supported ADITEC project (http://www.aditecproject.eu/; http://stm.sciencemag.org/content/4/128/128cm4.full) featured a work package on “Molecular signatures of immunity and immunogenicity,” aimed to identify key molecular mechanisms of innate and adaptive immunity during effector and memory stages of immune responses following vaccination. Specifically, technologies were developed to assess the human immune response to vaccination and infection at the level of the transcriptomic and proteomic response, T-cell and B-cell memory formation, cellular trafficking, and key molecular pathways of innate immunity, with emphasis on underlying mechanisms of protective immunity. This work intersected with other efforts in the ADITEC project. This review summarizes the main achievements of the work package.

Aging is an emerging worldwide threat to public health, even in the workplace, as it links with risk of illness and death. Bewildered inflammatory responses and stressful conditions associate with age-related disorders. Additionally, circadian rhythm disruption, a critical health issue in night-shift workers, correlates with premature aging. We investigated the hypothesis of a link between altered inflammatory response, detected by plasmatic long pentraxin 3 (PTX3), and biological aging, measured by leukocyte telomere length (LTL), attrition, and possibly induced by night-shift work. Within the framework of a cross-sectional study, such possible relationships were appraised by simultaneous equation model (SEM) technique among day and night-shift hospital workers. PTX3 levels, modulated by several aging conditions [i.e., body mass index (BMI) (beta = −0.22; p = 0.022), C-reactive protein (CRP) (beta = −0.07; p = 0.000), and cardiovascular diseases with hypertension included (CVD) (beta = −0.12; p = 0.000)], positively associate with LTL (coefficient = 0.15; p = 0.033). LTL, in turn is reduced by CVD (beta = −0.15; p = 0.000), binge drinking (beta = −0.10; p = 0.004), and CRP (beta = −0.05; p = 0.026). On the other hand, night-shift work, found to be remarkably free from aging risk factors [i.e., age (beta = −0.13; p = 0.017), BMI (beta = −0.17; p = 0.030), CVD (beta = −0.14; p = 0.000), and binge drinking (beta = −0.13; p = 0.000)], does associate almost significantly with reversed PTX3 (coefficient = −0.09; p = 0.089) and even with CRP (beta = 0.17; p = 0.000). In conclusion, the SEM analysis indicates that PTX3 is positively linked to LTL. The finding suggests a possible new role of this long pentraxin that, by orchestrating an efficient governance of inflammatory processes, may protect telomere from attrition, ensuring therefore the genetic stability of cells. The higher CRP levels among night-shift workers suggest that night-shift work is associated with increased systemic inflammation. This would make nocturnal workers more susceptible to premature aging.