Migrating Platelets Are Mechano-scavengers that Collect and Bundle Bacteria.

Blood platelets are critical for hemostasis and thrombosis and play diverse roles during immune responses. Despite these versatile tasks in mammalian biology, their skills on a cellular level are deemed limited, mainly consisting in rolling, adhesion, and aggregate formation. Here, we identify an unappreciated asset of platelets and show that adherent platelets use adhesion receptors to mechanically probe the adhesive substrate in their local microenvironment. When actomyosin-dependent traction forces overcome substrate resistance, platelets migrate and pile up the adhesive substrate together with any bound particulate material. They use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, exacerbating inflammatory tissue injury in sepsis. This assigns platelets a central role in innate immune responses and identifies them as potential targets to dampen inflammatory tissue damage in clinical scenarios of severe systemic infection.

C3d-Targeted factor H inhibits tissue complement in disease models and reduces glomerular injury without affecting circulating complement.

Complement-mediated diseases can be treated using systemic inhibitors. However, complement components are abundant in circulation, affecting systemic inhibitors' exposure and efficacy. Furthermore, because of complement's essential role in immunity, systemic treatments raise infection risk in patients. To address these challenges, we developed antibody fusion proteins combining the alternative-pathway complement inhibitor factor H (fH1-5) with an anti-C3d monoclonal antibody (C3d-mAb-2fH). Because C3d is deposited at sites of complement activity, this molecule localizes to tissue complement while minimizing circulating complement engagement. These fusion proteins bind to deposited complement in diseased human skin sections and localize to activated complement in a primate skin injury model. We further explored the pharmacology of C3d-mAb-2fH proteins in rodent models with robust tissue complement activation. Doses of C3d-mAb-2fH >1 mg/kg achieved >75% tissue complement inhibition in mouse and rat injury models while avoiding circulating complement blockade. Glomerular-specific complement inhibition reduced proteinuria and preserved podocyte foot-process architecture in rat membranous nephropathy, indicating disease-modifying efficacy. These data indicate that targeting local tissue complement results in durable and efficacious complement blockade in skin and kidney while avoiding systemic inhibition, suggesting broad applicability of this approach in treating a range of complement-mediated diseases.

A platelet-mediated system for shuttling blood-borne bacteria to CD8α+ dendritic cells depends on glycoprotein GPIb and complement C3.

The acquisition of pathogen-derived antigen by dendritic cells (DCs) is a key event in the generation of cytotoxic CD8(+) T cell responses. In mice, the intracellular bacterium Listeria monocytogenes is directed from the blood to splenic CD8α(+) DCs. We report that L. monocytogenes rapidly associated with platelets in the bloodstream in a manner dependent on GPIb and complement C3. Platelet association targeted a small but immunologically important portion of L. monocytogenes to splenic CD8α(+) DCs, diverting bacteria from swift clearance by other, less immunogenic phagocytes. Thus, an effective balance is established between maintaining sterility of the circulation and induction of antibacterial immunity by DCs. Other gram-positive bacteria also were rapidly tagged by platelets, revealing a broadly active shuttling mechanism for systemic bacteria.

Tirofiban potentiates agonist-induced platelet activation and degranulation, despite effectively inhibiting aggregation.

We aimed to investigate the effects of integrin αIIbß3 inhibitor tirofiban on hallmarks of platelet activation, degranulation, and aggregation during its use to analyze activated but non-complexed platelets via flow cytometry. To do so, we used washed platelets from healthy human donors. We combined aggregometry, an assay of platelet functionality, with flow cytometry and ELISA to detect and correlate, respectively, platelet aggregation, activation, and granule release. While tirofiban effectively inhibited agonist-induced platelet aggregation (thrombin receptor-activating peptide 6 (TRAP), convulxin (CVX), U46619 and IV.3), the surface expression of P-selectin and CD63 and granule release of RANTES were significantly increased, indicating that tirofiban enhances degranulation, uncoupled from aggregation. The results show that tirofiban alters the activation phenotype of platelets, something that should be considered when using tirofiban to enable flow cytometric analysis of activated but unaggregated platelet suspensions.

Pathogen clearance and immune adherence "revisited": Immuno-regulatory roles for CRIg.

Rapid elimination of microbes from the bloodstream, along with the ability to mount an adaptive immune response, are essential for optimal host-defense. Kupffer cells are strategically positioned in the liver sinusoids and efficiently capture circulating microbes from the hepatic artery and portal vein, thus preventing bacterial dissemination. In vivo and in vitro studies have probed how complement receptor of the immunoglobulin superfamily (CRIg), also referred to as Z39Ig and V-set and Ig domain-containing 4 (VSIG4), acts as a critical player in pathogen recognition and clearance. While recent data suggested that CRIg may bind bacterial cell wall components directly, the single transmembrane receptor is best known for its interaction with complement C3 opsonization products on the microbial surface. On Kupffer cells, CRIg must capture opsonized microbes against the shear forces of the blood flow. In vivo work reveals how immune adherence (IA), a process in which blood platelets or erythrocytes associate with circulating bacteria, plays a critical role in regulating pathogen capture by CRIg under flow conditions. In addition to its typical innate immune functions, CRIg was shown to directly and indirectly influence adaptive immune responses. Here, we review our current understanding of the diverse roles of CRIg in pathogen elimination, anti-microbial immunity and autoimmunity. In particular, we will explore how, through selective capturing by CRIg, an important balance is achieved between the immunological and clearance functions of liver and spleen.

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma.

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.

Old dogs-new tricks: immunoregulatory properties of C3 and C5 cleavage fragments.

The activation of the complement system by canonical and non-canonical mechanisms results in the generation of multiple C3 and C5 cleavage fragments including anaphylatoxins C3a and C5a as well as opsonizing C3b/iC3b. It is now well appreciated that anaphylatoxins not only act as pro-inflammatory mediators but as immunoregulatory molecules that control the activation status of cells and tissue at several levels. Likewise, C3b/iC3b is more than the opsonizing fragment that facilitates engulfment and destruction of targets by phagocytes. In the circulation, it also facilitates the transport and delivery of bacteria and immune complexes to phagocytes, through a process known as immune adherence, with consequences for adaptive immunity. Here, we will discuss non-classical immunoregulatory properties of C3 and C5 cleavage fragments. We highlight the influence of anaphylatoxins on Th2 and Th17 cell development during allergic asthma with a particular emphasis on their role in the modulation of CD11b+ conventional dendritic cells and monocyte-derived dendritic cells. Furthermore, we discuss the control of anaphylatoxin-mediated activation of dendritic cells and allergic effector cells by adaptive immune mechanisms that involve allergen-specific IgG1 antibodies and plasma or regulatory T cell-derived IL-10 production. Finally, we take a fresh look at immune adherence with a particular focus on the development of antibacterial cytotoxic T-cell responses.

Protein corona-mediated targeting of nanocarriers to B cells allows redirection of allergic immune responses.

BACKGROUND: Nanoparticle (NP)-based vaccines are attractive immunotherapy tools because of their capability to codeliver antigen and adjuvant to antigen-presenting cells. Their cellular distribution and serum protein interaction ("protein corona") after systemic administration and their effect on the functional properties of NPs is poorly understood. OBJECTIVES: We analyzed the relevance of the protein corona on cell type-selective uptake of dextran-coated NPs and determined the outcome of vaccination with NPs that codeliver antigen and adjuvant in disease models of allergy. METHODS: The role of protein corona constituents for cellular binding/uptake of dextran-coated ferrous nanoparticles (DEX-NPs) was analyzed both in vitro and in vivo. DEX-NPs conjugated with the model antigen ovalbumin (OVA) and immunostimulatory CpG-rich oligodeoxynucleotides were administered to monitor the induction of cellular and humoral immune responses. Therapeutic effects of this DEX-NP vaccine in mouse models of OVA-induced anaphylaxis and allergic asthma were assessed. RESULTS: DEX-NPs triggered lectin-induced complement activation, yielding deposition of activated complement factor 3 on the DEX-NP surface. In the spleen DEX-NPs targeted predominantly B cells through complement receptors 1 and 2. The DEX-NP vaccine elicited much stronger OVA-specific IgG2a production than coadministered soluble OVA plus CpG oligodeoxynucleotides. B-cell binding of the DEX-NP vaccine was critical for IgG2a production. Treatment of OVA-sensitized mice with the DEX-NP vaccine prevented induction of anaphylactic shock and allergic asthma accompanied by IgE inhibition. CONCLUSIONS: Opsonization of lectin-coated NPs by activated complement components results in selective B-cell targeting. The intrinsic B-cell targeting property of lectin-coated NPs can be exploited for treatment of allergic immune responses.

Minimally manipulated murine regulatory T cells purified by reversible Fab Multimers are potent suppressors for adoptive T-cell therapy.

The transfer of regulatory T cells, either freshly isolated, or modified, represents a promising therapeutic approach to dampen misdirected immune responses, like autoimmune diseases, chronic inflammatory syndromes and graft versus host disease. Clinical isolation of highly pure regulatory T cell (Treg) populations is still challenging and labeling reagents can influence their viability and functionality, potentially altering the potency of isolated Treg cell products. Here we show that reversible Fab multimer-based Treg purification can prevent conventional antibody label-induced interferences in vitro and in vivo. Remaining isolation reagents negatively interfere with Treg engraftment efficacy in C57BL/6 wild-type mice due to Fcγ-receptor- as well as IL-2 receptor-mediated mechanisms. Using a preclinical model for acute GvHD, we further show that purified 'label-freed' Tregs are protective at substantially lower cell numbers as compared to conventional nonreversible antibody staining, translating into significantly improved survival of mice treated with minimally manipulated Tregs. These findings have important clinical relevance for future Treg-based cell therapies.

Conduits mediate transport of low-molecular-weight antigen to lymph node follicles.

To track drainage of lymph-borne small and large antigens (Ags) into the peripheral lymph nodes and subsequent encounter by B cells and follicular dendritic cells, we used the approach of multiphoton intravital microscopy. We find a system of conduits that extend into the follicles and mediate delivery of small antigens to cognate B cells and follicular dendritic cells. The follicular conduits provide an efficient and rapid mechanism for delivery of small antigens and chemokines such as CXCL13 to B cells that directly contact the conduits. By contrast, large antigens were bound by subcapsular sinus macrophages and subsequently transferred to follicular B cells as previously reported. In summary, the findings identify a unique pathway for the channeling of small lymph-borne antigens and chemoattractants from the subcapsular sinus directly to the B cell follicles. This pathway could be used for enhancing delivery of vaccines or small molecules for improvement of humoral immunity.

Complement component C5 recruits neutrophils in the absence of C3 during respiratory infection with modified vaccinia virus Ankara.

Efficient leukocyte migration is important for an effective host response to viral infection and the development of adaptive immunity. The poxvirus strain modified vaccinia virus Ankara (MVA), a safe and efficient viral vector, rapidly induces chemokine expression and respiratory recruitment of leukocytes, which is unique among vaccinia viruses. In addition to chemokines, the complement system contributes to the attraction and activation of different types of leukocytes. Using a murine model of intranasal infection, we show in this study that MVA-induced neutrophil recruitment depends on complement component C5. Remarkably, we find that C5 mediates neutrophil recruitment to the lung, even in the absence of the central complement component C3. Our findings argue for complement C5 activation during MVA infection of the lung via a C3-independent pathway, which enables rapid recruitment of neutrophils.

The triggering receptor expressed on myeloid cells 2 inhibits complement component 1q effector mechanisms and exerts detrimental effects during pneumococcal pneumonia.

Phagocytosis and inflammation within the lungs is crucial for host defense during bacterial pneumonia. Triggering receptor expressed on myeloid cells (TREM)-2 was proposed to negatively regulate TLR-mediated responses and enhance phagocytosis by macrophages, but the role of TREM-2 in respiratory tract infections is unknown. Here, we established the presence of TREM-2 on alveolar macrophages (AM) and explored the function of TREM-2 in the innate immune response to pneumococcal infection in vivo. Unexpectedly, we found Trem-2(-/-) AM to display augmented bacterial phagocytosis in vitro and in vivo compared to WT AM. Mechanistically, we detected that in the absence of TREM-2, pulmonary macrophages selectively produced elevated complement component 1q (C1q) levels. We found that these increased C1q levels depended on peroxisome proliferator-activated receptor-δ (PPAR-δ) activity and were responsible for the enhanced phagocytosis of bacteria. Upon infection with S. pneumoniae, Trem-2(-/-) mice exhibited an augmented bacterial clearance from lungs, decreased bacteremia and improved survival compared to their WT counterparts. This work is the first to disclose a role for TREM-2 in clinically relevant respiratory tract infections and demonstrates a previously unknown link between TREM-2 and opsonin production within the lungs.

Involvement of Toso in activation of monocytes, macrophages, and granulocytes.

Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.

1,8-cineole (eucalyptol): A versatile phytochemical with therapeutic applications across multiple diseases.

1,8-cineole (Eucalyptol), a naturally occurring compound derived from botanical sources such as eucalyptus, rosemary, and camphor laurel, has a long history of use in traditional medicine and exhibits an array of biological properties, including anti-inflammatory, antioxidant, antimicrobial, bronchodilatory, analgesic, and pro-apoptotic effects. Recent evidence has also indicated its potential role in managing conditions such as Alzheimer's disease, neuropathic pain, and cancer. This review spotlights the health advantages of 1,8-cineole, as demonstrated in clinical trials involving patients with respiratory disorders, including chronic obstructive pulmonary disease, asthma, bronchitis, and rhinosinusitis. In addition, we shed light on potential therapeutic applications of 1,8-cineole in various conditions, such as depression, epilepsy, peptic ulcer disease, diarrhea, cardiac-related heart diseases, and diabetes mellitus. A comprehensive understanding of 1,8-cineole's pharmacodynamics and safety aspects as well as developing effective formulations, might help to leverage its therapeutic value. This thorough review sets the stage for future research on diverse health benefits and potential uses of 1,8-cineole in tackling complex medical conditions.

Purified complement C3b triggers phagocytosis and activation of human neutrophils via complement receptor 1.

The complement system provides vital immune protection against infectious agents by labeling them with complement fragments that enhance phagocytosis by immune cells. Many details of complement-mediated phagocytosis remain elusive, partly because it is difficult to study the role of individual complement proteins on target surfaces. Here, we employ serum-free methods to couple purified complement C3b onto E. coli bacteria and beads and then expose human neutrophils to these C3b-coated targets. We examine the neutrophil response using a combination of flow cytometry, confocal microscopy, luminometry, single-live-cell/single-target manipulation, and dynamic analysis of neutrophil spreading on opsonin-coated surfaces. We show that purified C3b can potently trigger phagocytosis and killing of bacterial cells via Complement receptor 1. Comparison of neutrophil phagocytosis of C3b- versus antibody-coated beads with single-bead/single-target analysis exposes a similar cell morphology during engulfment. However, bulk phagocytosis assays of C3b-beads combined with DNA-based quenching reveal that these are poorly internalized compared to their IgG1 counterparts. Similarly, neutrophils spread slower on C3b-coated compared to IgG-coated surfaces. These observations support the requirement of multiple stimulations for efficient C3b-mediated uptake. Together, our results establish the existence of a direct pathway of phagocytic uptake of C3b-coated targets and present methodologies to study this process.

Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

T cell-dependence of Lassa fever pathogenesis.

Lassa virus (LASV), the causative agent of Lassa fever (LF), is endemic in West Africa, accounting for substantial morbidity and mortality. In spite of ongoing research efforts, LF pathogenesis and mechanisms of LASV immune control remain poorly understood. While normal laboratory mice are resistant to LASV, we report that mice expressing humanized instead of murine MHC class I (MHC-I) failed to control LASV infection and develop severe LF. Infection of MHC-I knockout mice confirmed a key role for MHC-I-restricted T cell responses in controlling LASV. Intriguingly we found that T cell depletion in LASV-infected HHD mice prevented disease, irrespective of high-level viremia. Widespread activation of monocyte/macrophage lineage cells, manifest through inducible NO synthase expression, and elevated IL-12p40 serum levels indicated a systemic inflammatory condition. The absence of extensive monocyte/macrophage activation in T cell-depleted mice suggested that T cell responses contribute to deleterious innate inflammatory reactions and LF pathogenesis. Our observations in mice indicate a dual role for T cells, not only protecting from LASV, but also enhancing LF pathogenesis. The possibility of T cell-driven enhancement and immunopathogenesis should be given consideration in future LF vaccine development.

Impaired antibody response causes persistence of prototypic T cell-contained virus.

CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV), a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM) exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor gamma chain or Fc gamma receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell-controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy.