Staphylococcus epidermidis, a dominant human skin commensal that promotes microbial homeostasis from early life, can transition to an opportunistic pathogen under certain conditions, including invasive, biofilm-associated infections linked to medical devices. Neonatal exposure to skin commensals induces a lifelong immunological imprint in the skin, characterized by immunoregulatory responses. We therefore hypothesized that early life exposure to S. epidermidis influences immune responses to invasive biofilm-associated infections later in life. Using a murine model of biofilm-related S. epidermidis bone infection induced in adulthood, we show that mice previously colonized as neonates displayed substantially different immune responses to later-life invasive infection than those not colonized or those colonized as adults. Neonatal colonization led to greater numbers of NK cells and neutrophils than no colonization, along with reduced Tregs and Th1 cells, and consistent increase in immune checkpoint receptor PD-1+ Tregs, T effector, Th1 and Th2 cells across infected bone marrow, blood and spleen. These PD-1-related immune modulations were absent in the adult-colonized group, which had the highest numbers of Tregs, Th1 and Th2 cells of all three groups. These findings reveal that early exposure to commensal bacteria strongly impacts the response to invasive infection later in life. Notably, the response depends on the timing of previous exposure. Neonatal colonization drives T cell modulation, resembling neonatal immunity, while adult-colonization increases specific T cell abundance. These differences highlight the essential role of skin commensal colonization in shaping the quality of pathogen immunity to protect against invasive, biofilm-associated infection later in life.

Introduction Surgical site infections (SSIs) are significant complications that can occur after spine surgery. Compared with systemic infections such as sepsis, or localised musculoskeletal infections such as periprosthetic joint infection, the immune responses in postoperative infection of the spine are poorly understood. A deeper understanding may provide clinically relevant diagnostic or therapeutic options.Methods This study examined changes in immune cell profiles in the blood of patients who developed infections after spinal surgery (Infection group, I, n = 17) compared to control patients without infections (Non-infection, NI, n = 20). The two groups were matched by age, body mass index (BMI), and the invasiveness of the surgical procedure. Peripheral blood mononuclear cells (PBMCs) were collected after surgery and subjected to high-dimensional mass cytometry.Results Cluster analysis identified 46 immune cell clusters, 30 of which exhibited significant differences between the I and NI patients. Natural killer cells as well as myeloid dendritic cells were decreased in I patients compared to NI patients (p=0.0269; and p=0.0267, respectively). Conversely, Th17, CD69+ and HLA-DR+ CD4+ T cells were significantly increased in I patients compared to NI patients (p=0.0422, p=0.0267; p=0.0267, respectively). ROC curve analysis of immune cell counts demonstrated potential for differentiating NI from I patients.Discussion This study reveals that patients with SSI exhibit a significantly altered immune cell profile, with greatest differences observed in increased HLA-DR+ CD4+ T cells and decreased numbers of several innate immune cells. The diagnostic potential of these markers may prove clinically relevant, and further research into the impact of activation and exhaustion may yield future therapeutic strategies.

Fracture non-union represents a complex clinical challenge resulting from an incompletely understood interplay between mechanical and biological factors. Infection frequently contributes to non-union but many cases are misdiagnosed due to a lack of classical clinical symptoms. This study characterized peripheral blood mononuclear cells (PBMCs) from aseptic non-union (NU-AS, n = 24) and fracture-related infected non-union (NU-FRI, n = 20), and compare them to healed controls (H, n = 18). High-dimensional mass cytometry (CyTOF) revealed significant elevations of regulatory T cells (Tregs; p = 0.0028) and T helper 1 (Th1) cells (p = 0.0073), and reduced expression of the activation marker CD38 in CD4+ T cells (p = 0.0016) and Tregs (p = 0.0017) in NU compared to H. In a subgroup analysis between NU-AS and NU-FRI, monocyte and CD38+ Treg cell counts provided excellent diagnostic potential, with the combination achieving a sensitivity of 100% and a specificity of 91.7%. These findings highlight an important role of the activation marker CD38 in diagnosing chronic subclinical infection, which promises earlier identification of appropriate management of these patients.

1α25-dihydroxyvitamin D3, the active metabolite of vitamin D3 (VD3), is a modulator of inflammation well-known for its ability to promote anti-inflammatory and tolerogenic immune responses. It is therefore an attractive agent for the attenuation of inflammatory responses and the development of tolerogenic immunity in autoimmune diseases. To overcome VD3 toxicity and enhance its in vivo performance, nanoparticles (NPs) have emerged as a promising delivery platform. Therefore, in this study, we have developed VD3-loaded polymeric nanoparticles (VD3-NPs) as a therapeutical strategy for the treatment of autoimmune disorders. We demonstrate that VD3-NPs could successfully be generated and that they significantly inhibit secretion of IL-6, IL-10, IL-23, and TNFα in human whole blood cultures. We observed that poly(lactic-co-glycolic acid) (PLGA) NPs are efficiently taken up by neutrophils, monocytes and B cells, prompting further investigation into the effect of VD3-NPs on these subsets. Investigation into each of the immune cell subsets demonstrated that the VD3-NPs were able inhibit cytokine secretion by both monocytes and neutrophils. Moreover, VD3-NPs induced a tolerogenic phenotype in monocytes. In B cells, we observed that VD3-NPs impaired in vitro plasma B cell differentiation and suppressed antibody production. Together, our results validate for the first time in primary human cells the therapeutic potential of VD3 encapsulated in PLGA NPs, posing an attractive strategy for the treatment of autoimmune diseases.

Neutrophils have recently been demonstrated to play a variable role in cancer progression, likely due to adaptability to environmental signals that influence neutrophil phenotype and function. In the tumor microenvironment, tumor-derived prostaglandin E2 is well established as a major suppressor of immune cell function and facilitates tumor immune evasion. However, while prostaglandin E2 is widely regarded to signal through the E-prostanoid receptors type (EP) 2 and EP4 on neutrophils, the exact effects of targeting this axis therapeutically in the context of cancer remain largely unexplored. In this study, we investigated the effect of prostaglandin E2 signaling via EP2/4 in human neutrophils using specific EP2 and EP4 antagonists. We report that countering EP2/4 signaling yields an activated phenotype together with the acquisition of proinflammatory properties, including the augmented production of reactive oxygen species, enhanced phagocytosis, and increased production of IL-8. Given the phagocytic nature of neutrophils, we encapsulated EP2/4 antagonists into poly (lactic-co-glycolic acid) nanoparticles for the tailored delivery of these antagonists. Importantly, nanoparticles achieved robust phenotypical and functional activation of healthy neutrophils as well as cancer patient-derived neutrophils exposed to tumor-derived prostaglandin E2. Together, these results identify the role of EP2/4 in regulating activation of human neutrophils and illustrate the relevance of targeting this axis with nanoparticles in the context of cancer for the reactivation of tumor-exposed neutrophils. We propose that targeting this axis in neutrophils using nanoparticles can serve to attenuate tumor-induced suppression by steering them toward a proinflammatory state and thus potentially support antitumor immune responses.

In vivo targeting of dendritic cells (DCs) with nanocarriers containing tolerogenic adjuvants is an attractive strategy to dampen inflammation. Here, we used ex vivo skin vaccination to examine the effect of intradermal injection of liposomes loaded with the tolerogenic adjuvants all-trans retinoic acid (RA) and vitamin D3 (VD3). We investigated the effect of intradermal liposome injection on skin DCs and the skin DC-induced T cell response. Our study shows that intradermal injection of RA or VD3-loaded anionic phospholipid 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG) liposomes selectively induces CD14+ dermal DC (DDC) migration while reducing migration of CD1a dim DDCs. Migrated CD14+ DDCs displayed a partially immature phenotype. RA or VD3 liposome-treated CD1a dim DDCs exhibited reduced expression of maturation markers and induced expression of coinhibitory immunoglobulin-like transcript 3 (ILT3). VD3 liposome-treated CD14+ DDCs, as well as, CD1a dim DDCs, exhibited reduced expression of maturation markers, induction of coinhibitory molecules ILT3, and programmed death-ligand 1 (PD-L1). Migrated DCs from RA or VD3 liposome-injected skin differentiated naïve CD4+ T cells into FoxP3+ CD127 low and ICOS+ Tregs, expressing functional regulatory markers. Thus, our findings provide further substantiation for in vivo DC-modulating vaccines with tolerogenic liposomes as a putative clinical therapy for autoimmune diseases and allergies.

The active vitamin A metabolite, all-trans-retinoic acid (RA), primes precursor dendritic cells (DCs) into a mucosal phenotype with tolerogenic properties characterized by the expression of integrin CD103. CD103+ DCs can counteract pathogenic Th1 and Th17 in inflammatory bowel disease (IBD) or celiac disease (CD). Tolerogenic manipulation of DCs using nanoparticles carrying tolerogenic adjuvants and disease-specific antigens is a valuable treatment strategy to induce antigen-specific mucosal tolerance in vivo. Here, we investigated the effects of RA-loaded liposomes on human DC phenotype and function, including DC-driven T-cell development, both during the generation of monocyte-derived DCs (moDCs) as well as by priming immature moDCs. RA liposomes drove CD103+ DC differentiation as well as ALDH1A2 expression in DCs. Neutrophil-dependent Th17 cell development was reduced by RA-liposome-differentiated and RA-liposome-primed DCs. Moreover, RA liposome treatment shifted T-cell development toward a Th2 cell profile. Importantly, RA liposomes induced the development of IL-10-producing and FoxP3+ regulatory T cells (Tregs) of various Treg subsets, including ICOS+ Tregs, that were potent inhibitors of bystander memory T-cell proliferation. Taken together, RA-loaded liposomes could be a novel treatment avenue for IBD or CD patients.

Background:House dust mite extract-based allergen immunotherapy (AIT) to treat house dust mite allergy is substantially effective but still presents some safety and efficacy concerns that warrant improvement. Several major allergen-based approaches to increase safety and efficacy of AIT have been proposed. One of them is the use of the group 2 allergen, Der p 2.Objective:We sought to investigate the immunomodulatory effects of sialic acid-modified major allergen recombinant Der p 2 (sia-rDer p 2) on PBMCs from healthy volunteers.Methods:We activated PBMCs with anti-CD3/CD28 antibodies and incubated them at 37°C for 6 days in the presence or absence of either native rDer p 2 or α2-3 sialic acid-modified rDer p 2 (sia-rDer p 2). We assessed the changes in CD4+ T-cell activation and proliferation by flow cytometry and changes in T-lymphocyte cytokine production in cell culture supernatant by ELISA.Results:We observed that PBMCs treated with sia-rDer p 2 presented with a markedly decreased expression of CD69 and an increased abundance of LAG-3+ lymphocytes compared with cells treated with rDer p 2. Moreover, PBMCs treated with sia-rDer p 2 showed a reduced production of IL-4, IL-13, and IL-5 and displayed a higher IL-10/IL-5 ratio compared with rDer p 2-treated PBMCs.Conclusions:We demonstrate that sia-rDer p 2 might be a safer option than native rDer p 2 for Der p 2-specific AIT. This is most relevant in the early phase of AIT that is often characterized by heightened TH2 responses, because sia-rDer p 2 does not enhance the production of TH2 cytokines.

The molecular signature of cell-derived extracellular vesicles (EVs) from synovial fluid (SF) offers insights into the cells and molecular processes associated with joint disorders and can be exploited to define biomarkers. The EV-signature is determined by cargo molecules and the lesser-studied lipid bilayer. We here investigated the lipidome of SF-EVs in inflamed joints derived from Rheumatoid Arthritis (RA) and Spondyloarthritis (SpA) patients, two autoimmune-driven joint diseases, and compared these signatures to the lipid profile of equine SF-EVs obtained during induced acute synovitis. Since neutrophils are primary SF-infiltrating cells during these inflammatory joint diseases, we also analyzed how inflammatory stimuli alter the lipidomic profile of human and equine neutrophil-derived EVs (nEVs) in vitro and how these signatures relate to the lipidome signatures of SF-EVs from inflamed joints. We identified neutrophil stimulation intensity-dependent changes in the lipidomic profile of nEVs with elevated presence of dihexosylceramide (lactosylceramide), phosphatidylserine, and phosphatidylethanolamine ether-linked lipid classes in human nEVs upon full neutrophil activation. In horses, levels of monohexosylceramide (glucosylceramide) increased instead of dihexosylceramide, indicating species-specific differences. The lipid profiles of RA and SpA SF-EVs were relatively similar and showed a relative resemblance with stimulated human nEVs. Similarly, the lipidome of equine synovitis-derived SF-EVs closer resembled the one of stimulated equine nEVs. Hence, lipidome profiling can provide insights into the contribution of nEVs to the heterogeneous pool of SF-EVs, deepening our understanding of inflammatory joint diseases and revealing molecular changes in joint homeostasis, which can lead to the development of more precise disease diagnosis and treatment strategies.

Plant-produced Der p 2-bearing bioparticles activate Th1/Tregrelated activation patterns in dendritic cells irrespective of the allergic backgroundTo the Editor, Allergen immunotherapy (AIT) is the only disease-modifying treatment for allergic diseases with proven sustained efficacy, but long treatment duration, chronic exposure to aluminium salts and allergic side-effects are major drawbacks.AIT aims to redirect the underlying T helper type 2 (Th2)-skewed immune response towards an allergenspecific mixed T helper 1 (Th1)/regulatory T cell (Treg) response.

Pesticides are increasingly recognized to induce or accelerate the development of neurodegenerative diseases, which pose an increasing health burden to society. Pesticides in general, and (mixtures of) specific active substances in particular, have been associated with neurodegenerative diseases such as Parkinson's disease, both epidemiologically and experimentally. Current regulatory guidelines for safety assessment cannot fully capture neurodegenerative effects of chemical substances, primarily due to a lack of dedicated histopathological markers and functional behaviour parameters in both standard- and specific neurotoxicity studies. Given the widespread use of pesticides and societal burden of neurodegenerative diseases, there is an urgent need for improvements. Here we propose a tiered approach consisting of 1) short term adaptations to current guideline studies and 2) long term transition of chemical safety assessment to a strategy based on human biology using in vitro and in silico methods. and the Environment (RIVM), Antonie van Leeuwenhoeklaan 9, 3721

Background:House dust mite (HDM) is a major cause of respiratory allergic diseases. Dendritic cells (DCs) play a central role in orchestrating adaptive allergic immune responses. However, it remains unclear how DCs become activated by HDM. Biochemical functions of the major HDM allergens Der p 1 (cysteine protease) and Der p 2 (MD2-mimick) have been implicated to contribute to DC activation.Methods:We investigated the immune activating potential of HDM extract and its major allergens Der p 1 and Der p 2 using monocyte-derived DCs (moDCs). Maturation and activation markers were monitored by flow cytometry and cytokine production by ELISA. Allergen depletion and proteinase K digestion were used to investigate the involvement of proteins, and in particular of the major allergens. Inhibitors of spleen tyrosine kinase (Syk), Toll-like receptor 4 (TLR4) and of C-type lectin receptors (CLRs) were used to identify the involved receptors. The contribution of endotoxins in moDC activation was assessed by their removal from HDM extract.Results:HDM extract induced DC maturation and cytokine responses in contrast to the natural purified major allergens Der p 1 and Der p 2. Proteinase K digestion and removal of Der p 1 or Der p 2 did not alter the immune stimulatory capacity of HDM extract. Antibodies against the CLRs Dectin-1, Dectin-2, and DC-SIGN did not affect cytokine responses. In contrast, Syk inhibition partially reduced IL-6, IL-12 and completely blocked IL-10. Blocking TLR4 signaling reduced the HDM-induced IL-10 and IL-12p70 induction, but not IL-6, while endotoxin removal potently abolished the induced cytokine response.Conclusion:Our data strongly suggest that HDM-induced DC activation is neither dependent on Der p 1 nor Der p 2, but depend on Syk and TLR4 activation, which might suggest a crosstalk between Syk and TLR4 pathways. Our data highlight that endotoxins play a potent role in immune responses targeting HDM.

To the Editor: Since the early nineties several approaches have been tested to develop safe and effective allergen immunotherapy (AIT) for the treatment of peanut allergy, starting with subcutaneous AIT with aqueous extract, followed by hypo-allergenic major allergens produced in bacteria that were administered rectally. These approaches were abandoned, mainly because of too many severe side-effects. Other administration routes that are considered to be safer, like sublingual and epicutaneous, have not yet reached the market. The only treatment that did receive market authorization is oral immunotherapy (OIT). It is effective for desensitization, disappointing for sustained efficacy, and side-effects occur frequently.1 Furthermore, the most sensitive patients being at risk of severe life-threatening allergic reactions are unlikely to be helped with this approach, as side-effects may include severe anaphylaxis.2 Therefore, there is an urgent need to provide all patients, including the severest ones, with a safe and effective treatment. Recently, nano- and micro-particulate strategies, such as virus-like particles, have been proposed as potentially safe and effective approaches. For example, a virus-based particle expressing the peanut allergens Ara h 1 or 2, based on the Cucumber Mosaic Virus, was shown to be hypo-allergenic in a mouse model.3 Here we report the ground-breaking hypo-allergenicity of a novel non-virus-based microparticle produced in plants, that is, enveloped bioparticles (eBPs) expressing ~3000 copies of Ara h 2 on the surface of each particle. Ara h 2 eBPs were generated by transfection of Nicotiana benthamiana with Agrobacterium tumefaciens carrying Ara h 2 cDNA constructs followed by oligomerization and membrane sequences, as described before for Der p 2 eBPs.4 Quantification of Ara h 2 was performed by immuno-slot blot using an in-house polyclonal rabbit anti-nAra h 2 serum and by dot-blot using serum from an Ara h 2-sensitized patient (Figure 1A, B). Dilutions of Ara h 2-BPs were compared by densitometric scanning to a standard curve of titrated natural purified Ara h 2 (nAra h 2) that had been quantified by a protein assay (BCA). The number of Ara h 2 molecules per bioparticle was calculated based on the number of particles, determined by tunable resistive pulse sensing (TRSP), and the concentration of Ara h 2, determined by SLOT-BLOT.4 The eBP platform, first tested with house dust mite (Der p 2) and cat dander (Fel d 1) allergens, demonstrated significantly stronger immunogenicity than alum-adsorbed allergen, and close to a 1000-fold reduction in allergenicity shown by the basophil activation test (BAT) and the rat basophilic leukaemia (RBL) cell test.4, 5 In the present study, hypo-allergenicity of Ara h 2 eBPs was first evaluated by reduction of IgE binding using ImmunoCAP inhibition. A pool of sera from eight Ara h 2-sensitized patients was incubated with dilution series of either nAra h 2 or the Ara h 2 eBP, followed by quantification of IgE binding to the recombinant Ara h 2 ImmunoCAP (f423; Thermo Fisher Scientific, Uppsala, Sweden). The Ara h 2 concentration of the bioparticles on the X-axis in Figure 1C was directly based on the quantitative SLOT-BLOT (Figure 1A). Collection of sera was approved by the Institutional Review Board of the University of Colorado, Denver, all subjects or their guardians signed informed consent and, for minors, assent. The Ara h 2 eBP showed a 10,000-fold reduction in IgE binding potency compared to nAra h 2 (Figure 1C). Additionally, the degree of functional hypo-allergenicity was assessed using either CD34+ stem-cell-derived human mast cells or RBL cells which were loaded with human IgE from nine Ara h 2-sensitized patients, and subsequently incubated with soluble nAra h 2 or with Ara h 2 eBPs. Both assays demonstrated around a 10,000-fold reduction of β-hexosaminidase release, a measure for degranulation, for Ara h 2 eBPs compared to nAra h 2 (Figure 2A, B). In the case of the RBL assay, concentrations of Ara h 2 required to induce half-maximal release could be calculated, showing that the eBPs are >14,000-fold less allergenic than nAra h 2 (Figure 2C). To take patient-associated effector-cell properties into account, a BAT with basophils from five peanut allergic patients was performed, demonstrating a similar degree of hypo-allergenicity, using the established activation markers CD203c and CD63 (Figure 2D). Altogether, these data demonstrate a very substantial reduction in the capacity of Ara h 2 eBPs to induce effector cell driven allergic responses. Another aspect of hypo-allergenicity, rarely taken into account, is IgE-facilitated allergen presentation by B cells to allergen-specific Th2 cells, contributing to sustained allergen-specific Th2 responses.6 Here, we evaluated the capacity of Ara h 2 eBPs to form immune complexes with Ara h 2-specific IgE that can bind to the low-affinity IgE receptor (CD23) on CD23+ EBV-transformed B cells, serving as a surrogate read-out for the capacity of B cells to present allergens to allergen-specific T cells.7 Either nAra h 2 or Ara h 2 eBPs were pre-incubated with patient serum at 37°C, followed by addition of the EBV-transformed B cells at 4°C (to avoid internalization). When IgE forms a complex with Ara h 2, accessible IgE Fc regions present in the complex can be detected by flow cytometry on the B cells (CD23+IgE+ B cells). Soluble nAra h 2 pre-incubated with patient sera resulted in the presence of CD23+IgE+ B cells already at much lower concentrations of allergen than observed with the Ara h 2 eBPs, either after incubation with a patient serum pool of nine Ara h 2-sensitized patients or with individual sera from two different patients (Figure 2E). These results indicate that the eBPs have a > 10,000-fold reduced capacity to form IgE-Ara h 2 complexes and subsequently activate Th2 cell responses via IgE-facilitated allergen presentation by B cells. Together, these data demonstrate an impressive degree of hypo-allergenicity of Ara h 2 eBPs, at the level of IgE binding, allergic effector cell activation, and IgE-facilitated allergen-presentation. Interestingly, the IgE-binding capacity during ImmunoCAP inhibition assays was enhanced for Der p 2 eBPs and unaffected for Fel d 1 eBPs (unpublished data). These differences compared to Ara h 2 eBPs may be explained by the localization of dominant IgE epitopes, which either remain available or become shielded off in the one-directional positioning of the allergens on the surface of the particles. Nevertheless, all three allergen eBPs are exceptionally hypo-allergenic in functional assays with allergenic effector cells (mast cells, RBL, BAT).4, 5 This may be caused by insufficient FcεR cross-linking due to the restricted freedom of movement of the allergens on the eBPs, while soluble allergen can freely move around. Moreover, there is limited space for the 150–200 nm eBPs to bind to the surface of a ~ 1.5 μm large cell, decreasing effective FcεR cross-linking by steric hindrance.8 In conclusion, plant-produced Ara h 2 eBPs have a very promising safety profile that has great potential to be used as a safe subcutaneous treatment, possibly even in patients with the highest sensitivity. As a next step, a skin prick test study will have to establish whether the same degree of hypo-allergenicity is indeed confirmed in vivo. C.Castenmiller and R. van Ree conceptualized the study and wrote, revised, and edited the manuscript. C.Castenmiller, J.H. Akkerdaas, S. Versteeg, B.R. Blokhuis, M.E. Kirpas and M. Stigler designed experiments and/or acquired, interpreted, and analysed the data. L. Auger, R. Desgagnés, C. Martel, L. Mirande, B. Morel, J. Roberge, V. Stordeur, G. Tropper, L.P. Vézina, and V. Gomord contributed to the concept design, development, or manufacturing of the plant-derived bioparticle platform. S.C. Dreskin provided peanut allergic patient serum. E.C. de Jong, W.G. Shreffler, F. Redegeld, L. Aglas and R. van Ree supervised experiments. R. van Ree supervised the study. All authors critically reviewed the manuscript. This reported studies were embedded in the Siallergen consortium, which was supported by Health Holland. The work of the authors has further been supported by Angany Inc. (Quebec, Canada), the Austrian Science Funds (FWF Project P32189), the University of Salzburg priority program "Allergy-Cancer-BioNano Research Centre", and a grant from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, Bethesda, MD, USA (RO1AI165866 (SCD)). Charlotte Castenmiller, Lorenz Aglas, and Maria Stigler received contract research funding and study material from Angany Inc. Louis-P Vézina is a cofounder board member, and CEO of Angany Inc. Réjean Desgagnés, Caroline Martel, Lydia Auger, Joanie Roberge, Bertrand Morel, Virginie Stordeur, Lucie Mirande, Guy Tropper, and Véronique Gomord are employees of Angany Inc. Ronald van Ree received contract research funding and research material form Angany Inc. and besides received consulting fees and/or speaker's fees from Angany Inc. HAL Allergy BV, Citeq BV, ThermoFisher Scientific, Reacta Healthcare Ltd., Mission MightyMe, and AB Enzymes, and has stock options from Angany. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Neutrophils are important players in COVID-19, contributing to tissue damage by release of inflammatory mediators, including reactive oxygen species (ROS) and neutrophil elastase. Longitudinal studies on the effects of COVID-19 on neutrophil phenotype and function are scarce. Here, we longitudinally investigated the phenotype and degranulation of neutrophils in COVID-19 patients (28 non-hospitalized and 35 hospitalized patients) compared to 17 healthy donors (HDs). We assessed phenotype, degranulation, CXCL8 (IL-8) release and ROS generation within 8 days, at one or six month(s) after COVID-19 diagnosis. For degranulation and ROS production, we stimulated neutrophils, either with single-stranded RNA (ssRNA) and tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and N-Formylmethionyl-leucyl-phenylalanine (fMLP). During active COVID-19, neutrophils from hospitalized patients were more immature than from HDs and were impaired in degranulation and ROS generation, while neutrophils from non-hospitalized patients only demonstrated reduced CD66b+ granule release and ROS production. Baseline CD63 expression, indicative of primary granule release, and CXCL8 production by neutrophils from hospitalized patients were elevated for up to six months. These findings show that patients hospitalized due to COVID-19, but not non-hospitalized patients, demonstrated an aberrant neutrophil phenotype, degranulation, CXCL8 release and ROS generation that partially persists up to six months after infection. This article is protected by copyright. All rights reserved.

Human immunodeficiency virus type 1 (HIV-1) is able to infect a variety of cell types with differences in entry efficiency and replication kinetics determined by the host cell type or the viral phenotype. The phenotype of the virus produced from these various cell types, including infectivity, co-receptor usage and neutralisation sensitivity, may also be affected by the characteristics of the producing cell. This can be due to incorporation of variant cell-specific molecules or differences in post-translational modifications of the gp41/120 envelope. In this study we produced genetically identical virus strains from macrophages, CD4-enriched lymphocytes as well as Th1 and Th2 CD4+ cell lines and compared each different virus stock for their infectivity in various cell types and sensitivity to neutralisation. In order to study the effect of the producer host cell on the virus phenotype, virus stocks were normalised on infectivity and were sequenced to confirm env gene homogeneity. Virus production by Th1 or Th2 cells did not compromise infectivity of the variant cell types tested. We observed no difference in sensitivity to co-receptor blocking agents upon viral passage through Th1 and Th2 CD4+ cell lineages nor did this affect DC-SIGN-mediated viral capture as measured in a transfer assay to CD4+ lymphocytes. Virus produced by macrophages was comparably sensitive to CC-chemokine inhibition as was virus generated from the array of CD4+ lymphocytes. We identified that virus produced from macrophages was fourteen times more resistant to 2G12 neutralisation than virus produced from CD4+ lymphocytes. Macrophage-produced dual-tropic (R5/X4) virus was six times more efficiently transmitted to CD4+ cells than lymphocyte-derived HIV-1 (p<0.0001) after DCSIGN capture. These results provide further insights to what extent the host cell influences viral phenotype and thereby various aspects of HIV-1 pathogenesis but suggest that viruses generated from Th1 versus Th2 cells are consistent in phenotype.

Innate mononuclear phagocytic system (MPS) cells preserve mucosal immune homeostasis. We investigated their role at nasal mucosa following allergen challenge with house dust mite. We combined single-cell proteome and transcriptome profiling on nasal immune cells from nasal biopsies cells from 30 allergic rhinitis and 27 non-allergic subjects before and after repeated nasal allergen challenge. Biopsies of patients showed infiltrating inflammatory HLA-DRhi/CD14+ and CD16+ monocytes and proallergic transcriptional changes in resident CD1C+/CD1A+ conventional dendritic cells (cDC)2 following challenge. In contrast, non-allergic individuals displayed distinct innate MPS responses to allergen challenge: predominant infiltration of myeloid-derived suppressor cells (MDSC: HLA-DRlow/CD14+ monocytes) and cDC2 expressing inhibitory/tolerogenic transcripts. These divergent patterns were confirmed in ex vivo stimulated MPS nasal biopsy cells. Thus, we identified not only MPS cell clusters involved in airway allergic inflammation but also highlight novel roles for non-inflammatory innate MPS responses by MDSC to allergens in non-allergic individuals. Future therapies should address MDSC activity as treatment for inflammatory airway diseases.

Introduction:Nanomedicine provides a promising platform for manipulating dendritic cells (DCs) and the ensuing adaptive immune response. For the induction of regulatory responses, DCs can be targeted in vivo with nanoparticles incorporating tolerogenic adjuvants and auto-antigens or allergens.Methods:Here, we investigated the tolerogenic effect of different liposome formulations loaded with vitamin D3 (VD3). We extensively phenotyped monocyte-derived DCs (moDCs) and skin DCs and assessed DC-induced regulatory CD4+ T cells in coculture.Results:Liposomal VD3 primed-moDCs induced the development of regulatory CD4+ T cells (Tregs) that inhibited bystander memory T cell proliferation. Induced Tregs were of the FoxP3+ CD127low phenotype, also expressing TIGIT. Additionally, liposome-VD3 primed moDCs inhibited the development of T helper 1 (Th1) and T helper 17 (Th17) cells. Skin injection of VD3 liposomes selectively stimulated the migration of CD14+ skin DCs.Discussion:These results suggest that nanoparticulate VD3 is a tolerogenic tool for DC-mediated induction of regulatory T cell responses.

Spondyloarthritis (SpA) patients suffer from joint inflammation resulting in tissue damage, characterized by the presence of numerous neutrophils in the synovium and synovial fluid (SF). As it is yet unclear to what extent neutrophils contribute to the pathogenesis of SpA, we set out to study SF neutrophils in more detail. We analyzed the functionality of SF neutrophils of 20 SpA patients and 7 disease controls, determining ROS production and degranulation in response to various stimuli. In addition, the effect of SF on neutrophil function was determined. Surprisingly, our data show that SF neutrophils in SpA patients have an inactive phenotype, despite the presence of many neutrophil-activating stimuli such as GM-CSF and TNF in SF. This was not due to exhaustion as SF neutrophils readily responded to stimulation. Therefore, this finding suggests that one or more inhibitors of neutrophil activation may be present in SF. Indeed, when blood neutrophils from healthy donors were activated in the presence of increasing concentrations of SF from SpA patients, degranulation and ROS production were dose-dependently inhibited. This effect was independent of diagnosis, gender, age, and medication in the patients from which the SF was isolated. Treatment of SF with the enzyme hyaluronidase strongly reduced the inhibitory effect of SF on neutrophil activation, indicating that hyaluronic acid that is present in SF may be an important factor in preventing SF neutrophil activation. This finding provides novel insights into the role of soluble factors in SF regulating neutrophil function and may lead to the development of novel therapeutics targeting neutrophil activation via hyaluronic acid or associated pathways.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human myeloid cells. We previously showed that dendritic cell (DC)-driven Th17 cell differentiation of human naive CD4+ T cells requires presence of neutrophils, which is inhibited by SIRL-1 ligation. VSTM1-v2 is a soluble isoform of SIRL-1, which was previously proposed to function as a Th17 polarizing cytokine. Here, we investigated the effect of VSTM1-v2 on DC-driven Th17 cell development. Neutrophils induced DC-driven Th17 cell differentiation, which was not enhanced by VSTM1-v2. Similarly, we found no effect of VSTM1-v2 on cytokine-driven Th17 cell development. Thus, our results do not support a role for VSTM1-v2 in Th17 cell differentiation.