Retinoic acid-loaded liposomes induce human mucosal CD103+ dendritic cells that inhibit Th17 cells and drive regulatory T-cell development in vitro.

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.

Aberrant neutrophil degranulation in hospitalized patients with COVID-19 partially remains for 6 months.

Neutrophils are important players in COVID-19, contributing to tissue damage by release of inflammatory mediators, including 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 nonhospitalized and 35 hospitalized patients) compared with 17 healthy donors (HDs). We assessed phenotype, degranulation, CXCL8 (IL-8) release, and ROS generation within 8 days, at one or 6 month(s) after COVID-19 diagnosis. For degranulation and ROS production, we stimulated neutrophils, either with ssRNA and TNF or granulocyte-macrophage colony-stimulating factor and N-Formylmethionyl-leucyl-phenylalanine. 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 nonhospitalized 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 6 months. These findings show that patients hospitalized due to COVID-19, but not nonhospitalized patients, demonstrated an aberrant neutrophil phenotype, degranulation, CXCL8 release, and ROS generation that partially persists up to 6 months after infection.

Hyaluronic Acid in Synovial Fluid Prevents Neutrophil Activation in Spondyloarthritis.

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.

Plasmodium sporozoites induce regulatory macrophages.

Professional antigen-presenting cells (APCs), like macrophages (Mϕs) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoMϕs. Both MoDCs and MoMϕs readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoMϕs instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoMϕs show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFNγ) production by antigen specific CD8+ T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity.

Schistosoma mansoni soluble egg antigen (SEA) and recombinant Omega-1 modulate induced CD4+ T-lymphocyte responses and HIV-1 infection in vitro.

Parasitic helminths evade, skew and dampen human immune responses through numerous mechanisms. Such effects will likely have consequences for HIV-1 transmission and disease progression. Here we analyzed the effects that soluble egg antigen (SEA) from Schistosoma mansoni had on modulating HIV-1 infection and cytokine/chemokine production in vitro. We determined that SEA, specifically through kappa-5, can potently bind to DC-SIGN and thereby blocks DC-SIGN mediated HIV-1 trans-infection (p<0.05) whilst not interfering with cis-infection. DCs exposed to SEA whilst maturing under Th2 promoting conditions, will upon co-culture with naïve T-cells induce a T-cell population that was less susceptible to HIV-1 R5 infection (p<0.05) compared to DCs unexposed to SEA, whereas HIV-1 X4 virus infection was unaffected. This was not observed for DCs exposed to SEA while maturing under Th1 or Th1/Th2 (Tmix) promoting conditions. All T-cell populations induced by SEA exposed DCs demonstrate a reduced capacity to produce IFN-γ and MIP-1ß. The infection profile of T-cells infected with HIV-1 R5 was not associated with down-modulation of CCR5 cell surface expression. We further show that DCs maturing under Tmix conditions exposed to plant recombinant omega-1 protein (rω-1), which demonstrates similar functions to natural ω-1, induced T-cell populations that were less sensitive for HIV-1 R5 infection (p<0.05), but not for X4 virus infection. This inhibition associated again with a reduction in IFN-γ and MIP-1ß expression, but additionally correlated with reduced CCR5 expression. We have shown that SEA parasite antigens and more specifically rω-1 can modulate HIV-1 infectivity with the potential to influence disease course in co-infected individuals.

Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli.

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli.

Signal Inhibitory Receptor on Leukocytes-1 is highly expressed on lung monocytes, but absent on mononuclear phagocytes in skin and colon.

Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is expressed on human blood monocytes and granulocytes and inhibits myeloid effector functions. On monocytes, but not granulocytes, SIRL-1 expression is low or absent in individuals with the single nucleotide polymorphism (SNP) rs612529C. The expression of SIRL-1 in tissue and the influence of rs612529 hereon is currently unknown. Here, we used flow cytometry to determine SIRL-1 expression on immune cells in human blood and three barrier tissues; skin, colon and lung. SIRL-1 was expressed by virtually all neutrophils and eosinophils in these tissues. In contrast, SIRL-1 was not expressed by monocyte-derived cells in skin and colon, whereas it was highly expressed by lung classical monocytes. Lung monocytes from individuals with a rs612529C allele had decreased SIRL-1 expression, consistent with the genotype association in blood. Within the different monocyte subsets in blood and lung, SIRL-1 expression was highest in classical monocytes and lowest in nonclassical monocytes. SIRL-1 was not expressed by dendritic cells in blood and barrier tissues. Together, these results indicate that SIRL-1 is differentially expressed on phagocyte subsets in blood and barrier tissues, and that its expression on monocytes is genotype- and tissue-specific. Immune regulation of monocytes by SIRL-1 may be of particular importance in the lung.

The Nature of Antibacterial Adaptive Immune Responses against Staphylococcus aureus Is Dependent on the Growth Phase and Extracellular Peptidoglycan.

Staphylococcus aureus has evolved different strategies to evade the immune response, which play an important role in its pathogenesis. The bacteria express and shed various cell wall components and toxins during different stages of growth that may affect the protective T cell responses to extracellular and intracellular S. aureus However, if and how the dendritic cell (DC)-mediated T cell response against S. aureus changes during growth of the bacterium remain elusive. In this study, we show that exponential-phase (EP) S. aureus bacteria were endocytosed very efficiently by human DCs, and these DCs strongly promoted production of the T cell polarizing factor interleukin-12 (IL-12). In contrast, stationary-phase (SP) S. aureus bacteria were endocytosed less efficiently by DCs, and these DCs produced small amounts of IL-12. The high level of IL-12 production induced by EP S. aureus led to the development of a T helper 1 (Th1) cell response, which was inhibited after neutralization of IL-12. Furthermore, preincubation with the staphylococcal cell wall component peptidoglycan (PGN), characteristically shed during the exponential growth phase, modulated the DC response to EP S. aureus PGN preincubation appeared to inhibit IL-12p35 expression, leading to downregulation of IL-12 and an increase of IL-23 production by DCs, enhancing Th17 cell development. Taken together, our data indicate that exponential-phase S. aureus bacteria induce a stronger IL-12-dependent Th1 cell response than stationary-phase S. aureus and that this Th1 cell response shifted toward a Th17 cell response in the presence of PGN.

Microbes and asthma: Opportunities for intervention.

The worldwide incidence and prevalence of asthma continues to increase. Asthma is now understood as an umbrella term for different phenotypes or endotypes, which arise through different pathophysiologic pathways. Understanding the many factors contributing to development of the disease is important for the identification of novel therapeutic targets for the treatment of certain asthma phenotypes. The hygiene hypothesis has been formulated to explain the increasing prevalence of allergic disease, including asthma. This hypothesis postulates that decreased exposure at a young age to certain infectious agents as a result of improved hygiene, increased antibiotic use and vaccination, and changes in lifestyle and dietary habits is associated with changes in the immune system, which predispose subjects to allergy. Many microbes, during their coevolution with human subjects, developed mechanisms to manipulate the human immune system and to increase their chances of survival. Improving models of asthma, as well as choosing adequate end points in clinical trials, will lead to a more complete understanding of the underlying mechanisms, thus providing an opportunity to devise primary and secondary interventions at the same time as identifying new molecular targets for treatment. This article reports the discussion and conclusion of a workshop under the auspices of the Netherlands Lung Foundation to extend our understanding of how modulation of the immune system by bacterial, parasitic, and viral infections might affect the development of asthma and to map out future lines of investigation.

Acetylcholine-producing T cells in the intestine regulate antimicrobial peptide expression and microbial diversity.

The cholinergic anti-inflammatory pathway reduces systemic tumor necrosis factor (TNF) via acetylcholine-producing memory T cells in the spleen. These choline acetyltransferase (ChAT)-expressing T cells are also found in the intestine, where their function is unclear. We aimed to characterize these cells in mouse and human intestine and delineate their function. We made use of the ChAT-enhanced green fluorescent protein (eGFP) reporter mice. CD4Cre mice were crossed to ChATfl/fl mice to achieve specific deletion of ChAT in CD4+ T cells. We observed that the majority of ChAT-expressing T cells in the human and mouse intestine have characteristics of Th17 cells and coexpress IL17A, IL22, and RORC The generation of ChAT-expressing T cells was skewed by dendritic cells after activation of their adrenergic receptor ß2 To evaluate ChAT T cell function, we generated CD4-specific ChAT-deficient mice. CD4ChAT-/- mice showed a reduced level of epithelial antimicrobial peptides lysozyme, defensin A, and ang4, which was associated with an enhanced bacterial diversity and richness in the small intestinal lumen in CD4ChAT-/- mice. We conclude that ChAT-expressing T cells in the gut are stimulated by adrenergic receptor activation on dendritic cells. ChAT-expressing T cells may function to mediate the host AMP secretion, microbial growth and expansion.

MicroRNA-146a regulates survival and maturation of human plasmacytoid dendritic cells.

During microbial infections, plasmacytoid dendritic cells (pDCs) are a main source of type I interferons α/ß (IFN-α/-ß). Nucleic acids from microbes are sensed by Toll-like receptors 7/9 (TLR7/9), which are selectively expressed in pDCs. Activated pDCs also produce proinflammatory cytokines and upregulate costimulatory molecules. Together, this equips pDCs with the ability to prime T, B, and NK cells and conventional DCs, thereby initiating adaptive immune responses. To avoid deleterious effects to the host, tight regulation of pDC activation is required. Despite data linking aberrant activation of pDCs with autoimmune diseases, little is known about mechanisms controlling pDC activation. Here, we investigated the role of microRNA-146a (miR-146a) in TLR pathway regulation in human pDCs. MiR-146a expression was induced upon TLR7/9 signaling. Furthermore, ectopic miR-146a expression effectively impaired TLR-mediated signaling in pDCs as TLR-induced nuclear factor-κB activation was reduced. This consequently diminished the production of proinflammatory cytokines and reduced pDC survival. Moreover, miR-146a-expressing pDCs had decreased ability to induce CD4(+) T-cell proliferation likely due to reduced expression levels of major histocompatibility complex class II and costimulatory molecules. Our data unravel the crucial immunomodulatory role of miR-146a in pDCs and may add to our understanding of aberrant responses in autoimmune diseases.

IgG opsonization of bacteria promotes Th17 responses via synergy between TLRs and FcγRIIa in human dendritic cells.

Dendritic cells (DCs) are essential in inducing adaptive immune responses against bacteria by expressing cytokines that skew T-cell responses toward protective Th17 cells. Although it is widely recognized that induction of these cytokines by DCs involves activation of multiple receptors, it is still incompletely characterized which combination of receptors specifically skews Th17-cell responses. Here we have identified a novel role for FcγRIIa in promoting human Th17 cells. Activation of DCs by bacteria opsonized by serum IgG strongly promoted Th17 responses, which was FcγRIIa-dependent and coincided with enhanced production of selected cytokines by DCs, including Th17-promoting IL-1ß and IL-23. Notably, FcγRIIa stimulation on DCs did not induce cytokine production when stimulated individually, but selectively amplified cytokine responses through synergy with TLR2, 4, or 5. Importantly, this synergy is mediated at 2 different levels. First, TLR-FcγRIIa costimulation strongly increased transcription of pro-IL-1ß and IL-23p19. Second, FcγRIIa triggering induced activation of caspase-1, which cleaves pro-IL-1ß into its bioactive form and thereby enhanced IL-1ß secretion. Taken together, these data identified cross-talk between TLRs and FcγRIIa as a novel mechanism by which DCs promote protective effector Th17-cell responses against bacteria.

Sialic acid-modified der p 2 allergen exerts immunomodulatory effects on human PBMCs.

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.

Selective C-Rel activation via Malt1 controls anti-fungal T(H)-17 immunity by dectin-1 and dectin-2.

C-type lectins dectin-1 and dectin-2 on dendritic cells elicit protective immunity against fungal infections through induction of T(H)1 and T(H)-17 cellular responses. Fungal recognition by dectin-1 on human dendritic cells engages the CARD9-Bcl10-Malt1 module to activate NF-κB. Here we demonstrate that Malt1 recruitment is pivotal to T(H)-17 immunity by selective activation of NF-κB subunit c-Rel, which induces expression of T(H)-17-polarizing cytokines IL-1ß and IL-23p19. Malt1 inhibition abrogates c-Rel activation and T(H)-17 immunity to Candida species. We found that Malt1-mediated activation of c-Rel is similarly essential to induction of T(H)-17-polarizing cytokines by dectin-2. Whereas dectin-1 activates all NF-κB subunits, dectin-2 selectively activates c-Rel, signifying a specialized T(H)-17-enhancing function for dectin-2 in anti-fungal immunity by human dendritic cells. Thus, dectin-1 and dectin-2 control adaptive T(H)-17 immunity to fungi via Malt1-dependent activation of c-Rel.

CD5 costimulation induces stable Th17 development by promoting IL-23R expression and sustained STAT3 activation.

IL-17-producing CD4(+) T helper (Th17) cells are important for immunity against extracellular pathogens and in autoimmune diseases. The factors that drive Th17 development in human remain a matter of debate. Here we show that, compared with classic CD28 costimulation, alternative costimulation via the CD5 or CD6 lymphocyte receptors forms a superior pathway for human Th17-priming. In the presence of the Th17-promoting cytokines IL-1ß, IL-6, IL-23, and transforming growth factor-ß (TGF-ß), CD5 costimulation induces more Th17 cells that produce higher amounts of IL-17, which is preceded by prolonged activation of signal transducer and activator of transcription 3 (STAT3), a key regulator in Th17 differentiation, and enhanced levels of the IL-17-associated transcription factor retinoid-related orphan receptor-γt (ROR-γt). Strikingly, these Th17-promoting signals critically depend on CD5-induced elevation of IL-23 receptor (IL-23R) expression. The present data favor the novel concept that alternative costimulation via CD5, rather than classic costimulation via CD28, primes naive T cells for stable Th17 development through promoting the expression of IL-23R.

Cutting edge: virus selectively primes human langerhans cells for CD70 expression promoting CD8+ T cell responses.

The two outermost compartments of skin are populated by different Ag-presenting dendritic cell types. Epidermal Langerhans cells (LCs) are evolutionarily adapted to the continuous presence of harmless skin commensals by the selective lack of cell surface TLRs that sense bacteria. In this article, we analyze the ability of LCs and dermal dendritic cells (DDCs) to respond to virus infection. Live virus and intracellular TLR3-agonist dsRNA commit LCs more effectively than DDCs to stimulate naive CD8(+) T cell expansion and their differentiation into effector cells. This potent CD8(+) T cell-promoting capacity of LCs is causally related to high levels of virus-induced CD70 expression but not to IL-12 production. These data suggest a remarkable specialization of LCs in the induction of pathogen class-specific adaptive immunity. Whereas LCs ignore bacteria, they are superior to DDCs to initiate effective CD70-mediated CD8(+) T cells in response to virus stimulation.