TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

Investigating pyroptosis as a mechanism of L. major cell-to-cell spread in the human BLaER1 infection model.

Leishmania is the causative agent of the tropical neglected disease leishmaniasis and infects macrophages as its definitive host cell. In order to sustain and propagate infections, Leishmania parasites have to complete cycles of exit and re-infection. Yet, the mechanism driving the parasite spread to other cells remains unclear. Recent studies reported pro-inflammatory monocytes as replicative niche of Leishmania major and showed prolonged expression of IL-1ß at the site of infection, indicating an activation of the NLRP3 inflammasome and pointing toward pyroptosis as a possible mechanism of parasite spread. To address the species-specific inflammasome activation of human cells, we characterized the BLaER1 monocytes as a model for L. major infection. We found that BLaER1 monocytes support infection and activation by Leishmania parasites to the same extent as primary human macrophages. Harnessing the possibilities of this infection model, we first showed that BLaER1 GSDMD-/- cells, which carry a deletion of the pore-forming protein gasdermin D, are more resistant to pyroptotic cell death and, concomitantly, display a strongly delayed release of intracellular parasite. Using that knockout in a co-incubation assay in comparison with wild-type BLaER1 cells, we demonstrate that impairment of the pyroptosis pathway leads to lower rates of parasite spread to new host cells, thus, implicating pyroptotic cell death as a possible exit mechanism of L. major in pro-inflammatory microenvironments.

Leishmania major drives host phagocyte death and cell-to-cell transfer depending on intracellular pathogen proliferation rate.

The virulence of intracellular pathogens relies largely on the ability to survive and replicate within phagocytes but also on release and transfer into new host cells. Such cell-to-cell transfer could represent a target for counteracting microbial pathogenesis. However, our understanding of the underlying cellular and molecular processes remains woefully insufficient. Using intravital 2-photon microscopy of caspase-3 activation in the Leishmania major-infected (L. major-infected) live skin, we showed increased apoptosis in cells infected by the parasite. Also, transfer of the parasite to new host cells occurred directly without a detectable extracellular state and was associated with concomitant uptake of cellular material from the original host cell. These in vivo findings were fully recapitulated in infections of isolated human phagocytes. Furthermore, we observed that high pathogen proliferation increased cell death in infected cells, and long-term residency within an infected host cell was only possible for slowly proliferating parasites. Our results therefore suggest that L. major drives its own dissemination to new phagocytes by inducing host cell death in a proliferation-dependent manner.

The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5-/-, or MyD88-/- mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1ß, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NFκB, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NFκB, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88-, MAPK-, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.

Distinct single-component adjuvants steer human DC-mediated T-cell polarization via Toll-like receptor signaling toward a potent antiviral immune response.

The COVID-19 pandemic highlights the importance of efficient and safe vaccine development. Vaccine adjuvants are essential to boost and tailor the immune response to the corresponding pathogen. To allow for an educated selection, we assessed the effect of different adjuvants on human monocyte-derived dendritic cells (DCs) and their ability to polarize innate and adaptive immune responses. In contrast to commonly used adjuvants, such as aluminum hydroxide, Toll-like receptor (TLR) agonists induced robust phenotypic and functional DC maturation. In a DC-lymphocyte coculture system, we investigated the ensuing immune reactions. While monophosphoryl lipid A synthetic, a TLR4 ligand, induced checkpoint inhibitors indicative for immune exhaustion, the TLR7/8 agonist Resiquimod (R848) induced prominent type-1 interferon and interleukin 6 responses and robust CTL, B-cell, and NK-cell proliferation, which is particularly suited for antiviral immune responses. The recently licensed COVID-19 vaccines, BNT162b and mRNA-1273, are both based on single-stranded RNA. Indeed, we could confirm that the cytokine profile induced by lipid-complexed RNA was almost identical to the pattern induced by R848. Although this awaits further investigation, our results suggest that their efficacy involves the highly efficient antiviral response pattern stimulated by the RNAs' TLR7/8 activation.

The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies.

Since the approval of the first monoclonal antibody (mAb) in 1986, a huge effort has been made to guarantee safety and efficacy of therapeutic mAbs. As of July 2021, 118 mAbs are approved for the European market for a broad range of clinical indications. In order to ensure clinical efficacy and safety aspects, (pre-)clinical experimental approaches evaluate the respective modes of action (MoA). In addition to antigen-specificity including binding affinity and -avidity, MoA comprise Fc-mediated effector functions such as antibody dependent cellular cytotoxicity (ADCC) and the closely related antibody dependent cellular phagocytosis (ADCP). For this reason, a variety of cell-based assays have been established investigating effector functions of therapeutic mAbs with different effector/target-cell combinations and several readouts including Fcγ receptor (FcγR)-mediated lysis, fluorescence, or luminescence. Optimized FcγR-mediated effector functions regarding clinical safety and efficacy are addressed with modification strategies such as point mutations, altered glycosylation patterns, combination of different Fc subclasses (cross isotypes), and Fc-truncation of the mAb. These strategies opened the field for a next generation of therapeutic mAbs. In conclusion, it is of major importance to consider FcγR-mediated effector functions for the efficacy of therapeutic mAbs.

Human monocyte-derived type 1 and 2 macrophages recognize Ara h 1, a major peanut allergen, by different mechanisms.

Evidence has suggested that major peanut allergen Ara h 1 activates dendritic cells (DCs) via interaction with DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin), a C-type lectin receptor, and contributes to development of peanut allergy. Since macrophages, as well as DCs, play a crucial role in innate immunity, we investigated whether natural Ara h 1 (nAra h 1) activates two different subsets of macrophages, human monocyte derived macrophage type 1 (hMDM1: pro-inflammatory model) and type 2 (hMDM2: anti-inflammatory model). hMDM1 and hMDM2 predominantly produced pro-inflammatory cytokines (IL-6 and TNF-α) and an anti-inflammatory cytokine (IL-10) in response to nAra h 1, respectively. hMDM2 took up nAra h 1 and expressed DC-SIGN at higher levels than hMDM1. However, small interfering RNA knockdown of DC-SIGN did not suppress nAra h 1 uptake and nAra h 1-mediated cytokine production in hMDM2. Inhibitors of scavenger receptor class A type I (SR-AI) suppressed the response of hMDM2, but not of hMDM1, suggesting that SR-AI is a major receptor in hMDM2 for nAra h 1 recognition and internalization. nAra h 1 appears to exert stimulatory capacity on DC and macrophages via different receptors. This study advances our understanding how a major peanut allergen interacts with innate immunity.

Corrigendum: Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

[This corrects the article DOI: 10.3389/fimmu.2020.569331.].

Enhanced activation of blood neutrophils and monocytes in patients with Ethiopian localized cutaneous leishmaniasis in response to Leishmania aethiopica Neutrophil activation in Ethiopian cutaneous leishmaniasis.

Recent studies suggest an essential role of the innate immune effector cells neutrophils and monocytes in protection or disease progression in the early course of Leishmania infection. In areas endemic for cutaneous leishmaniasis in Ethiopia most individuals are exposed to bites of infected sandflies. Still only a minor ratio of the inhabitants develops symptomatic disease. Neutrophils, followed by monocytes, are the first cells to be recruited to the site of Leishmania infection, the initial response of neutrophils to parasites appears to be crucial for the protective response and disease outcome. Our working hypothesis is that neutrophils and/or monocytes in localized cutaneous leishmaniasis (LCL) patients may have defects in function of innate immune cell that contribute to failure to parasite clearance that lead to establishment of infection. The response of cells in Ethiopian LCL patients and healthy controls to Leishmania aethiopica and to the Toll like receptor (TLR) agonists lipopolysaccharide (LPS) and macrophage activating lipopeptide-2 (MALP-2) was investigated by assessing the cell surface expression of CD62L (on neutrophil and monocyte) and CD66b (only on neutrophil), as well as reactive oxygen species (ROS) production by using whole blood-based assays in vitro. No impaired response of neutrophils and monocytes to the microbial constituents LPS and MALP-2 was observed. Neutrophils and monocytes from LCL patients responded stronger to Leishmania aethiopica in the applied whole blood assays than cells from healthy individuals. These experimental findings do not support the hypothesis regarding a possible dysfunction of neutrophils and monocytes in cutaneous leishmaniasis. On the contrary, these cells react stronger in LCL patients as compared to healthy controls. The differential response to L. aethiopica observed between LCL patients and healthy controls have the potential to serve as biomarker to develop FACS based diagnostic/ prognostic techniques for LCL.

Enhanced production of pro-inflammatory cytokines and chemokines in Ethiopian cutaneous leishmaniasis upon exposure to Leishmania aethiopica.

The clinical course and outcome of cutaneous leishmaniasis (CL) vary due to the infecting Leishmania species and host genetic makeup that result in different immune responses against the parasites. The host immune response to Leishmania aethiopica (L.aethiopica), the causative agent of CL in Ethiopia, is poorly understood. To contribute to the understanding of the protective immune response in CL due to L.aethiopica, we characterized the cytokine response to L. aethiopica in patients with the localized form of CL (LCL) and age-and sex-matched apparently healthy controls. By applying a whole blood based in vitro culture we found enhanced release of TNF, IL-6, MCP-1 or CCL2, IP-10 or CXCL10, MIP-1ß or CCL4 and IL-8 or CXCL8- but not of IL-10CL patients in response to L. aethiopica compared to the controls. No difference was observed between LCL cases and controls in the secretion of these cytokines and chemokines in whole blood cultures treated with the TLR-ligands LPS, MALP-2 or polyI: C. The observed increased secretion of the pro-inflammatory cytokines/chemokines reflects an enhanced response against the parasites by LCL patients as compared to healthy controls rather than a generally enhanced ability of blood leukocytes from LCL patients to respond to microbial constituents. Our findings suggest that the enhanced production of pro-inflammatory cytokines/chemokines is associated with localized cutaneous leishmaniasis caused by L.aethiopica.

[Therapeutic vaccination for tumors and neurodegenerative diseases]. / Therapeutische Immunisierungen gegen Tumore und neurodegenerative Erkrankungen.

Therapeutic vaccines are intended for the treatment of established diseases by harnessing the patient's own immune system. In this article we discuss therapeutic areas that are of relevance for therapeutic vaccination, i.e., oncology and neurodegenerative diseases. Clinical and regulatory aspects related to the manufacture and clinical use of actively personalized cancer vaccines are thoroughly reviewed. This applies to the regulatory classification of genomic sequencing approaches to identify tumor-specific mutations, combination therapies with checkpoint inhibitors, clinical study designs, and the use of suitable adjuvants and drug substances. Huge amounts of data (big data) are increasingly being generated in the area of personalized therapies; we briefly address the impact and usability of big data in regulatory procedures.

[Checkpoint inhibitors for cancer therapy]. / Checkpointinhibitoren in der Tumortherapie.

In recent years, a breakthrough in tumor therapy was achieved with the development of checkpoint inhibitors. Checkpoint inhibitors activate the immune defense against tumors by overcoming the inhibitory effect of specific cell surface proteins acting as control points, the so-called checkpoints. This article provides an overview of the mode of action of approved checkpoint inhibitors and the status of current clinical development.The previously approved checkpoint inhibitors, monoclonal antibodies directed against the checkpoints CTLA­4 and PD-1/PD-L1, are used in various tumor entities (including lung, kidney, and urothelial carcinoma; head and neck cancer; melanoma; and Hodgkin lymphoma). For the first time, long-term survival has been achieved in some of these patients with advanced tumors. Unfortunately, this efficacy can be observed only in a small proportion of the treated patients, depending on the tumor indication. Improved efficacy is envisioned by patient selection via predictive biomarkers and the development of combination therapies. Mandatory testing of the expression level of the predictive PD-L1 biomarker is already required in some indications to select patients with an enhanced benefit/risk relationship.

In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges.

The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo-electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.

Unveiling Interindividual Variability of Human Fibroblast Innate Immune Response Using Robust Cell-Based Protocols.

The LabEx Milieu Interieur (MI) project is a clinical study centered on the detailed characterization of the baseline and induced immune responses in blood samples from 1,000 healthy donors. Analyses of these samples has lay ground for seminal studies on the genetic and environmental determinants of immunologic variance in a healthy cohort population. In the current study we developed in vitro methods enabling standardized quantification of MI-cohort-derived primary fibroblasts responses. Our results show that in vitro human donor cohort fibroblast responses to stimulation by different MAMPs analogs allows to characterize individual donor immune-phenotype variability. The results provide proof-of-concept foundation to a new experimental framework for such studies. A bio-bank of primary fibroblast lines was generated from 323 out of 1,000 healthy individuals selected from the MI-study cohort. To study inter-donor variability of innate immune response in primary human dermal fibroblasts we chose to measure the TLR3 and TLR4 response pathways, both receptors being expressed and previously studied in fibroblasts. We established high-throughput automation compatible methods for standardized primary fibroblast cell activation, using purified MAMPS analogs, poly I:C and LPS that stimulate TLR3 and TLR4 pathways respectively. These results were in turn compared with a stimulation method using infection by HSV-1 virus. Our "Add-only" protocol minimizes high-throughput automation system variability facilitating whole process automation from cell plating through stimulation to recovery of cell supernatants, and fluorescent labeling. Images were acquired automatically by high-throughput acquisition on an automated high-content imaging microscope. Under these methodological conditions standardized image acquisition provided for quantification of cellular responses allowing biological variability to be measured with low system noise and high biological signal fidelity. Optimal for automated analysis of immuno-phenotype of primary human cell responses our method and experimental framework as reported here is highly compatible to high-throughput screening protocols like those necessary for chemo-genomic screening. In context of primary fibroblasts derived from donors enrolled to the MI-clinical-study our results open the way to assert the utility of studying immune-phenotype characteristics relevant to a human clinical cohort.

Cathelicidin Contributes to the Restriction of Leishmania in Human Host Macrophages.

In cutaneous Leishmaniasis the parasitic control in human host macrophages is still poorly understood. We found an increased expression of the human cathelicidin CAMP in skin lesions of Ethiopian patients with cutaneous leishmaniasis. Vitamin D driven, Cathelicidin-type antimicrobial peptides (CAMP) play an important role in the elimination of invading microorganisms. Recombinant cathelicidin was able to induce cell-death characteristics in Leishmania in a dose dependent manner. Using human primary macrophages, we demonstrated pro-inflammatory macrophages (hMDM1) to express a higher level of human cathelicidin, both on gene and protein level, compared to anti-inflammatory macrophages (hMDM2). Activating the CAMP pathway using Vitamin D in hMDM1 resulted in a cathelicidin-mediated-Leishmania restriction. Finally, a reduction of cathelicidin in hMDM1, using a RNA interference (RNAi) approach, increased Leishmania parasite survival. In all, these data show the human cathelicidin to contribute to the innate immune response against Leishmaniasis in a human primary cell model.

HIF1A and NFAT5 coordinate Na+-boosted antibacterial defense via enhanced autophagy and autolysosomal targeting.

Infection and inflammation are able to induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of mouse macrophages and augments their antibacterial and antiparasitic activity. While Na+-boosted host defense against the protozoan parasite Leishmania major is mediated by increased expression of the leishmanicidal NOS2 (nitric oxide synthase 2, inducible), the molecular mechanisms underpinning this enhanced antibacterial defense of mouse macrophages with high Na+ (HS) exposure are unknown. Here, we provide evidence that HS-increased antibacterial activity against E. coli was neither dependent on NOS2 nor on the phagocyte oxidase. In contrast, HS-augmented antibacterial defense hinged on HIF1A (hypoxia inducible factor 1, alpha subunit)-dependent increased autophagy, and NFAT5 (nuclear factor of activated T cells 5)-dependent targeting of intracellular E. coli to acidic autolysosomal compartments. Overall, these findings suggest that the autolysosomal compartment is a novel target of Na+-modulated cell autonomous innate immunity. Abbreviations: ACT: actins; AKT: AKT serine/threonine kinase 1; ATG2A: autophagy related 2A; ATG4C: autophagy related 4C, cysteine peptidase; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1; BMDM: bone marrow-derived macrophages; BNIP3: BCL2/adenovirus E1B interacting protein 3; CFU: colony forming units; CM-H2DCFDA: 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester; CTSB: cathepsin B; CYBB: cytochrome b-245 beta chain; DAPI: 4,6-diamidino-2-phenylindole; DMOG: dimethyloxallyl glycine; DPI: diphenyleneiodonium chloride; E. coli: Escherichia coli; FDR: false discovery rate; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; GO: gene ontology; HIF1A: hypoxia inducible factor 1, alpha subunit; HUGO: human genome organization; HS: high salt (+ 40 mM of NaCl to standard cell culture conditions); HSP90: heat shock 90 kDa proteins; LDH: lactate dehydrogenase; LPS: lipopolysaccharide; Lyz2/LysM: lysozyme 2; NFAT5/TonEBP: nuclear factor of activated T cells 5; MΦ: macrophages; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MIC: minimum inhibitory concentration; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NaCl: sodium chloride; NES: normalized enrichment score; n.s.: not significant; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; NS: normal salt; PCR: polymerase chain reaction; PGK1: phosphoglycerate kinase 1; PHOX: phagocyte oxidase; RFP: red fluorescent protein; RNA: ribonucleic acid; ROS: reactive oxygen species; sCFP3A: super cyan fluorescent protein 3A; SBFI: sodium-binding benzofuran isophthalate; SLC2A1/GLUT1: solute carrier family 2 (facilitated glucose transporter), member 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like kinase 1; v-ATPase: vacuolar-type H+-ATPase; WT: wild type.

Type-1 interferons prolong the lifespan of neutrophils by interfering with members of the apoptotic cascade.

Polymorphonuclear Neutrophils (PMNs) are metabolically highly active phagocytes, present in abundant numbers in the circulation. These active cells take the onus of clearing invading pathogens by crowding at inflammatory sites in huge numbers. Though PMNs are extremely short living and die upon spontaneous apoptosis, extended lifespan has been observed among those cells arrive at the inflammation sites or tackle intracellular infections or face any microbial challenges. The delay/inhibition of spontaneous apoptosis of these short-living cells at the inflammatory core rather helps in combating pathogens. Like many candidates, type-1 interferons (type-1 IFNs) is a group of cytokines predominant at the inflammation site. Although there are some isolated reports, a systematic study is still lacking which addresses the impact of the predominant type of interferon on the spontaneous apoptosis of neutrophils. Here in, we have observed that exposure of these IFNs (IFN-ß, IFN-α & IFN-ω etc) on human neutrophils prevents the degradation of the Bfl1, an important anti-apoptotic partner in the apoptotic cascade. Treatment showed a significant reduction in the release of cytochrome-C in the cytosol, a critical regulator in the intrinsic apoptotic pathway. We also noticed a reduction in the conversion of procaspase -3 to active caspase-3, a crucial executioner caspase towards initiation of apoptosis. Taken together our results show that exposure to interferon interferes with apoptotic pathways of neutrophils and thereby delay its spontaneous apoptosis. These findings would help us further deciphering specific roles if these inflammatory agents are causing any immune-metabolomic changes on PMNs at the inflammatory and infection core.

Anti-Tumor Necrosis Factor α Therapeutics Differentially Affect Leishmania Infection of Human Macrophages.

Tumor necrosis factor α (TNFα) drives the pathophysiology of human autoimmune diseases and consequently, neutralizing antibodies (Abs) or Ab-derived molecules directed against TNFα are essential therapeutics. As treatment with several TNFα blockers has been reported to entail a higher risk of infectious diseases such as leishmaniasis, we established an in vitro model based on Leishmania-infected human macrophages, co-cultured with autologous T-cells, for the analysis and comparison of anti-TNFα therapeutics. We demonstrate that neutralization of soluble TNFα (sTNFα) by the anti-TNFα Abs Humira®, Remicade®, and its biosimilar Remsima® negatively affects infection as treatment with these agents significantly reduces Leishmania-induced T-cell proliferation and increases the number of infected macrophages. By contrast, we show that blockade of sTNFα by Cimzia® does not affect T-cell proliferation and infection rates. Moreover, compared to Remicade®, treatment with Cimzia® does not impair the expression of cytolytic effector proteins in proliferating T-cells. Our data demonstrate that Cimzia® supports parasite control through its conjugated polyethylene glycol (PEG) moiety as PEGylation of Remicade® improves the clearance of intracellular Leishmania. This effect can be linked to complement activation, with levels of complement component C5a being increased upon treatment with Cimzia® or a PEGylated form of Remicade®. Taken together, we provide an in vitro model of human leishmaniasis that allows direct comparison of different anti-TNFα agents. Our results enhance the understanding of the efficacy and adverse effects of TNFα blockers and they contribute to evaluate anti-TNFα therapy for patients living in countries with a high prevalence of leishmaniasis.

Dectin-1 Positive Dendritic Cells Expand after Infection with Leishmania major Parasites and Represent Promising Targets for Vaccine Development.

Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c+ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1+ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1+ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1+ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4+ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1-/- BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies.