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.

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.

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.

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.

[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.

LC3-associated phagocytosis in microbial pathogenesis.

Phagocytosis is essential for uptake and elimination of pathogenic microorganisms. Autophagy is a highly conserved mechanism for incorporation of cellular constituents to replenish nutrients by degradation. Recently, parts of the autophagy machinery - above all microtubule-associated protein 1 light chain 3 (LC3) - were found to be specifically recruited to phagosomal membranes resulting in phagosome-lysosome fusion and efficient degradation of internalized cargo in a process termed LC3-associated phagocytosis (LAP). Many pathogenic bacterial, fungal and parasitic microorganisms reside within LAP-targeted single-membrane phagosomes or vacuoles after infection of host cells. In this minireview we describe the state of knowledge on the interaction of pathogens with LAP or LAP-like pathways and report on various pathogens that have evolved strategies to circumvent degradation in LAP compartments.

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.

In silico prediction of Leishmania major-specific CD8+ epitopes.

Infections with Leishmania (L.) major induce protective IFN-γ-dependent Th1/Tc1 immunity in C57BL/6 mice as well as in immunocompetent humans. Even though antigen-specific immunity provides lifelong immunity against reinfection, a vaccine against this pathogen does not yet exist. Here, we compared the results obtained from in silico predictions of murine CD8-specific L. major peptides using the algorithm SYFPEITHI with the number and predicted affinity of known proteins/peptides. Our results indicate that the majority of "immunodominant" epitopes of L. major have not been identified so far; thus, computer-based prediction algorithms may aid the development of an effective vaccine.

Recombinant human IgA1 and IgA2 autoantibodies to type VII collagen induce subepidermal blistering ex vivo.

Subepidermal autoimmune blistering dermatoses (AIBD) are prototypic autoantibody-mediated diseases. In epidermolysis bullosa acquisita (EBA), an autoimmune disease with severe and chronic skin blistering, autoantibodies are directed against type VII collagen. IgG is the predominant autoantibody isotype of EBA, the pathogenicity of which has been demonstrated in a variety of in vivo and ex vivo disease models. In contrast, there is not much evidence for the pathogenicity of IgA, which may appear as the only autoantibody isotype in some EBA patients. To investigate the pathogenic potential of IgA autoantibodies, we generated chimeric V gene-matched human IgA1, IgA2, and control IgG1 autoantibodies directed against type VII collagen. Immobilized immune complexes containing the rIgA1 and rIgA2 autoantibodies induced the dose-dependent release of reactive oxygen species from neutrophil granulocytes, a precondition for blister formation. Moreover, both rIgA1 and rIgA2 induced leukocyte-dependent dermal-epidermal separation in cryosections of human skin. In contrast with rIgG1, neither rIgA1 nor rIgA2 was capable of inducing complement deposition at the dermal-epidermal junction. Because complement activation is a prerequisite for blister induction, this lack of function compared with IgG1 may be compensated for by the stronger activation of neutrophil granulocytes by both IgA1 and IgA2. For IgG-mediated AIBD, immunoadsorption therapy is a convenient treatment modality for the removal of pathogenic autoantibodies, particularly in treatment-resistant cases. The results of this study show the pathogenic potential of IgA autoantibodies and support the development of adsorber matrices for IgA-mediated AIBD.

Mechanisms of apoptosis inhibition in Chlamydia pneumoniae-infected neutrophils.

The obligatory intracellular bacterium Chlamydia pneumoniae (C. pneumoniae) can survive and multiply in neutrophil granulocytes. Since neutrophils are short living cells, inhibition of neutrophil apoptosis appears to play a major role in the productive infection of neutrophils by C. pneumoniae. In the present study, we have investigated which survival pathways and which events of the apoptotic process are modulated in C. pneumoniae-infected neutrophils. All infection experiments were carried out using primary human neutrophils in vitro. We show that infection with C. pneumoniae activates PI3K/Akt as well as the ERK1/2 and p38 MAP kinases and present evidence that activation of the PI3K/Akt and ERK1/2 pathways are essential to initiate the apoptosis delay in C. pneumoniae-infected neutrophils. Both the PI3K/Akt and ERK1/2 pathways are involved in the maintained expression of the anti-apoptotic protein Mcl-1. In addition, we also showed that the PI3K/Akt pathway leads to the activation of NF-κB-dependent release of IL-8 by infected neutrophils. Infection with C. pneumoniae activates the PI3K/Akt and ERK1/2 MAPK survival pathways in neutrophils, induces the NF-κB dependent release of IL-8 and leads to the maintenance of Mcl-1 expression in neutrophils.

Abelson tyrosine kinase controls phagosomal acidification required for killing of Mycobacterium tuberculosis in human macrophages.

The mechanisms that regulate the acidification of intracellular compartments are key to host defense against pathogens. In this paper, we demonstrate that Abl tyrosine kinase, a master switch for cell growth and trafficking of intracellular organelles, controls the acidification of lysosomes in human macrophages. Pharmacological inhibition by imatinib and gene silencing of Abelson (Abl) tyrosine kinase reduced the lysosomal pH in human macrophages by increasing the transcription and expression of the proton pumping enzyme vacuolar-type H(+)-adenosine triphosphatase. Because lysosomal acidification is required for antimicrobial activity against intracellular bacteria, we determined the effect of imatinib on the growth of the major human pathogen Mycobacterium tuberculosis. Imatinib limited the multiplication of M. tuberculosis, and growth restriction was dependent on acidification of the mycobacterial compartment. The effects of imatinib were also active in vivo because circulating monocytes from imatinib-treated leukemia patients were more acidic than monocytes from control donors. Importantly, sera from imatinib-treated patients triggered acidification and growth restriction of M. tuberculosis in macrophages. In summary, our results identify the control of phagosomal acidification as a novel function of Abl tyrosine kinase and provide evidence that the regulation occurs on the level of the vacuolar-type H(+)-adenosine triphosphatase. Given the efficacy of imatinib in a mouse model of tuberculosis and our finding that orally administered imatinib increased the ability of human serum to trigger growth reduction of intracellular M. tuberculosis, clinical evaluation of imatinib as a complementary therapy of tuberculosis, in particular multidrug or extremely drug-resistant disease, is warranted.

Potent anti-coronaviral activity of pateamines and new insights into their mode of action.

Pateamines, derived from the sponge Mycale hentscheli, function as inhibitors of the RNA helicase eIF4A and exhibit promising antiviral and anticancer properties. eIF4A plays a pivotal role in unwinding stable RNA structures within the 5'-UTR of selected mRNAs, facilitating the binding of the 43S preinitiation complex during translation initiation. Pateamines function by clamping RNA substrates onto the eIF4A surface, effectively preventing eIF4A from carrying out the unwinding step. Rocaglates, a compound class isolated from plants of the genus Aglaia, target the same binding pocket on eIF4A, and based on structural data, a similar mode of action has been proposed for pateamines and rocaglates. In this study, we conducted a detailed characterization of pateamines' binding mode and assessed their antiviral activity against human pathogenic coronaviruses (human coronavirus 229E (HCoV-229E), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)). Our findings reveal significant differences in the binding behavior of pateamines compared to rocaglates when interacting with an eIF4A-RNA complex. We also observed that pateamines do not depend on the presence of a polypurine tract in the RNA substrate for efficient RNA clamping, as it is the case for rocaglates. Most notably, pateamines demonstrate potent antiviral activity against coronaviruses in the low nanomolar range. Consequently, pateamines broaden our toolbox for combating viruses that rely on the host enzyme eIF4A to conduct their viral protein synthesis, indicating a possible future treatment strategy against new or re-emerging pathogenic viruses.

Leishmania major parasite stage-dependent host cell invasion and immune evasion.

Leishmania pathogenesis is primarily studied using the disease-inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease-relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage-specific DsRed protein expression. We found parasite stage-specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote-specific complement receptor 3-mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1-mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage-specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease-propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigote-driven immune response in leishmaniasis.

Hypoxia abrogates antichlamydial properties of IFN-γ in human fallopian tube cells in vitro and ex vivo.

IFN-γ has an important role in the adaptive immune response against intracellular pathogens. In urogenital tract (UGT) infections with the obligate intracellular pathogen Chlamydia trachomatis, IFN-γ-mediated control of chlamydial growth implies the JAK-STAT signaling cascades and subsequent induction of the indoleamine 2,3-dioxygenase (IDO). As oxygen concentrations in the UGT are low under physiological conditions (O(2) < 5%) and further decrease during an inflammatory process, we wondered whether antibacterial properties of IFN-γ are maintained under hypoxic conditions. Using primary cells that were isolated from human fallopian tubes and an ex vivo human fallopian tube model (HFTM), we found that even high IFN-γ concentrations (200 units/mL) were not sufficient to limit growth of C. trachomatis under hypoxia. Reduced antibacterial activity of IFN-γ under hypoxia was restricted to the urogenital serovars D and L(2), but was not observed with the ocular serovar A. Impaired effectiveness of IFN-γ on chlamydial growth under hypoxia was accompanied by reduced phosphorylation of Stat-1 on Tyr701 and diminished IDO activity. This study shows that IFN-γ effector functions on intracellular C. trachomatis depend on the environmental oxygen supply, which could explain inadequate bacterial clearance and subsequent chronic infections eventually occurring in the UGT of women.

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 protective Th1 response in mice is induced in the T-cell zone only three weeks after infection with Leishmania major and not during early T-cell activation.

The protozoan parasite Leishmania spp. causes clinical pictures ranging in severity from spontaneously healing skin ulcers to systemic disease. The immune response associated with healing involves the differentiation of IFNγ-producing Th1 cells, whereas the non-healing phenotype is associated with IL4-producing Th2 cells. The widespread assumption has been that the T-cell differentiation that leads to a healing or non-healing phenotype is established at the time of T-cell activation early after infection. By selectively analyzing the expression of cytokine genes in the T-cell zones of lymph nodes of resistant (Th1) C57BL/6 mice and susceptible (Th2) BALB/c mice during an infection with Leishmania major in vivo, we show that the early T-cell response does not differ between C57BL/6 mice and BALB/c mice. Instead, Th1/Th2 polarization appears suddenly 3 weeks after infection. At the same time point, the number of parasites increases in lymph nodes of both mouse strains, but about 100-fold more in susceptible BALB/c mice. We conclude that the protective Th1 response in C57BL/6 mice is facilitated by the capacity of their innate effector cells to keep parasite numbers at low levels.

Phagocytosis of apoptotic cells by neutrophil granulocytes: diminished proinflammatory neutrophil functions in the presence of apoptotic cells.

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-gamma results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-alpha and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-alpha, which was independent of NF-kappaB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.