T-cells of invasive candidiasis patients show patterns of T-cell-exhaustion suggesting checkpoint blockade as treatment option.

OBJECTIVE: Recent data imply that strengthening host immunity by checkpoint inhibition improves outcome in invasive fungal infections (IFI), particularly in candidiasis. METHODS: To assess T-cell exhaustion in this context, we compared peripheral blood mononuclear cells (PBMCs) and serum samples of patients with invasive Candida albicans infection (IC, n = 21) to PBMCs or tumor-infiltrating lymphocytes (TILs) from cancer patients (n = 14) and PBMCs of healthy controls (n = 20). Type and differentiation of lymphocytes and expression of 29 immune-regulatory molecules were analyzed by flow cytometry. C. albicans specific responses were assessed by FluoroSpot (n = 8) and antibody measurement (n = 14). RESULTS: Fractions and phenotypes of lymphocyte subsets in PBMCs of IC patients were similar compared to PBMCs of controls, while they were different in TILs. PBMCs of patients with IC showed increased expression of immune-checkpoint molecules. The pattern of upregulated molecules was similar to TILs, but not present in PBMCs of control cancer patients. Fractions of T-cells expressing PD-1 and TIGIT were higher in IC patients that died. FluoroSpot analysis showed a Candida-specific IFN-y or IL-2 response in 5/8 patients, enhanced by addition of nivolumab in vitro. CONCLUSIONS: Together with preclinical data and preliminary evidence of clinical efficacy in mucormycosis, our results support clinical evaluation of immune-checkpoint inhibition in IFI treatment. TRIAL REGISTRATION: NCT04533087; retrospectively registered on August 31, 2020.

Publisher Correction: Tumor-necrosis factor impairs CD4+ T cell-mediated immunological control in chronic viral infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

LL37:DNA complexes provide antimicrobial activity against intracellular bacteria in human macrophages.

As part of the innate host response neutrophils release neutrophil extracellular traps (NETs), protein:DNA complexes that contain a number of antimicrobial peptides (AMPs), such as cathelicidin. Human cathelicidin in its active form, LL37, has potent antimicrobial activity against bacteria. However, whether LL37 derived from NETs contributes to antimicrobial activity against intracellular pathogens remains unclear. Here, we report that NETs induced by mycobacteria contain cathelicidin. Human macrophages internalized NET-bound cathelicidin, which is transported to lysosomal compartments. Furthermore, using a model of in vitro-generated LL37:DNA complexes we found that LL37 derived from such complexes attacks mycobacteria in macrophage phagolysosomes resulting in antimicrobial activity. Taken together, our results suggest a mechanism by which LL37 in complex with DNA contributes to host defence against intracellular bacteria in human macrophages.

Tumor-necrosis factor impairs CD4(+) T cell-mediated immunological control in chronic viral infection.

Persistent viral infections are characterized by the simultaneous presence of chronic inflammation and T cell dysfunction. In prototypic models of chronicity--infection with human immunodeficiency virus (HIV) or lymphocytic choriomeningitis virus (LCMV)--we used transcriptome-based modeling to reveal that CD4(+) T cells were co-exposed not only to multiple inhibitory signals but also to tumor-necrosis factor (TNF). Blockade of TNF during chronic infection with LCMV abrogated the inhibitory gene-expression signature in CD4(+) T cells, including reduced expression of the inhibitory receptor PD-1, and reconstituted virus-specific immunity, which led to control of infection. Preventing signaling via the TNF receptor selectively in T cells sufficed to induce these effects. Targeted immunological interventions to disrupt the TNF-mediated link between chronic inflammation and T cell dysfunction might therefore lead to therapies to overcome persistent viral infection.

High Intracellular Concentrations of Posaconazole Do Not Impact on Functional Capacities of Human Polymorphonuclear Neutrophils and Monocyte-Derived Macrophages In Vitro.

Posaconazole is a commonly used antifungal for the prophylaxis and treatment of invasive fungal infections. We previously demonstrated that the intracellular concentration of posaconazole in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) was greatly increased compared to the plasma concentration. As these professional phagocytes are crucial to combat fungal infections, we set out to investigate if and how, beneficial or deleterious, this high loading of intracellular posaconazole impacts the functional capacities of these cells. Here, we show that high intracellular concentrations of posaconazole do not significantly impact PMN and monocyte-derived macrophage function in vitro In particular, killing capacity and cytoskeletal features of PMN, such as migration, are not affected, indicating that these cells serve as vehicles for posaconazole to the site of infection. Moreover, since posaconazole as such slowed the germination of Aspergillus fumigatus conidia, infected neutrophils released less reactive oxygen species (ROS). Based on these findings, we propose that the delivery of posaconazole by neutrophils to the site of Aspergillus species infection warrants control of the pathogen and preservation of tissue integrity at the same time.

The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron-sulfur cluster biogenesis and oxidative stress defence.

The complex multiprotein systems for the assembly of protein-bound iron-sulfur (Fe-S) clusters are well defined in Gram-negative model organisms. However, little is known about Fe-S cluster biogenesis in other bacterial species. The ISC (iron-sulfur cluster) operon of Mycobacterium tuberculosis lacks several genes known to be essential for the function of this system in other organisms. However, the cysteine desulfurase IscSMtb (Rv number Rv3025c; Mtb denotes M. tuberculosis) is conserved in this important pathogen. The present study demonstrates that deleting iscSMtb renders the cells microaerophilic and hypersensitive to oxidative stress. Moreover, the ∆iscSMtb mutant shows impaired Fe-S cluster-dependent enzyme activity, clearly indicating that IscSMtb is associated with Fe-S cluster assembly. An extensive interaction network of IscSMtb with Fe-S proteins was identified, suggesting a novel mechanism of sulfur transfer by direct interaction with apoproteins. Interestingly, the highly homologous IscS of Escherichia coli failed to complement the ∆iscSMtb mutant and showed a less diverse protein-interaction profile. To identify a structural basis for these observations we determined the crystal structure of IscSMtb, which mirrors adaptations made in response to an ISC operon devoid of IscU-like Fe-S cluster scaffold proteins. We conclude that in M. tuberculosis IscS has been redesigned during evolution to compensate for the deletion of large parts of the ISC operon.

Incorporation of antigens into viral capsids augments immunogenicity of adeno-associated virus vector-based vaccines.

Genetic modification of adeno-associated virus (AAV) capsids has previously been exploited to redirect viral tropism. Here we demonstrate that engineering of AAV capsids as scaffolds for antigen display augments antigen-specific immunogenicity. Combining antigen display with vector-mediated overexpression resulted in a single-shot prime-boost vaccine. This new class of vaccines induced immune responses significantly faster and an IgG antibody pool of higher avidity than conventional vectors, highlighting the potency of capsid modification in vaccine development.

Intracellular concentrations of micafungin in different cellular compartments of the peripheral blood.

Whilst micafungin serum concentrations are well studied, little is known about its concentrations within cellular compartments of the peripheral blood. Hence, in this study blood samples were collected from patients receiving micafungin (n=26). These samples were separated by double-discontinuous Ficoll-Hypaque density gradient centrifugation. Intracellular concentrations within the obtained cells, i.e. peripheral blood mononuclear cells (PBMCs), polymorphonuclear leukocytes (PMNs) and red blood cells (RBCs), were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Within PBMCs and PMNs, the intracellular micafungin concentration was significantly increased compared with the concentration in plasma (P<0.001). The intracellular concentration within RBCs did not significantly differ from the plasma concentration. Micafungin reaches high concentrations in human PBMCs and PMNs and is present in RBCs. In vitro data showed that intracellular uptake of micafungin by PBMCs depends on the albumin concentration of the surrounding medium, but only at non-physiological protein concentrations.

Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors.

UNLABELLED: Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB. CONCLUSION: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV.

MHC class I chain-related protein A shedding in chronic HIV-1 infection is associated with profound NK cell dysfunction.

Natural killer (NK) cells play a critical role in host defense against viral infections. However chronic HIV-1 infection is associated with an accumulation of dysfunctional NK cells, that poorly control viral replication. The underlying mechanisms for this NK cell mediated dysfunction are not understood. Certain tumors evade NK cell mediated detection by dampening NK cell activity through the downregulation of NKG2D, via the release of soluble NKG2D-ligands, resulting in a potent suppression of NK cell function. Here we show that chronic HIV-1 infection is associated with a specific defect in NKG2D-mediated NK cell activation, due to reduced expression and transcription of NKG2D. Reduced NKG2D expression was associated with elevated levels of the soluble form of the NKG2D-ligand, MICA, in patient sera, likely released by HIV+CD4+ T cells. Thus, like tumors, HIV-1 may indirectly suppress NK cell recognition of HIV-1-infected CD4+ T cells by enhancing NKG2D-ligand secretion into the serum resulting in a profound impairment of NK cell function.

Nucleoside-free boosted double PI regimen: significant CD4+ T-cell recovery in patients with poor immunologic response despite virologic suppression.

Antiretroviral therapy in human immunodeficiency virus infection is occasionally associated with poor immunologic responses despite full suppression of viral replication. As some combinations of nucleoside analogues (NA) have been associated with paradoxical depletion of CD4(+) T- cells, we postulated that depleting the antiretroviral regimen of NAs would improve quantitative immunological parameters. In a longitudinal prospective study we quantified CD4(+) T-cells after removing NAs from antiretroviral therapy. The NA for regimen consisted of atazanavir (300 mg qd), saquinavir (1000 mg bid), and ritonavir (100mg qd) in 14 patients with immunologic failure despite undetectable plasma HIV-RNA (CD4(+) T-cells < 250 cells/microL (<17%) HIV RNA, <= 50 copies/mL). Additionally, we assessed the state of immunologic activation markers (CD38(+)HLA-DR(+) on CD4(+) and CD8(+) T-cells) by flow cytometry. The regimen was well tolerated. During the 48 week study CD4(+) T-cell counts improved significantly (mean and +/- SEM [standard error of mean], baseline: 174/microL (12.4%) [15, 5.8%], week 24: 232/microL (14%) [26, 5.3%], week 48: 267/microL (15.4%) [34, 4.3%]) with preservation of full viral suppression (p<0.05). Activation parameters of CD4(+) T-cells, but not of CD8(+) T-cells, decreased significantly. This treatment strategy may represent an option for patients with poor immunologic responses to antiretroviral therapy despite undetectable viremia.

Identification of three cytotoxic early proteins of mycobacteriophage L5 leading to growth inhibition in Mycobacterium smegmatis.

Mycobacteriophage L5 is a temperate phage with a broad host range among the fast- and slow-growing mycobacteria such as Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium ulcerans. L5 switches off host protein synthesis during the early stage of lytic growth, as was previously shown by protein expression profiling. Also, lethal genetic elements have been identified in L5 based on the fact that transformants could not be obtained with these genes. Using an inducible mycobacterial shuttle vector, we have identified three ORFs within an early operon of mycobacteriophage L5 which encode gene products (gp) toxic to the host M. smegmatis when expressed. These ORFs, coding for gp77, gp78 and gp79, presumably function as shut-off genes during early stages of phage replication. There is evidence that cell division is affected by one of the proteins (gp79). The transcription of the cytotoxic polypeptides is directed by a promoter situated in ORF83 and transcription control is achieved through the phage repressor gp71, which is shown by co-expression of this protein. The findings presented here should provide useful tools for the molecular genetics of mycobacteria. Further analysis of these and other mycobacteriophage-derived toxic polypeptides, together with the identification of their cellular targets, might provide a tool for the rapid identification of promising drug targets in emerging and re-emerging mycobacterial pathogens.

Bacterial translocation increases phagocytic activity of polymorphonuclear leucocytes in portal hypertension: priming independent of liver cirrhosis.

AIM: Bacterial translocation (BT) to mesenteric lymph nodes (MLN) in cirrhosis has been linked to impaired host defence. Phagocytosis by polymorphonuclear leucocytes (PMNLs) is the primary event in the killing of bacteria but has not been investigated in relation to the presence of BT. METHODS: Mesenteric lymph nodes were harvested sterile and assessed for BT by culture techniques. Study groups included ascitic cirrhotic rats (LC), healthy controls (Con) as well as portal-vein-ligated (PVL) rats 2 days (acute PVL with and without norfloxacin) or 3 weeks after surgery (chronic PVL). PMNLs were isolated from systemic blood and the capacity to phagocytose opsonized Escherichia coli was evaluated by FACS analysis. RESULTS: No BT was observed in Con and chronic PVL animals but 11/20 LC (55%) and six out of six acute PVL (100%) presented with BT. In the presence of BT, PMNL from PVL as well as LC rats showed significantly increased phagocytic activity as compared with controls. In contrast, PMNL from animals without BT, whether PVL or LC, exhibited phagocytic activity similar to those from control rats. The number of PMNLs involved in the phagocytic process was significantly increased only in portal-hypertensive rats with but not without BT as compared with controls. Norfloxacin did prevent BT in acute PVL animals, thereby correcting the increase in phagocytic capacity in PMNL. CONCLUSIONS: Cirrhosis per se is not associated with alterations of the phagocytic capacity of PMNL. The occurrence of BT, however, increases the phagocytic capacity of PMNL, being observed likewise in prehepatic portal hypertension, indicating an in vivo'priming' of PMNL by BT independent of cirrhosis.

[Lymph node tuberculosis as primary manifestation of Hodgkin's disease]. / Lymphknotentuberkulose als Erstmanifestation eines Morbus Hodgkin.

HISTORY AND FINDINGS ON ADMISSION: A 63-year-old female patient was admitted to the authors' hospital for further diagnostic work-up for suspected reactivation of a previously successfully treated lymph node tuberculosis, which had been diagnosed 1 year prior to the current admission. The clinical signs consisted of worsening of the patient's general condition, negacervical lymphadenopathy, night sweats, dyspnea, and superficial inflammation of the right mamma. FINDINGS: A contrast-enhanced CT scan of the neck, thorax and abdomen revealed a generalized enlargement of the cervical, axillar, mediastinal and retroperitoneal lymph nodes, multiple intrapulmonary nodular lesions with a diameter of up to 6 mm, and a substantial right-sided pleural effusion. COURSE OF DISEASE: Under the assumption of reactivation of a lymph node tuberculosis, the patient was initially treated with an extended tuberculostatic therapy. Because of disease progression another lymph node biopsy was performed revealing Hodgkin's disease of mixed-cellularity type with a partly histiocytic necrotizing, partly tuberculoid reaction. The biopsy was negative for acid-fast bacilli. Thereupon initiated chemotherapy according to the ABVD protocol led to a rapid amelioration of the clinical symptoms. CONCLUSION: In the clinical setting of suspected or confirmed lymph node tuberculosis malignant lymphoma should always be considered. This consideration is particular important since Hodgkin's disease is typically associated with a cellular immunosuppression predisposing the subject to tuberculosis.

A designed TLR4/MD-2 complex to capture LPS.

The family of Toll-like receptors (TLRs) is involved in the defense of an organism to microbial attack. TLR4-induced signaling is involved in infectious diseases, chronic inflammatory diseases and sepsis; therefore, we aimed at modulating TLR4-signaling via ligand-binding soluble receptors. Because recognition of microbial structures shows some species-specific traits, we specifically selected the mouse model for later in vivo studies. We first prepared the N-terminally Flag-tagged mouse (m) recombinant (r) soluble (s) fusion proteins mrsTLR4-IgGFc (T4Fc) and mrsMD-2 in Drosophila melanogaster Schneider 2 (S2) cells. The function of these molecules was tested by inhibition of synthesis of pro-inflammatory cytokines after stimulation of mouse macrophage RAW 264.7 cells with purified lipopolysaccharide (LPS). T4Fc alone had no inhibitory activity; however, a T4Fc/MD-2 complex blocked LPS activity. By analogy with 'cytokine traps', we then prepared a designer molecule (LPS-Trap) by fusing MD-2 to the C-terminus of soluble TLR4 via a flexible linker. LPS-Trap significantly inhibited TNF production by LPS-stimulated RAW 264.7 cells. Thus, the T4Fc/MD-2 complex as well as the LPS-Trap blocked LPS activity in vitro and might thus represent a new therapeutic option in sepsis by neutralization of TLR4-activating ligands.

Blockade of TNF does not alter oxygen burst and phagocytosis of human neutrophils in patients with rheumatoid arthritis.

Clinical trials evaluating tumor necrosis factor alpha (TNF-alpha) binding agents in patients with rheumatoid arthritis (RA) have demonstrated significant efficacy in reducing symptoms of disease and slowing radiographic progression. However, infectious complications are the most severe and common adverse effects of anti-TNF therapy. The functional capacities of neutrophils (PMNs) as the first line of defense in bacterial and fungal infections are enhanced by soluble TNF as a potent neutrophil primer. The aim of this study was to assess the influence of in vivo TNF blockade on oxygen burst (OB) and phagocytosis of human neutrophils. PMNs were derived from 20 patients with RA on anti-TNF-alpha therapy and 13 patients using conventional DMARDs. By flow cytometry we measured OB upon stimulation with Escherichia coli and N-formyl-1-methionyl-1-leucyl-phenylalanine (FMLP) with and without priming with granulocyte-colony stimulating factor (G-CSF) and/or TNF-alpha using dihydrorhodamine (DHR) 123. Phagocytosis of fluorescein isothiocyanate (FITC)-labeled E. coli was also assessed by flow cytometry. Thirty-three healthy volunteers served as controls. Upon stimulation with E. coli and FMLP, there was no significant difference in OB between the two patient groups and healthy controls. Priming was effective in all groups. Phagocytosis of E. coli by PMNs was equally effective in controls and patients independent from the treatment regimen. These data show that OB, phagocytosis and responsiveness to priming with TNF and G-CSF of PMNs are not impaired in patients with RA treated with anti-TNF agents in comparison with patients on conventional DMARDs or healthy controls. Thus, the infectious complications observed in patients with TNF blockade cannot be explained by functional impairment of PMNs; however, the neutralization of TNF as a potent primer of neutrophil response may increase the susceptibility for infections in these patients.

Current treatment strategies for patients with Hodgkin's lymphoma and HIV infection.

Hodgkin's lymphoma is the most common non-AIDS-defining tumor diagnosed in HIV-infected patients. Although the introduction of highly active antiretroviral therapy (HAART) led to a decreased incidence of several malignancies among HIV-infected patients, the incidence of HIV-associated Hodgkin's lymphoma (HIV-HL) has been persistent in recent years. Its unusually aggressive tumor behavior includes a higher frequency of unfavorable histologic subtypes, high stage and extranodal involvement by the time of presentation and poor therapeutic outcome, in comparison with Hodgkin's lymphoma outside the HIV setting. Treatment of HIV-HL is challenging considering the underlying immunodeficiency caused by HIV itself and may increase the risk of opportunistic infections by inducing further immunosuppression. To address this delicate vulnerability of the HIV-infected host, tailored regimens, which are less aggressive than standard regimens for HIV-negative hosts, have been applied to achieve tumor control. The introduction of HAART has opened a new perspective in the treatment of HIV-associated malignancies. The improved control of HIV infection and the subsequently improved survival rates of HIV-infected patients has changed the goal from tumor control to cure and new treatment approaches with more potent regimens need to be evaluated to improve survival and quality of life in HIV-HL.