Crosstalk between sentinel and helper macrophages permits neutrophil migration into infected uroepithelium.

The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. Here, we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C(-) macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C(+) macrophages. Such Ly6C(+) macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C(-) macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia.

CD8+ T Cells Orchestrate pDC-XCR1+ Dendritic Cell Spatial and Functional Cooperativity to Optimize Priming.

Adaptive cellular immunity is initiated by antigen-specific interactions between T lymphocytes and dendritic cells (DCs). Plasmacytoid DCs (pDCs) support antiviral immunity by linking innate and adaptive immune responses. Here we examined pDC spatiotemporal dynamics during viral infection to uncover when, where, and how they exert their functions. We found that pDCs accumulated at sites of CD8+ T cell antigen-driven activation in a CCR5-dependent fashion. Furthermore, activated CD8+ T cells orchestrated the local recruitment of lymph node-resident XCR1 chemokine receptor-expressing DCs via secretion of the XCL1 chemokine. Functionally, this CD8+ T cell-mediated reorganization of the local DC network allowed for the interaction and cooperation of pDCs and XCR1+ DCs, thereby optimizing XCR1+ DC maturation and cross-presentation. These data support a model in which CD8+ T cells upon activation create their own optimal priming microenvironment by recruiting additional DC subsets to the site of initial antigen recognition.

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2.

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.

Integration of Scanning Electrochemical Microscopy and Scanning Electrochemical Cell Microscopy in a Bifunctional Nanopipette toward Simultaneous Mapping of Activity and Selectivity in Electrocatalysis.

Scanning electrochemical microscopy (SECM) and scanning electrochemical cell microscopy (SECCM) were integrated in a single bifunctional probe for simultaneous mapping of the oxygen reduction current and the oxidation current of the produced H2O2. The dual probe is fabricated from a double-barrel θ capillary, comprising one open barrel filled with the electrolyte and another filled with pyrolytic carbon. Pt is deposited with a gas injection system (GIS) at the end of the carbon barrel. The probe integrates the advantages of both SECM and SECCM by forming an electrochemical droplet cell that embeds the Pt working electrode of the carbon barrel directly into the electrolyte meniscus formed upon sample contact from the electrolyte barrel. The versatility of the dual probe is demonstrated by mapping the oxygen reduction reaction (ORR) current and the H2O2 oxidation current of a Pt microstrip on a gold substrate. This allows simultaneous localized electrochemical measurements, highlighting the potential of the dual probe for broader applications in characterizing the electrocatalytic properties of materials.

Differential impact of high-salt levels in vitro and in vivo on macrophage core functions.

The consumption of processed food is on the rise leading to huge intake of excess dietary salt, which strongly correlates with development of hypertension, often leading to cardiovascular diseases such as stroke and heart attack, as well as activation of the immune system. The effect of salt on macrophages is especially interesting as they are able to sense high sodium levels in tissues leading to transcriptional changes. In the skin, macrophages were shown to influence lymphatic vessel growth which, in turn, enables the transport of excess salt and thereby prevents the development of high blood pressure. Furthermore, salt storage in the skin has been linked to the onset of pro-inflammatory effector functions of macrophages in pathogen defence. However, there is only little known about the mechanisms which are involved in changing macrophage function to salt exposure. Here, we characterize the response of macrophages to excess salt both in vitro and in vivo. Our results validate and strengthen the notion that macrophages exhibit chemotactic migration in response to salt gradients in vitro. Furthermore, we demonstrate a reduction in phagocytosis and efferocytosis following acute salt challenge in vitro. While acute exposure to a high-salt diet in vivo has a less pronounced impact on macrophage core functions such as phagocytosis, our data indicate that prolonged salt challenge may exert a distinct effect on the function of macrophages. These findings suggest a potential role for excessive salt sensing by macrophages in the manifestation of diseases related to high-salt diets and explicitly highlight the need for in vivo work to decipher the physiologically relevant impact of excess salt on tissue and cell function.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

NCX1 represents an ionic Na+ sensing mechanism in macrophages.

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.

Stability Investigations on a Pt@HGS Catalyst as a Model Material for Fuel Cell Applications: The Role of the Local pH.

Increasing the resistance of catalysts against electrochemical degradation is one of the key requirements for the wider use of Proton Exchange Membrane Fuel Cells (PEMFCs). Here, we study the degradation of one entity of a highly stable catalyst, Pt@HGS, on a nanoelectrode under accelerated mass transport conditions. We find that the catalyst degrades more rapidly than expected based on previous ensemble measurements. Corroborated by identical location transmission electron microscopy and catalyst layer experiments, we deduce that locally different pH values are likely the reason for this difference in stability. Ultimately, this work provides insights into the actual conditions present in a PEMFC and raises questions about the applicability of accelerated stress tests usually performed to evaluate catalyst stability, particularly when they are performed in half-cell setups under inert gas.

Single-entity Electrochemistry Unveils Dynamic Transformation during Tandem Catalysis of Cu2 O and Co3 O4 for Converting NO3 - to NH3.

Electrochemically converting nitrate to ammonia is an essential and sustainable approach to restoring the globally perturbed nitrogen cycle. The rational design of catalysts for the nitrate reduction reaction (NO3 RR) based on a detailed understanding of the reaction mechanism is of high significance. We report a Cu2 O+Co3 O4 tandem catalyst which enhances the NH3 production rate by ≈2.7-fold compared to Co3 O4 and ≈7.5-fold compared with Cu2 O, respectively, however, most importantly, we precisely place single Cu2 O and Co3 O4 cube-shaped nanoparticles individually and together on carbon nanoelectrodes provide insight into the mechanism of the tandem catalysis. The structural and phase evolution of the individual Cu2 O+Co3 O4 nanocubes during NO3 RR is unveiled using identical location transmission electron microscopy. Combining single-entity electrochemistry with precise nano-placement sheds light on the dynamic transformation of single catalyst particles during tandem catalysis in a direct way.

Scalable Synthesis of Multi-Metal Electrocatalyst Powders and Electrodes and their Application for Oxygen Evolution and Water Splitting.

Multi-metal electrocatalysts provide nearly unlimited catalytic possibilities arising from synergistic element interactions. We propose a polymer/metal precursor spraying technique that can easily be adapted to produce a large variety of compositional different multi-metal catalyst materials. To demonstrate this, 11 catalysts were synthesized, characterized, and investigated for the oxygen evolution reaction (OER). Further investigation of the most active OER catalyst, namely CoNiFeMoCr, revealed a polycrystalline structure, and operando Raman measurements indicate that multiple active sites are participating in the reaction. Moreover, Ni foam-supported CoNiFeMoCr electrodes were developed and applied for water splitting in flow-through electrolysis cells with electrolyte gaps and in zero-gap membrane electrode assembly (MEA) configurations. The proposed alkaline MEA-type electrolyzers reached up to 3 A cm-2 , and 24 h measurements demonstrated no loss of current density of 1 A cm-2 .

Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell-licensed DCs.

Cross-priming allows dendritic cells (DCs) to induce cytotoxic T cell (CTL) responses to extracellular antigens. DCs require cognate 'licensing' for cross-priming, classically by helper T cells. Here we demonstrate an alternative mechanism for cognate licensing by natural killer T (NKT) cells recognizing microbial or synthetic glycolipid antigens. Such licensing caused cross-priming CD8alpha(+) DCs to produce the chemokine CCL17, which attracted naive CTLs expressing the chemokine receptor CCR4. In contrast, DCs licensed by helper T cells recruited CTLs using CCR5 ligands. Thus, depending on the type of antigen they encounter, DCs can be licensed for cross-priming by NKT cells or helper T cells and use at least two independent chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this can potentially be exploited to improve vaccinations.

Redox Replacement of Silver on MOF-Derived Cu/C Nanoparticles on Gas Diffusion Electrodes for Electrocatalytic CO2 Reduction.

Bimetallic tandem catalysts have emerged as a promising strategy to locally increase the CO flux during electrochemical CO2 reduction, so as to maximize the rate of conversion to C-C-coupled products. Considering this, a novel Cu/C-Ag nanostructured catalyst has been prepared by a redox replacement process, in which the ratio of the two metals can be tuned by the replacement time. An optimum Cu/Ag composition with similarly sized particles showed the highest CO2 conversion to C2+ products compared to non-Ag-modified gas-diffusion electrodes. Gas chromatography and in-situ Raman measurements in a CO2 gas diffusion cell suggest the formation of top-bound linear adsorbed *CO followed by consumption of CO in the successive cascade steps, as evidenced by the increasingνC-H bands. These findings suggest that two mechanisms operate simultaneously towards the production of HCO2 H and C-C-coupled products on the Cu/Ag bimetallic surface.

Probing the Local Reaction Environment During High Turnover Carbon Dioxide Reduction with Ag-Based Gas Diffusion Electrodes.

Discerning the influence of electrochemical reactions on the electrode microenvironment is an unavoidable topic for electrochemical reactions that involve the production of OH- and the consumption of water. That is particularly true for the carbon dioxide reduction reaction (CO2 RR), which together with the competing hydrogen evolution reaction (HER) exert changes in the local OH- and H2 O activity that in turn can possibly affect activity, stability, and selectivity of the CO2 RR. We determine the local OH- and H2 O activity in close proximity to a CO2 -converting Ag-based gas diffusion electrode (GDE) with product analysis using gas chromatography. A Pt nanosensor is positioned in the vicinity of the working GDE using shear-force-based scanning electrochemical microscopy (SECM) approach curves, which allows monitoring changes invoked by reactions proceeding within an otherwise inaccessible porous GDE by potentiodynamic measurements at the Pt-tip nanosensor. We show that high turnover HER/CO2 RR at a GDE lead to modulations of the alkalinity of the local electrolyte, that resemble a 16 m KOH solution, variations that are in turn linked to the reaction selectivity.

Single Particle Nanoelectrochemistry Reveals the Catalytic Oxygen Evolution Reaction Activity of Co3 O4 Nanocubes.

Co3 O4 nanocubes are evaluated concerning their intrinsic electrocatalytic activity towards the oxygen evolution reaction (OER) by means of single-entity electrochemistry. Scanning electrochemical cell microscopy (SECCM) provides data on the electrocatalytic OER activity from several individual measurement areas covering one Co3 O4 nanocube of a comparatively high number of individual particles with sufficient statistical reproducibility. Single-particle-on-nanoelectrode measurements of Co3 O4 nanocubes provide an accelerated stress test at highly alkaline conditions with current densities of up to 5.5 A cm-2 , and allows to derive TOF values of up to 2.8×104  s-1 at 1.92 V vs. RHE for surface Co atoms of a single cubic nanoparticle. Obtaining such high current densities combined with identical-location transmission electron microscopy allows monitoring the formation of an oxy(hydroxide) surface layer during electrocatalysis. Combining two independent single-entity electrochemistry techniques provides the basis for elucidating structure-activity relations of single electrocatalyst nanoparticles with well-defined surface structure.

Single-Entity Electrocatalysis of Individual "Picked-and-Dropped" Co3 O4 Nanoparticles on the Tip of a Carbon Nanoelectrode.

Nano-electrochemical tools to assess individual catalyst entities are critical to comprehend single-entity measurements. The intrinsic electrocatalytic activity of an individual well-defined Co3 O4 nanoparticle supported on a carbon-based nanoelectrode is determined by employing an efficient SEM-controlled robotic technique for picking and placing a single catalyst particle onto a modified carbon nanoelectrode surface. The stable nanoassembly is microscopically investigated and subsequently electrochemically characterized. The hexagonal-shaped Co3 O4 nanoparticles demonstrate size-dependent electrochemical activity and exhibit very high catalytic activity with a current density of up to 11.5 A cm-2 at 1.92 V (vs. RHE), and a turnover frequency of 532±100 s-1 at 1.92 V (vs. RHE) towards catalyzing the oxygen evolution reaction.

B-Cu-Zn Gas Diffusion Electrodes for CO2 Electroreduction to C2+ †Products at High Current Densities.

Electroreduction of CO2 to multi-carbon products has attracted considerable attention as it provides an avenue to high-density renewable energy storage. However, the selectivity and stability under high current densities are rarely reported. Herein, B-doped Cu (B-Cu) and B-Cu-Zn gas diffusion electrodes (GDE) were developed for highly selective and stable CO2 conversion to C2+  products at industrially relevant current densities. The B-Cu GDE exhibited a high Faradaic efficiency of 79 % for C2+  products formation at a current density of -200 mA cm-2 and a potential of -0.45 V vs. RHE. The long-term stability for C2+ formation was substantially improved by incorporating an optimal amount of Zn. Operando Raman spectra confirm the retained Cu+ species under CO2 reduction conditions and the lower overpotential for *OCO formation upon incorporation of Zn, which lead to the excellent conversion of CO2 to C2+ products on B-Cu-Zn GDEs.

A Metal-Organic Framework derived Cux Oy Cz Catalyst for Electrochemical CO2 Reduction and Impact of Local pH Change.

Developing highly efficient and selective electrocatalysts for the CO2 reduction reaction to produce value-added chemicals has been intensively pursued. We report a series of Cux Oy Cz nanostructured electrocatalysts derived from a Cu-based MOF as porous self-sacrificial template. Blending catalysts with polytetrafluoroethylene (PTFE) on gas diffusion electrodes (GDEs) suppressed the competitive hydrogen evolution reaction. 25 to 50 wt % teflonized GDEs exhibited a Faradaic efficiency of ≈54 % for C2+ products at -80 mA cm-2 . The local OH- ions activity of PTFE-modified GDEs was assessed by means of closely positioning a Pt-nanoelectrode. A substantial increase in the OH- /H2 O activity ratio due to the locally generated OH- ions at increasing current densities was determined irrespective of the PTFE amount.

Ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma.

Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

Needle-type organic electrochemical transistor for spatially resolved detection of dopamine.

In this work, the advantages of carbon nanoelectrodes (CNEs) and orgonic electrochemical transistors (OECTs) were merged to realise nanometre-sized, spearhead OECTs based on single- and double-barrel CNEs functionalised with a conducting polymer film. The needle-type OECT shows a high aspect ratio that allows its precise positioning by means of a macroscopic handle and its size is compatible with single-cell analysis. The device was characterised with respect to its electrolyte-gated behaviour and was employed as electrochemical sensor for the proof-of-concept detection of dopamine (DA) over a wide concentration range (10-12-10-6 M). Upon application of fixed drain and gate voltages (Vd = - 0.3 V, Vg = - 0.9 V, respectively), the nano-sized needle-type OECT sensor exhibited a linear response in the low pM range and from 0.002 to 7 µM DA, with a detection limit of 1 × 10-12 M. Graphical abstract.

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.