Dendritic cells Trigger IFN-γ secretion by NK cells independent of IL-12 and IL-18.

It is commonly believed that IL-12 produced by DCs in response to pathogens is the first signal that stimulates the production of IFN-γ by NK cells. However, IL-12 production by DCs in response to bacterial LPS depends on either engagement of CD40 by CD40L on activated T cells or IFN-γ from NK cells. This suggests that during the primary immune response, NK cells produce IFN-γ before IL-12 production by DCs. Here, using single-cell measurements, cell sorting and mouse lines deficient in IL-12, IL-23, type I IFN receptor and the IL-18 receptor, we show that a subset of BM-derived DCs characterized by low expression of MHC class II (MHCIIlow ) stimulates IFN-γ production by NK cells. The expression of Toll-like Receptor (TLR) 4 on DCs but not NK cells was required for such NK-derived IFN-γ. In addition, soluble factor(s) produced by LPS-activated MHCIIlow DCs were sufficient to induce IFN-γ production by NK cells independent of IL-12, IL-23, and IL-18. This response was enhanced in the presence of a low dose of IL-2. These results delineate a previously unknown pathway of DC-mediated IFN-γ production by NK cells, which is independent of commonly known cytokines.

The subunits of IL-12, originating from two distinct cells, can functionally synergize to protect against pathogen dissemination in vivo.

Cytokines are typically single gene products, except for the heterodimeric interleukin (IL)-12 family. The two subunits (IL-12p40 and IL-12p35) of the prototype IL-12 are known to be simultaneously co-expressed in activated myeloid cells, which secrete the fully active heterodimer to promote interferon (IFN)γ production in innate and adaptive cells. We find that chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, generate functional IL-12. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism is sufficient to propel local T cell differentiation in sites distal to the initial infection and helps control systemic dissemination of a pathogen, although not parasite burden, at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival.

Bone Marrow-Derived Dendritic Cell Cultures from RAG-/- Mice Include IFN-γ-Producing NK Cells.

Dendritic cells (DCs) play a key role in the initiation of an immune response and are known as "professional" APCs because of their ability to activate naive T cells. A widely used method to generate DCs in vitro is to culture bone marrow (BM) cells or blood monocytes in the presence of GM-CSF and IL-4. In this study, we show that a small population of NK cells residing in the BM of RAG-/-, but not RAG-/- γc chain-/- mice, remain in the DC culture and is the source of IFN-γ produced after stimulation with LPS. These cells, which may represent early promoters of LPS-induced responses, have to be taken into account when interpreting experiments using BM-derived DCs.

PD-1 Expression on NK Cells in Malaria-Exposed Individuals Is Associated with Diminished Natural Cytotoxicity and Enhanced Antibody-Dependent Cellular Cytotoxicity.

Natural killer (NK) cells are key effector cells of innate resistance capable of destroying tumors and virus-infected cells through cytotoxicity and rapid cytokine production. The control of NK cell responses is complex and only partially understood. PD-1 is an inhibitory receptor that regulates T cell function, but a role for PD-1 in regulating NK cell function is only beginning to emerge. Here, we investigated PD-1 expression on NK cells in children and adults in Mali in a longitudinal analysis before, during, and after infection with Plasmodium falciparum malaria. We found that NK cells transiently upregulate PD-1 expression and interleukin-6 (IL-6) production in some individuals during acute febrile malaria. Furthermore, the percentage of PD-1 expressing NK cells increases with age and cumulative malaria exposure. Consistent with this, NK cells of malaria-naive adults upregulated PD-1 following P. falciparum stimulation in vitro Additionally, functional in vitro studies revealed that PD-1 expression on NK cells is associated with diminished natural cytotoxicity but enhanced antibody-dependent cellular cytotoxicity (ADCC). These data indicate that PD-1+ NK cells expand in the context of chronic immune activation and suggest that PD-1 may contribute to skewing NK cells toward enhanced ADCC during infections such as malaria.

Introducing an affordable catalyst for biohydrogen production in microbial electrolysis cells.

This study reports the fabrication of a new cathode electrode assembly using polyaniline (PANI) and graphene on a stainless steel mesh (SSM) as an alternative for the conventional expensive cathode of microbial electrolysis cells (MECs). With respect to the previous efforts to propose an efficient and cost-effective alternative for platinum (Pt) catalysts and cathode electrodes, the present study investigates the assessment of different catalysts to elucidate the potential of the modified SSM cathode electrode for larger-scale MECs. In the case of feeding dairy wastewater to the MEC, the maximum hydrogen production rate and COD removal were obtained by SSM/PANI/graphene cathode and had the values 0.805 m3 H2 m-3 anolyte day-1 and 82%, respectively, at the applied potential of 1 V. These values were only 20% and 7% lower than those of the MEC with Pt on the carbon cloth cathode, respectively. The coulombic efficiencies of SSM/Pt and SSM/PANI/graphene were seen to be 64.48% and 56.67%, respectively. It was also concluded that the fabrication cost of the modified cathode was 50% lower than the conventional cathodes with Pt on the carbon cloth. Finally, the evaluation of the modified cathode performance was achieved based on Fourier transform infrared spectroscopy, linear sweep voltammetry, scanning electron microscopy, and atomic force microscopy.

Novel microfluidic graphene oxide-protein amperometric biosensor for detecting sulfur compounds.

Sulfur compounds are essential for many industries and organisms; however, they cause serious respiratory problems in human beings. Therefore, determination of sulfur concentration is of paramount importance. The research approach in the field of detecting contaminants has led to smaller systems that provide faster and more effective ways for diagnosis purposes. In this study, a novel portable amperometric graphene oxide-protein biosensor platform is investigated. The main characteristic of this structure is the implementation of a microfluidic configuration. With albumin metalloprotein as the biorecognition element, graphene oxide was synthesized and characterized by transmission electron microscopy and Fourier-transform infrared spectroscopy (FTIR). Albumin protein was stabilized on the surface of graphene oxide by the application of the N-(3-dimethylamionpropyl)-N-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide method. The stabilization was confirmed by FTIR and electrochemistry analyses. The calibration curve of sulfur concentration was determined. When the graphene oxide-protein complex was stabilized by nephion on the surface of the microfluidic system, the response time reduced to 50 Sec, which is a relatively faster response among the similar studies and validated the significant effect of the microfluidic system. The nanosystem had an optimized pH of 7.4 and exhibited high sensitivity in determining sulfide. The results confirm that the portable graphene oxide-protein nanosystem has a fast and accurate response in detecting sulfide.

Cutting Edge: Quantitative Determination of CD40L Threshold for IL-12 and IL-23 Production from Dendritic Cells.

Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/µm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.

Mechanisms by which Porphyromonas gingivalis evades innate immunity.

The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines.

Making many from few: IL-12p40 as a model for the combinatorial assembly of heterodimeric cytokines.

How dendritic cells (DCs) gather information from the local milieu at a site of infection or injury and communicate this to influence adaptive immunity is not well understood. We and others have reported that soon after microbial encounter, DCs secrete the p40 subunit of IL-12, by itself, in a monomeric form. Based on recent data that this p40 monomer subsequently associates with p35 released from other cells to generate functional IL-12, we proposed that p40 can function as a DC-derived probe which samples the composition of the local milieu by looking for other binding partners. In this opinion, we discuss how such a sampling function might generate an elaborate combinatorial "code" of heterodimeric cytokines, capable of conveying location-specific information to cells downstream of DC activation, including NK and T cells.

Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly.

IL-12p40 partners with the p35 and p19 polypeptides to generate the heterodimeric cytokines IL-12 and IL-23, respectively. These cytokines play critical and distinct roles in host defense. The assembly of these heterodimers is thought to take place within the cell, resulting in the secretion of fully functional cytokines. Although the p40 subunit alone can also be rapidly secreted in response to inflammatory signals, its biological significance remains unclear. In this article, we show that the secreted p40 monomer can generate de novo IL-12-like activities by combining extracellularly with p35 released from other cells. Surprisingly, an unbiased proteomic analysis reveals multiple such extracellular binding partners for p40 in the serum of mice after an endotoxin challenge. We biochemically validate the binding of one of these novel partners, the CD5 Ag-like glycoprotein, to the p40 monomer. Nevertheless, the assembled p40-CD5L heterodimer does not recapitulate the biological activity of IL-12. These findings underscore the plasticity of secreted free p40 monomer, suggesting that p40 functions as an adaptor that is able to generate multiple de novo composites in combination with other locally available polypeptide partners after secretion.

Lipopolysaccharide-activated dendritic cells: "exhausted" or alert and waiting?

LPS-activated dendritic cells (DCs) are thought to follow a set program in which they secrete inflammatory cytokines (such as IL-12) and then become refractory to further stimulation (i.e., "exhausted"). In this study, we show that mouse DCs do indeed lose their responsiveness to LPS, but nevertheless remain perfectly capable of making inflammatory cytokines in response to signals from activated T cells and to CD40-ligand and soluble T cell-derived signals. Furthermore, far from being rigidly programmed by the original activating stimulus, the DCs retained sufficient plasticity to respond differentially to interactions with Th0, Th1, Th2, and Th17 T cells. These data suggest that LPS activation does not exhaust DCs but rather primes them for subsequent signals from T cells.