Regulatory T-cell dysfunction and cutaneous exposure to Staphylococcus aureus underlie eczema in DOCK8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8)-deficient patients have severe eczema, elevated IgE, and eosinophilia, features of atopic dermatitis (AD). OBJECTIVE: We sought to understand the mechanisms of eczema in DOCK8 deficiency. METHODS: Skin biopsy samples were characterized by histology, immunofluorescence microscopy, and gene expression. Skin barrier function was measured by transepidermal water loss. Allergic skin inflammation was elicited in mice by epicutaneous sensitization with ovalbumin (OVA) or cutaneous application of Staphylococcus aureus. RESULTS: Skin lesions of DOCK8-deficient patients exhibited type 2 inflammation, and the patients' skin was colonized by Saureus, as in AD. Unlike in AD, DOCK8-deficient patients had a reduced FOXP3:CD4 ratio in their skin lesions, and their skin barrier function was intrinsically intact. Dock8-/- mice exhibited reduced numbers of cutaneous T regulatory (Treg) cells and a normal skin barrier. Dock8-/- and mice with an inducible Dock8 deletion in Treg cells exhibited increased allergic skin inflammation after epicutaneous sensitization with OVA. DOCK8 was shown to be important for Treg cell stability at sites of allergic inflammation and for the generation, survival, and suppressive activity of inducible Treg cells. Adoptive transfer of wild-type, but not DOCK8-deficient, OVA-specific, inducible Treg cells suppressed allergic inflammation in OVA-sensitized skin of Dock8-/- mice. These mice developed severe allergic skin inflammation and elevated serum IgE levels after topical exposure to Saureus. Both were attenuated after adoptive transfer of WT but not DOCK8-deficient Treg cells. CONCLUSION: Treg cell dysfunction increases susceptibility to allergic skin inflammation in DOCK8 deficiency and synergizes with cutaneous exposure to Saureus to drive eczema in DOCK8 deficiency.

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis.

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

DOCK8 is essential for neutrophil mediated clearance of cutaneous S. aureus infection.

DOCK8 deficient patients are susceptible to skin infection with Staphylococcus aureus which is normally cleared by neutrophils. We examined the mechanism of this susceptibility in mice. Dock8-/- mice had delayed clearance of S. aureus from skin mechanically injured by tape stripping. The numbers and viability of neutrophils in infected but not in uninfected, tape stripped skin were significantly reduced in Dock8-/- mice compared to WT controls. This is despite comparable numbers of circulating neutrophils, and normal to elevated cutaneous expression of Il17a and IL-17A inducible neutrophil attracting chemokines Cxcl1, Cxcl2 and Cxcl3. DOCK8 deficient neutrophils were significantly more susceptible to cell death upon in vitro exposure to S. aureus and exhibited reduced phagocytosis of S. aureus bioparticles but had a normal respiratory burst. Impaired neutrophil survival in infected skin and defective neutrophil phagocytosis likely underlie the susceptibility to cutaneous S. aureus infection in DOCK8 deficiency.

Azolation of Benzylic C-H Bonds via Photoredox-Catalyzed Carbocation Generation.

A visible-light photoredox-catalyzed method is reported that enables the coupling between benzylic C-H substrates and N-H azoles. Classically, medicinally relevant N-benzyl azoles are produced via harsh substitution conditions between the azole and a benzyl electrophile in the presence of strong bases at high temperatures. Use of C-H bonds as the alkylating partner streamlines the preparation of these important motifs. In this work, we report the use of N-alkoxypyridinium salts as a critically enabling reagent for the development of a general C(sp3)-H azolation. The platform enables the alkylation of electron-deficient, -neutral, and -rich azoles with a range of C-H bonds, most notably secondary and tertiary partners. Moreover, the protocol is mild enough to tolerate benzyl electrophiles, thus offering an orthogonal approach to existing SN2 and cross-coupling methods.

The IL-4Rα Q576R polymorphism is associated with increased severity of atopic dermatitis and exaggerates allergic skin inflammation in mice.

BACKGROUND: Atopic dermatitis (AD) is characterized by TH2-dominated skin inflammation and systemic response to cutaneously encountered antigens. The TH2 cytokines IL-4 and IL-13 play a critical role in the pathogenesis of AD. The Q576->R576 polymorphism in the IL-4 receptor alpha (IL-4Rα) chain common to IL-4 and IL-13 receptors alters IL-4 signaling and is associated with asthma severity. OBJECTIVE: We sought to investigate whether the IL-4Rα R576 polymorphism is associated with AD severity and exaggerates allergic skin inflammation in mice. METHODS: Nighttime itching interfering with sleep, Rajka-Langeland, and Eczema Area and Severity Index scores were used to assess AD severity. Allergic skin inflammation following epicutaneous sensitization of mice 1 or 2 IL-4Rα R576 alleles (QR and RR) and IL-4Rα Q576 (QQ) controls was assessed by flow cytometric analysis of cells and quantitative RT-PCR analysis of cytokines in skin. RESULTS: The frequency of nighttime itching in 190 asthmatic inner-city children with AD, as well as Rajka-Langeland and Eczema Area and Severity Index scores in 1116 White patients with AD enrolled in the Atopic Dermatitis Research Network, was higher in subjects with the IL-4Rα R576 polymorphism compared with those without, with statistical significance for the Rajka-Langeland score. Following epicutaneous sensitization of mice with ovalbumin or house dust mite, skin infiltration by CD4+ cells and eosinophils, cutaneous expression of Il4 and Il13, transepidermal water loss, antigen-specific IgE antibody levels, and IL-13 secretion by antigen-stimulated splenocytes were significantly higher in RR and QR mice compared with QQ controls. Bone marrow radiation chimeras demonstrated that both hematopoietic cells and stromal cells contribute to the mutants' exaggerated allergic skin inflammation. CONCLUSIONS: The IL-4Rα R576 polymorphism predisposes to more severe AD and increases allergic skin inflammation in mice.

Emerging tools for understanding the human microbiome.

Recent advances in sequencing technologies, experimental protocols and approaches in data generation and analysis have enabled us to investigate the human microbiome at an unprecedented level of resolution. The current chapter aims to provide an understanding of the different computational and bioinformatic strategies adopted to answer the different questions of a typical microbiome investigation and how the upstream DNA sequencing methodologies can affect this. The chapter enlist the state-of-the-art in metagenomic data analysis along with the available strategies to perform an integrated investigation of the human microbiome along with other data layers.

Basophil-derived IL-4 promotes cutaneous Staphylococcus aureus infection.

Superficial cutaneous Staphylococcus aureus (S. aureus) infection in humans can lead to soft tissue infection, an important cause of morbidity and mortality. IL-17A production by skin TCRγδ+ cells in response to IL-1 and IL-23 produced by epithelial and immune cells is important for restraining S. aureus skin infection. How S. aureus evades this cutaneous innate immune response to establish infection is not clear. Here we show that mechanical injury of mouse skin by tape stripping predisposed mice to superficial skin infection with S. aureus. Topical application of S. aureus to tape-stripped skin caused cutaneous influx of basophils and increased Il4 expression. This basophil-derived IL-4 inhibited cutaneous IL-17A production by TCRγδ+ cells and promoted S. aureus infection of tape-stripped skin. We demonstrate that IL-4 acted on multiple checkpoints that suppress the cutaneous IL-17A response. It reduced Il1 and Il23 expression by keratinocytes, inhibited IL-1+IL-23-driven IL-17A production by TCRγδ+ cells, and impaired IL-17A-driven induction of neutrophil-attracting chemokines by keratinocytes. IL-4 receptor blockade is shown to promote Il17a expression and enhance bacterial clearance in tape-stripped mouse skin exposed to S. aureus, suggesting that it could serve as a therapeutic approach to prevent skin and soft tissue infection.

Combined immunodeficiency with autoimmunity caused by a homozygous missense mutation in inhibitor of nuclear factor 𝛋B kinase alpha (IKKα).

Inhibitor of nuclear factor kappa B kinase alpha (IKKα) is critical for p100/NF-κB2 phosphorylation and processing into p52 and activation of the noncanonical NF-κB pathway. A patient with recurrent infections, skeletal abnormalities, absent secondary lymphoid structures, reduced B cell numbers, hypogammaglobulinemia, and lymphocytic infiltration of intestine and liver was found to have a homozygous p.Y580C mutation in the helix-loop-helix domain of IKKα. The mutation preserves IKKα kinase activity but abolishes the interaction of IKKα with its activator NF-κB­inducing kinase and impairs lymphotoxin-ß­driven p100/NF-κB2 processing and VCAM1 expression. Homozygous IKKαY580C/Y580C mutant mice phenocopy the patient findings; lack marginal zone B cells, germinal centers, and antigen-specific T cell response to cutaneous immunization; have impaired Il17a expression; and are susceptible to cutaneous Staphylococcus aureus infection. In addition, these mice demonstrate a severe reduction in medullary thymic epithelial cells, impaired thymocyte negative selection, a restricted TCRVß repertoire, a selective expansion of potentially autoreactive T cell clones, a decreased frequency of regulatory T cells, and infiltration of liver, pancreas, and lung by activated T cells coinciding with organ damage. Hence, this study identifies IKKα deficiency as a previously undescribed cause of primary immunodeficiency with associated autoimmunity.

Easy access to secondary and tertiary alcohols via metal-free and light mediated radical carbonyl allylation.

Here we report a strategy for carbonyl addition with unactivated alkenes using an organic photocatalyst on both aldehyde and ketone substrates. This protocol grants us a good alternative to the traditional Barbier-Grignard allylation that exhibits poor functional group tolerance. With this method the stoichiometric use of metals can be avoided, high atom economy can be achieved and fewer by-products are generated.

Mast cell-derived IL-13 downregulates IL-12 production by skin dendritic cells to inhibit the TH1 cell response to cutaneous antigen exposure.

BACKGROUND: Atopic dermatitis (AD) is characterized by a skin barrier defect aggravated by mechanical injury inflicted by scratching, a TH2 cell-dominated immune response, and susceptibility to viral skin infections that are normally restrained by a TH1 cell response. The signals leading to a TH2 cell-dominated immune response in AD are not completely understood. OBJECTIVE: Our aim was to determine the role of IL-13 in initiation of the TH cell response to cutaneously encountered antigens. METHODS: Wild-type, Il13-/-, Il1rl1-/-, and Il4ra-/- mice, as well as mice with selective deficiency of IL-13 in mast cells (MCs) were studied; in addition, dendritic cells (DCs) purified from the draining lymph nodes of tape-stripped and ovalbumin (OVA)-sensitized skin were examined for their ability to polarize naive OVA-TCR transgenic CD4+ T cells. Cytokine expression was examined by reverse-transcriptase quantitative PCR, intracellular flow cytometry, and ELISA. Contact hypersensitivity to dinitrofluorobenzene was examined. RESULTS: Tape stripping caused IL-33-driven upregulation of Il13 expression by skin MCs. MC-derived IL-13 acted on DCs from draining lymph nodes of OVA-sensitized skin to selectively suppress their ability to polarize naive OVA-TCR transgenic CD4+ T cells into IFN-γ-secreting cells. MC-derived IL-13 inhibited the TH1 cell response in contact hypersensitivity to dinitrofluorobenzene. IL-13 suppressed IL-12 production by mouse skin-derived DCs in vitro and in vivo. Scratching upregulated IL13 expression in human skin, and IL-13 suppressed the capacity of LPS-stimulated human skin DCs to express IL-12 and promote IFN-γ secretion by CD4+ T cells. CONCLUSION: Release of IL-13 by cutaneous MCs in response to mechanical skin injury inhibits the TH1 cell response to cutaneous antigen exposure in AD.

Ligand-Protected Ultrasmall Pd Nanoclusters Supported on Metal Oxide Surfaces for CO Oxidation: Does the Ligand Activate or Passivate the Pd Nanocatalyst?

Herein, we report on the synthesis of ultrasmall Pd nanoclusters (∼2 nm) protected by L-cysteine [HOCOCH(NH2 )CH2 SH] ligands (Pdn (L-Cys)m ) and supported on the surfaces of CeO2 , TiO2 , Fe3 O4 , and ZnO nanoparticles for CO catalytic oxidation. The Pdn (L-Cys)m nanoclusters supported on the reducible metal oxides CeO2 , TiO2 and Fe3 O4 exhibit a remarkable catalytic activity towards CO oxidation, significantly higher than the reported Pd nanoparticle catalysts. The high catalytic activity of the ligand-protected clusters Pdn (L-Cys)m is observed on the three reducible oxides where 100 % CO conversion occurs at 93-110 °C. The high activity is attributed to the ligand-protected Pd nanoclusters where the L-cysteine ligands aid in achieving monodispersity of the Pd clusters by limiting the cluster size to the active sub-2-nm region and decreasing the tendency of the clusters for agglomeration. In the case of the ceria support, a complete removal of the L-cysteine ligands results in connected agglomerated Pd clusters which are less reactive than the ligand-protected clusters. However, for the TiO2 and Fe3 O4 supports, complete removal of the ligands from the Pdn (L-Cys)m clusters leads to a slight decrease in activity where the T100% CO conversion occurs at 99 °C and 107 °C, respectively. The high porosity of the TiO2 and Fe3 O4 supports appears to aid in efficient encapsulation of the bare Pdn nanoclusters within the mesoporous pores of the support.

Heterogeneous catalysis by ultra-small bimetallic nanoparticles surpassing homogeneous catalysis for carbon-carbon bond forming reactions.

Palladium catalyzed cross-coupling reactions represent a significant advancement in contemporary organic synthesis as these reactions are of strategic importance in the area of pharmaceutical drug discovery and development. Supported palladium-based catalysts are highly sought-after in carbon-carbon bond forming catalytic processes to ensure catalyst recovery and reuse while preventing product contamination. This paper reports the development of heterogeneous Pd-based bimetallic catalysts supported on fumed silica that have high activity and selectivity matching those of homogeneous catalysts, eliminating the catalyst's leaching and sintering and allowing efficient recycling of the catalysts. Palladium and base metal (Cu, Ni or Co) contents of less than 1.0 wt% loading are deposited on a mesoporous fumed silica support (surface area SABET = 350 m2 g-1) using strong electrostatic adsorption (SEA) yielding homogeneously alloyed nanoparticles with an average size of 1.3 nm. All bimetallic catalysts were found to be highly active toward Suzuki cross-coupling (SCC) reactions with superior activity and stability for the CuPd/SiO2 catalyst. A low CuPd/SiO2 loading (Pd: 0.3 mol%) completes the conversion of bromobenzene and phenylboronic acid to biphenyl in 30 minutes under ambient conditions in water/ethanol solvent. In contrast, monometallic Pd/SiO2 (Pd: 0.3 mol%) completes the same reaction in three hours under the same conditions. The combination of Pd with the base metals helps in retaining the Pd0 status by charge donation from the base metals to Pd, thus lowering the activation energy of the aryl halide oxidative addition step. Along with its exceptional activity, CuPd/SiO2 exhibits excellent recycling performance with a turnover frequency (TOF) of 280 000 h-1 under microwave reaction conditions at 60 °C. Our study demonstrates that SEA is an excellent synthetic strategy for depositing ultra-small Pd-based bimetallic nanoparticles on porous silica for SCC. This avenue not only provides highly active and sintering-resistant catalysts but also significantly lowers Pd contents in the catalysts without compromising catalytic activity, making the catalysts very practical for large-scale applications.

Regulatory T cells do not suppress rather activate human basophils by IL-3 and STAT5-dependent mechanisms.

Basophils play an important role in orienting Th2 immune response, and in the pathogenesis of allergic and inflammatory disorders. However, the mechanism by which basophils are kept in check remains unclear and hence we explored the role of regulatory T cells (Treg cells) in this process. We demonstrate that human Treg cells do not suppress rather induce activation of basophils, and promote Th2 responses by IL-3 and STAT5-dependent mechanism.

DOCK8 is essential for LFA-1-dependent positioning of T follicular helper cells in germinal centers.

T follicular helper (Tfh) cell migration into germinal centers (GCs) is essential for the generation of GC B cells and antibody responses to T cell-dependent (TD) antigens. This process requires interactions between lymphocyte function-associated antigen 1 (LFA-1) on Tfh cells and ICAMs on B cells. The mechanisms underlying defective antibody responses to TD antigens in DOCK8 deficiency are incompletely understood. We show that mice selectively lacking DOCK8 in T cells had impaired IgG antibody responses to TD antigens, decreased GC size, and reduced numbers of GC B cells. However, they developed normal numbers of Tfh cells with intact capacity for driving B cell differentiation into a GC phenotype in vitro. Notably, migration of DOCK8-deficient T cells into GCs was defective. Following T cell receptor (TCR)/CD3 ligation, DOCK8-deficient T cells had impaired LFA-1 activation and reduced binding to ICAM-1. Our results therefore indicate that DOCK8 is important for LFA-1-dependent positioning of Tfh cells in GCs, and thereby the generation of GC B cells and IgG antibody responses to TD antigen.

Green Synthesis of Oxide-Supported Pd Nanocatalysts by Laser Methods for Room-Temperature Carbon-Carbon Cross-Coupling Reactions.

This work reports the design and development of a new class of highly active Pd nanocatalysts supported on substoichiometric oxides. These novel catalysts are generated by green laser synthesis methods to generate high-surface-area substoichiometric oxide nanoparticles followed by photoreduction in aqueous solutions to deposit highly active Pd nanocatalysts within the surface defects of the oxides. The laser methods eliminate the use of toxic chemicals, including hazardous solvents and chemical reducing agents, and allow efficient reduction of the Pd ions in aqueous solutions aided by the photogenerated electrons from the semiconductor support. The Pd catalysts incorporated within these oxides exhibit high activity for carbon-carbon bond-forming reactions. The Pd/TiO2 catalyst with 0.3 mol % Pd achieves 100% conversion in the reaction between bromobenzene and benzeneboronic acid to the biphenyl product within 240 minutes at room temperature without any external heating. With a catalyst loading of 0.3 mol % Pd in the microwave-assisted reaction between bromobenzene and benzeneboronic acid at 60 °C, 92 and 83% conversions to the biphenyl product are achieved within 5 min of reaction time using the Pd/TiO2 and Pd/ZnO catalysts, respectively. The results demonstrate a remarkable catalytic activity of the substoichiometric oxide-supported Pd catalysts with turnover frequencies (TOF, h-1) of 24 000, 10 000, and 3200 achieved under mirowave-assisted reactions at 60 °C for the 0.03 mol% Pd of the Pd/TiO2, Pd/ZnO, and Pd/ZrO2 catalysts, respectively. The high activity and good reusability of these nanocatalysts are attributed to the optimum catalyst-support interaction between the small Pd nanoparticles and the surface defects of the substoichiometric oxide support prepared by the laser vaporization-controlled condensation method.

Therapeutic normal IgG intravenous immunoglobulin activates Wnt-ß-catenin pathway in dendritic cells.

Therapeutic normal IgG intravenous immunoglobulin (IVIG) is a well-established first-line immunotherapy for many autoimmune and inflammatory diseases. Though several mechanisms have been proposed for the anti-inflammatory actions of IVIG, associated signaling pathways are not well studied. As ß-catenin, the central component of the canonical Wnt pathway, plays an important role in imparting tolerogenic properties to dendritic cells (DCs) and in reducing inflammation, we explored whether IVIG induces the ß-catenin pathway to exert anti-inflammatory effects. We show that IVIG in an IgG-sialylation independent manner activates ß-catenin in human DCs along with upregulation of Wnt5a secretion. Mechanistically, ß-catenin activation by IVIG requires intact IgG and LRP5/6 co-receptors, but FcγRIIA and Syk are not implicated. Despite induction of ß-catenin, this pathway is dispensable for anti-inflammatory actions of IVIG in vitro and for mediating the protection against experimental autoimmune encephalomyelitis in vivo in mice, and reciprocal regulation of effector Th17/Th1 and regulatory T cells.

Adjusting for age improves identification of gut microbiome alterations in multiple diseases.

Interaction between disease-microbiome associations and ageing has not been explored in detail. Here, using age/region-matched sub-sets, we analysed the gut microbiome differences across five major diseases in a multi-cohort dataset constituting more than 2500 individuals from 20 to 89 years old. We show that disease-microbiome associations display specific age-centric trends. Ageing-associated microbiome alterations towards a disease-like configuration occur in colorectal cancer patients, thereby masking disease signatures. We identified a microbiome disease response shared across multiple diseases in elderly subjects that is distinct from that in young/middle-aged individuals, but also a novel set of taxa consistently gained in disease across all age groups. A subset of these taxa was associated with increased frailty in subjects from the ELDERMET cohort. The relevant taxa differentially encode specific functions that are known to have disease associations.

The human body is an ecosystem made up of both human cells and trillions of microbes, and the largest microbial community is in the gut. This community of gut microbes helps harvest nutrients from our food, modulates our immune system, and even affects our mood. Infectious and chronic diseases appear to cause changes in the make-up of the gut microbiome, while microbiome changes may increase the risk of some non-infectious diseases. Learning more about these disease-linked changes in the gut microbiome may therefore help scientists to develop new tests and treatments. To do this, scientists need to understand which microbes play a role in individual diseases, if risk-related microbes are gained or helpful microbes lost in patients with particular diseases, and if certain changes in gut microbes occur across many diseases. Ageing also changes the gut microbes. This may happen because older individuals eat a less complex diet and are likely to take many medications that may alter the microbes in their gut. Because of this, age may affect changes in gut microbes associated with diseases. This highlights the need for studies that tease apart the importance of ageing-related and disease-related changes in the gut microbiome. Now, Ghosh et al. show that gut microbe changes linked to diseases may vary with a person's age. The analysis compared the gut microbiomes of more than 2,500 individuals aged 20 to 89. This included individuals with inflammatory bowel disease, colorectal cancer, type 2 diabetes, intestinal polyps and liver cirrhosis. The study revealed that younger people gradually gain disease-associated gut microbes, while older people tend to lose the gut microbes usually found in a healthy gut. Ghosh et al. also identified a set of gut microbes that were gained in many diseases and across age-groups. This set of microbes was also associated with frailty in elderly people. The characteristics of the microbes in this set are all known to have detrimental effects on human health. This analysis shows how important it is to control for age and other factors that may skew the results of microbiome projects. Future studies are needed to understand why these gut microbe changes occur and what the consequences of these changes are for a person's health and the course of their disease. This may lead to the development of treatment strategies that help promote a healthy gut microbiome and fight disease throughout life.