CD169+ Skin Macrophages Function as a Specialized Subpopulation in Promoting Psoriasis-like Skin Disease in Mice.

Skin macrophages are critical to maintain and restore skin homeostasis. They serve as major producers of cytokines and chemokines in the skin, participating in diverse biological processes such as wound healing and psoriasis. The heterogeneity and functional diversity of macrophage subpopulations endow them with multifaceted roles in psoriasis development. A distinct subpopulation of skin macrophages, characterized by high expression of CD169, has been reported to exist in both mouse and human skin. However, its role in psoriasis remains unknown. Here, we report that CD169+ macrophages exhibit increased abundance in imiquimod (IMQ) induced psoriasis-like skin lesions. Specific depletion of CD169+ macrophages in CD169-ditheria toxin receptor (CD169-DTR) mice inhibits IMQ-induced psoriasis, resulting in milder symptoms, diminished proinflammatory cytokine levels and reduced proportion of Th17 cells within the skin lesions. Furthermore, transcriptomic analysis uncovers enhanced activity in CD169+ macrophages when compared with CD169- macrophages, characterized by upregulated genes that are associated with cell activation and cell metabolism. Mechanistically, CD169+ macrophages isolated from IMQ-induced skin lesions produce more proinflammatory cytokines and exhibit enhanced ability to promote Th17 cell differentiation in vitro. Collectively, our findings highlight the crucial involvement of CD169+ macrophages in psoriasis development and offer novel insights into the heterogeneity of skin macrophages in the context of psoriasis.

CD169+ subcapsular sinus macrophage-derived microvesicles are associated with light zone follicular dendritic cells.

Follicular dendritic cells (FDCs) are a specialized type of stromal cells that exclusively reside in B-cell follicles. When inflammation occurs, the FDC network is reorganized to support germinal center (GC) polarization into the light zone (LZ) and dark zone (DZ). Despite the indispensable role of FDCs in supporting humoral responses, the FDC regulatory requirements remain incompletely defined. In this study, we unexpectedly observed an accumulation of CD169+ subcapsular sinus macrophage (SSM)-derived microvesicles (MVs) in the B-cell zone, which were tightly associated with the FDC network. Interestingly, a selective deposition of CD169+ MVs was detected in both GC LZ FDCs in secondary follicles and on predetermined LZ FDCs in primary follicles. The ablation of CD169+ MVs, resulting from SSM depletion, resulted in significantly decreased expression of LZ-related genes in FDCs. In addition, we found that CD169+ MVs could colocalize with fluorescently tagged antigen-containing immune complexes (ICs), supporting a possible role of CD169+ MVs in transporting antigens to the FDC network. Thus, our data reveal intimate crosstalk between FDCs and SSMs located outside B-cell follicles via SSM-released MVs, providing a novel perspective on the mechanisms underlying the regulation of FDC maturation and polarization.

A functional motif of long noncoding RNA Nron against osteoporosis.

Long noncoding RNAs are widely implicated in diverse disease processes. Nonetheless, their regulatory roles in bone resorption are undefined. Here, we identify lncRNA Nron as a critical suppressor of bone resorption. We demonstrate that osteoclastic Nron knockout mice exhibit an osteopenia phenotype with elevated bone resorption activity. Conversely, osteoclastic Nron transgenic mice exhibit lower bone resorption and higher bone mass. Furthermore, the pharmacological overexpression of Nron inhibits bone resorption, while caused apparent side effects in mice. To minimize the side effects, we further identify a functional motif of Nron. The delivery of Nron functional motif to osteoclasts effectively reverses bone loss without obvious side effects. Mechanistically, the functional motif of Nron interacts with E3 ubiquitin ligase CUL4B to regulate ERα stability. These results indicate that Nron is a key bone resorption suppressor, and the lncRNA functional motif could potentially be utilized to treat diseases with less risk of side effects.

SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.

Salmonella typhimurium (S. typhimurium) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1ß. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD+) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation. Methods: We initially tested IL-1ß production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium-induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis. Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo. Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation. Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.

Polarizing Graphene Quantum Dots toward Long-Acting Intracellular Reactive Oxygen Species Evaluation and Tumor Detection.

The evaluation of intracellular reactive oxygen species (ROS) would greatly deepen the understanding of cell metabolism/proliferation and tumor detection. However, current long-acting level tracking techniques for intracellular ROS remain unsuited to practical applications. To solve this problem, we synthesized cyclotriphosphazene-doped graphene quantum dots (C-GQDs) whose quantum yield is highly sensitive to ROS (increased by 400% from 0.12 to 0.63). Electron cloud polarization of oxidized cyclotriphosphazene rings in C-GQDs is confirmed to account for this novel optical property by density functional theory calculations and experimental results. In combination with excellent biological stability, C-GQDs achieve a long-acting evaluation of intracellular ROS level (more than 72 h) with an accuracy of 98.3%. In addition, recognition rates exceeding 90% are demonstrated to be feasible for eight kinds of tumor cell lines cultured with C-GQDs, which can also be expanded to in vivo detection. C-GQDs also show a high recognition rate (82.33%) and sensitivity (79.65%) for tumor cells in blood samples.

PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome.

The innate immune sensor NLRP3 assembles an inflammasome complex with NEK7 and ASC to activate caspase-1 and drive the maturation of proinflammatory cytokines IL-1ß and IL-18. NLRP3 inflammasome activity must be tightly controlled, as its over-activation is involved in the pathogenesis of inflammatory diseases. Here, we show that NLRP3 inflammasome activation is suppressed by a centrosomal protein Spata2. Spata2 deficiency enhances NLRP3 inflammasome activity both in the macrophages and in an animal model of peritonitis. Mechanistically, Spata2 recruits the deubiquitinase CYLD to the centrosome for deubiquitination of polo-like kinase 4 (PLK4), the master regulator of centrosome duplication. Deubiquitination of PLK4 facilitates its binding to and phosphorylation of NEK7 at Ser204. NEK7 phosphorylation in turn attenuates NEK7 and NLRP3 interaction, which is required for NLRP3 inflammasome activation. Pharmacological or shRNA-mediated inhibition of PLK4, or mutation of the NEK7 Ser204 phosphorylation site, augments NEK7 interaction with NLRP3 and causes increased NLRP3 inflammasome activation. Our study unravels a novel centrosomal regulatory pathway of inflammasome activation and may provide new therapeutic targets for the treatment of NLRP3-associated inflammatory diseases.

Fate Mapping via Ms4a3-Expression History Traces Monocyte-Derived Cells.

Most tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes, and their functions compared to embryonic RTMs remain unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation, and disease are highly debated. Here, we identified Ms4a3 as a specific gene expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3TdT reporter, Ms4a3Cre, and Ms4a3CreERT2 fate-mapping models. These models traced efficiently monocytes and granulocytes, but no lymphocytes or tissue dendritic cells. Using these models, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

PD-1 blockade augments humoral immunity through ICOS-mediated CD4+ T cell instruction.

Successful applications of PD-1/PD-L1 blockade in multiple cancers highlight the efficacy of immunotherapy mediated by enhancing CD8+ T cell immunity both in mouse and human. How PD-1 blockade affects humoral immunity remains unclear. Herein we demonstrated that treatment of anti-PD-1 antibody led to the increase in both total IgG and OVA-specific IgG in OVA-immunized mice. However, no effect was observed on Ab affinity maturation. Accumulation of germinal center (GC) and memory B cells was observed in the spleens together with elevated percentages of plasma cells in the spleens and bone marrow. More interestingly, dramatic infiltration of CD4+ T cells was apparent in GCs after PD-1 blockade with a significant increase in the expression of ICOS. When CD4+ T cells and B cells from OVA-immunized mice were co-cultured with neutralizing anti-PD-1 Ab in vitro, PD-1 blockade recapitulated the up-regulation of ICOS expression on CD4+ T cells with the activation of ERK signaling. Suppression of ERK activation not only reduced ICOS expression on CD4+ T cells but also attenuated IgG production upon PD-1 blockade. Taken together, PD-1 blockade enhances humoral immunity. This process partially relies on more accumulation of CD4+ T cells in GCs with the up-regulation of ICOS expression and the promotion of B cell terminal differentiation. The regulatory pattern of PD-1 blockade illustrated here provides a new mechanism of how immune checkpoint molecules regulating humoral immune responses.

Metabolic control of regulatory T cell stability and function by TRAF3IP3 at the lysosome.

Metabolic programs are crucial for regulatory T (T reg) cell stability and function, but the underlying mechanisms that regulate T reg cell metabolism are elusive. Here, we report that lysosomal TRAF3IP3 acts as a pivotal regulator in the maintenance of T reg cell metabolic fitness. T reg-specific deletion of Traf3ip3 impairs T reg cell function, causing the development of inflammatory disorders and stronger antitumor T cell responses in mice. Excessive mechanistic target of rapamycin complex 1 (mTORC1)-mediated hyper-glycolytic metabolism is responsible for the instability of TRAF3IP3-deficient T reg cells. Mechanistically, TRAF3IP3 restricts mTORC1 signaling by recruiting the serine-threonine phosphatase catalytic subunit (PP2Ac) to the lysosome, thereby facilitating the interaction of PP2Ac with the mTORC1 component Raptor. Our results define TRAF3IP3 as a metabolic regulator in T reg cell stability and function and suggest a lysosome-specific mTORC1 signaling mechanism that regulates T reg cell metabolism.

SENP3 maintains the stability and function of regulatory T cells via BACH2 deSUMOylation.

Regulatory T (Treg) cells are essential for maintaining immune homeostasis and tolerance, but the mechanisms regulating the stability and function of Treg cells have not been fully elucidated. Here we show SUMO-specific protease 3 (SENP3) is a pivotal regulator of Treg cells that functions by controlling the SUMOylation and nuclear localization of BACH2. Treg cell-specific deletion of Senp3 results in T cell activation, autoimmune symptoms and enhanced antitumor T cell responses. SENP3-mediated BACH2 deSUMOylation prevents the nuclear export of BACH2, thereby repressing the genes associated with CD4+ T effector cell differentiation and stabilizing Treg cell-specific gene signatures. Notably, SENP3 accumulation triggered by reactive oxygen species (ROS) is involved in Treg cell-mediated tumor immunosuppression. Our results not only establish the role of SENP3 in the maintenance of Treg cell stability and function via BACH2 deSUMOylation but also clarify the function of SENP3 in the regulation of ROS-induced immune tolerance.

Anisotropic thermal conductive properties of cigarette filter-templated graphene/epoxy composites.

Herein, a cigarette filter-templated graphene/epoxy composite was prepared with enhanced thermal conductive properties. The through-plane thermal conductivity of the epoxy composite was up to 1.2 W mK-1, which was 4 times that of it in the in-plane (0.298 W mK-1) after only 5 filtration cycles. The thermal conductive anisotropy and improvement in the through-plane thermal conductivity of the epoxy composite were attributed to the particular structure of cigarette filter-templated graphene in the epoxy matrix. The unique structure formed effective conductive pathways in the composite to improve the thermal transportation properties. The excellent thermal transportation properties allow the epoxy composite to be used as an efficient heat dissipation material for thermal management applications.

A Tunable Diffusion-Consumption Mechanism of Cytokine Propagation Enables Plasticity in Cell-to-Cell Communication in the Immune System.

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 µm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors.

AUTOIMMUNE DISEASE. Patients with LRBA deficiency show CTLA4 loss and immune dysregulation responsive to abatacept therapy.

Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.

Novel sinomenine derivative 1032 improves immune suppression in experimental autoimmune encephalomyelitis.

Sinomenine (SIN) is an alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It is widely used as an immunosuppressive drug for treating autoimmune diseases. Due to its poor efficiency, the large-dose treatment presents some side effects and limits its further applications. In this study, we used chemical modification to improve the therapeutic effect of SIN in vitro and in vivo. A new derivative of sinomenine, named 1032, demonstrates significantly improved immunosuppressive activity over that of its parent natural compound (SIN). In an experimental autoimmune encephalomyelitis (EAE) model, 1032 significantly reduced encephalitogenic T cell responses and induced amelioration of EAE, which outcome was related to its selective inhibitory effect on the production of IL-17. By contrast, SIN treatment only led to a moderate alleviation of EAE severity and the expression level of IL-17 was not significantly reduced. Furthermore, 1032 exhibited suppression of Th17, but not Treg, cell differentiation, a result probably related to its inhibitory effect on IkappaB-alpha degradation as well as on IL-6 and TNF-alpha secretion in BMDCs. We speculate that 1032 as a novel anti-inflammatory agent may target DC to block IL-6 production, which in turn would terminate Th17 cell development. Thus, SIN derivative 1032 presents considerable potential in new drug development for treating autoimmune and inflammatory disease.

Requirement for the basic helix-loop-helix transcription factor Dec2 in initial TH2 lineage commitment.

How naive CD4(+) T cells commit to the T helper type 2 (T(H)2) lineage is poorly understood. Here we show that the basic helix-loop-helix transcription factor Dec2 was selectively expressed in T(H)2 cells. CD4(+) T cells from Dec2-deficient mice showed defective T(H)2 differentiation in vitro and in vivo in an asthma model and in response to challenge with a parasite antigen. Dec2 promoted expression of interleukin 4 (IL-4), IL-5 and IL-13 during early T(H)2 differentiation and directly bound to and activated transcription of genes encoding the transcription factors JunB and GATA-3. As GATA-3 induces Dec2 expression, our findings also indicate a feed-forward regulatory circuit during T(H)2 differentiation.

Differential expression of neutrophilic granule proteins between Th1 and Th2 cells.

T helper cell type 1 (Th1) and 2 (Th2) play central roles in immune regulation. To identify the novel genes differentially expressed between Th1 and Th2 cells, CD4+ T cells were isolated from DO11.10 transgenic mice and induced under Th1 or Th2 conditions. Microarray showed differential expression of neutrophilic granule proteins (NGP) between Th1 and Th2 cells. NGP was first identified as a myeloid-specific granule protein with homology to the cystatin superfamily. Here we confirmed greater expression of NGP in Th2 cells by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction analysis. We also showed that the expression of NGP mRNA had a peak expression after 5 d culture under Th2- but not Th1-biasing conditions. Antibody against NGP was prepared, and in concert with the results of mRNA analysis, the level of NGP protein in Th2 cells detected by Western blot analysis was also higher than that in Th1 cells. GFP-NGP fusion proteins overexpressed in HeLa cells were localized to the cytoplasm. These results suggest NGP is a novel marker distinguishing Th2 from Th1 cells and maybe a novel cytokine secreted by Th2 cells.

Expression of intracellular domain of epidermal growth factor receptor and generation of its monoclonal antibody.

To prepare monoclonal antibody specific to epidermal growth factor receptor (EGFR) intracellular domain, its gene was amplified from total RNA of A431 cell by RT-PCR. Then the gene was cloned into prokaryotic vector pET30a(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni2+-NTA agarose. Then the anti-EGFR monoclonal antibody (mAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked immunoabsorbent assay (ELISA). Totally 4 hybridoma clones were obtained and these mAbs were IgG1 (3 clones) and IgG2a (1 clone), respectively. Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line. The mAbs will be useful in the study of EGFR-mediated signal transduction.