A Robust In Vitro Culture Model and Generation of Memory Monocytes.

Innate monocytes can be trained or reprogrammed to adopt distinct memory states, such as low-grade inflammation and immune exhaustion, bearing fundamental relevance to the pathogenesis of both acute diseases such as sepsis as well as chronic diseases such as atherosclerosis. Therefore, it is critically important to develop a regimen for generating memory monocytes in vitro in order to better define key monocyte memory states with diverse potentials for proliferation, differentiation, and activation, as well as underlying mechanisms. Here, we describe an efficient in vitro system to propagate a large number of highly purified murine memory monocytes through sustaining bone marrow-derived monocytes with macrophage colony-stimulating factor (M-CSF, 10 ng/mL)-containing medium, together with other polarization agents such as lipopolysaccharide (LPS) for a 5-day period. This method can yield high-purity monocytes, capable of exhibiting dynamic memory behaviors upon training with various polarizing agents.

A Robust In Vitro Co-culture Model for Studying the Intercellular Communication of Neutrophils.

Communication among neutrophils plays critical roles during various phases of inflammatory responses, with clinical relevance to both acute and chronic inflammatory diseases. Despite its significance, underlying mechanisms are not well understood, due to the lack of an effective in vitro system to properly address this important question. Here we report a robust in vitro method to culture primary murine neutrophils derived from bone marrow, amenable for well-controlled studies of both neutrophil activation and intercellular communication among co-cultured neutrophils. This protocol can generate primary neutrophils with high purity and survival for an extended culture period, suitable for further phenotypic and functional analyses.

Isolation and Single-Cell Transcriptomic Analysis of Murine Regulatory B Cells.

Regulatory B (Breg) cells have been demonstrated to play an important role in the inhibition of a wide range of immunological responses, and they are absent or malfunction in autoimmune diseases like lupus. Breg cells can control immunological responses and keep the immune system in a balanced state by releasing immunosuppressive cytokines such as transforming growth factor-beta (TGF-ß) and interleukin-10 (IL-10), which in turn promote regulatory T (Treg) cells and reduce effector T cell responses. Breg cells have also been linked to the modulation of cancer immunity. Due to their immunosuppressive role, in the context of cancer, Breg cells aid in tumor immune evasion and promote tumor progression. Nonetheless, it has been established that Breg cells are involved in both cancer immunity and autoimmunity, and their characterizations beyond surface markers, for example, on the transcriptomic level, are essential for our understanding of Breg biology in health and disease. In this chapter, using lupus-prone MRL/lpr mice, we describe a Breg cell isolation protocol for the purpose of single-cell RNA sequencing analysis.

Tollip deficiency exaggerates airway type 2 inflammation in mice exposed to allergen and influenza A virus: role of the ATP/IL-33 signaling axis.

Toll-interacting protein (Tollip) is a negative regulator of the pro-inflammatory response to viruses, including influenza A virus (IAV). Genetic variation of Tollip has been associated with reduced airway epithelial Tollip expression and poor lung function in patients with asthma. Whether Tollip deficiency exaggerates type 2 inflammation (e.g., eosinophils) and viral infection in asthma remains unclear. We sought to address this critical, but unanswered question by using a Tollip deficient mouse asthma model with IAV infection. Further, we determined the underlying mechanisms by focusing on the role of the ATP/IL-33 signaling axis. Wild-type and Tollip KO mice were intranasally exposed to house dust mite (HDM) and IAV with or without inhibitors for IL-33 (i.e., soluble ST2, an IL-33 decoy receptor) and ATP signaling (i.e., an antagonist of the ATP receptor P2Y13). Tollip deficiency amplified airway type 2 inflammation (eosinophils, IL-5, IL-13 and mucins), and the release of ATP and IL-33. Blocking ATP receptor P2Y13 decreased IL-33 release during IAV infection in HDM-challenged Tollip KO mice. Furthermore, soluble ST2 attenuated airway eosinophilic inflammation in Tollip KO mice treated with HDM and IAV. HDM challenges decreased lung viral load in wild-type mice, but Tollip deficiency reduced the protective effects of HDM challenges on viral load. Our data suggests that during IAV infection, Tollip deficiency amplified type 2 inflammation and delayed viral clearance, in part by promoting ATP signaling and subsequent IL-33 release. Our findings may provide several therapeutic targets, including ATP and IL-33 signaling inhibition for attenuating excessive airway type 2 inflammation in human subjects with Tollip deficiency and IAV infection.

TRAM deletion attenuates monocyte exhaustion and alleviates sepsis severity.

Monocyte exhaustion characterized by immune-suppressive features can develop during sepsis and contribute to adverse patient outcomes. However, molecular mechanisms responsible for the establishment of immune-suppressive monocytes with reduced expression of immune-enhancing mediators such as CD86 during sepsis are not well understood. In this study, we identified that the TLR4 intracellular adaptor TRAM plays a key role in mediating the sustained reduction of CD86 expression on exhausted monocytes and generating an immune-suppressive monocyte state. TRAM contributes to the prolonged suppression of CD86 through inducing TAX1BP1 as well as SARM1, collectively inhibiting Akt and NFκB. TRAM deficient mice are protected from cecal slurry-induced experimental sepsis and retain immune-competent monocytes with CD86 expression. Our data reveal a key molecular circuitry responsible for monocyte exhaustion and provide a viable target for rejuvenating functional monocytes and treating sepsis.

Tollip Inhibits IL-33 Release and Inflammation in Influenza A Virus-Infected Mouse Airways.

Respiratory influenza A virus (IAV) infection continues to pose significant challenges in healthcare of human diseases including asthma. IAV infection in mice was shown to increase IL-33, a key cytokine in driving airway inflammation in asthma, but how IL-33 is regulated during viral infection remains unclear. We previously found that a genetic mutation in Toll-interacting protein (Tollip) was linked to less airway epithelial Tollip expression, increased neutrophil chemokines, and lower lung function in asthma patients. As Tollip is involved in maintaining mitochondrial function, and mitochondrial stress may contribute to extracellular ATP release and IL-33 secretion, we hypothesized that Tollip downregulates IL-33 secretion via inhibiting ATP release during IAV infection. Wild-type and Tollip knockout (KO) mice were infected with IAV and treated with either an ATP converter apyrase or an IL-33 decoy receptor soluble ST2 (sST2). KO mice significantly lost more body weight and had increased extracellular ATP, IL-33 release, and neutrophilic inflammation. Apyrase treatment reduced extracellular ATP levels, IL-33 release, and neutrophilic inflammation in Tollip KO mice. Excessive lung neutrophilic inflammation in IAV-infected Tollip KO mice was reduced by sST2, which was coupled with less IL-33 release. Our data suggest that Tollip inhibits IAV infection, potentially by inhibiting extracellular ATP release and reducing IL-33 activation and lung inflammation. In addition, sST2 may serve as a potential therapeutic approach to mitigate respiratory viral infection in human subjects with Tollip deficiency.

Innate Neutrophil Memory Dynamics in Disease Pathogenesis.

Neutrophils, the most abundant leukocytes in circulation and the first responders to infection and inflammation, closely modulate both acute and chronic inflammatory processes. Resting neutrophils constantly patrol vasculature and migrate to tissues when challenges occur. When infection and/or inflammation recede, tissue neutrophils will be subsequently cleaned up by macrophages which collectively contribute to the resolution of inflammation. While most studies focus on the anti-microbial function of neutrophils including phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation, recent research highlighted additional contributions of neutrophils beyond simply controlling infectious agents. Neutrophils with resolving characteristics may alter the activities of neighboring cells and facilitate inflammation resolution, modulate long-term macrophage and adaptive immune responses, therefore having important impacts on host pathophysiology. The focus of this chapter is to provide an updated assessment of recent progress in the emerging field of neutrophil programming and memory in the context of both acute and chronic diseases.

Application of chromium catalysis technology to compound-specific hydrogen isotope analysis of natural gas samples.

An easy-to-use and efficient approach for the catalytic pyrolysis of hydrocarbon gas here is proposed and tested, which allowed the precise measurement of δ2H. This method requires the reaction tube filled with twisted chromium wire, establishing a gas chromatography-Chromium wire/high temperature conversion-isotope ratio mass spectrometer (GC‒Cr/HTC‒IRMS) system. The approach described here has many remarkable advantages over other techniques. In comparison to traditional carbon-coating pyrolysis technique, this proposed method greatly reduces the pyrolysis temperature of the hydrocarbon gas, especially methane, with temperatures reduced from 1450 to 1250 °C, while still ensuring excellent analytical precision. Therefore, hydrogen isotope analysis of natural gas can be carried out at a lower cracking temperature for a longer time, with the advantage of increasing the service life of the HTC furnace. Meanwhile, no pretreatment or activation is required while putting the Cr/HTC system to use. Further, the accuracy of δ2H values obtained using this new method is better than using the traditional method. Our experiments demonstrate that the test errors of this novel method and the conventional method are within ±5.5‰ and ±6.5‰, respectively.

Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Implantable optical fibers for immunotherapeutics delivery and tumor impedance measurement.

Immune checkpoint blockade antibodies have promising clinical applications but suffer from disadvantages such as severe toxicities and moderate patient-response rates. None of the current delivery strategies, including local administration aiming to avoid systemic toxicities, can sustainably supply drugs over the course of weeks; adjustment of drug dose, either to lower systemic toxicities or to augment therapeutic response, is not possible. Herein, we develop an implantable miniaturized device using electrode-embedded optical fibers with both local delivery and measurement capabilities over the course of a few weeks. The combination of local immune checkpoint blockade antibodies delivery via this device with photodynamic therapy elicits a sustained anti-tumor immunity in multiple tumor models. Our device uses tumor impedance measurement for timely presentation of treatment outcomes, and allows modifications to the delivered drugs and their concentrations, rendering this device potentially useful for on-demand delivery of potent immunotherapeutics without exacerbating toxicities.

LYSMD3: A mammalian pattern recognition receptor for chitin.

Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, it is unclear how mammals recognize chitin and the principal receptor(s) on epithelial cells that sense chitin remain to be determined. In this study, we show that LYSMD3 is expressed on the surface of human airway epithelial cells and demonstrate that LYSMD3 is able to bind chitin, as well as ß-glucan, on the cell walls of fungi. Knockdown or knockout of LYSMD3 also sharply blunts the production of inflammatory cytokines by epithelial cells in response to chitin and fungal spores. Competitive inhibition of the LYSMD3 ectodomain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 also blocks chitin signaling. Our study reveals LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and establishes its role in epithelial cell inflammatory responses to chitin and fungi.

A resolving role for neutrophil CD11d in facilitating neutrophil survival and macrophage efferocytosis during sepsis?

Discussion on neutrophil CD11d's potential role in facilitating neutrophil survival and macrophage efferocytosis during sepsis.

Fusobacterium nucleatum host-cell binding and invasion induces IL-8 and CXCL1 secretion that drives colorectal cancer cell migration.

Fusobacterium nucleatum is implicated in accelerating colorectal cancer (CRC) and is found within metastatic CRC cells in patient biopsies. Here, we found that bacterial invasion of CRC cells and cocultured immune cells induced a differential cytokine secretion that may contribute to CRC metastasis. We used a modified galactose kinase markerless gene deletion approach and found that F. nucleatum invaded cultured HCT116 CRC cells through the bacterial surface adhesin Fap2. In turn, Fap2-dependent invasion induced the secretion of the proinflammatory cytokines IL-8 and CXCL1, which are associated with CRC progression and promoted HCT116 cell migration. Conditioned medium from F. nucleatum-infected HCT116 cells caused naïve cells to migrate, which was blocked by depleting CXCL1 and IL-8 from the conditioned medium. Cytokine secretion from HCT116 cells and cellular migration were attenuated by inhibiting F. nucleatum host-cell binding and entry using galactose sugars, l-arginine, neutralizing membrane protein antibodies, or fap2 deletion. F. nucleatum also induces the mobilization of immune cells in the tumor microenvironment. However, in neutrophils and macrophages, the bacterial-induced secretion of cytokines was Fap2 independent. Thus, our findings show that F. nucleatum both directly and indirectly modulates immune and cancer cell signaling and migration. Because increased IL-8 and CXCL1 production in tumors is associated with increased metastatic potential and cell seeding, poor prognosis, and enhanced recruitment of tumor-associated macrophages and fibroblasts, we propose that inhibition of host-cell binding and invasion, potentially through vaccination or novel galactoside compounds, could be an effective strategy for reducing F. nucleatum-associated CRC metastasis.

The Use of Noble Gas Isotopes in Detecting Methane Contamination of Groundwater in Shale Gas Development Areas: An Overview of Technology and Methods.

Groundwater contamination by stray gas (mainly methane) in areas of shale-gas development has captured publics, political and scientific attention. However, the sources and potential mechanisms of groundwater contamination are still debated. Noble gases can provide useful information on fluid migration for discerning the scale, conditions, and physical mechanisms. In this study, details about analytical technology and theoretical approach of noble gases in tracing groundwater contaminations are presented. In addition, applications of noble-gases isotopes for determining contamination sources and potential pathways are explored and reviewed. Recent developments are discussed and highlighted with focusing on new utilities of noble-gas isotope parameters in evaluating groundwater contamination. Some usages of indicators (4He/20Ne, CH4/36Ar, 4He/CH4, etc.) are discussed through specific research articles. And it is a new trend to make comprehensive use of multiple geochemical parameters to determine the occurrence, source, and process of methane pollution in groundwater.

Neutrophils Deficient in Innate Suppressor IRAK-M Enhances Anti-tumor Immune Responses.

Tumor-associated immune-suppressive neutrophils are prevalent in various cancers, including colorectal cancer. However, mechanisms of immune-suppressive neutrophils are not well understood. We report that a key innate suppressor, IRAK-M (interleukin-1 receptor-associated kinase M), is critically involved in the establishment of immune-suppressive neutrophils. In contrast to the wild-type (WT) neutrophils exhibiting immune-suppressive signatures of CD11bhighPD-L1highCD80low, IRAK-M-deficient neutrophils are rewired with reduced levels of inhibitory molecules PD-L1 and CD11b, as well as enhanced expression of stimulatory molecules CD80 and CD40. The reprogramming of IRAK-M-deficient neutrophils is mediated by reduced activation of STAT1/3 and enhanced activation of STAT5. As a consequence, IRAK-M-deficient neutrophils demonstrate enhanced capability to promote, instead of suppress, the proliferation and activation of effector T cells both in vitro and in vivo. Functionally, we observed that the transfusion of IRAK-M-/- neutrophils can potently render an enhanced anti-tumor immune response in the murine inflammation-induced colorectal cancer model. Collectively, our study defines IRAK-M as an innate suppressor for neutrophil function and reveals IRAK-M as a promising target for rewiring neutrophils in anti-cancer immunotherapy.

EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity.

Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.

Interleukin-1ß-induced IRAK1 ubiquitination is required for TH-GM-CSF cell differentiation in T cell-mediated inflammation.

Accumulating evidence suggests granulocyte macrophage-colony stimulating factor (GM-CSF) can function as an inflammatory mediator, but whether GM-CSF-producing CD4+ T cells (TH-GM-CSF) are a distinct T helper cell subset is lacking. Herein we demonstrate that interleukin (IL)-1ß exclusively drives differentiation of naïve CD4+ T cells into TH-GM-CSF cells via inducing ubiquitination of IL-1 receptor-associated kinase 1 (IRAK1) and subsequent activation of the transcription factor NF-kappaB (NF-κB), independent of RAR-related orphan receptor gamma (RORγt) required for TH17 differentiation. In vivo, TH-GM-CSF cells are present in murine Citrobacter Rodentium infections and mediate colitis following adoptive transfer of CD4+ T cells into Rag1-/- mice via GM-CSF-induced macrophage activation. The TH-GM-CSF cell phenotype is stable and distinct from the TH17 genetic program, but IL-1ß can convert pre-formed TH17 cells into TH-GM-CSF cells, thereby accounting for previously reported associations between IL-17 and GM-CSF. Together, our results newly identify IL-1ß/NF-κB-dependent TH-GM-CSF cells as a unique T helper cell subset and highlight the importance of CD4+ T cell-derived GM-CSF induced macrophage activation as a previously undescribed T cell effector mechanism.

Enhanced tumor immune surveillance through neutrophil reprogramming due to Tollip deficiency.

Although the importance of the tumor immune environment for the modulation of tumorigenesis and tumor regression is becoming increasingly clear, most of the research related to tumor-immune therapies has focused on adaptive immune cells, while the role and regulation of innate leukocytes such as neutrophils remains controversial and less defined. Here we observed that the selective deletion of Tollip, a key innate immune-cell modulator, led to enhanced tumor immune surveillance in a chemically induced colorectal cancer model. Tollip-deficient neutrophils significantly elevated T cell activation through enhanced expression of the costimulatory molecule CD80, and reduced expression of the inhibitory molecule PD-L1. Mechanistically, Tollip deficiency increased STAT5 and reduced STAT1, the transcription factors responsible for the expression of CD80 and PD-L1, respectively. Through adoptive transfer, we demonstrate that Tollip-deficient neutrophils, but not Tollip-deficient monocytes, are sufficient to drive enhanced tumor immune surveillance and reduced colorectal cancer burden in vivo. Our data reveal a strategy for the reprogramming of neutrophil functions conducive for the enhancement of the antitumor immune environment.