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Macroautophagy/autophagy regulates inflammation via multiple mechanisms, including lysosomal degradation of specific cellular components. Certain autophagy gene "cassettes" also participate in non-canonical processes to mediate important biological activities. While select autophagy genes in myeloid cells have been implicated in protecting mice in models of cytokine storm syndromes (CSS), a more extensive genetic analysis of the autophagy pathway for this disorder has not been reported to date. We determined that multiple canonical autophagy genes in the myeloid compartment protected against fatal disease from both intravenous TNF and intraperitoneal LPS, with the notable exception that Atg14 was dispensable for the latter. Serum cytokine analyses and genetic crosses further revealed distinct mechanisms contribute to the hypersensitivity of autophagy gene-deficient mice in these CSS models. Surprisingly, TNF was dispensable for the increased mortality of myeloid 5-deficient mice challenged with LPS. Tissue-specific ablation of Atg5 in cells expressing ITGAX/CD11c and LYZ2/LYSM, but not S100A8/MRP8, defined a myeloid subset that protected against TNF, while protection against LPS was conferred by Atg5 in a distinct subset of LYZ2-expressing cells. Together, this study identifies autophagy gene sets and specific cell types that protect against fatal inflammation due to CSS, highlighting important differences in two commonly used murine models of the disorder. ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BECN1: beclin 1, autophagy related; CASP1: caspase 1; CASP4/CASP11: caspase 4, apoptosis-related cysteine peptidase; CIM: conditionally immortalized macrophage; CLP: cecal ligation and puncture; CSS: cytokine storm syndrome; DC: dendritic cell; IFNG/IFNγ: interferon gamma; IFNGR1: interferon gamma receptor 1; ip: intraperitoneal; iv: intravenous; IL12/p70: interleukin 12, p70 heterodimer; IL18: Interleukin 18; ITGAX/CD11c: integrin alpha X; LAP: LC3-associated phagocytosis; LPS: lipopolysaccharide; LYZ2/LYSM: lysozyme 2; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; RB1CC1/FIP200: RB1-inducible coiled-coil 1; S100A8/MRP8: S100 calcium binding protein A8 (calgranulin A); TICAM1/TRIF: TIR domain containing adaptor molecule 1; TLR4: toll-like receptor 4; TNF: tumor necrosis factor. [ABSTRACT FROM AUTHOR]

The absorptive surface of the small intestine is covered by a layer of mucus secreted by goblet cells. The secreted mucins and thickness of the adherent layer influence nutrient digestion and absorption processes as well as the functionality of the mucosa. In this study, methods for the analysis of mucin synthesis and dynamics in the chick small intestine are described. A fragment of chicken mucin cDNA was isolated and characterized; this fraction had 60% homology to human mucin MUC-5AC. The thickness of the mucus adherent layer and the relative amounts of mucin glycoprotein and mRNA were also examined in the small intestines of control and starved chicks. Relative amounts of intestinal mucin mRNA and protein increased in the duodenum and jejunum of starved chicks, and mucus adherent layer thickness decreased throughout the small intestine. In starved chicks, higher mRNA expression and protein concentrations with lower amounts of adherent mucus may be related to a higher rate of degradation of the mucus layer, a lower rate of mucus secretion, or an altered rate of mucin turnover. It thus appears that starvation alters mucus dynamics in the small intestine, and this may affect intestinal digestive function and defense. KEY WORDS: * chick * mucin * small intestine * starvation * mucus adherent layer

Human carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs) are a family of receptors that mediate intercellular interactions. Pathogenic bacteria have ligands that bind CEACAMs on human cells. Neisseria gonorrhoeae (Gc) encodes numerous unique outer membrane opacity‐associated (Opa) proteins that are ligands for one or more CEACAMs. CEACAMs that are expressed on epithelial cells facilitate Gc colonization, while those expressed on neutrophils affect phagocytosis and consequent intracellular survival of Gc. Since Opa protein expression is phase‐variable, variations in receptor tropism affect how individual bacteria within a population interact with host cells. Here we report the development of a rapid, quantitative method for collecting and analyzing fluorescence intensity data from thousands of cells in a population using imaging flow cytometry to detect N‐CEACAM bound to the surface of Opa‐expressing Gc. We use this method to confirm previous findings regarding Opa‐CEACAM interactions and to examine the receptor–ligand interactions of Gc expressing other Opa proteins, as well as for other N‐CEACAM proteins. © 2020 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Polymorphisms in the IRGM gene are associated with susceptibility to tuberculosis in humans. A murine ortholog of Irgm , Irgm1 , is also essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. Multiple processes have been associated with IRGM1 activity that could impact the host response to Mtb infection, including roles in autophagy-mediated pathogen clearance and expansion of activated T cells. However, what IRGM1-mediated pathway is necessary to control Mtb infection in vivo and the mechanistic basis for this control remains unknown. We dissected the contribution of IRGM1 to immune control of Mtb pathogenesis in vivo and found that Irgm1 deletion leads to higher levels of IRGM3-dependent type I interferon signaling. The increased type I interferon signaling precludes T cell expansion during Mtb infection. The absence of Mtb-specific T cell expansion in Irgm1 -/- mice results in uncontrolled Mtb infection in neutrophils and alveolar macrophages, which directly contributes to susceptibility to infection. Together, our studies reveal that IRGM1 is required to promote T cell-mediated control of Mtb infection in neutrophils, which is essential for the survival of Mtb-infected mice. These studies also uncover new ways type I interferon signaling can impact T H 1 immune responses.

Neutrophils are professional phagocytes that are important for innate host defenses against pathogens and resolution of inflammation. Traditionally, the phagocytic capacity of neutrophils was quantified by enumeration of cells containing either internalized or bound bacteria or other cargo from a series of microscopic images. Here we describe an imaging flow cytometry-based protocol and analysis method for quantifying the binding and uptake of Neisseria gonorrhoeae by primary adherent human neutrophils. Imaging flow cytometry combines the capacity for quantitative, high-throughput analysis of tens of thousands of cells per condition, with the imaging power of fluorescence microscopy. Here, all bacteria are labeled with Tag-it Violet™ and bound bacteria are differentially stained with a DyLight™ 650-conjugated antibody. Images are analyzed using spot count and other algorithms. Outputs include the percent of neutrophils associated with bacteria, the percent of neutrophils with internalized bacteria, and the percent of internalized bacteria. This basic protocol can be adapted to a variety of particle types and can be used for multiplex analysis in combination with staining for different neutrophil surface and intracellular markers.