Protocol for long-term monocultures of murine macrophages derived from distinct adult tissues.

Tissue-resident macrophages (TRMs) constitute the first line of defense against infection in all organs and perform organ-specific functions during tissue homeostasis. Here, we present a protocol for long-term monocultures of murine macrophages from different adult organs, including the brain, liver, peritoneal cavity, and lung. We describe steps for tissue preparation and the use of a combination of organotypic conditions to maintain a TRM-like identity in vitro, resulting in an ideal screening platform for a wide range of assays and readouts. For complete details on the use and execution of this protocol, please refer to Aktories et al.1.

An improved organotypic cell culture system to study tissue-resident macrophages ex vivo.

Tissue-resident macrophages (TRMs) perform organ-specific functions that are dependent on factors such as hematopoietic origin, local environment, and biological influences. A diverse range of in vitro culture systems have been developed to decipher TRM functions, including bone marrow-derived macrophages (BMDMs), induced pluripotent stem cell (iPSC)-derived TRMs, or immortalized cell lines. However, despite the usefulness of such systems, there are notable limitations. Attempts to culture primary macrophages often require purification of cells and lack a high cell yield and consistent phenotype. Here, we aimed to address these limitations by establishing an organotypic primary cell culture protocol. We obtained long-term monocultures of macrophages derived from distinct organs without prior purification using specific growth factors and tissue normoxic conditions that largely conserved a TRM-like identity in vitro. Thus, this organotypic system offers an ideal screening platform for primary macrophages from different organs that can be used for a wide range of assays and readouts.

Microglia in a Dish-Which Techniques Are on the Menu for Functional Studies?

Microglia build the first line of defense in the central nervous system (CNS) and play central roles during development and homeostasis. Indeed, they serve a plethora of diverse functions in the CNS of which many are not yet fully described and more are still to be discovered. Research of the last decades unraveled an implication of microglia in nearly every neurodegenerative and neuroinflammatory disease, making it even more challenging to elucidate molecular mechanisms behind microglial functions and to modulate aberrant microglial behavior. To understand microglial functions and the underlying signaling machinery, many attempts were made to employ functional in vitro studies of microglia. However, the range of available cell culture models is wide and they come with different advantages and disadvantages for functional assays. Here we aim to provide a condensed summary of common microglia in vitro systems and discuss their potentials and shortcomings for functional studies in vitro.

Inverse control of Rab proteins by Yersinia ADP-ribosyltransferase and glycosyltransferase related to clostridial glucosylating toxins.

We identified a glucosyltransferase (YGT) and an ADP-ribosyltransferase (YART) in Yersinia mollaretii, highly related to glucosylating toxins from Clostridium difficile, the cause of antibiotics-associated enterocolitis. Both Yersinia toxins consist of an amino-terminal enzyme domain, an autoprotease domain activated by inositol hexakisphosphate, and a carboxyl-terminal translocation domain. YGT N-acetylglucosaminylates Rab5 and Rab31 at Thr52 and Thr36, respectively, thereby inactivating the Rab proteins. YART ADP-ribosylates Rab5 and Rab31 at Gln79 and Gln64, respectively. This activates Rab proteins by inhibiting GTP hydrolysis. We determined the crystal structure of the glycosyltransferase domain of YGT (YGTG) in the presence and absence of UDP at 1.9- and 3.4-Å resolution, respectively. Thereby, we identified a previously unknown potassium ion-binding site, which explains potassium ion-dependent enhanced glycosyltransferase activity in clostridial and related toxins. Our findings exhibit a novel type of inverse regulation of Rab proteins by toxins and provide new insights into the structure-function relationship of glycosyltransferase toxins.