Piezo1 agonist restores meningeal lymphatic vessels, drainage, and brain-CSF perfusion in craniosynostosis and aged mice.

Skull development coincides with the onset of cerebrospinal fluid (CSF) circulation, brain-CSF perfusion, and meningeal lymphangiogenesis, processes essential for brain waste clearance. How these processes are affected by craniofacial disorders such as craniosynostosis are poorly understood. We report that raised intracranial pressure and diminished CSF flow in craniosynostosis mouse models associate with pathological changes to meningeal lymphatic vessels that affect their sprouting, expansion, and long-term maintenance. We also show that craniosynostosis affects CSF circulatory pathways and perfusion into the brain. Further, craniosynostosis exacerbates amyloid pathology and plaque buildup in Twist1+/-:5xFAD transgenic Alzheimer's disease models. Treating craniosynostosis mice with Yoda1, a small molecule agonist for Piezo1, reduces intracranial pressure and improves CSF flow, in addition to restoring meningeal lymphangiogenesis, drainage to the deep cervical lymph nodes, and brain-CSF perfusion. Leveraging these findings, we show that Yoda1 treatments in aged mice with reduced CSF flow and turnover improve lymphatic networks, drainage, and brain-CSF perfusion. Our results suggest that CSF provides mechanical force to facilitate meningeal lymphatic growth and maintenance. Additionally, applying Yoda1 agonist in conditions with raised intracranial pressure and/or diminished CSF flow, as seen in craniosynostosis or with ageing, is a possible therapeutic option to help restore meningeal lymphatic networks and brain-CSF perfusion.

TGFB1 induces fetal reprogramming and enhances intestinal regeneration.

The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.

Piezo1 agonist restores meningeal lymphatic vessels, drainage, and brain-CSF perfusion in craniosynostosis and aged mice.

Skull development coincides with the onset of cerebrospinal fluid (CSF) circulation, brain-CSF perfusion, and meningeal lymphangiogenesis, processes essential for brain waste clearance. How these processes are affected by craniofacial disorders such as craniosynostosis are poorly understood. We report that raised intracranial pressure and diminished CSF flow in craniosynostosis mouse models associates with pathological changes to meningeal lymphatic vessels that affect their sprouting, expansion, and long-term maintenance. We also show that craniosynostosis affects CSF circulatory pathways and perfusion into the brain. Further, craniosynostosis exacerbates amyloid pathology and plaque buildup in Twist1 +/- :5xFAD transgenic Alzheimer's disease models. Treating craniosynostosis mice with Yoda1, a small molecule agonist for Piezo1, reduces intracranial pressure and improves CSF flow, in addition to restoring meningeal lymphangiogenesis, drainage to the deep cervical lymph nodes, and brain-CSF perfusion. Leveraging these findings, we show Yoda1 treatments in aged mice with reduced CSF flow and turnover improve lymphatic networks, drainage, and brain-CSF perfusion. Our results suggest CSF provides mechanical force to facilitate meningeal lymphatic growth and maintenance. Additionally, applying Yoda1 agonist in conditions with raised intracranial pressure and/or diminished CSF flow, as seen in craniosynostosis or with ageing, is a possible therapeutic option to help restore meningeal lymphatic networks and brain-CSF perfusion.

TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming.

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.

Exploiting vita-PAMPs in vaccines.

Live attenuated vaccines elicit stronger protective immunity than dead vaccines. Distinct PAMPs designated as vita-PAMPs signify microbial viability to innate immune cells. Two vita-PAMPs have been characterized: cyclic-di-adenosine-monophosphate (c-di-AMP) and prokaryotic messenger RNA (mRNA). c-di-AMP produced by live Gram-positive bacteria elicits augmented production of STING-dependent type-I interferon, whereas prokaryotic mRNA from live bacteria is detected by TLR8 enabling discrimination of live from dead bacteria. Bacterial mRNA from live Gram-negative bacteria triggers a heightened type-I interferon and NLRP3 inflammasome response. By mobilizing unique viability-associated innate responses, vita-PAMPs mobilize adaptive immunity that best elicits protection, including follicular T helper cell and antibody responses. Here, we review the molecular mechanisms that confer the unique adjuvanticity of vita-PAMPs and discuss their applications in vaccine design.

Sensing Microbial Viability through Bacterial RNA Augments T Follicular Helper Cell and Antibody Responses.

Live vaccines historically afford superior protection, yet the cellular and molecular mechanisms mediating protective immunity remain unclear. Here we found that vaccination of mice with live, but not dead, Gram-negative bacteria heightened follicular T helper cell (Tfh) differentiation, germinal center formation, and protective antibody production through the signaling adaptor TRIF. Complementing the dead vaccine with an innate signature of bacterial viability, bacterial RNA, recapitulated these responses. The interferon (IFN) and inflammasome pathways downstream of TRIF orchestrated Tfh responses extrinsically to B cells and classical dendritic cells. Instead, CX3CR1+CCR2- monocytes instructed Tfh differentiation through interleukin-1ß (IL-1ß), a tightly regulated cytokine secreted upon TRIF-dependent IFN licensing of the inflammasome. Hierarchical production of IFN-ß and IL-1ß dictated Tfh differentiation and elicited the augmented humoral responses characteristic of live vaccines. These findings identify bacterial RNA, an innate signature of microbial viability, as a trigger for Tfh differentiation and suggest new approaches toward vaccine formulations for coordinating augmented Tfh and B cell responses.

Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs.

Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4+ T-cell activation. A common 'suppression of inflammation' signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4+ T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.

Apoptosis in response to microbial infection induces autoreactive TH17 cells.

Microbial infections often precede the onset of autoimmunity. How infections trigger autoimmunity remains poorly understood. We investigated the possibility that infection might create conditions that allow the stimulatory presentation of self peptides themselves and that this might suffice to elicit autoreactive T cell responses that lead to autoimmunity. Self-reactive CD4(+) T cells are major drivers of autoimmune disease, but their activation is normally prevented through regulatory mechanisms that limit the immunostimulatory presentation of self antigens. Here we found that the apoptosis of infected host cells enabled the presentation of self antigens by major histocompatibility complex class II molecules in an inflammatory context. This was sufficient for the generation of an autoreactive TH17 subset of helper T cells, prominently associated with autoimmune disease. Once induced, the self-reactive TH17 cells promoted auto-inflammation and autoantibody generation. Our findings have implications for how infections precipitate autoimmunity.

Aging-like phenotype and defective lineage specification in SIRT1-deleted hematopoietic stem and progenitor cells.

Aging hematopoietic stem cells (HSCs) exhibit defective lineage specification that is thought to be central to increased incidence of myeloid malignancies and compromised immune competence in the elderly. Mechanisms underlying these age-related defects remain largely unknown. We show that the deacetylase Sirtuin (SIRT)1 is required for homeostatic HSC maintenance. Differentiation of young SIRT1-deleted HSCs is skewed toward myeloid lineage associated with a significant decline in the lymphoid compartment, anemia, and altered expression of associated genes. Combined with HSC accumulation of damaged DNA and expression patterns of age-linked molecules, these have striking overlaps with aged HSCs. We further show that SIRT1 controls HSC homeostasis via the longevity transcription factor FOXO3. These findings suggest that SIRT1 is essential for HSC homeostasis and lineage specification. They also indicate that SIRT1 might contribute to delaying HSC aging.

The TRPM4 channel controls monocyte and macrophage, but not neutrophil, function for survival in sepsis.

A favorable outcome following acute bacterial infection depends on the ability of phagocytic cells to be recruited and properly activated within injured tissues. Calcium (Ca(2+)) is a ubiquitous second messenger implicated in the functions of many cells, but the mechanisms involved in the regulation of Ca(2+) mobilization in hematopoietic cells are largely unknown. The monovalent cation channel transient receptor potential melastatin (TRPM) 4 is involved in the control of Ca(2+) signaling in some hematopoietic cell types, but the role of this channel in phagocytes and its relevance in the control of inflammation remain unexplored. In this study, we report that the ablation of the Trpm4 gene dramatically increased mouse mortality in a model of sepsis induced by cecal ligation and puncture. The lack of the TRPM4 channel affected macrophage population within bacteria-infected peritoneal cavities and increased the systemic level of Ly6C(+) monocytes and proinflammatory cytokine production. Impaired Ca(2+) mobilization in Trpm4(-/-) macrophages downregulated the AKT signaling pathway and the subsequent phagocytic activity, resulting in bacterial overgrowth and translocation to the bloodstream. In contrast, no alteration in the distribution, function, or Ca(2+) mobilization of Trpm4(-/-) neutrophils was observed, indicating that the mechanism controlling Ca(2+) signaling differs among phagocytes. Our results thus show that the tight control of Ca(2+) influx by the TRPM4 channel is critical for the proper functioning of monocytes/macrophages and the efficiency of the subsequent response to infection.

Dynamic imaging of the effector immune response to listeria infection in vivo.

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites.

The calcium-activated nonselective cation channel TRPM4 is essential for the migration but not the maturation of dendritic cells.

Dendritic cell (DC) maturation and migration are events critical for the initiation of immune responses. After encountering pathogens, DCs upregulate the expression of costimulatory molecules and subsequently migrate to secondary lymphoid organs. Calcium (Ca(2+)) entry governs the functions of many hematopoietic cell types, but the role of Ca(2+) entry in DC biology remains unclear. Here we report that the Ca(2+)-activated nonselective cation channel TRPM4 was expressed in and controlled the Ca(2+) homeostasis of mouse DCs. The absence of TRPM4, which elicited Ca(2+) overload, did not influence DC maturation but did considerably impair chemokine-dependent DC migration. Our results establish TRPM4-regulated Ca(2+) homeostasis as crucial for DC mobility but not maturation and emphasize that DC maturation and migration are independently regulated.