BACKGROUND: Bacillus Calmette-Guerin (BCG) therapy is an established immunotherapy for non-muscle invasive bladder cancer (NMIBC); however, the response variability of patients remains a challenge, necessitating insight into immune cell function. Previous studies established that a preexisting Th2 immune microenvironment correlates with a positive BCG therapy outcome. Therefore, in this study, we explored the role of mast cells (MCs) and eosinophils in bladder cancer as a potential predicting tool for BCG immunotherapy response. METHODS: We investigated the effect of MCs and eosinophils on bladder cancer cell viability together with their chemotactic migration towards cancer cells in vitro. The effect of BCG on these immune cells was also evaluated. Moreover, we performed an orthotopic model of bladder cancer in MC- and eosinophil-deficient mice. Finally, to evaluate whether these immune cells predict the therapy response, 26 patient biopsies pre-BCG treatment were analyzed for MC and eosinophil numbers in the tissue and sequenced for gene expression. RESULTS: Eosinophils, but not MCs, reduced bladder cancer cell viability in vitro and inhibited tumor growth in vivo. However, addition of BCG did not increase these effects in vitro. Patient biopsy analysis and mRNA sequencing showed that neither cell type predicted long-term therapy responsiveness. Gene expression analysis suggested that extracellular matrix and epithelial-to-mesenchymal transition factors could influence BCG therapy outcomes. CONCLUSION: Even though eosinophils exhibit anti-tumorigenic effects in bladder cancer, neither MCs nor eosinophils were predictive of the long-term BCG therapy response. However, our findings implicate that matrix-related factors may modulate BCG therapy responses.

Chronic respiratory diseases such as asthma and chronic obstructive pulmonary disease afflict millions of individuals globally and are significant sources of disease mortality. While the molecular mechanisms underlying such diseases are unclear, environmental and social factors, such as cigarette smoke and obesity, increase the risk of disease development. Yet, not all smokers or obese individuals will develop chronic respiratory diseases. The mitogen-activated protein kinase p38α is abnormally active in such maladies, but its contribution, if any, to disease etiology is unknown. To assess whether p38α activation per se in the lung could impose disease symptoms, we generated a transgenic mouse model allowing controllable expression of an intrinsically active variant, p38α(D176A+F327S), specifically in lung alveolar type 2 pneumocytes. Sustained expression of p38α(D176A+F327S) did not appear to induce obvious pathological outcomes or to exacerbate inflammatory outcomes in mice challenged with common respiratory disease triggers. However, mice expressing p38α(D176A+F327S) in alveolar type 2 cells and fed with a high-fat diet exhibited increased numbers of airway eosinophils and lymphocytes, upregulated levels of proinflammatory cytokines and chemokines including interleukin-1β and eotaxin, as well as a reduction in levels of leptin and adiponectin within the lung. Neither high-fat diet nor p38α(D176A+F327S) alone induced such outcomes. Perhaps in obese individuals with associated respiratory diseases, elevated p38α activity which happens to occur is the factor that promotes their development.

BACKGROUND: ATAD3A, a nuclear gene encoding the ATAD3A protein, has diverse roles in mitochondrial processes, encompassing mitochondrial dynamics, mitochondrial DNA maintenance, metabolic pathways and inter-organellar interactions. Pathogenic variants in this gene cause neurological diseases in humans with recognizable genotype-phenotype correlations. Yet, gaps in knowledge remain regarding the underlying pathogenesis. METHODS: To further investigate the gene function and its implication in health and disease, we utilized CRISPR/Cas9 genome editing to generate a knockout model of the zebrafish ortholog gene, atad3. We characterized the phenotype of the null model, performed mitochondrial and functional tests, and compared the transcriptome of null embryos to their healthy siblings. RESULTS: Analysis of atad3-null zebrafish embryos revealed microcephaly, small eyes, pericardial edema and musculature thinning, closely mirroring the human rare disease phenotype. Larvae exhibited delayed hatching and embryonic lethality by 13 days post-fertilization (dpf). Locomotor activity, ATP content, mitochondrial content, and mitochondrial activity were all reduced in the mutant embryos. Transcriptome analysis at 3 dpf via RNA-sequencing indicated decline in most mitochondrial pathways, accompanied by a global upregulation of cytosolic tRNA synthetases, presumably secondary to mitochondrial stress and possibly endoplasmic reticulum (ER)-stress. Differential expression of select genes was corroborated in fibroblasts from an affected individual. CONCLUSIONS: The atad3-null zebrafish model emerges as a reliable representation of human ATAD3A-associated disorders, with similarities in differentially expressed pathways and processes. Furthermore, our study underscores mitochondrial dysfunction as the primary underlying pathogenic mechanism in ATAD3A-associated disorders and identifies potential readouts for therapeutic studies.

BACKGROUND: Chicken embryos emerge from their shell by the piercing movement of the hatching muscle. Although considered a key player during hatching, with activity that imposes a substantial metabolic demand, data are still limited. The study provides a bioenergetic and transcriptomic analyses during the pre-post-hatching period. METHODS: Weight and morphology alongside content determination of creatine and glycogen were analysed. Transcriptome identified differentially expressed genes and enriched biological processes associated with hatching muscle development, catabolism, and energy provision. Using gene set enrichment, we followed the dynamics of gene-sets involved in energy pathways of oxidative phosphorylation, protein catabolism, glycolysis/gluconeogenesis, and glycogen metabolism. RESULTS: Results show several significant findings: (A) Creatine plays a crucial role in the energy metabolism of the hatching muscle, with its concentration remaining stable while glycogen concentration is depleted at hatch and placement. (B) The hatching muscle has the capacity for de-novo creatine synthesis, as indicated by the expression of related genes (AGAT, GAMT). (C) Transcriptome provided insights into genes related to energy pathways under conditions of pre-hatch oxygen and post-hatch glucose limitations (oxidative phosphorylation: NDUF, MT-ND, SDH, UQCR, COX, MT-CO, ATP5, MT-ATP; glycolysis/gluconeogenesis: FBP,G6PC, PFKM; glycogen metabolism: PPP1, PYGL, GYG1). (D) The post-hatch upregulation of protein catabolic processes genes (PSMA, RNF, UBE, FBX), which align with the muscle's weight dynamics, indicates a functional shift from movement during hatching to lifting the head during feeding. CONCLUSIONS: There is a dynamic metabolic switch in the hatching muscle during embryo-to-hatchling transition. When glycogen concentration depletes, energy supply is maintained by creatine and its de-novo synthesis. Understanding the hatching muscle's energy dynamics is crucial, for reducing hatching failures in endangered avian species, and in domesticated chickens.

Demyelination and axonal injury in chronic-progressive Multiple Sclerosis (MS) are presumed to be driven by a neurotoxic bystander effect of meningeal-based myeloid infiltrates. There is an unmet clinical need to attenuate disease progression in such forms of CNS-compartmentalized MS. The failure of systemic immune suppressive treatments has highlighted the need for neuroprotective and repair-inducing strategies. Here, we examined whether direct targeting of CNS myeloid cells and modulating their toxicity may prevent irreversible tissue injury in chronic immune-mediated demyelinating disease. To that end, we utilized the experimental autoimmune encephalomyelitis (EAE) model in Biozzi mice, a clinically relevant MS model. We continuously delivered intracerebroventricularly (ICV) a retinoic acid receptor alpha agonist (RARα), as a potent regulator of myeloid cells, in the chronic phase of EAE. We assessed disease severity and performed pathological evaluations, functional analyses of immune cells, and single-cell RNA sequencing on isolated spinal CD11b+ cells. Although initiating treatment in the chronic phase of the disease, the RARα agonist successfully improved clinical outcomes and prevented axonal loss. ICV RARα agonist treatment inhibited pro-inflammatory pathways and shifted CNS myeloid cells toward neuroprotective phenotypes without affecting peripheral infiltrating myeloid cell phenotypes, or peripheral immunity. The treatment regulated cell-death pathways across multiple myeloid cell populations and suppressed apoptosis, resulting in paradoxically marked increased neuroinflammatory infiltrates, consisting mainly of microglia and CNS / border-associated macrophages. This work establishes the notion of bystander neurotoxicity by CNS immune infiltrates in chronic demyelinating disease. Furthermore, it shows that targeting compartmentalized neuroinflammation by selective regulation of CNS myeloid cell toxicity and survival reduces irreversible tissue injury, and may serve as a novel disease-modifying approach.

Triple-negative breast cancer (TNBC) is an aggressive subtype that accounts for 10-15 % of breast cancer. Current treatment of high-risk early-stage TNBC includes neoadjuvant chemo-immune therapy. However, the substantial variation in immune response prompts an urgent need for new immune-targeting agents. This requires a comprehensive understanding of TNBC's tumor microenvironment. We recently demonstrated that Galectin-3 (Gal-3) binding protein/Gal-3 complex secreted by TNBC cells induces immunosuppression, through inhibiting CD45 signaling in T cells. Here, we further investigated the interaction between secreted Gal-3 and T cells in TNBC. Using CRISPR/Cas9 gene editing of the TNBC MDA-MB-231 cell-line, we obtained Gal-3 negative((neg)) clones. We studied these in an in-vitro model, co-cultured with peripheral blood mononuclear cells (PBMC) to imitate immune-tumor interaction, and in an in-vivo model, when implanted in mice. Gal-3(neg) tumors in mice had decelerated tumor growth after PBMC inoculation. In contrast, the Gal-3 positive((pos)) tumors continued growing despite PBMC inoculation, and tumor T regulatory cell (CD4/FoxP3+) infiltration increased. RNA sequencing of T cells from women with TNBC with elevated plasma levels of Gal-3 revealed significantly lower expression of oxidative phosphorylation genes than in T cells from healthy women. Similarly, in our in-vitro model, the decreased expression of oxidative phosphorylation genes and mitochondrial dysfunction resulted in a significant increase in CD8 intracellular reactive oxygen species. Consequently, T exhausted cells (CD8/PD1/Tim3/Lag3+) significantly increased in PBMC co-cultured with Gal-3(pos) TNBCs. To conclude, we revealed a novel TNBC-related Gal-3 suppressor mechanism that involved upregulation of CD4 T regulatory and of CD8 T exhausted cells.

Due to its capacity to drive osteoclast differentiation, the receptor activator of nuclear factor kappa-β ligand (RANKL) is believed to exert a pathological influence in periodontitis. However, RANKL was initially identified as an activator of dendritic cells (DCs), expressed by T cells, and exhibits diverse effects on the immune system. Hence, it is probable that RANKL, acting as a bridge between the bone and immune systems, plays a more intricate role in periodontitis. Using ligature-induced periodontitis (LIP), rapid alveolar bone loss was detected that was later halted even though the ligature was still present. This late phase of LIP was also linked with immunosuppressive conditions in the gingiva. Further investigation revealed that the ligature prompted an immediate migration of RANK-expressing Langerhans cells (LCs) and EpCAM DCs, the antigen-presenting cells (APCs) of the gingival epithelium, to the lymph nodes, followed by an expansion of T regulatory (Treg) cells in the gingiva. Subsequently, the ligatured gingiva was repopulated by monocyte-derived RANK-expressing EpCAM DCs, while gingival epithelial cells upregulated RANKL expression. Blocking RANKL signaling with monoclonal antibodies significantly reduced the frequencies of Treg cells in the gingiva and prevented gingival immunosuppression. In addition, RANKL signaling facilitated the differentiation of LCs from bone marrow precursors. To further investigate the role of RANKL, we used K14-RANKL mice, in which RANKL is overexpressed by gingival epithelial cells. The elevated RANKL expression shifted the steady-state frequencies of LCs and EpCAM DCs within the epithelium, favoring LCs over EpCAM DCs. Following ligature placement, heightened levels of Treg cells were observed in the gingiva of K14-RANKL mice, and alveolar bone loss was significantly reduced. These findings suggest that RANKL-RANK interactions between gingival epithelial cells and APCs are crucial for suppressing gingival inflammation, highlighting a protective immunological role for RANKL in periodontitis that was overlooked due to its osteoclastogenic activity.

Demyelination and axonal injury in chronic-progressive Multiple Sclerosis (MS) are presumed to be driven by a neurotoxic bystander effect of meningeal-based myeloid infiltrates. There is an unmet clinical need to attenuate disease progression in such forms of CNS-compartmentalized MS. The failure of systemic immune suppressive treatments has highlighted the need for neuroprotective and repair-inducing strategies. Here, we examined whether direct targeting of CNS myeloid cells and modulating their toxicity may prevent irreversible tissue injury in chronic immune-mediated demyelinating disease. To that end, we utilized the experimental autoimmune encephalomyelitis (EAE) model in Biozzi mice, a clinically relevant MS model. We continuously delivered intracerebroventricularly (ICV) a retinoic acid receptor alpha agonist (RARα), as a potent regulator of myeloid cells, in the chronic phase of EAE. We assessed disease severity and performed pathological evaluations, functional analyses of immune cells, and single-cell RNA sequencing on isolated spinal CD11b+ cells. Although initiating treatment in the chronic phase of the disease, the RARα agonist successfully improved clinical outcomes and prevented axonal loss. ICV RARα agonist treatment inhibited pro-inflammatory pathways and shifted CNS myeloid cells toward neuroprotective phenotypes without affecting peripheral infiltrating myeloid cell phenotypes, or peripheral immunity. The treatment regulated cell-death pathways across multiple myeloid cell populations and suppressed apoptosis, resulting in paradoxically marked increased neuroinflammatory infiltrates, consisting mainly of microglia and CNS / border-associated macrophages. This work establishes the notion of bystander neurotoxicity by CNS immune infiltrates in chronic demyelinating disease. Furthermore, it shows that targeting compartmentalized neuroinflammation by selective regulation of CNS myeloid cell toxicity and survival reduces irreversible tissue injury, and may serve as a novel disease-modifying approach.

Maternal caloric restriction during pregnancy significantly impacts kidney development, influencing susceptibility to chronic kidney disease in adulthood. This study explores DNA methylation changes in nephron progenitor cells resulting from caloric restriction and their implications for kidney health. Global DNA hypomethylation is observed in nephron progenitors from caloric-restricted embryos, with specific genomic regions displaying distinct methylation patterns, including hypomethylation and hypermethylation. Differentially methylated regions exhibit enhanced chromatin accessibility, indicating biological relevance. Hypomethylated regions are enriched for genes associated with developmental processes, reflecting changes in gene expression and highlighting their functional relevance in kidney development. The study also reveals that supplementing methionine, an essential amino acid, restores disrupted DNA methylation patterns, particularly in enhancer regions, emphasizing methionine's critical role in regulating nephron progenitor cell epigenetics and ensuring proper kidney development. The intricate relationship between maternal nutrition, dynamic DNA methylation, and kidney development is highlighted, emphasizing the enduring impact of early-life nutritional challenges on kidney function. This research elucidates epigenetic mechanisms as mediators for the lasting effects of maternal caloric restriction on kidney health. The study contributes valuable insights into the origins of chronic kidney diseases during early developmental stages, offering potential interventions to mitigate adverse outcomes.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as ``second-hand'' EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

Senescent cells can influence the function of tissues in which they reside, and their propensity for disease. A portion of adult human pancreatic beta cells express the senescence marker p16, yet it is unclear whether they are in a senescent state, and how this affects insulin secretion. We analyzed single-cell transcriptome datasets of adult human beta cells, and found that p16-positive cells express senescence gene signatures, as well as elevated levels of beta-cell maturation genes, consistent with enhanced functionality. Senescent human beta-like cells in culture undergo chromatin reorganization that leads to activation of enhancers regulating functional maturation genes and acquisition of glucose-stimulated insulin secretion capacity. Strikingly, Interferon-stimulated genes are elevated in senescent human beta cells, but genes encoding senescence-associated secretory phenotype (SASP) cytokines are not. Senescent beta cells in culture and in human tissue show elevated levels of cytoplasmic DNA, contributing to their increased interferon responsiveness. Human beta-cell senescence thus involves chromatin-driven upregulation of a functional-maturation program, and increased responsiveness of interferon-stimulated genes, changes that could increase both insulin secretion and immune reactivity.

This study sheds light on the central role of adenine nucleotide translocase 2 (ANT2) in the pathogenesis of obesity-induced CKD. Our data demonstrate that ANT2 depletion in renal proximal tubule cells (RPTCs) leads to a shift in their primary metabolic program from fatty acid oxidation to aerobic glycolysis, resulting in mitochondrial protection, cellular survival, and preservation of renal function. These findings provide new insights into the underlying mechanisms of obesity-induced CKD and have the potential to be translated toward the development of targeted therapeutic strategies for this debilitating condition.

OBJECTIVE: Epidemiological studies highlight an association between pancreatic ductal adenocarcinoma (PDAC) and oral carriage of the anaerobic bacterium , a species highly linked to periodontal disease. We analysed the potential for to promote pancreatic cancer development in an animal model and probed underlying mechanisms.

DESIGN: We tracked bacterial translocation from the oral cavity to the pancreas following administration to mice. To dissect the role of in PDAC development, we administered bacteria to a genetically engineered mouse PDAC model consisting of inducible acinar cell expression of mutant ( /LSL-G12D; Ptf1a-CreER, iKC mice). These mice were used to study the cooperative effects of mutation and on the progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. The direct effects of on acinar cells and PDAC cell lines were studied in vitro.

RESULTS: migrated from the oral cavity to the pancreas in mice and can be detected in human PanIN lesions. Repetitive administration to wild-type mice induced pancreatic acinar-to-ductal metaplasia (ADM), and altered the composition of the intrapancreatic microbiome. In iKC mice, accelerated PanIN to PDAC progression. In vitro, infection induced acinar cell ADM markers SOX9 and CK19, and intracellular bacteria protected PDAC cells from reactive oxygen species-mediated cell death resulting from nutrient stress.

CONCLUSION: Taken together, our findings demonstrate a causal role for in pancreatic cancer development in mice.

The interplay between neural progenitors and stem cells (NPSCs), and their extracellular matrix (ECM) is a crucial regulatory mechanism that determines their behavior. Nonetheless, how the ECM dictates the state of NPSCs remains elusive. The hindbrain is valuable to examine this relationship, as cells in the ventricular surface of hindbrain boundaries (HBs), which arise between any two neighboring rhombomeres, express the NPSC marker Sox2, while being surrounded with the membrane-bound ECM molecule chondroitin sulphate proteoglycan (CSPG), in chick and mouse embryos. CSPG expression was used to isolate HB Sox2+ cells for RNA-sequencing, revealing their distinguished molecular properties as typical NPSCs, which express known and newly identified genes relating to stem cells, cancer, the matrisome and cell cycle. In contrast, the CSPG- non-HB cells, displayed clear neural-differentiation transcriptome. To address whether CSPG is significant for hindbrain development, its expression was manipulated in vivo and in vitro. CSPG manipulations shifted the stem versus differentiation state of HB cells, evident by their behavior and altered gene expression. These results provide further understanding of the uniqueness of hindbrain boundaries as repetitive pools of NPSCs in-between the rapidly growing rhombomeres, which rely on their microenvironment to maintain their undifferentiated state during development.

is a Gram-negative anaerobic bacterium strongly associated with periodontal disease. Toll-like receptor 2 (TLR2) is indispensable for the host response to , but escapes from immune clearance via TLR2-dependent activation of phosphoinositide-3-kinase (PI3K). To probe the TLR2-dependent escape pathway of , we analyzed the TLR2 interactome induced following infection or activation by a synthetic lipopeptide TLR2/1 agonist on human macrophages overexpressing TLR2. Interacting proteins were stabilized by cross-linking and then immunoprecipitated and analyzed by mass spectrometry. In total, 792 proteins were recovered and network analysis enabled mapping of the TLR2 interactome at baseline and in response to infection. The infection-induced TLR2 interactome included the poly (ADP-ribose) polymerase family member mono-ADP-ribosyltransferase protein 9 (PARP9) and additional members of the PARP9 complex (DTX3L and NMI). PARP9 and its complex members are highly upregulated in macrophages exposed to or to the synthetic TLR2/1 ligand PamCys-Ser-(Lys) (PAM). Consistent with its known role in virally induced interferon production, PARP9 knockdown blocked type I interferon (IFN-I) production in response to and reduced inflammatory cytokine production. We found that drives signal transducer and activation of transcription (STAT) 1 (S727) phosphorylation through TLR2-PARP9, explaining PARP9's role in the induction of IFN-I downstream of TLR2. Furthermore, PARP9 knockdown reduced PI3K activation by , leading to improved macrophage bactericidal activity. In summary, PARP9 is a novel TLR2 interacting partner that enables IFN-I induction and immune escape in macrophages downstream of TLR2 sensing.