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The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death

Matsuzawa-Ishimoto, Yu; Yao, Xiaomin; Koide, Akiko; Ueberheide, Beatrix M; Axelrad, Jordan E; Reis, Bernardo S; Parsa, Roham; Neil, Jessica A; Devlin, Joseph C; Rudensky, Eugene; Dewan, M Zahidunnabi; Cammer, Michael; Blumberg, Richard S; Ding, Yi; Ruggles, Kelly V; Mucida, Daniel; Koide, Shohei; Cadwell, Ken
Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.
PMID: 36198790
ISSN: 1476-4687
CID: 5351622

Interleukin-17 governs hypoxic adaptation of injured epithelium

Konieczny, Piotr; Xing, Yue; Sidhu, Ikjot; Subudhi, Ipsita; Mansfield, Kody P; Hsieh, Brandon; Biancur, Douglas E; Larsen, Samantha B; Cammer, Michael; Li, Dongqing; Landén, Ning Xu; Loomis, Cynthia; Heguy, Adriana; Tikhonova, Anastasia N; Tsirigos, Aristotelis; Naik, Shruti
Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt+ (RORγt+) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.
PMID: 35709248
ISSN: 1095-9203
CID: 5268732

Metabolic reprogramming of tumor-associated macrophages by collagen turnover promotes fibrosis in pancreatic cancer

LaRue, Madeleine M; Parker, Seth; Puccini, Joseph; Cammer, Michael; Kimmelman, Alec C; Bar-Sagi, Dafna
SignificanceThe highly desmoplastic and immunosuppressive microenvironment of pancreatic tumors is a major determinant of the aggressive nature and therapeutic resistance of pancreatic cancer. Therefore, improving our understanding of the mechanisms that regulate the composition and function of the pancreatic tumor microenvironment is critical for the design of intervention strategies for this devastating malignancy. This study identifies a modality for the reprogramming of tumor-associated macrophages involving collagen scavenging followed by a metabolic switch toward a profibrotic paracrine phenotype. These findings establish a molecular framework for the elucidation of regulatory processes that could be harnessed to mitigate the stroma-dependent protumorigenic effects in pancreatic cancer.
PMID: 35412885
ISSN: 1091-6490
CID: 5201902

chinmo-mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche

Tseng, Chen-Yuan; Burel, Michael; Cammer, Michael; Harsh, Sneh; Flaherty, Maria Sol; Baumgartner, Stefan; Bach, Erika A
Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, we show that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo-/- GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. Our results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution.
PMID: 34942115
ISSN: 1878-1551
CID: 5109062

APOL1 variant-expressing endothelial cells exhibit autophagic dysfunction and mitochondrial stress

Blazer, Ashira; Qian, Yingzhi; Schlegel, Martin Paul; Algasas, Huda; Buyon, Jill P; Cadwell, Ken; Cammer, Michael; Heffron, Sean P; Liang, Feng-Xia; Mehta-Lee, Shilpi; Niewold, Timothy; Rasmussen, Sara E; Clancy, Robert M
Polymorphisms in the Apolipoprotein L1 (APOL1) gene are common in ancestrally African populations, and associate with kidney injury and cardiovascular disease. These risk variants (RV) provide an advantage in resisting Trypanosoma brucei, the causal agent of African trypanosomiasis, and are largely absent from non-African genomes. Clinical associations between the APOL1 high risk genotype (HRG) and disease are stronger in those with comorbid infectious or immune disease. To understand the interaction between cytokine exposure and APOL1 cytotoxicity, we established human umbilical vein endothelial cell (HUVEC) cultures representing each APOL1 genotype. Untreated HUVECs were compared to IFNÉ£-exposed; and APOL1 expression, mitochondrial function, lysosome integrity, and autophagic flux were measured. IFNÉ£ increased median APOL1 expression across all genotypes 22.1 (8.3 to 29.8) fold (p=0.02). Compared to zero risk variant-carrying HUVECs (0RV), HUVECs carrying 2 risk variant copies (2RV) showed both depressed baseline and maximum mitochondrial oxygen consumption (p<0.01), and impaired mitochondrial networking on MitoTracker assays. These cells also demonstrated a contracted lysosomal compartment, and an accumulation of autophagosomes suggesting a defect in autophagic flux. Upon blocking autophagy with non-selective lysosome inhibitor, hydroxychloroquine, autophagosome accumulation between 0RV HUVECs and untreated 2RV HUVECs was similar, implicating lysosomal dysfunction in the HRG-associated autophagy defect. Compared to 0RV and 2RV HUVECs, HUVECs carrying 1 risk variant copy (1RV) demonstrated intermediate mitochondrial respiration and autophagic flux phenotypes, which were exacerbated with IFNÉ£ exposure. Taken together, our data reveal that IFNÉ£ induces APOL1 expression, and that each additional RV associates with mitochondrial dysfunction and autophagy inhibition. IFNÉ£ amplifies this phenotype even in 1RV HUVECs, representing the first description of APOL1 pathobiology in variant heterozygous cell cultures.
PMCID:9551299
PMID: 36238153
ISSN: 1664-8021
CID: 5361182

Monocyte-derived S1P in the lymph node regulates immune responses

Baeyens, Audrey; Bracero, Sabrina; Chaluvadi, Venkata S; Khodadadi-Jamayran, Alireza; Cammer, Michael; Schwab, Susan R
The lipid chemoattractant sphingosine 1-phosphate (S1P) guides cells out of tissues, where the concentration of S1P is relatively low, into circulatory fluids, where the concentration of S1P is high1. For example, S1P directs the exit of T cells from lymph nodes, where T cells are initially activated, into lymph, from which T cells reach the blood and ultimately inflamed tissues1. T cells follow S1P gradients primarily using S1P receptor 1 (ref. 1). Recent studies have described how S1P gradients are established at steady state, but little is known about the distribution of S1P in disease or about how changing levels of S1P may affect immune responses. Here we show that the concentration of S1P increases in lymph nodes during an immune response. We found that haematopoietic cells, including inflammatory monocytes, were an important source of this S1P, which was an unexpected finding as endothelial cells provide S1P to lymph1. Inflammatory monocytes required the early activation marker CD69 to supply this S1P, in part because the expression of CD69 was associated with reduced levels of S1pr5 (which encodes S1P receptor 5). CD69 acted as a 'stand-your-ground' signal, keeping immune cells at a site of inflammation by regulating both the receptors and the gradients of S1P. Finally, increased levels of S1P prolonged the residence time of T cells in the lymph nodes and exacerbated the severity of experimental autoimmune encephalomyelitis in mice. This finding suggests that residence time in the lymph nodes might regulate the differentiation of T cells, and points to new uses of drugs that target S1P signalling.
PMID: 33658712
ISSN: 1476-4687
CID: 4801642

STAT3 Inhibitor OPB-51602 Is Cytotoxic to Tumor Cells Through Inhibition of Complex I and ROS Induction

Brambilla, Lara; Lahiri, Tanaya; Cammer, Michael; Levy, David E
STAT3 is a transcription factor involved in several cellular activities including inflammation, proliferation, and survival, but it also plays a non-transcriptional role in modulating mitochondrial metabolism. Given its diverse functions in human cancers, it is an emerging therapeutic target. Here we show that OPB-51602, a small molecule inhibitor of STAT3, is highly toxic in a STAT3-dependent manner. Specifically, drug toxicity depends on mitochondrial STAT3 as tumor cells expressing only a mitochondrially restricted form of STAT3 are sensitive to the compound, whereas STAT3-null cells are protected. OPB-51602 inhibited complex I activity and led to increased ROS production, which in turn induced mitophagy, actin rearrangements, and cell death. Cells undergoing reduced oxidative phosphorylation or expressing NDI1 NADH dehydrogenase from Saccharomyces cerevisiae, which bypasses mammalian complex I, were resistant to OPB-51602 toxicity. These results show that targeting mitochondrial STAT3 function causes synthetic lethality through complex I inhibition that could be exploited for cancer chemotherapy.
PMCID:7708861
PMID: 33305182
ISSN: 2589-0042
CID: 4709362

Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer

Hollinshead, Kate E R; Parker, Seth J; Eapen, Vinay V; Encarnacion-Rosado, Joel; Sohn, Albert; Oncu, Tugba; Cammer, Michael; Mancias, Joseph D; Kimmelman, Alec C
Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive fibrosis and hypovascularization, resulting in significant intratumoral hypoxia (low oxygen) that contributes to its aggressiveness, therapeutic resistance, and high mortality. Despite oxygen being a fundamental requirement for many cellular and metabolic processes, and the severity of hypoxia in PDAC, the impact of oxygen deprivation on PDAC biology is poorly understood. Investigating how PDAC cells survive in the near absence of oxygen, we find that PDAC cell lines grow robustly in oxygen tensions down to 0.1%, maintaining mitochondrial morphology, membrane potential, and the oxidative metabolic activity required for the synthesis of key metabolites for proliferation. Disrupting electron transfer efficiency by targeting mitochondrial respiratory supercomplex assembly specifically affects hypoxic PDAC proliferation, metabolism, and in vivo tumor growth. Collectively, our results identify a mechanism that enables PDAC cells to thrive in severe, oxygen-limited microenvironments.
PMID: 33027658
ISSN: 2211-1247
CID: 4626982

3-Dimensional organization and dynamics of the microsporidian polar tube invasion machinery

Jaroenlak, Pattana; Cammer, Michael; Davydov, Alina; Sall, Joseph; Usmani, Mahrukh; Liang, Feng-Xia; Ekiert, Damian C; Bhabha, Gira
Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidia species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s-1, and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.
PMID: 32946515
ISSN: 1553-7374
CID: 4593522

An Intestinal Organoid-Based Platform That Recreates Susceptibility to T Cell-Mediated Tissue Injury

Matsuzawa-Ishimoto, Yu; Hine, Ashley; Shono, Yusuke; Rudensky, Eugene; Lazrak, Amina; Yeung, Frank; Neil, Jessica A; Yao, Xiaomin; Chen, Ying-Han; Heaney, Thomas; Schuster, Samantha L; Zwack, Erin E; Axelrad, Jordan Eric; Hudesman, David; Tsai, Jennifer Jia-Ying; Nichols, Katherine B; Dewan, M Zahidunnabi; Cammer, Michael; Beal, Allison; Hoffman, Sandra; Geddes, Brad; Bertin, John; Liu, Chen; Torres, Victor J; Loke, P'ng; van den Brink, Marcel Rm; Cadwell, Ken
A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT). We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that co-cultured allogeneic T cells kill Atg16L1 mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.
PMID: 32232483
ISSN: 1528-0020
CID: 4370252