Generated Bone Marrow Chimeras Research Articles

The Tetraspanin CD53 Promotes the Clonal Advantage of Dnmt3a-Deficient Hematopoietic Stem Cells

Clonal hematopoiesis involves the selective expansion of HSCs with mutations, most commonly in genes such as the epigenetic modifiers TET2 and DNMT3A, and confers an increased risk of hematopoietic malignancies, cardiovascular disease and all-cause mortality. Studies show that Tet2- or Dnmt3a-mutant HSCs expand relative to their WT counterparts in response to inflammatory stress, indicating that under these conditions, mutant HSCs have a resistance to inflammatory cytokines and selective advantage. It has been reported, and confirmed in our lab with confocal microscopy on purified HSCs, that expression of the tetraspanin CD53 is markedly increased in HSCs lacking Dnmt3a or Tet2 compared to WT. While normally expressed at low levels, WT HSCs transiently upregulate CD53 in response to inflammatory stimuli. We recently reported that this transient elevation in CD53 expression protects the function of stressed HSCs by promoting their return to quiescence. Given this role of CD53, we hypothesize that the elevated CD53 in mutant HSCs contributes to their clonal advantage. To test the possible role of CD53 in the expansion of mutant HSCs, we first used lentivirus to enforce expression in WT HSCs and tag them with GFP. Lethally irradiated recipient mice were transplanted with minority populations of sorted CD53-overexpressing HSPCs together with majority populations of WT competitor bone marrow. These experiments revealed that HSCs overexpressing CD53 had superior multilineage engraftment to empty vector control HSCs in primary competitive transplants (10% vs 0.2% GFP+ at 12 weeks). Consistently, serial re-plating assays show that CD53-overexpressing HSCs form a greater number of colonies week to week in Methocult. Next, to address the function of CD53 in mutant HSCs, we assessed HSC function in Dnmt3aflx/flx;Vav-iCre and Dnmt3aflx/flx;Vav-iCre;Cd53-/- mice. HSC frequencies at baseline were comparable between groups, and they engrafted similarly in primary competitive transplants into lethally irradiated recipient mice. However, in secondary transplants, Dnmt3aflx/flx;Vav-iCre;Cd53- /- HSCs exhibited lower overall engraftment compared to Dnmt3aflx/flx;Vav-iCre HSCs (46% vs. 73% chimerism at 16 weeks post-transplant). In support of this finding, Dnmt3aflx/flx;Vav- iCre;Cd53-/- HSCs showed mitigated serial replating capacity in Methocult (2 vs 5+ re-platings). To evaluate CD53's role in mutant HSC clonal expansion in response to inflammatory stress, we generated bone marrow chimeras with either Dnmt3aflx/flx;Vav-iCre or Dnmt3aflx/flx;Vav-iCre;Cd53-/- HSCs, along with WT HSCs, and treated them with IFNg daily for 30 days. While Dnmt3aflx/flx;Vav-iCre HSCs expanded with treatment over WT HSCs, Dnmt3aflx/flx;Vav-iCre;Cd53-/- HSCs did not. Together, these data indicate a critical role for CD53 in promoting mutant HSC clonal expansion. Similar studies with Tet2-mutant mice are ongoing, and other studies underway aim to understand the mechanism by which elevated CD53 supports mutant HSCs.

Impact of PARP Inhibitor and Platinum Therapy on Clonal Hematopoiesis

Poly (ADP-ribose) polymerase inhibitors (PARPi) are a promising new class of targeted therapy used in a variety of solid tumors with homologous recombination deficiencies (HRD). Data from clinical trials and observational studies have linked PARPi and platinum-based agents to therapy-related myeloid neoplasia (tMN). However, interpretation of these findings is limited by confounding variables including germline mutation status and prior therapeutic exposure. We and others have shown that the oncologic therapies known to confer an elevated risk of leukemia also promote the expansion of clonal hematopoiesis (CH) due to mutations of genes in the DNA damage response (DDR) pathway, including TP53, PPM1D and CHEK2. However, the extent to which CH is associated with targeted therapies such as PARPi is not clear. We hypothesized that exposure to PARPi confers a competitive advantage to hematopoietic stem/progenitor cells (HSPCs) containing mutations in DDR pathway genes. Moreover, we hypothesized that in patients with germline mutations in HRD genes, such as BRCA1/BRCA2genotoxic stress induced by PARPi or cytotoxic agents is increased, further enhancing the fitness of HSPCs carrying DDR gene mutations . To test these hypotheses, we performed longitudinal studies on 942 peripheral blood samples collected from 418 patients (ages 32 to 94) before and after treatment with PARPi (n = 100), carboplatin (n = 142), and untreated individuals (n = 176). Patients with lung, ovarian, uterine, prostate, and breast cancer who received PARPi or carboplatin on clinical trials or through standard of care were included. Patients were treated with a variety of PARPi including Olaparib (n=48), Talazoparib (n=33), Rucaparib (n = 13) and Niraparib (n = 6.) Of the 418 patients, 45 individuals harbored an HRD germline mutation. Samples were sequenced at a depth of 18,946x using a UMI-based panel including 9 common CH genes ( DNMT3A, TET2, ASXL1, TP53, PPM1D, SRSF2, SF3B1 and JAK2). Both carboplatin and PARPi treatment significantly increased the growth rate of CH mutations, driven almost entirely by mutations in DDR genes (Figure 1). Patients undergoing carboplatin treatment had significantly higher rates of mutational growth compared to those undergoing PARPi therapy (p-value = 5e -17). The strongest PARP-trapping member, Talazoparib, had the largest effect on the growth rate of CH mutations in DDR genes (p-value = 3e -9) but still lower compared to carboplatin. Surprisingly, DDR CH expansion was largely abrogated in patients harboring a pathogenic HRD germline variant for both carboplatin (p-value = 3e -5) and PARPi (p-value = 7e -10). To validate these findings, we developed a mouse model of TP53-mutatedCH by transplanting a limited number of Trp53 R172Hbone marrow cells along with congenic wildtype bone marrow into irradiated recipients. The percentage of Trp53 R172Hcells in the blood was measured by flow cytometry at baseline (6 weeks after transplantation) and after treatment with vehicle alone, Talazoparib or Olaparib daily, or cisplatin weekly, for 4 weeks. As expected, treatment with cisplatin resulted in a significant expansion of TP53-mutated cells (Figure 2). A non-significant trend to expansion of TP53-mutated cells also was observed after talazoparib, but not olaparib. We next modeled the effect of germline HRD mutations by generating bone marrow chimeras containing heterozygous Brca1-mutated HSPCs competed with Brca1-mutated HSPCs also carrying Trp53 R172H. Consistent with the human CH data, no expansion of TP53-mutated cells was observed after treatment with either cisplatin or Talazoparib, indeed there was a small, but significant decrease (Figure 2). Collectively, these data support the hypothesis that mutations in DDR genes provide a fitness advantage to HSPCs following PARP inhibitor and platinum therapy. However, compared to platinum therapy, PARPi shows less selective pressure on DDR CH. Importantly, these data strongly argue against our initial hypothesis that germline HRD variants enhance this fitness advantage. Indeed, our data suggest the opposite, with the fitness advantage of DDR gene-mutated HSPCs lost in cells with a heterozygous Brca1-mutations. These data suggest that PARPi therapy may have less of an impact on leukemia risk compared to carboplatin and in fact may show synergistic effects with HRD in blocking the competitive advantage of DDR CH during genotoxic stress.

Abstract PR017: Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is soon to be the second deadliest types of cancer, with a 5-year survival of only 10%. The unique features of PDAC are the expansion of cancer-associated fibroblasts (CAFs), presence of a dense fibrous stroma with high bundled collagen I and immunosuppression, representing a challenge for therapies. Ways to overcome these unique pro-tumor features will be key for the development of better therapeutic strategies for PDAC. Herein we report that, in addition to the identified ectopic expression of NGL-1 in PDAC cells, it is also expressed in the stroma (CAFs and immune cells) of PDAC patients, inversely correlating with overall survival. Stromal NGL-1 was important for tumorigenesis in vivo, as NGL-1 knockout mice (KO) orthotopically allografted with pancreatic cancer cells presented less tumor burden. Further analysis showed that the tumors from KO mice presented more CD8+ T cells and less immunosuppressive cytokines, such as TGFβ. Single cell RNAseq analysis of these tumors showed decreased expression of pro-tumor factors, such as immune checkpoint molecules in T cells and TGFβ related genes across different cellular compartments (epithelial, immune and fibroblasts). In accordance with these results, these tumors presented a limited amount of desmoplastic bundled collagen, suggestive of a TGFβ-deficient environment. In order to further dissect between the NGL-1-dependent contributions of immune vs. local stroma (e.g., CAFs) cells, we generated bone marrow chimeras and performed orthotopic injections to generate tumors. The loss of NGL-1 in each cellular compartment alone failed to phenocopy the full body loss of NGL-1, suggesting that NGL-1 in both immune cells and local stromal are important for tumorigenesis. Functionally, NGL-1 deficient CAFs failed to support the survival of starved PDAC cells in vitro, downregulated important myofibroblastic molecules such as p-smad, and produced less immunosuppressive cytokines, suggesting a role for NGL-1 in key pro-tumor features of CAFs. In fact, the fibroblastic NGL-1 dependent immunomodulatory effects were confirmed with human CD8+ T cells from healthy donors, which lost their cytotoxic profile in the presence of conditioned media (CM) from NGL-1+ CAFs, but were able to keep this profile when exposed to CM from NGL-1 deficient CAFs. Bone marrow derived macrophages from KO mice produced less pro-tumor cytokines and CD8+ T cells lacking NGL-1 proliferated more than those from wild type animals, when stimulated in vitro. Mechanistically, while immune cells and CAFs deficient in NGL-1 are both tumor suppressive, the latter can regain pro-tumor functions in response to TGFβ, explaining the need for a global modulation of NGL-1 expression for an anti-tumor effect. Finally, NGL-1 KO mice orthotopically allografted with PDAC cells responded better (smaller tumors) to chemotherapeutical regimen (FOLFIRINOX) compared to WT animals. All these results point to NGL-1 as a potential new target to modulate immunosuppression and tumorigenesis in pancreatic cancer. Citation Format: Debora Barbosa Vendramini Costa, Ralph Francescone, Janusz Franco-Barraza, Tiffany Luong, Esteban Martinez, Stephen Sykes, Nina Steele, Marina Pasca di Magliano, Dmitry Zhigarev, Charline Ogier, Huamin Wang, Igor Astsaturov, Kerry Campbell, Edna Cukierman. Stromal Netrin G1 ligand (NGL-1): A new modulator of tumorigenesis and immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR017.

Inflammation depletes humoral immunity by limiting plasma cell access to the bone marrow survival niche

Abstract The survival of plasma cells (PCs) is directly correlated to the longevity of prophylactic titers against pathogens. The bone marrow contains hematopoietic and non-hematopoietic cells that contribute to the maintenance of PCs. Studies have shown that pathogens contain a variety of mechanisms that result in the depletion of bone marrow PCs and antibody titers. Most, if not all, forms of infection induce inflammation, however, the role of inflammation in bone marrow PC depletion is unclear. Here, we show that acute Tumor Necrosis Factor Alpha (TNFa) treatment can deplete pre-existing antibody titers in a variety of models. We also found that recombinant TNFa treatment limits PC retention in the bone marrow via adoptive transfer experiments. To determine if PC retention in the bone marrow was mediated in a cell-intrinsic or − extrinsic manner, we generated bone marrow chimeras with wild-type and TNFa receptor knockout mice. Results showed that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells regulates PC retention in the bone marrow, indicating that this mechanism is cell-extrinsic. Another cell-extrinsic mechanism that we identified is TNFa regulation of syndecan-1, a cell surface proteoglycan found to regulate PC survival and motility. These findings demonstrate that inflammation limits PC access to the bone marrow survival niche, ultimately leading to the reduction of antibody titers and long-term humoral protection. Supported by grants from the NIH (R01 HLI141491) and the Eric Heyer M.D. Ph.D Translational Research Grant

Research Highlights

Research Highlights

Tumor Necrosis Factor Alpha inhibits humoral immunity by regulating the antibody secreting cell bone marrow niche

Abstract Humoral immunity is a critical component of the immune memory, and its longevity is partly dependent on the survival of antibody-secreting cells (ASC). Previous studies have shown that several types of infections can deplete pre-existing antibody titers, but the exact mechanism of this process is unclear. Most, if not all, forms of infection induce inflammation including Tumor Necrosis Factor Alpha (TNFa). Here we show that TNFa-mediated inflammation depletes pre-existing antibody titers by limiting ASC retention in the bone marrow (BM) survival niche. Acute recombinant TNFa (rTNFa) treatment induces ASC egress from the BM into the blood and limits engraftment of adoptively transferred ASCs into the BM of recipient mice. To determine if the TNFa-mediated ASC egress from the BM was cell-intrinsic or –extrinsic, we generated BM chimeras with wild-type and TNFa receptor knockout mice. We found that TNFa signaling through TNFa receptor 1 in non-hematopoietic cells induces ASC egress from the BM in a cell-extrinsic manner. Treatment with rTNFa did not induce ASC death in the BM, but it increased glucose uptake and expression of CXCR4, a receptor involved in ASC retention in the BM. In contrast, mobilized ASCs in the blood had reduced glucose uptake, CD98 expression, and CD93 expression suggesting that ASC metabolism is highly dependent on the BM niche. Our findings demonstrate that inflammation alters ASC physiology in both cell-intrinsic and extrinsic manners, leading to reduced antibody titers and long-term immune memory.

Type I interferon-induced β-catenin promotes hematopoietic stem cell activation in persistent viral infection in mice

Abstract Quiescent hematopoietic stem cells (HSCs) become activated and exit their bone marrow niches in response to infections or sterile inflammation to initiate extramedullary hematopoiesis at peripheral sites. While these processes have been described in acute infections, the impact of persistent viral infections on HSC activation and mobilization remains less clear. Our results demonstrate that an active persistent lymphocytic choriomeningitis virus (LCMV) clone 13 infection resulted in type I interferon-dependent stabilization of β-catenin in HSCs, promoting their activation, mobilization and functional exhaustion that was sustained in time and could be seen even after transfer to uninfected hosts. To further examine the functional role of β-catenin in HSC activation, we generated bone marrow chimeras to follow β-catenin-deficient (Ctnnb1−/−) and wild type HSC response to LCMV Cl13 in the same environment. Ctnnb1−/− HSCs and multipotent progenitor cells failed to expand in response to the infection, while still retaining their ability to reconstitute secondary hosts. Moreover, through the course of the infection, Ctnnb1−/− cells produced myeloid progeny at a similar level to the wild type. These findings lead us to propose that β-catenin is required for HSC activation by interferons in the early phase of the infection; however, it appears dispensable for LCMV-induced emergency myelopoiesis.

TGF-β Signaling Contributes to the Clonal Dominance of Jak2V617F Hematopoietic Stem/Progenitor Cells

Increased TGF-β expression in the bone marrow microenvironment is present in most cases of myeloproliferative neoplasms (MPNs), however, its contribution to disease pathogenesis is not well understood. Multiple prior studies show that TGF-β suppresses the proliferation of hematopoietic stem/progenitor cells (HSPCs) in vitro. However, its impact on HSCs in vivo is less clear, with conflicting data showing that TGF-β suppresses or induces HSPC proliferation, depending upon the dose of TGF-β and cell context. Here, we explore the hypothesis that HSPCs carrying a Jak2V617F mutation are resistant to the suppressive effects of TGF-β, conferring a fitness advantage that contributes to their expansion in MPNs. To test this hypothesis, we first assessed TGF-β signaling in wildtype (WT) and Jak2V617F HSPCs. Whereas TGF-β1 induced robust Smad2/3 phosphorylation in WT phenotypic hematopoietic stem cells (HSCs), it was barely detectable in Jak2V617F HSCs. Interestingly, this phenotype was lost in lineage-restricted progenitors, where TGF-β1-induced Smad2/3 phosphorylation was comparable in WT and Jak2V617F cells. These data suggest that TGF-β signaling, at least Smad2/3 phosphorylation, is selectively loss in Jak2V617F HSCs but not lineage-restricted progenitors. Competitive repopulation assays (CRAs) were performed using WT and Jak2V617F HSPCs in which Tgfbr2 was deleted to abrogate TGF-β signaling in HSCs. We first established bone marrow chimeras containing equal amounts of WT and UBC-CreERT2; Tgfbr2f/f HSPCs (hereafter referred to as TR2KO cells). Tamoxifen was given 6 weeks after transplantation to activate Cre expression and delete Tgfbr2. Multilineage donor chimerism of the Tgfbr2-deleted cells increased over time, suggesting that TGF-β signaling negatively regulates HSPC repopulating activity. However, when competing Jak2V617F cells with Jak2V617F TR2KO cells, the TR2KO compartment no longer had competitive advantage, suggesting that Jak2 mutated HSPC are insensitive to the suppressive effect of TGF-β. To extend these findings, CRAs were established that included the following 4 cohorts: 1) WT vs. Jak2V617F; 2) TR2KO vs. Jak2V617F; 3) TR2KO vs. Jak2V617FTR2KO; and 4) WT vs. Jak2V617FTR2KO. In each case, a 5:1 ratio of competitor to Jak2-mutated cells was used and tamoxifen was given 6 weeks after transplantation to induce both Cre-mediated deletion of Tgfbr2 and activation of the Jak2V617F allele. In cohort 1, as expected, donor chimerism with Jak2 mutated cells increased over time (Figure 1A, black). Consistent with this finding, Jak2 mutant chimerism in HSPCs was increased at 12-14 weeks (Figure 1B). In cohort 2 (orange), the loss of TGF-β signaling in WT HSPCs completely abrogated the competitive advantage of Jak2V617F HSPCs. In fact, the TR2KO cells outcompeted Jak2V617F HSPCs. In cohort 3 (red), we asked whether loss of TGF-β signaling in both WT and Jak2V617F HSPCs "leveled the playing field". Indeed, no expansion of Jak2 mutant peripheral blood leukocytes or HSPCs was observed in these mice. Finally, in cohort 4 (blue), we observed that loss of TGF-β signaling in Jak2 mutant cells had little impact on their repopulating activity. To assess the impact of TGF-β signaling on WT and Jak2V617F HSPC proliferation, we generated bone marrow chimeras containing either WT vs. Jak2V617F HSPCs or TR2KO vs. Jak2V617FTR2KO HSPCs. Of note, in both types of bone marrow chimeras, the presence of Jak2 mutant cells is expected to increase the local level of endogenous TGF-β. As expected, in the WT vs. Jak2V617F chimeras, the Jak2V617F HSPCs were less quiescent than WT cells. Surprisingly, loss of TGF-β signaling resulted in an increase in HSC quiescence in both TR2KO and Jak2V617F TR2KO HSCs. Of note, the loss of TGF-β signaling again "leveled the playing field" with a similar percentage of quiescent TR2KO vs. Jak2V617FTR2KO HSCs. Collectively, these data suggest that attenuated TGF-β signaling in Jak2V617F HSPCs plays a key role in the pathogenesis of MPN. Specifically, the data support a model in which increased local production of TGF-β by Jak2V617F hematopoietic cells selectively inhibits WT HSPCs, thereby promoting the expansion of Jak2V617F HSPCs. Targeting TGF-β signaling early in the course of MPN or in persons with Jak2V617F clonal hematopoiesis may prevent or delay clonal progression. Disclosures No relevant conflicts of interest to declare.

Comparison of cesium and X-ray irradiation for generation of bone marrow chimeras

Abstract Bone marrow chimeras are mice which have been irradiated with ionizing radiation, thus killing their bone marrow and all bone marrow derived cells, followed by injection of bone marrow from another mouse. Generation of bone marrow chimeras has been a useful tool to interrogate stromal and hematopoietic contributions to immune function. Cesium sourced irradiators have been demonstrated to be effective in generating bone marrow chimeras and have long historic use in academia and industry. However, the increasing cost to maintain a cesium irradiator and greater security measures set in place for using a radioactive source material has led to a need for utilizing an alternative technology – X-ray irradiation. To our knowledge, limited number of publications compare these two modalities. In our study, we evaluated the X-ray irradiator by analyzing and comparing the reconstitution profile of specific immune cell subsets in the blood and spleens of mice irradiated with a cesium source versus mice irradiated with an X-ray source. Our data suggest that the two modalities provide functionally comparable degrees of chimerism in mice. Thereafter, we transitioned to using the X-ray irradiator and evaluated the effects of irradiation and reconstitution on tumor implantation. Our data shows that there is delayed tumor growth after total body irradiation. In summary, we have established a protocol for an alternative irradiation modality, confirmed comparable levels of chimerism to traditional generation of bone marrow chimeras, and observed delayed tumor growth following irradiation and reconstitution.

Tbet provides survival advantage to Tregs during Type 1 inflammation

Abstract Regulatory T-cells (Tregs) comprise subgroups with transcriptional programs similar to helper T-cell lineages. To visualize Tbet expression by Tregs during a T helper 1 (Th1) driven inflammation induced by Complete Freund’s Adjuvant in vivo, we imaged Tregs in the lymph nodes of a Tbet.ZsGreen-Foxp3.RFP mouse. While the majority of conventional Foxp3+ Tregs, resided within the T-cell zone, Tbet+Foxp3+ Tregs localized to the interfollicular zone that also contained the recently activated Th1 cells. To understand whether Tbet serves merely as a marker of activation or plays an actual role in Treg function, we generated bone marrow chimeras from Tbet+/+ and Tbet−/− mice. The peripheral Treg pool in chimeric mice was equally constituted by the Tbet+/+ and Tbet−/− cells. However, when the mice were challenged with Plasmodium chabaudi, a parasite that initiates a strong systemic Th1 response, the Treg compartment was significantly dominated by Tbet+/+ Tregs that displayed increased Tbet expression over time. While Tbet+/+ and Tbet−/− Tregs displayed similar activation status and proliferation, Tbet−/− Treg were more prone to apoptosis. Furthermore, Tbet−/− Tregs expressed lower levels of the ATP ectonucleotidase CD39 that was more pronounced during the peak of infection. Pharmacological inhibition of the activity of CD39 increased apoptosis of Tbet+/+ but not Tbet−/− Tregs in an ATP enriched culture in-vitro, suggesting a positive correlation between the availability of Tbet, CD39 activity and viability. Taken together, Tbet may preserve the ability of Tregs to cope with elevated toxic extracellular ATP at the inflammation site by tuning their CD39 expression or activity.

IRF3 signaling in stromal cells contributes to sepsis pathogenesis

Abstract Interferon regulatory factor 3 is a transcription factor that induces type 1 interferons and related inflammatory genes following activation of pattern recognition receptors. Our prior report demonstrates that IRF3-KO mice are substantially protected from sepsis in a lethal mouse model of cecal ligation and puncture (CLP). Here, we demonstrate that IRF3 remains relevant in the context of a CLP model incorporating clinical treatments (lactated ringer’s solution and antibiotics). 92.3% of IRF3-KO mice survived sepsis in this model, compared to 61.5% of wild-type mice. Septic IRF3-KO mice also had an improved disease score and lower levels of serum cytokines (IL-6 and IL-12), relative to wild-type counterparts. To examine if IRF3 exerts its effect within leukocytes, we generated bone marrow chimeras. We found that chimeras with IRF3-KO leukocytes did not exhibit a significant survival advantage over chimeras with wild-type leukocytes. However, chimeras with IRF3-KO stroma exhibited substantially improved survival relative to chimeras with wild-type stroma, paired with lower disease score and serum cytokines (IL-6 and IL-12). In vitro experiments with thioglycollate-elicited macrophages from IRF3-KO and wild-type mice demonstrate that IRF3 does not directly reduce the amount of IL-6 produced in response to cecal bacteria. However, wild-type monocytes that are adoptively transferred into septic hosts produce less IL-6 when the host is IRF3-KO vs wild-type. These experiments reveal the surprising finding that IRF3 exerts its major effect on sepsis pathogenesis through its presence in stromal cells, with indirect effects on leukocytes.

Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity.

Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.

Short-Term Effects of Microglia-Specific Mitochondrial Dysfunction on Amyloidosis in Transgenic Models of Alzheimer's Disease.

Reduction of mitochondrial activity is a subtle and early event in the pathogenesis of Alzheimer's disease. Mitochondrial damage and consequentially enhanced production of reactive oxygen species is particularly occurring in the vicinity of amyloid plaques. Since all cells are affected by mitochondrial damage, analyses of cell type-specific effects are challenging. To study the impact of mitochondrial alterations on microglial activity in a homogeneous genetic background, we generated bone marrow chimeras of irradiated 46-days-old APP-transgenic mice. For reconstitution, bone marrow from CX3CR1-eGFP mice with mitochondria of either non-obese diabetic or C57BL/6J animals was utilized. Successful reconstitution was evident in 100-day-old animals, by the presence of eGFP-positive cells in liver and spleen. In the brain, one-third of IBA1-positive microglia cells were newly recruited eGFP-expressing cells. Although donor-derived microglia were equally located in the proximity of amyloid plaques, no difference was observed in either the amyloid level, total number, or microglial coverage of plaques. These results indicate that during this brief and early phase of amyloid deposition, beneficial mitochondrial alterations in the newly recruited third of microglial cells were not sufficient to affect the amyloidosis in APP-transgenic mice.

MicroRNA signature of central nervous system-infiltrating dendritic cells in an animal model of multiple sclerosis.

Innate immune cells are integral to the pathogenesis of several diseases of the central nervous system (CNS), including multiple sclerosis (MS). Dendritic cells (DCs) are potent CD11c+ antigen-presenting cells that are critical regulators of adaptive immune responses, particularly in autoimmune diseases such as MS. The regulation of DC function in both the periphery and CNS compartment has not been fully elucidated. One limitation to studying the role of CD11c+ DCs in the CNS is that microglia can upregulate CD11c during inflammation, making it challenging to distinguish bone marrow-derived DCs (BMDCs) from microglia. Selective expression of microRNAs (miRNAs) has been shown to distinguish populations of innate cells and regulate their function within the CNS during neuro-inflammation. Using the experimental autoimmune encephalomyelitis (EAE) murine model of MS, we characterized the expression of miRNAs in CD11c+ cells using a non-biased murine array. Several miRNAs, including miR-31, were enriched in CD11c+ cells within the CNS during EAE, but not LysM+ microglia. Moreover, to distinguish CD11c+ DCs from microglia that upregulate CD11c, we generated bone marrow chimeras and found that miR-31 expression was specific to BMDCs. Interestingly, miR-31-binding sites were enriched in mRNAs downregulated in BMDCs that migrated into the CNS, and a subset was confirmed to be regulated by miR-31. Finally, miR-31 was elevated in DCs migrating through an invitro blood-brain barrier. Our findings suggest miRNAs, including miR-31, may regulate entry of DCs into the CNS during EAE, and could potentially represent therapeutic targets for CNS autoimmune diseases such as MS.

Endothelial Cell IQGAP1 is Required to Support Efficient Leukocyte Transmigration both In Vitro and In Vivo

Inflammation is the body's fundamental response to tissue insult and damage. When operating correctly, it allows the organism to fight infection, repair injuries, and return the body to homeostasis. When the response is incorrectly directed, whether insufficient, excessive, or against an improper target, it contributes to and even directly causes numerous human pathologies. One of the critical components of inflammation is the recruitment of leukocytes to the target site. This process is a carefully choreographed series of sequential steps that leads to the net efflux of leukocytes out of the blood stream and into tissue. In the culminating and putative committed stage, the leukocyte squeezes between adjacent endothelial cells in a process commonly referred to as transendothelial migration (TEM). TEM is a critical step that requires several intercellular interactions and thus represents an attractive therapeutic target for regulating aberrant inflammation. During TEM, endothelial cells direct the lateral border recycling compartment (LBRC), a sub‐junctional membrane network, to the site of leukocyte migration to facilitate its passage. All manipulations that affect the function and delivery of the LBRC substantially reduce leukocyte efflux.To better understand the molecular components and function of the LBRC, we recently conducted a screen for LBRC‐interacting proteins and identified IQGAP1. IQGAP1 is a large multi‐domain cytoplasmic protein that is reported to bind several signaling proteins in other systems. Knockdown of endothelial cell IQGAP1 disrupted the directed movement of the LBRC and substantially reduced leukocyte TEM. To determine which domains of IQGAP1 are required for its function in TEM, we expressed a series of truncated constructs and examined their ability to rescue the knockdown of endogenous IQGAP1. We found that the actin‐binding domain of IQGAP1 is required for its localization to endothelial borders. Furthermore, both the actin‐binding domain and the IQ‐motifs are required for its function in TEM. We extended these studies in vivo by generating bone marrow chimeras, in which IQGAP1 knockout animals were reconstituted with wild‐type leukocytes. These studies show that IQGAP1 in endothelium is required for efficient TEM in vivo in two different inflammatory models. Together these findings detail a novel function for IQGAP1 in TEM that involves both its actin‐binding domain and IQ motifs which together coordinate the directed movement of the LBRC to the site of TEM.Support or Funding InformationFunding Support: R01HL046849, R01HL064774This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chlamydia and Lipids Engage a Common Signaling Pathway That Promotes Atherogenesis

BackgroundRecent studies indicate that Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) signaling promote the development of high fat diet–induced atherosclerosis in hypercholesterolemic mice. ObjectivesThe authors investigated the role of TLR4/MyD88 signaling in hematopoietic and stromal cells in the development and infection-mediated acceleration of atherosclerosis. MethodsThe authors generated bone marrow chimeras between wild-type and Tlr4–/– mice, as well as wild-type and Myd88–/– mice. All mice were on the Apoe–/– background and fed high fat diet. The authors infected the chimeric mice with C. pneumoniae (CP) and fed them high fat diet. ResultsAortic sinus plaques and lipid content were significantly reduced in Apoe–/– mice that received Tlr4–/–or Myd88–/– bone marrow compared with control animals despite similar cholesterol levels. Similarly, Tlr4 or Myd88 deficiency in stromal cells also led to a reduction in the lesion area and lipid in aortic sinus plaques. MyD88 expression only in CD11c+ dendritic cells (myeloid cells) in cells was sufficient in otherwise MyD88-deficient mice to induce CP infection–mediated acceleration of atherosclerosis, underlining the key role of MyD88 in CD11c+ dendritic cells (myeloid cells). Whereas CP infection markedly accelerated atherosclerosis in TLR4- or MyD88-positive chimeras, CP infection had a minimal effect on atherosclerosis in TLR4- or MyD88-deficient mice (either in the hematopoietic or stromal cell compartments). ConclusionsThe authors show that both CP infection and metabolic stress associated with dyslipidemia use the same innate immune response pathway, utilizing TLR4/MyD88 signaling, with similar relative contributions in bone marrow–derived hematopoietic cells and in stromal cells. Further studies are required to understand this intricate and complex cross talk among innate and adaptive immune systems in various conditions to more effectively design dendritic cell–mediated atheroprotective vaccines and other therapeutic strategies.

Non-Hematopoietic β-Arrestin1 Confers Protection Against Experimental Colitis.

β-Arrestins are multifunctional scaffolding proteins that modulate G protein-coupled receptor (GPCR)-dependent and -independent cell signaling pathways in various types of cells. We recently demonstrated that β-arrestin1 (β-arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)-induced colitis in mice. Since DSS-induced colitis is in part dependent on gut epithelial injury, we examined the role of β-arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β-arr1 in SW480 cells enhanced epithelial cell death via a caspase-3-dependent process. To understand the in vivo relevance and potential cell type-specific role of β-arr1 in colitis development, we generated bone marrow chimeras with β-arr1 deficiency in either the hematopoietic or non-hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS-induced colitis. Similar to our previous findings, β-arr1 deficiency in the hematopoietic compartment protected mice from DSS-induced colitis. However, consistent with the role of β-arr1 in epithelial apoptosis in vitro, non-hematopoietic β-arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β-arr1 on TNF-α-mediated NFκB and MAPK pathways. Our results demonstrate that β-arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF-α in IECs. Together, our results show that β-arr1-dependent signaling in hematopoietic and non-hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β-arr1 in epithelial cells inhibits TNF-α-induced cell death pathways.

Neuropsychiatric systemic lupus erythematosus persists despite attenuation of systemic disease in MRL/lpr mice.

BackgroundSystemic lupus erythematosus (SLE) is a prototypical autoimmune disease marked by both B and T cell hyperactivity which commonly affects the joints, skin, kidneys, and brain. Neuropsychiatric disease affects about 40 % of SLE patients, most frequently manifesting as depression, memory deficits, and general cognitive decline. One important and yet unresolved question is whether neuropsychiatric SLE (NPSLE) is a complication of systemic autoimmunity or whether it is primarily driven by brain-intrinsic factors.MethodsTo dissect the relative contributions of the central nervous system from those of the hematopoietic compartment, we generated bone marrow chimeras between healthy control (MRL/+) and lupus-prone MRL/Tnfrsf6lpr/lpr mice (MRL/+ → MRL/lpr), as well as control chimeras. After bone marrow reconstitution, mice underwent extensive behavioral testing, analysis of brain tissue, and histological assessment.ResultsDespite transfer of healthy MRL/+ bone marrow and marked attenuation of systemic disease, we found that MRL/+ → MRL/lpr mice had a behavioral phenotype consisting of depressive-like behavior and visuospatial memory deficits, comparable to MRL/lpr → MRL/lpr control transplanted mice and the behavioral profile previously established in MRL/lpr mice. Moreover, MRL/+ → MRL/lpr chimeric mice displayed increased brain RANTES expression, neurodegeneration, and cellular infiltration in the choroid plexus, as well as blood brain barrier disruption, all in the absence of significant systemic autoimmunity.ConclusionsChimeric MRL/+ → MRL/lpr mice displayed no attenuation of the behavioral phenotype found in MRL/lpr mice, despite normalized serum autoantibodies and conserved renal function. Therefore, neuropsychiatric disease in the MRL/lpr lupus-prone strain of mice can occur absent any major contributions from systemic autoimmunity.

Neuropsychiatric lupus occurs independently of systemic autoimmunity (BA6P.120)

Abstract Neuropsychiatric disease is a common manifestation of systemic lupus erythematosus (SLE). Potential mechanisms underlying neuropsychiatric SLE (NPSLE) include thrombosis, complement deposition, brain-reactive autoantibodies, and cytokine mediated inflammation. However, it is not clear whether the neuropsychiatric manifestations of SLE are secondary to systemic disease, or a primary pathogenic process in the brain. In order to distinguish between central nervous system (CNS) and hematopoietic contributions, we generated bone marrow chimeras between lupus-prone MRL/lpr and congenic control MRL/+ mice. We monitored systemic disease progression, performed extensive behavioral testing, and analyzed brain tissue for evidence of inflammation. MRL/lpr (donor) → MRL/lpr (recipient) chimeric mice showed significantly elevated autoantibodies and marked proteinuria while MRL/+→MRL/+ and MRL/+→MRL/lpr did not, indicating abrogation of the MRL/lpr systemic disease phenotype. Surprisingly, MRL/+→MRL/lpr mice displayed a behavioral phenotype consistent with MRL/lpr→MRL/lpr mice, including depression-like behavior and increased spatial memory deficits. In contrast, MRL/+→MRL/+ mice behaved normally. Additionally, there was elevated RANTES expression, cellular infiltration, and neurodegeneration in MRL/lpr→MRL/lpr and MRL/+→MRL/lpr chimeric mice. The data presented herein indicate that the MRL/lpr CNS is sufficient to mediate NPSLE development absent significant hematopoietic contribution.

A transgenic mouse model correlates anomalous immune dynamics in human sickle cell disease (HUM1P.256)

Abstract Sickle cell disease, SCD, is an inherited blood disorder characterized by hardened, sticky C-shaped RBCs is a major cause of morbidity and mortality. It is the most prevalent genetic disease affecting 1/500 African Americans and 1/36000 Hispanics Americans, most residing in southeastern Virginia. Severe, frequent infections occur as a result of immune dysregulation associated with disease progression, impairing both innate and adaptive immunity. APCs such as DCs, B cells and macrophages express MHC II bind antigen that is recognized by TCR expressed on CD4+ Th cells. Thus, the interaction between receptor-ligand clusters on Th and APCs led to the activation of naïve CD4+ Th cells, stimulating the release of cytokines that influenced the activity of many cell types, including the NKT, CD8+ CTLs and the APCs that activated them. A “humanized” sickle mouse model of SCD was created by generating bone marrow chimeras using sickling mice donors and Pepboy recipients. We recently found that the transition from SPF to ‘dirty’, conventional, housing significantly dysregulated the immune system. These changes were characterized by a hyper-inflammatory state with the activation of innate-immune response, upregulation of TLR4, and loss of antigen-presentation machinery, loss of MHC II expression, with disease progression. Results indicate the loss of MHC II expression on APCs from various tissue of sickle mouse with the disease progression.