Wild-type Hosts Research Articles

T Cell-Specific STAT1 Expression Promotes Lytic Replication and Supports the Establishment of Gammaherpesvirus Latent Reservoir in Splenic B Cells.

ABSTRACTGammaherpesviruses establish lifelong infections in most vertebrate species, including humans and rodents, and are associated with cancers, including B cell lymphomas. While type I and II interferon (IFN) systems of the host are critical for the control of acute and chronic gammaherpesvirus infection, the cell type-specific role(s) of IFN signaling during infection is poorly understood and is often masked by the profoundly altered viral pathogenesis in the hosts with global IFN deficiencies. STAT1 is a critical effector of all classical IFN responses along with its involvement in other cytokine signaling pathways. In this study, we defined the effect of T cell-specific STAT1 deficiency on the viral and host parameters of infection with murine gammaherpesvirus 68 (MHV68). MHV68 is a natural rodent pathogen that, similar to human gammaherpesviruses, manipulates and usurps B cell differentiation to establish a lifelong latent reservoir in B cells. Specifically, germinal center B cells host the majority of latent MHV68 reservoir in the lymphoid organs, particularly at the peak of viral latency. Unexpectedly, T cell-specific STAT1 expression, while limiting the overall expansion of the germinal center B cell population during chronic infection, rendered these B cells more effective at hosting the latent virus reservoir. Further, T cell-specific STAT1 expression in a wild type host limited circulating levels of IFNγ, with corresponding increases in lytic MHV68 replication and viral reactivation. Thus, our study unveils an unexpected proviral role of T cell-specific STAT1 expression during gammaherpesvirus infection of a natural intact host.

Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6

BackgroundBacterial cells often harbor multiple plasmids, but the interaction between these plasmids is poorly understood. Pseudomonas putida ND6, one of the typical naphthalene-degrading bacteria, contains two annotated plasmids, pND6-1 and pND6-2. The conjugative plasmid pND6-2 can be used as a helper to mobilize the naphthalene-degrading plasmid pND6-1. The role of pND6-1 in functions other than naphthalene degradation has never been explored. Therefore, it is of great significance to study the possible effect of pND6-1 on the transfer rate or maintenance of pND6-2. ResultsTo investigate the interaction between pND6-1 and pND6-2, we successfully cured plasmid pND6-1 using the incompatibility method, generated the pND6-1-null strain ND6dp1, and first classified it into the IncP-7 group by curing experimental evidence. Subsequently, the pND6-1-cured mutant showed an insignificant increase in cell growth compared with the wild ND6, indicating that a lower cost was imposed on the host by pND6-1. The influences of pND6-1 on the stability and conjugation of plasmid pND6-2 were subsequently explored. The results showed that the presence of pND6-1 enhanced the stability of pND6-2 in natural host ND6 and heterologous host P. putida KT2440. In addition, the presence of pND6-1 in wild-type hosts promoted intra-specific and inter-specific conjugative transfer efficiency of pND6-2 by 3.9–6.3-fold with P. putida, P. resinovans, and P. fluorescens as the recipients, respectively. ConclusionsCollectively, these results suggest that the plasmid pND6-1 improves the pND6-2 plasmid's stability and transfer rate in the ND6 strain with the selected recipient strains.How to cite: Wang S, Liu M, Wang D, et al. Plasmid pND6-1 enhances the stability and conjugative transfer of co-resident companion plasmid pND6-2 in the naphthalene-degradative Pseudomonas putida strain ND6. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.08.002.

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2–/– mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone–like B cells when transferred into Rag2–/– lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Host CLIC4 expression in the tumor microenvironment is essential for breast cancer metastatic competence.

The TGF-β-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-β pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence.

Abstract 252: STAT3 signaling in the tumor microenvironment promotes primary gastric cancer growth and metastasis formation

Abstract Tumor cell intrinsic activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling has been shown to promote stemness, tumorigenesis and cancer therapy resistance. Therefore, Stat3 inhibitors are being developed and tested pre-clinically and in clinical trials for multiple solid cancers. Importantly, STAT3 signaling also occurs in the non-tumor cells within the tumor microenvironment (TME). The effect of Stat3 signaling within the TME on primary gastric cancers (GC), metastasis formation and therapy responses is unknown. Here, we describe a novel genetically defined, tumor organoid-driven, transplantable, orthotopic gastric cancer model that represents gastric carcinomas with associated metastasis formation in immune competent recipient mice. Utilizing our murine GC organoid model in subcutaneous allograft experiments, we showed that established GC tumors respond to pharmacological Stat3 inhibition. This anti-tumor effect is primarily driven by inhibition of the Stat3 signaling in the TME as tumor growth was increased in Stat3 high TME recipients, while tumor growth was inhibited in Stat3-signaling reduced Stat3+/- mice. Furthermore, Crisp-Cas9 -mediated Stat3 knockout in the GC organoids did not alter their in vitro growth potential. Stat3KO GC allograft tumor growth was not changed compared to Stat3WT GC tumors grown in wildtype host mice. Collectively, this suggests that inhibition of Stat3 signaling within the TME but not the tumor cell intrinsic Stat3 reduction confer anti-tumor responses. Through gastric serosa transplantation of the murine GC organoids into Stat3-elevated gp130FF mutant mice, we confirmed that high Stat3 signaling within the TME promotes distal metastasis formation to the liver. Taken together, we show that Stat3 signaling represents a therapy target, that predominantly acts through the TME. Better understanding of the signaling within the TME in primary and metastatic GC lesions will allow identification of novel tumor cell and TME-centric therapy targets. Combination of therapies targeting tumor cell intrinsic vulnerabilities with novel pro-tumorigenic TME -targeting agents could help to improve responses against primary and metastatic disease and therefore improve outcome for the worst prognosis patients. Citation Format: Moritz Eissmann, Anne Huber, Amr Allam, Christine Dijkstra, Matthias Ernst. STAT3 signaling in the tumor microenvironment promotes primary gastric cancer growth and metastasis formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 252.

NLRP6 negatively regulates type 2 immune responses in mice.

Inflammasomes are large protein complexes that assemble in the cytosol in response to danger such as tissue damage or infection. Following activation, inflammasomes trigger cell death and the release of biologically active forms of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammasome is required for IL-18 secretion by intestinal epithelial cells, macrophages, and T cells, contributing to homeostasis and self-defense against pathogenic microbes. However, the involvement of NLRP6 in type 2 lung inflammation remains elusive. Wild-type (WT) and Nlrp6-/- mice were used. Birch pollen extract (BPE)-induced allergic lung inflammation, eosinophil recruitment, Th2-related cytokine and chemokine production, airway hyperresponsiveness, and lung histopathology, Th2 cell differentiation, GATA3, and Th2 cytokines expression, were determined. Nippostrongylus brasiliensis (Nb) infection, worm count in intestine, type 2 innate lymphoid cell (ILC2), and Th2 cells in lungs were evaluated. We demonstrate in Nlrp6-/- mice that a mixed Th2/Th17 immune responses prevailed following birch pollen challenge with increased eosinophils, ILC2, Th2, and Th17 cell induction and reduced IL-18 production. Nippostrongylus brasiliensis infected Nlrp6-/- mice featured enhanced early expulsion of the parasite due to enhanced type 2 immune responses compared to WT hosts. In vitro, NLRP6 repressed Th2 polarization, as shown by increased Th2 cytokines and higher expression of the transcription factor GATA3 in the absence of NLRP6. Exogenous IL-18 administration partially reduced the enhanced airways inflammation in Nlrp6-/- mice. In summary, our data identify NLRP6 as a negative regulator of type 2 immune responses.

HoxB8‐conditional Neutrophil Progenitor Transplant Provides a Therapeutic Benefit for S. aureusPulmonary Infection in a Murine Model of Chronic Granulomatous Disease (CGD)

CGD is a congenital disease marked by neutrophil dysfunction leading to increased infectious susceptibility. Despite prophylactic treatment with antimicrobial agents, outcomes for CGD patients are troubling with a typical lifespan of 30‐40 years due to breakthrough infections. As we strive to stay ahead of the impacts of drug‐resistant microbes, it is reasonable to consider cellular therapeutics which replace immune cells to treat CGD and other immunodeficiencies. We hypothesize that a conditionally‐immortalized neutrophil progenitor cell line has a translational application as a therapy for immune compromised hosts. This cell line was first generated by Wang, et al. through inducing ectopic expression of the transcription factor HoxB8 in murine bone marrow stem cells. Induction of HoxB8 leads to exponential growth of cells, while withdrawal leads to differentiation into neutrophil‐like cells. We have found that unconditioned transplant of HoxB8 progenitors lead to robust engraftment in the bone marrow. Engrafted progenitor cells differentiate in vivointo donor‐derived neutrophils found circulating in the peripheral blood. Donor neutrophils are also recruited to the lungs of mice infected with Staphylococcus aureus. Using a novel fluorescent killing assay, we demonstrate that donor‐derived neutrophils can kill pulmonary S. aureus both in vitro and in vivo with comparable efficiency to wild type host neutrophils. Strikingly, our results find that treatment with HoxB8‐conditional progenitors improves survival of CGD animals infected with S. aureus from 0% to >50%. This work demonstrates the potential of conditionally‐immortalized neutrophil progenitors as a novel cellular therapy in the context of neutrophil dysfunction.

PD-1H/VISTA expressed on host myeloid cells regulates acute graft-versus-host disease (aGVHD)

Abstract Programmed Death-1 Homolog (PD-1H, also known as VISTA) is a co-inhibitory molecule of the immunoglobulin superfamily and broadly found in hematopoietic cells. We previously demonstrated that PD-1H agonists prevent aGVHD in a murine MHC mismatched bone marrow (BM) transplantation model. aGVHD occurs when donor T cells are primed and activated by host antigen-presenting cells (APCs). PD-1H agonism suppressed donor allogeneic T cell activation leading to the suppression of aGVHD. However, it remains unknown whether PD-1H agonists also suppress host myeloid APCs, in which PD-1H is highly expressed. To test this, we adoptively transferred wildtype (WT) C57B/L6 (B6) BM into either PD-1H knock-out (KO) or WT BALB/c mice as hosts. PD-1H KO host mice had more severe aGVHD clinical scores and a worse survival rate when compared to WT host mice. To determine whether PD-1H agonism impacts host immune cells, we transferred B6 PD-1H KO BM into BALB/c WT host mice and treated host recipients with or without PD-1H agonists. Consistent with prior data, host mice treated with PD-1H agonists suppressed aGVHD and extended survival compared with isotype control treated mice. To test which cell subsets expressing PD-1H in host mice regulate aGVHD, we transferred WT BALB/c BM into myeloid lineage specific KO of PD-1H or WT littermate hosts. Interestingly, the myeloid lineage specific KO mice host had significantly worse survival rates and worse aGVHD symptoms compared with WT littermates. Our studies indicate that PD-1H expression on host myeloid cells regulates aGVHD in addition to donor T cell PD-1H. Finding new mechanisms of PD-1H agonism on aGVHD enables development of future therapeutic approaches for aGVHD treatment, such as myeloid APC suppression. Supported by an Edward P. Evans Foundation Young Investigator Award, a Conquer Cancer Foundation American Society of Clinical Oncology Career Development Grant, and an American Cancer Society Clinician Scientist Development Grant

Cyp11a2 Is Essential for Oocyte Development and Spermatogonial Stem Cell Differentiation in Zebrafish.

Cytochrome P45011A1, encoded by Cyp11a1, converts cholesterol to pregnenolone (P5), the first and rate-limiting step in steroidogenesis. In zebrafish, cyp11a1 is maternally expressed and cyp11a2 is considered the ortholog of Cyp11a1 in mammals. A recent study has shown that depletion of cyp11a2 resulted in steroidogenic deficiencies and the mutants developed into males with feminized secondary sexual characteristics. Here, we independently generated cyp11a2 mutants in zebrafish and showed that the mutants can develop into males and females in the juvenile stage, but finally into infertile males with defective mating behavior in the adult stage. In the developing ovaries, the cyp11a2 mutation led to stage I oocyte apoptosis and final sex reversal, which could be partially rescued by treatment with P5 but not estradiol. In the developing testes, depletion of cyp11a2 resulted in dysfunction of Sertoli cells and lack of functional Leydig cells. Spermatogonial stem cells (SSCs) in the mutant testes underwent active self-renewal but no differentiation, resulting in a high abundance of SSCs in the testis, as revealed by immunofluorescence staining with Nanos2 antibody. The high abundance and differentiation competence of SSCs in the mutant testes were verified by a novel testicular cell transplantation method developed in this study, by transplanting mutant testicular cells into germline-depleted wild-type (WT) fish. The transplanted mutant SSCs efficiently differentiated into functional spermatids in WT hosts. Overall, our study demonstrates the functional importance of cyp11a2 in early oogenesis and differentiation of SSCs.

Role of mesonephric contribution to mouse testicular development revisited

The role of the mesonephros in testicular development was re-evaluated by growing embryonic day 11.5 (E11.5) mouse testes devoid of mesonephros for 8–21 days in vivo under the renal capsule of castrated male athymic nude mice. This method provides improved growth conditions relative to previous studies based upon short-term (4–7 days) organ culture. Meticulous controls involved wholemount examination of dissected E11.5 mouse testes as well as serial sections of dissected E11.5 mouse testes which were indeed shown to be devoid of mesonephros. As expected, grafts of E11.5 mouse testes with mesonephros attached formed seminiferous tubules and also contained mesonephric derivatives. Grafts of E11.5 mouse testes without associated mesonephros also formed seminiferous tubules and never contained mesonephric derivatives. The consistent absence of mesonephric derivatives in grafts of E11.5 mouse testes grafted alone is further proof of the complete removal of the mesonephros from the E11.5 mouse testes. The testicular tissues that developed in grafts of E11.5 mouse testes alone contained canalized seminiferous tubules composed of Sox9-positive Sertoli cells as well as GENA-positive germ cells. The seminiferous tubules were surrounded by α-actin-positive myoid cells, and the interstitial space contained 3βHSD-1-positive Leydig cells. Grafts of E11.5 GFP mouse testes into wild-type hosts developed GFP-positive vasculature indicating that E11.5 mouse testes contain vascular precursors. These results indicate that the E11.5 mouse testis contains precursor cells for Sertoli cells, Leydig cells, myoid cells and vasculature whose development and differentiation are independent of cells migrating from the E11.5 mesonephros.

Deubiquitinating Enzyme USP8 Is Essential for Skeletogenesis by Regulating Wnt Signaling.

Disturbance in a differentiation program of skeletal stem cells leads to indecorous skeletogenesis. Growing evidence suggests that a fine-tuning of ubiquitin-mediated protein degradation is crucial for skeletal stem cells to maintain their stemness and osteogenic potential. Here, we demonstrate that the deubiquitinating enzyme (DUB) ubiquitin-specific protease 8 (USP8) stabilizes the Wnt receptor frizzled 5 (FZD5) by preventing its lysosomal degradation. This pathway is essential for Wnt/β-catenin signaling and the differentiation of osteoprogenitors to mature osteoblasts. Accordingly, deletion of USP8 in osteoprogenitors (Usp8Osx) resulted in a near-complete blockade in skeletal mineralization, similar to that seen in mice with defective Wnt/β-catenin signaling. Likewise, transplanting USP8-deficient osteoprogenitors under the renal capsule in wild-type secondary hosts did not to induce bone formation. Collectively, this study unveils an essential role for the DUB USP8 in Wnt/β-catenin signaling in osteoprogenitors and osteogenesis during skeletal development.

Patch grafting, strategies for transplantation of organoids into solid organs such as liver

Epithelial cell therapies have been at an impasse because of inefficient methods of transplantation to solid organs. Patch grafting strategies were established enabling transplantation of ≥107th organoids/patch of porcine GFP+ biliary tree stem/progenitors into livers of wild type hosts. Grafts consisted of organoids embedded in soft (~100 Pa) hyaluronan hydrogels, both prepared in serum-free Kubota's Medium; placed against target sites; covered with a silk backing impregnated with more rigid hyaluronan hydrogels (~700 Pa); and use of the backing to tether grafts with sutures or glue to target sites. Hyaluronan coatings (~200–300 Pa) onto the serosal surface of the graft served to minimize adhesions with neighboring organs. The organ's clearance of hyaluronans enabled restoration of tissue-specific paracrine and systemic signaling, resulting in return of normal hepatic histology, with donor parenchymal cells uniformly integrated amidst host cells and that had differentiated to mature hepatocytes and cholangiocytes. Grafts containing donor mature hepatocytes, partnered with endothelia, and in the same graft biomaterials as for stem/progenitor organoids, did not engraft. Engraftment occurred if porcine liver-derived mesenchymal stem cells (MSCs) were co-transplanted with donor mature cells. RNA-seq analyses revealed that engraftment correlated with expression of matrix-metalloproteinases (MMPs), especially secreted isoforms that were found expressed strongly by organoids, less so by MSCs, and minimally, if at all, by adult cells.Engraftment with patch grafting strategies occurred without evidence of emboli or ectopic cell distribution. It was successful with stem/progenitor organoids or with cells with a source(s) of secreted MMP isoforms and offers significant potential for enabling cell therapies for solid organs.

Ulk1 Kinase Mediates Clearance of Free α-Globin in β-Thalassemia

Normal erythroid maturation is highly regulated to maximize the production of HbA heterotetramer (α2β2) and minimize the accumulation of potentially toxic free α- or β-globin subunits. In β-thalassemia, β-globin gene (HBB) mutations cause buildup of free α-globin, which forms intracellular precipitates that impair erythroid cell maturation and viability. Endogenous mechanisms exist to detoxify free α-globin, as evidenced by clinical observations that most individuals with HBB haploinsufficiency (β-thalassemia trait) are asymptomatic. Previously, we showed that free α-globin is eliminated in erythroid precursors by the ubiquitin-proteasome (UPS) system and by a lysosomal-dependent process presumed to be autophagy (Khandros et al., Blood 2012;119:5265). Our current study investigates the latter.Atg5 is required for basal autophagy and autophagosome formation via conjugation with Atg12 in most cell types. We investigated whether Atg5 played a selective role in eliminating free α-globin during unbalanced globin chain synthesis. β-thalassemic mice (HbbTh3/+) carrying a conditional Atg5 allele (Atg5fl) in which exon 3 is flanked by loxP sites were interbred with mice expressing erythroid-specific Cre recombinase (EpoR-Cre). Loss of Atg5 in β-thalassemic erythroid precursors inhibited autophagy, as evidenced by the accumulation of the markers p62/SQSTM1 and LC3. However, there was minimal effect on the β-thalassemic phenotype as measured by RBC count, Hb, hematocrit, reticulocyte count, and spleen weight. Moreover, loss of Atg5 did not alter the level of insoluble α-globin in β-thalassemic RBC fractions, as assessed by triton-acetic acid-urea (TAU) gel electrophoresis to resolve the α- and β-globin chains. Thus, Atg5, a key component of canonical autophagy, is not required for the elimination of free α-globin in β-thalassemia.The Ulk1 (unc-51−like kinase 1) protein kinase participates in canonical and alternative autophagy pathways and mediates mitophagy during erythropoiesis. To determine the role of Ulk1 in a-globin degradation, we introduced a null allele into HbbTh3/+ mice. Ulk1−/− HbbTh3/+ mice were born at normal Mendelian ratio, but exhibited a high rate of perinatal death. Only 20% of double-mutant mice survived for 30 days (n=5), compared to 70% of Ulk1+/+ HbbTh3/+ mice (n=13) (P <0.05). To examine the consequences of Ulk1 loss in β-thalassemic erythroblasts, we transplanted whole bone marrow cells from double mutants and controls (CD45.2) into lethally irradiated wild-type hosts (CD45.1; >87% engraftment after 30 days). Loss of Ulk1 exacerbated the β-thalassemic phenotype, as evidence by reduced Hb (10.85 ± 0.18 vs 10.21 ± 0.18 g/dL, n=14 vs 8; P <0.01), reduced RBC number (7.37 ± 0.13 vs 6.35 ± 0.09 × 106/mL; P <0.0001), increased reticulocyte count (17.99 ± 2.76 vs 39.41 ± 2.14%; P <0.0001), and increased spleen weight (0.24 ± 0.01 vs 0.34 ± 0.03 g; P <0.01). Moreover, insoluble α-globin was increased by approximately 2-fold in Ulk1−/− HbbTh3/+ reticulocytes, ascompared to Ulk1+/+ HbbTh3/+ cells (Figure). To assess the turnover of newly synthesized free α-globin, we pulse-labeled reticulocytes with 35S amino acids and chased with or without a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine or bafilomycin A1). Analysis of control (Ulk1+/+ HbbTh3/+) reticulocytesshowed that newly synthesized insoluble free α-globin was cleared by separate proteasomal and lysosomal pathways. In marked contrast, the lysosomal-dependent α-globin degradation pathway was completely eliminated in Ulk1−/− HbbTh3/+ reticulocytes. Thus, we conclude that Ulk1 mediates the degradation of free α-globin in β-thalassemia.Our findings illustrate a new mechanism through which protein quality-control pathways can modulate the severity of β-thalassemia by eliminating unstable free α-globin, and they open up possibilities for new therapeutic approaches, as Ulk1 activity can be modulated pharmacologically. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Virus-Host Interaction Gets Curiouser and Curiouser. PART II: Functional Transcriptomics of the E. coli DksA-Deficient Cell upon Phage P1vir Infection.

The virus–host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage–host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA− hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.

LATERAL ORGAN BOUNDARIES DOMAIN 25 functions as a key regulator of haustorium development in dodders.

Parasitic plants reduce crop yield worldwide. Dodder (Cuscuta campestris) is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, LATERAL ORGAN BOUNDARIES DOMAIN 25 (CcLBD25), as highly expressed in prehaustoria and haustoria. Gene coexpression networks from different tissue types and laser-capture microdissection RNA-sequencing data indicated that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing by generating transgenic tomato (Solanum lycopersicum) hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had reduced biomass compared with C. campestris growing on wild-type (WT) hosts, suggesting that parasites growing on transgenic plants were stressed due to insufficient nutrient acquisition. We developed an in vitro haustorium system to assay the number of prehaustoria produced on strands from C. campestris. Cuscuta campestris grown on CcLBD25 RNAi tomatoes produced fewer prehaustoria than those grown on WT tomatoes, indicating that down-regulating CcLBD25 may affect haustorium initiation. Cuscuta campestris haustoria growing on CcLBD25 RNAi tomatoes exhibited reduced pectin digestion and lacked searching hyphae, which interfered with haustorium penetration and formation of vascular connections. The results of this study elucidate the role of CcLBD25 in haustorium development and might contribute to developing parasite-resistant crops.

Repression of MUC1 Promotes Expansion and Suppressive Function of Myeloid-Derived Suppressor Cells in Pancreatic and Breast Cancer Murine Models.

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-β, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.

Variability of plasmid fitness effects contributes to plasmid persistence in bacterial communities

Plasmid persistence in bacterial populations is strongly influenced by the fitness effects associated with plasmid carriage. However, plasmid fitness effects in wild-type bacterial hosts remain largely unexplored. In this study, we determined the fitness effects of the major antibiotic resistance plasmid pOXA-48_K8 in wild-type, ecologically compatible enterobacterial isolates from the human gut microbiota. Our results show that although pOXA-48_K8 produced an overall reduction in bacterial fitness, it produced small effects in most bacterial hosts, and even beneficial effects in several isolates. Moreover, genomic results showed a link between pOXA-48_K8 fitness effects and bacterial phylogeny, helping to explain plasmid epidemiology. Incorporating our fitness results into a simple population dynamics model revealed a new set of conditions for plasmid stability in bacterial communities, with plasmid persistence increasing with bacterial diversity and becoming less dependent on conjugation. These results help to explain the high prevalence of plasmids in the greatly diverse natural microbial communities.

Stroma-derived Notch signals drive transdifferentiation of follicular B cells into marginal zone B cells during lymphopenia

Abstract In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain poorly understood. To evaluate the homeostasis of the mature B cell pool during lymphopenia, we developed an adoptive transfer model of follicular B (FoB) cells into Rag2−/− mouse recipients. We report that highly purified FoB cells transdifferentiate into marginal zone (MZ)-like B cells when transferred into Rag2−/− lymphopenic hosts, but not into wild type hosts. In lymphopenic spleens, transferred B cells gradually lost their FoB phenotype and acquired multiple characteristics of MZ B cells, as judged by their cell surface phenotype (sIgMhi sIgDlow CD21hi CD1dhi CD23low), expression of integrins and chemokine receptors, positioning close to the marginal sinus, and functional ability to rapidly generate plasma cells. Initiation of FoB to MZ B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by Ccl19-Cre+ fibroblastic stromal cells, a pathway also required for MZ B cell development in steady-state conditions. We postulate that stromal Notch ligands are critical to regulate the size and composition of the splenic B cell pool via interactions with Notch2 receptors in B cells. Thus, FoB cells are not locked in their FoB cell fate, but are are endowed with plastic transdifferentiation potential in response to increased stromal Dll1 availability in lymphopenic environments.

STAT1 protects CD4 T cells from deletion by NK cells during influenza infection and promotes Th1 identity that can be enhanced through STAT4 to improve their anti-viral impact

Abstract Most CD4 T cells responding to influenza A virus (IAV) fit prototypical Th1 criteria including high expression of the transcription factor T-bet, the Th1 ‘master regulator’, and strong IFNg production. Optimal Th1 development in vitro also requires cytokine-dependent activation of the transcription factors STAT4 and STAT1, but how these pathways impact anti-viral responses is not clear. Here, to focus on the roles of CD4 T cell-intrinsic STAT4 and STAT1, we transferred STAT-deficient (STAT−/−) or wildtype CD4 T cells recognizing virus to congenic wildtype hosts, challenged with IAV, and analyzed donor effector responses in the infected lungs. STAT4−/− and wildtype cells reached similar numbers with no differences seen in their Th1 identity. In contrast, STAT1−/− donor cells were barely detected in host mice but reached similar numbers to wildtype donors when NK cells were depleted. Analysis of STAT1−/− cells in NK cell-depleted hosts revealed severely compromised Th1 identity that mirrored T-bet−/− donor cell responses. While these findings suggest that full Th1-polarization during IAV infection does not require STAT4, treatment of mice with IL-12 dramatically increases Th1 attributes in wildtype but not STAT4−/−CD4 T cells. This indicates that the wildtype IAV-primed CD4 T cell response is not strongly Th1-polarized. Finally, priming CD4 T cells in vitro with STAT4- and STAT1-activating cytokines promotes effector cells better able to combat IAV when transferred to unprimed mice than when only STAT1 activating signals are present. Enhancing Th1 identity through harnessing CD4 T cell-intrinsic STAT4 activation can thus improve their anti-viral impact during infection, with important implications for vaccine design.

Transcriptomics analysis of Toxoplasma gondii-infected mouse macrophages reveals coding and noncoding signatures in the presence and absence of MyD88

BackgroundToxoplasma gondii is a globally distributed protozoan parasite that establishes life-long asymptomatic infection in humans, often emerging as a life-threatening opportunistic pathogen during immunodeficiency. As an intracellular microbe, Toxoplasma establishes an intimate relationship with its host cell from the outset of infection. Macrophages are targets of infection and they are important in early innate immunity and possibly parasite dissemination throughout the host. Here, we employ an RNA-sequencing approach to identify host and parasite transcriptional responses during infection of mouse bone marrow-derived macrophages (BMDM). We incorporated into our analysis infection with the high virulence Type I RH strain and the low virulence Type II strain PTG. Because the well-known TLR-MyD88 signaling axis is likely of less importance in humans, we examined transcriptional responses in both MyD88+/+ and MyD88−/− BMDM. Long noncoding (lnc) RNA molecules are emerging as key regulators in infection and immunity, and were, therefore, included in our analysis.ResultsWe found significantly more host genes were differentially expressed in response to the highly virulent RH strain rather than with the less virulent PTG strain (335 versus 74 protein coding genes for RH and PTG, respectively). Enriched in these protein coding genes were subsets associated with the immune response as well as cell adhesion and migration. We identified 249 and 83 non-coding RNAs as differentially expressed during infection with RH and PTG strains, respectively. Although the majority of these are of unknown function, one conserved lncRNA termed mir17hg encodes the mir17 microRNA gene cluster that has been implicated in down-regulating host cell apoptosis during T. gondii infection. Only a minimal number of transcripts were differentially expressed between MyD88 knockout and wild type cells. However, several immune genes were among the differences. While transcripts for parasite secretory proteins were amongst the most highly expressed T. gondii genes during infection, no differentially expressed parasite genes were identified when comparing infection in MyD88 knockout and wild type host BMDM.ConclusionsThe large dataset presented here lays the groundwork for continued studies on both the MyD88-independent immune response and the function of lncRNAs during Toxoplasma gondii infection.