Gabriela Pacholczyk

Bovine Blastocyst Development from Oocytes Injected with Freeze-Dried Spermatozoa

Abstract Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4°C until use. After 22–24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 μM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.

Cutting Edge: DNA Sensing via the STING Adaptor in Myeloid Dendritic Cells Induces Potent Tolerogenic Responses

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αβ). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b+ DCs were the only cells to express IFN-β, whereas CD11b+ non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-β expression by CD11b+ DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.

STING Promotes the Growth of Tumors Characterized by Low Antigenicity via IDO Activation

Cytosolic DNA sensing is an important process during the innate immune response that activates the stimulator of interferon genes (STING) adaptor and induces IFN-I. STING incites spontaneous immunity during immunogenic tumor growth and accordingly, STING agonists induce regression of therapy-resistant tumors. However DNA, STING agonists, and apoptotic cells can also promote tolerogenic responses via STING by activating immunoregulatory mechanisms such as indoleamine 2,3 dioxygenase (IDO). Here, we show that IDO activity induced by STING activity in the tumor microenvironment (TME) promoted the growth of Lewis lung carcinoma (LLC). Although STING also induced IDO in tumor-draining lymph nodes (TDLN) during EL4 thymoma growth, this event was insufficient to promote tumorigenesis. In the LLC model, STING ablation enhanced CD8(+) T-cell infiltration and tumor cell killing while decreasing myeloid-derived suppressor cell infiltration and IL10 production in the TME. Depletion of CD8(+) T cells also eliminated the growth disadvantage of LLC tumors in STING-deficient mice, indicating that STING signaling attenuated CD8(+) T-cell effector functions during tumorigenesis. In contrast with native LLC tumors, STING signaling neither promoted growth of neoantigen-expressing LLC, nor did it induce IDO in TDLN. Similarly, STING failed to promote growth of B16 melanoma or to induce IDO activity in TDLN in this setting. Thus, our results show how STING-dependent DNA sensing can enhance tolerogenic states in tumors characterized by low antigenicity and how IDO inhibition can overcome this state by attenuating tumor tolerance. Furthermore, our results reveal a greater complexity in the role of STING signaling in cancer, underscoring how innate immune pathways in the TME modify tumorigenesis in distinct tumor settings, with implications for designing effective immunotherapy trials. Cancer Res; 76(8); 2076-81. ©2016 AACR.

Activation of the STING Adaptor Attenuates Experimental Autoimmune Encephalitis

Cytosolic DNA sensing activates the stimulator of IFN genes (STING) adaptor to induce IFN type I (IFN-αβ) production. Constitutive DNA sensing to induce sustained STING activation incites tolerance breakdown, leading to autoimmunity. In this study, we show that systemic treatments with DNA nanoparticles (DNPs) induced potent immune regulatory responses via STING signaling that suppressed experimental autoimmune encephalitis (EAE) when administered to mice after immunization with myelin oligodendrocyte glycoprotein (MOG), at EAE onset, or at peak disease severity. DNP treatments attenuated infiltration of effector T cells into the CNS and suppressed innate and adaptive immune responses to myelin oligodendrocyte glycoprotein immunization in spleen. Therapeutic responses were not observed in mice treated with cargo DNA or cationic polymers alone, indicating that DNP uptake and cargo DNA sensing by cells with regulatory functions was essential for therapeutic responses to manifest. Intact STING and IFN-αβ receptor genes, but not IFN-γ receptor genes, were essential for therapeutic responses to DNPs to manifest. Treatments with cyclic diguanylate monophosphate to activate STING also delayed EAE onset and reduced disease severity. Therapeutic responses to DNPs were critically dependent on IDO enzyme activity in hematopoietic cells. Thus, DNPs and cyclic diguanylate monophosphate attenuate EAE by inducing dominant T cell regulatory responses via the STING/IFN-αβ/IDO pathway that suppress CNS-specific autoimmunity. These findings reveal dichotomous roles for the STING/IFN-αβ pathway in either stimulating or suppressing autoimmunity and identify STING-activating reagents as a novel class of immune modulatory drugs.

Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line

Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models.

Use of Cryopreserved Pronuclear Embryos for the Production of Transgenic Mice

Abstract A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

Virus Infections Incite Pain Hypersensitivity by Inducing Indoleamine 2,3 Dioxygenase.

Increased pain sensitivity is a comorbidity associated with many clinical diseases, though the underlying causes are poorly understood. Recently, chronic pain hypersensitivity in rodents treated to induce chronic inflammation in peripheral tissues was linked to enhanced tryptophan catabolism in brain mediated by indoleamine 2,3 dioxygenase (IDO). Here we show that acute influenza A virus (IAV) and chronic murine leukemia retrovirus (MuLV) infections, which stimulate robust IDO expression in lungs and lymphoid tissues, induced acute or chronic pain hypersensitivity, respectively. In contrast, virus-induced pain hypersensitivity did not manifest in mice lacking intact IDO1 genes. Spleen IDO activity increased markedly as MuLV infections progressed, while IDO1 expression was not elevated significantly in brain or spinal cord (CNS) tissues. Moreover, kynurenine (Kyn), a tryptophan catabolite made by cells expressing IDO, incited pain hypersensitivity in uninfected IDO1-deficient mice and Kyn potentiated pain hypersensitivity due to MuLV infection. MuLV infection stimulated selective IDO expression by a discreet population of spleen cells expressing both B cell (CD19) and dendritic cell (CD11c) markers (CD19+ DCs). CD19+ DCs were more susceptible to MuLV infection than B cells or conventional (CD19neg) DCs, proliferated faster than B cells from early stages of MuLV infection and exhibited mature antigen presenting cell (APC) phenotypes, unlike conventional (CD19neg) DCs. Moreover, interactions with CD4 T cells were necessary to sustain functional IDO expression by CD19+ DCs in vitro and in vivo. Splenocytes from MuLV-infected IDO1-sufficient mice induced pain hypersensitivity in uninfected IDO1-deficient recipient mice, while selective in vivo depletion of DCs alleviated pain hypersensitivity in MuLV-infected IDO1-sufficient mice and led to rapid reduction in splenomegaly, a hallmark of MuLV immune pathogenesis. These findings reveal critical roles for CD19+ DCs expressing IDO in host responses to MuLV infection that enhance pain hypersensitivity and cause immune pathology. Collectively, our findings support the hypothesis elevated IDO activity in non-CNS due to virus infections causes pain hypersensitivity mediated by Kyn. Previously unappreciated links between host immune responses to virus infections and pain sensitivity suggest that IDO inhibitors may alleviate heightened pain sensitivity during infections.

Generation of C57BL/6 knockout mice using C3H × BALB/c blastocysts

The International Mouse Knockout Consortium aims to generate a knockout mouse for every single gene on a C57BL/6 background. Our ability to generate such mice is hampered by the poor economics of producing blastocysts to achieve germline transmission of C57BL/6 embryonic stem (ES) cells. We demonstrate superior utility of (C3H x BALB/c)F1 blastocysts compared with BALB/c blastocysts, with blastocyst numbers and germline transmission from subsequent chimeras at a rate 2- to 3-fold higher than that produced with BALB/c blastocysts.

Cutting edge: DNA sensing via the STING adaptor in myeloid dendritic cells s tolerogenic responses

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αβ). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b+ DCs were the only cells to express IFN-β, whereas CD11b+ non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-β expression by CD11b+ DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.

DNA sensing via the Stimulator of Interferon Genes (STING) adaptor in myeloid dendritic cells induces potent tolerogenic responses

Cytosolic DNA sensing via the stimulator of IFN genes (STING) adaptor incites autoimmunity by inducing type I IFN (IFN-αβ). In this study, we show that DNA is also sensed via STING to suppress immunity by inducing IDO. STING gene ablation abolished IFN-αβ and IDO induction by dendritic cells (DCs) after DNA nanoparticle (DNP) treatment. Marginal zone macrophages, some DCs, and myeloid cells ingested DNPs, but CD11b+ DCs were the only cells to express IFN-β, whereas CD11b+ non-DCs were major IL-1β producers. STING ablation also abolished DNP-induced regulatory responses by DCs and regulatory T cells, and hallmark regulatory responses to apoptotic cells were also abrogated. Moreover, systemic cyclic diguanylate monophosphate treatment to activate STING induced selective IFN-β expression by CD11b+ DCs and suppressed Th1 responses to immunization. Thus, previously unrecognized functional diversity among physiologic innate immune cells regarding DNA sensing via STING is pivotal in driving immune responses to DNA.