Transplanting fecal material from wild‐type mice fed black raspberries alters the immune system of recipient mice

AbstractWell-adapted pathogens must evade clearance by the host immune system and the study of how they do this has revealed myriad complex strategies and mechanisms. Classical bordetellae are very closely related subspecies that are known to modulate adaptive immunity in a variety of ways, permitting them to either persist for life or repeatedly infect the same host. Exploring the hypothesis that exposure to immune cells would cause bordetellae to induce expression of important immunomodulatory mechanisms, we identified a putative regulator of an immunomodulatory pathway. The deletion of btrS in B. bronchiseptica did not affect colonization or initial growth in the respiratory tract of mice, its natural host, but did increase activation of the inflammasome pathway, and recruitment of inflammatory cells. The mutant lacking btrS recruited many more B and T cells into the lungs, where they rapidly formed highly organized and distinctive Bronchial Associated Lymphoid Tissue (BALT) not induced by any wild type Bordetella species, and a much more rapid and strong antibody response than observed with any of these species. Immunity induced by the mutant was measurably more robust in all respiratory organs, providing completely sterilizing immunity that protected against challenge infections for many months. Moreover, the mutant induced sterilizing immunity against infection with other classical bordetellae, including B. pertussis and B. parapertussis, something the current vaccines do not provide. These findings reveal profound immunomodulation by bordetellae and demonstrate that by disrupting it much more robust protective immunity can be generated, providing a pathway to greatly improve vaccines and preventive treatments against these important pathogens.

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Creation and Preliminary Characterization of a Leptin Knockout Rat

Endocrinology ◽

10.1210/en.2012-1462 ◽

2012 ◽

Vol 153 (11) ◽

pp. 5622-5628 ◽

Cited By ~ 25

Author(s):

Sergio Vaira ◽

Chang Yang ◽

Aaron McCoy ◽

Kelly Keys ◽

Shurong Xue ◽

...

Keyword(s):

Bone Mineral Density ◽

Immune System ◽

Insulin Sensitivity ◽

Bone Metabolism ◽

Bone Volume ◽

Wild Type ◽

Mineral Density ◽

Trabecular Bone Mineral Density ◽

Zinc Finger Nuclease Technology

Abstract Leptin, a cytokine-like hormone secreted mainly by adipocytes, regulates various pathways centered on food intake and energy expenditure, including insulin sensitivity, fertility, immune system, and bone metabolism. Here, using zinc finger nuclease technology, we created the first leptin knockout rat. Homozygous leptin null rats are obese with significantly higher serum cholesterol, triglyceride, and insulin levels than wild-type controls. Neither gender produced offspring despite of repeated attempts. The leptin knockout rats also have depressed immune system. In addition, examination by microcomputed tomography of the femurs of the leptin null rats shows a significant increase in both trabecular bone mineral density and bone volume of the femur compared with wild-type littermates. Our model should be useful for many different fields of studies, such as obesity, diabetes, and bone metabolism-related illnesses.

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The Immune Phenotype of Three Drosophila Leukemia Models

G3 Genes|Genome|Genetics ◽

10.1534/g3.117.039487 ◽

2017 ◽

Vol 7 (7) ◽

pp. 2139-2149 ◽

Cited By ~ 7

Author(s):

Badrul Arefin ◽

Martin Kunc ◽

Robert Krautz ◽

Ulrich Theopold

Keyword(s):

Immune System ◽

Tumor Suppressors ◽

Natural Infection ◽

Fruit Fly ◽

Innate Immune ◽

Infection Model ◽

Tissue Formation ◽

Wild Type ◽

Behavioral Abnormalities ◽

Drosophila Models

Abstract Many leukemia patients suffer from dysregulation of their immune system, making them more susceptible to infections and leading to general weakening (cachexia). Both adaptive and innate immunity are affected. The fruit fly Drosophila melanogaster has an innate immune system, including cells of the myeloid lineage (hemocytes). To study Drosophila immunity and physiology during leukemia, we established three models by driving expression of a dominant-active version of the Ras oncogene (RasV12) alone or combined with knockdowns of tumor suppressors in Drosophila hemocytes. Our results show that phagocytosis, hemocyte migration to wound sites, wound sealing, and survival upon bacterial infection of leukemic lines are similar to wild type. We find that in all leukemic models the two major immune pathways (Toll and Imd) are dysregulated. Toll–dependent signaling is activated to comparable extents as after wounding wild-type larvae, leading to a proinflammatory status. In contrast, Imd signaling is suppressed. Finally, we notice that adult tissue formation is blocked and degradation of cell masses during metamorphosis of leukemic lines, which is akin to the state of cancer-dependent cachexia. To further analyze the immune competence of leukemic lines, we used a natural infection model that involves insect-pathogenic nematodes. We identified two leukemic lines that were sensitive to nematode infections. Further characterization demonstrates that despite the absence of behavioral abnormalities at the larval stage, leukemic larvae show reduced locomotion in the presence of nematodes. Taken together, this work establishes new Drosophila models to study the physiological, immunological, and behavioral consequences of various forms of leukemia.

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The Maternal Immune Response to in Utero Hematopoietic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v114.22.64.64 ◽

2009 ◽

Vol 114 (22) ◽

pp. 64-64

Author(s):

Tippi MacKenzie ◽

Erin Jarvis ◽

Amar Nijagal ◽

Tom Le ◽

Marta Wegorzewska ◽

...

Keyword(s):

Immune Response ◽

Stem Cell ◽

Immune System ◽

B Cells ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

In Utero ◽

Wild Type ◽

Hematopoietic Stem ◽

Adaptive Immune

Abstract Abstract 64 In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising treatment strategy for many congenital hematopoietic disorders such as immunodeficiencies. However, clinical applications have been hampered by lack of engraftment, possibly secondary to a host immune response. This has been a conundrum in the field, since the fetus can also be tolerized to allogeneic cells in some circumstances. We hypothesized that it is the maternal immune response which limits engraftment of in utero transplanted cells. Methods: Fetal BALB/c mice at 14 days' gestation were transplanted with age-matched fetal liver (FL) cells (2.5 × 106 cells/fetus) from allogeneic C57B6 mice and levels of circulating donor cell chimerism were determined serially starting at 4 weeks after in utero transplantation. Rates of engraftment (number of chimeric pups/number of surviving pups) and levels of chimerism (donor CD45 cells/total CD45 cells) were compared to controls in which animals were transplanted with congenic cells (C57B6 (CD45.2) fetal hosts transplanted with C57B6 (CD45.1) FL). In order to determine the role of the maternal adaptive immune system, immunodeficient BALB/c.Rag−/− mothers (deficient in T and B cells) were bred to wild type BALB/c males, such that the fetuses (BALB/c.Rag+/−) would be immunocompetent. These fetuses were transplanted with C57B6 FL and rates of engraftment and levels of chimerism in these transplants were compared to those in wild type allogeneic transplants. In order to determine whether the maternal influence is caused by maternal lymphocytes trafficking into the fetus, C57B6 (CD45.2) females were bred to C57B6 (CD45.1) males, such that the fetal cells (CD45.1+/CD45.2+) could be distinguished from maternal cells (CD45.1−/CD45.2+). Fetal blood and tissues were examined for the presence of maternal cells by flow cytometry at various gestational ages. Results: The rate of engraftment after IUHSCTx in control animals transplanted with congenic cells was 14/16 (88%) and average levels of chimerism were 9.9±8.4%. In contrast, the rate of engraftment in wild-type BALB/c fetuses transplanted with allogeneic B6 cells was 11/25 (44%; p<0.05 compared to congenic), and levels of chimerism were 21±19 (p=NS), confirming there is an adaptive immune response to fetal stem cell transplantation. As expected, chimeric animals were tolerant to the donor strain by mixed lymphocyte reaction while injected, non-chimeric animals were sensitized. However, in the absence of a maternal adaptive immune system, rates of chimerism (in immunocompetent BALB/c.Rag+/− pups) increased to 100% (n=10, p<0.05 compared to wild type allogeneic) and levels of chimerism were significantly higher (44±18, p<0.05). Levels of chimerism in engrafted animals declined over time after allogeneic transplantation but not after congenic transplantation, indicating there is a second, late phase immune response to allogeneic cells. However, chimerism levels did not decline in the BALB/c.Rag+/− recipients, suggesting that the maternal immune system has long-lasting effects on the success of fetal transplantation, perhaps by priming the host immune system. In our analysis of maternal/fetal cellular trafficking, we detected maternal lymphocytes in the blood of midgestation fetuses (14±7% at E12.5–E14.5, n=9) which declined gradually and was undetectable after birth. Lineage analysis demonstrated that 45±15 % of maternal cells are Gr-1+ granulocytes and 21±15% are B cells. Trafficking of maternal cells into the fetus was increased following fetal manipulation (injection of PBS < injection of allogeneic HSC). Conclusions: There is an adaptive immune response which limits early engraftment after in utero transplantation of allogeneic cells and leads to a gradual decline in levels of chimerism in engrafted animals. However, in the selective absence of maternal T and B cells, all fetuses transplanted with allogeneic FL cells show long-term, multilineage engraftment and demonstrate donor-specific tolerance. These results indicate that the maternal immune system plays a significant role in the success of fetal HSC transplantation. Cellular trafficking between the mother and fetus may be a mechanism by which maternal lymphocytes encounter cells transplanted into the fetus. Our findings have clinical implications in that the success of IUHSCTx may be improved by harvesting cells from the mother or HLA-matching cells to the mother. Disclosures: No relevant conflicts of interest to declare.

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A Critical Role for Innate Immunity In Allogeneic Hematopoietic Cell Rejection Via the TLR4/TRIF Pathway

Blood ◽

10.1182/blood.v116.21.77.77 ◽

2010 ◽

Vol 116 (21) ◽

pp. 77-77

Author(s):

Hong Xu ◽

Jun Yan ◽

Ziqiang Zhu ◽

Yiming Huang ◽

Yujie Wen ◽

...

Keyword(s):

Bone Marrow ◽

Innate Immunity ◽

T Cells ◽

Immune System ◽

T Cell ◽

Innate Immune System ◽

Critical Role ◽

Innate Immune ◽

Wild Type

Abstract Abstract 77 Adaptive immunity, especially T cells, has long been believed to be the dominant immune barrier in allogeneic transplantation. Targeting host T cells significantly reduces conditioning for bone marrow cell (BMC) engraftment. Innate immunity has been recently shown to pose a significant barrier in solid organ transplantation, but has not been addressed in bone marrow transplantation (BMT). Using T cell deficient (TCR-β/δ−/−) or T and B cell deficient (Rag−/−) mice, we found that allogeneic BMC rejection occurred early before the time required for T cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. Therefore, we hypothesized that by controlling both innate and adaptive immunity, the donor BMC would have a window of advantage to engraft. Survival of BMC in vivo was significantly improved by depleting recipient macrophages and/or NK cells, but not neutrophils. Moreover, depletion of macrophages and NK cells in combination with co-stimulatory blockade with anti-CD154 and rapamycin as a novel form of conditioning resulted in 100% allogeneic engraftment without any irradiation and T cell depletion. Donor chimerism remained stable and durable up to 6 months. Moreover, specific Vβ5½ and Vβ11 clonal deletion was detected in host CD4+ T cells in chimeras, indicating central tolerance to donor alloantigens. Whether and how the innate immune system recognizes or responds to allogeneic BMCs remains unknown. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors. The adaptor MyD88 transmits signals emanating from all TLR, except TLR3 while TRIF specifically mediates TLR3 and TLR4 signaling via type 1 IFN. To further determine the innate signaling pathways in allogeneic BMC rejection, B6 background (H2b) MyD88−/− and TRIF−/− mice were conditioned with anti-CD154/rapamycin plus 100 cGy total body irradiation and transplanted with 15 × 106 BALB/c (H2d) BMC. Only 33.3% of MyD88−/− recipients engrafted at 1 month, resembling outcomes for wild-type B6 mice. In contrast, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month, significantly higher than in MyD88−/− and wild-type B6 controls (P < 0.005). To determine the mechanism of innate signaling in BMC rejection, we examined whether TRIF linked TLR3 or TLR4 is the key pattern recognition receptor involved in BMC recognition. To this end, TLR3−/− and TLR4−/− mice were transplanted with BALB/c BMC with same conditioning. None of the TLR3−/− mice engrafted. In contrast, engraftment was achieved in 100% of TLR4−/− mice up to 6 months follow up. Taken together, these results suggest that rejection of allogeneic BMC is uniquely dependent on the TLR4/TRIF signaling pathway. Thus, our results clearly demonstrate a previously unappreciated role for innate immunity in allogeneic BMC rejection. Our current findings are distinct from prior reports demonstrating a critical role of MyD88 in rejection of allogeneic skin grafts and lung, and may reflect unique features related to BMC. The findings of the role of innate immunity in BMC rejection would lead to revolutionary changes in our understanding and management of BMT. This would be informative in design of more specific innate immune targeted conditioning proposals in BMT to avoid the toxicity. Disclosures: Bozulic: Regenerex LLC: Employment. Ildstad:Regenerex LLC: Equity Ownership.

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Systemic Metabolite Changes in Wild-type C57BL/6 Mice Fed Black Raspberries

Nutrition and Cancer ◽

10.1080/01635581.2017.1263748 ◽

2017 ◽

Vol 69 (2) ◽

pp. 299-306 ◽

Cited By ~ 14

Author(s):

Pan Pan ◽

Chad W. Skaer ◽

Hsin-Tzu Wang ◽

Michael A. Kreiser ◽

Steven M. Stirdivant ◽

...

Keyword(s):

Wild Type ◽

Black Raspberries

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Infection of lymphocytes by a virus that aborts cytotoxic T lymphocyte activity and establishes persistent infection.

Journal of Experimental Medicine ◽

10.1084/jem.174.1.203 ◽

1991 ◽

Vol 174 (1) ◽

pp. 203-212 ◽

Cited By ~ 35

Author(s):

P Borrow ◽

A Tishon ◽

M B Oldstone

Keyword(s):

Immune System ◽

T Lymphocytes ◽

T Lymphocyte ◽

Persistent Infection ◽

Infectious Virus ◽

Cytotoxic T Lymphocyte ◽

Lymphoid Tissues ◽

Wild Type ◽

Ctl Response ◽

Adult Mice

For viruses to establish persistent infections in their hosts, they must possess some mechanism for evading clearance by the immune system. When inoculated into adult immunocompetent mice, wild-type lymphocytic choriomeningitis virus (LCMV ARM) induces a CD8(+)-mediated cytotoxic T lymphocyte (CTL) response that clears the infection within 7-14 d (CTL+ [P-]). By contrast, variant viruses isolated from lymphoid tissues of persistently infected mice fail to induce a CTL response and are thus able to establish a persistent infection in adult mice (CTL- [P+]). This report compares the interaction of CTL+ (P-) and CTL- (P+) viruses with cells of the immune system. Both types of virus initially bind to 2-4% of CD4+ and CD8+ T lymphocytes and replicate within cells of both subsets. The replication of CTL- (P+) and CTL+ (P-) viruses in lymphocytes in vivo is similar for the first 5 d after initiating infection. Thereafter, in mice infected with CTL- (P+) variants, lymphocytes retain viral genetic information, and infectious virus can be recovered throughout the animals' lives. In contrast, when adult mice are infected with wild-type CTL+ (P-) LCMV ARM, virus is not recovered from lymphocytes for greater than 7 d after infection. A CD8(+)-mediated anti-LCMV CTL response is induced in such mice. Clearance of infected lymphocytes is produced by these LCMV-specific CTLs, as shown by their ability to lyse lymphocytes expressing LCMV determinants in vitro and the fact that depletion of CD8+ lymphocytes before infection with CTL+ (P-) viruses results in levels of infected lymphocytes similar to those found in undepleted CTL- (P+)-infected mice. Hence, CTL-mediated lysis of T lymphocytes carrying infectious virus is a critical factor determining whether virus persists or the infection is terminated.

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The Tetraspanin CD81 Facilitates Tumor Metastasis By Modulating Immune Suppression

Blood ◽

10.1182/blood.v124.21.4136.4136 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4136-4136

Author(s):

Felipe Vences Catalan ◽

Ranjani Rajapaksa ◽

Minu K. Srivastava ◽

Aurelien Marabelle ◽

Shoshana Levy

Keyword(s):

T Cells ◽

Immune System ◽

Tumor Metastasis ◽

Conflicts Of Interest ◽

Lung Metastases ◽

Suppressor Cells ◽

Immune Functions ◽

Wild Type ◽

Primary Tumor Site ◽

Tumor Bearing

Abstract Tumor metastasis is the major cause of cancer mortality. While immune cells are aimed at halting metastases, the proliferating cancer cells at the primary tumor site recruit cells that counteract the adaptive anti-tumor immune response. Indeed, it is well established that tumors induce the accumulation and proliferation of immune suppressive cells, such as regulatory T cells (Tregs) and Myeloid Derived Suppressor cells (MDSC). The tetraspanin CD81 belongs to a family of proteins that play an important role in the immune system as evident by impaired immune functions of DCs, B and T cells in several tetraspanin-deficient mice (CD37, CD81, CD151, and CD82). While many studies have addressed the function of CD81 in infection and in the immune system, few have focused on its role in tumorigenesis and metastasis. Here we report that the growth of subcutaneously implanted breast cancer tumors (4T1) is slightly impaired in CD81 knockout (CD81KO) compared to wild type mice. Moreover, CD81KO mice develop very few lung metastases compared to their wild type and heterozygous counterparts. Both wild type and CD81KO tumor-bearing mice show substantial increases in Tregs, and MDSCs. However, these Tregs and MDSCs differ functionally - those derived from wild type mice are effective suppressors of T cell proliferation. By contrast, Tregs and MDSCs derived from tumor-bearing CD81KO mice do not suppress the proliferation of Teff cells. Thus, it is highly likely that while Teff cells are greatly restrained by Tregs and MDSCs in wild type tumor-bearing mice, the impairment of both suppressive cell populations in CD81KO mice enables the anti-tumor function thereby opposing metastases. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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The Impact of Broadly Relevant Novel Discoveries on Student Project Ownership in a Traditional Lab Course Turned CURE

CBE—Life Sciences Education ◽

10.1187/cbe.19-06-0113 ◽

2019 ◽

Vol 18 (4) ◽

pp. ar57 ◽

Cited By ~ 6

Author(s):

Katelyn M. Cooper ◽

Joseph N. Blattman ◽

Taija Hendrix ◽

Sara E. Brownell

Keyword(s):

Immune System ◽

Mutant Strain ◽

Student Outcomes ◽

Scientific Community ◽

Undergraduate Research ◽

Wild Type ◽

Research Experiences ◽

Undergraduate Research Experiences ◽

The Impact ◽

Insight Into

Course-based undergraduate research experiences (CUREs) have been shown to lead to multiple student benefits, but much is unknown about how CUREs lead to specific student outcomes. In this study, we examined the extent to which students making “broadly relevant novel discoveries” impacted student project ownership by comparing the experiences of students in a CURE and a traditional lab course. The CURE and traditional lab were similar in most aspects; students were exposed to an identical curriculum taught by the same instructor. However, there was one major difference between the two types of courses: the type of data that the students produced. Students in the traditional lab characterized the immune system of wild-type mice, thereby confirming results already known to the scientific community, while students in the CURE characterized the immune system of a mutant strain of mice, which produced broadly relevant novel discoveries. Compared with traditional lab students, CURE students reported higher cognitive and emotional ownership over their projects. Students’ perceptions of collaboration and making broadly relevant novel discoveries were significantly and positively related to their cognitive and emotional ownership. This work provides insight into the importance of integrating opportunities for broadly relevant novel discoveries in lab courses.

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Proteolytically Inactivatable Factor VIII is Less Immunogenic than Factor VIII in a Murine Hemophilia A Model.

Blood ◽

10.1182/blood.v114.22.27.27 ◽

2009 ◽

Vol 114 (22) ◽

pp. 27-27

Author(s):

Shannon Meeks ◽

Ernest T Parker ◽

Amy L. Dunn ◽

John F Healey ◽

Pete Lollar

Keyword(s):

Immune Response ◽

Immune System ◽

Factor Viii ◽

Binding Site ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Foreign Protein ◽

C2 Domain ◽

Wild Type ◽

Antibody Titers

Abstract Abstract 27 Patients with hemophilia A have a congenital deficiency of the factor VIII (fVIII) protein due to a mutation in the fVIII gene that frequently leads to absence of detectable expression of fVIII. Accordingly, the therapeutic replacement fVIII protein potentially is recognized as non-self by the immune system. Thirty percent of patients with severe hemophilia A develop detectable inhibitory anti-fVIII antibodies (inhibitors). Additionally, greater than 90 percent of hemophilia A mice treated with human fVIII develop inhibitors using dosing schedule that mimics use in humans. Because fVIII is an immunologically foreign protein, it might be expected that a hemophilia A patient would make a fVIII inhibitor. However, intravenous injection of soluble proteins in either humans or rodents usually results in tolerance rather than a humoral immune response. One major difference between fVIII and other proteins is that it is released from its large carrier protein von Willebrand factor (VWF) and is potentially exposed to the immune system at sites of active hemostasis and inflammation. Heat-inactivated, denatured fVIII, which maintains all T-cell epitopes but lacks several B-cell epitopes, is less immunogenic than native fVIII, suggesting that fVIII-dependent thrombin generation along the intrinsic pathway of blood coagulation may provide co-stimulatory signals necessary for the immune response (Skupsky BS, Zhang A, Scott DW Blood 2008; 112:1220a). We constructed a B domain-deleted human fVIII mutant, designated fVIIIi, which contains alanine substitutions at two critical thrombin cleavage sites, Arg372 and Arg1689, and purified it to homogeneity. FVIIIi does not develop procoagulant activity and is not released from VWF in response to thrombin. Therefore fVIIIi is less likely than wild-type fVIII to be exposed to the immune system at sites of active hemostasis and inflammation. Additionally, VWF binds to the immunodominant fVIII C2 domain and potentially hides part of fVIII from the immune system. FVIIIi was antigenically intact judging from intact binding to a panel of11 mouse anti-fVIII monoclonal antibodies whose epitope specificity was represented by all five domains of BDD fVIII. The immunogenicity of wild-type fVIII and fVIIIi was compared in a murine hemophilia A model in which groups of 25 mice received 8 weekly injections of physiologic doses of fVIII. Plasma was collected weekly for total anti-fVIII antibody titers by ELISA and one week following the last injection for total anti-fVIII antibody titers, inhibitor titers by Bethesda assay and for epitope mapping. Mice treated with fVIIIi had significantly lower levels of inhibitory as well as total anti-fVIII antibodies than mice treated with wild-type fVIII. Domain mapping using single human domain hybrid human/porcine molecules as ELISA antigens revealed that hemophilia A mice broadly recognized all fVIII domains in response to either wild-type or fVIIIi, although fVIIIi produced less anti-light chain antibodies. Mice in both the wild-type fVIII and fVIIIi groups produced antibodies that recognized the phospholipid-binding site of the C2 domain, even though this site overlaps the VWF binding site on fVIII. There was no difference in the isotype spectrum of the antibodies made to fVIII or fVIIIi. This study indicates that inactivatable fVIII is less immunogenic than native fVIII and suggests that the immunogenicity of fVIII is related either to its interaction with VWF or to events triggered by activation of the coagulation mechanism. Disclosures: No relevant conflicts of interest to declare.

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