Katie Crump
- Courses3
- Reviews8
- School: J. Sargeant Reynolds Community College
- Campus:
- Department: Biology
- Email address: Join to see
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Location:
1651 E Parham Rd
Richmond, VA - 23228 - Dates at J. Sargeant Reynolds Community College: November 2015 - November 2016
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Biography
J. Sargeant Reynolds Community College - Biology
Assistant Professor at Nova Southeastern University
Biotechnology
Katie
Crump, Ph.D.
Miami/Fort Lauderdale Area
Research training focused on identifying novel pathways and regulators of inflammation during antimicrobial responses with special considerations to epigenetics, redox signaling, and the Toll-like receptor 9 pathway as potential alternative therapeutic approaches for limiting various infectious diseases.
Additionally, extensive teaching training focused on creative and innovative teaching methods, course, and curriculum design for the improvement of higher education life sciences for all students to reflect the changing diversity and demand of growing interdisciplinary nature of scientific jobs and academia.
Experience
Nova Southeastern University
Assistant Professor
Katie worked at Nova Southeastern University as a Assistant Professor
Virginia Commonwealth University Health System
Postdoctoral Fellow
Postdoctoral IRACDA Fellow in the School of Dentistry, Department of Periodontics at Virginia Commonwealth University
Received IRACDA Postdoctoral Fellowship to study the novel inflammatory pathway TLR9 in periodontal inflammation under the guidance of Dr. S. Esra Sahingur.
Communicated key discoveries at professional conferences: AADR.
Mentored dental students, graduate students, and undergraduates in inflammation, cell signaling, chronic disease, and murine models for periodontal inflammation.Virginia Commonwealth University Health System
Postdoctoral Fellow
Post-Doctoral IRACDA Fellow in the School of Dentistry, The Philips Institute for Oral Health Research at Virginia Commonwealth University.
Research Focus: The Role of Mn and Oxidative Stress On Streptococcus sanguinis pathogenesis.
-Certificate in Preparing Future Faculty Members
-Teaching internshipJ Sargeant Reynolds Community College
Adjunct Professor
Adjunct Professor teaching General Microbiology in the evenings
Virginia Union University
Adjunct Faculty Member-Department of Natural Sciences
Adjunct Faculty Member Co-Teaching General Microbiology Lecture and Laboratory Course for Biology Majors
Wake Forest Baptist Health
Ph.D.
Ph.D. in Microbiology and Immunology
Research Focus: Redox Regulation of the Humoral Immune Response
-Mentored junior students
-Intellectually designed and performed experiments relating to my research
-Published work in a peer reviewed journal
-Ruth L. Kirschstein National Research Service Award Recipient
Education
Virginia Commonwealth University
Preparing Future Faculty Certificate
Appalachian State University
Bachelor of Science (B.S.)
Biology, General
Graduated Summa Cum Laude with HonorsWake Forest University School of Medicine
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
Publications
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis
Journal Of Biological Chemistry
Full Author Byline: Rhodes DV, Crump KE, Makhlynets O, Snyder M, Ge X, Xu P, Stubbe J, Kitten T. Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a Mn transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y*) in vitro whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y*) cofactor requires NrdI, and that MnIII2-Y* is more active than FeIII2-Y* with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent, adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y*-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why Mn is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR, and suggests NrdF and NrdI may serve as promising new antimicrobial targets.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis
Journal Of Biological Chemistry
Full Author Byline: Rhodes DV, Crump KE, Makhlynets O, Snyder M, Ge X, Xu P, Stubbe J, Kitten T. Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a Mn transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y*) in vitro whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y*) cofactor requires NrdI, and that MnIII2-Y* is more active than FeIII2-Y* with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent, adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y*-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why Mn is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR, and suggests NrdF and NrdI may serve as promising new antimicrobial targets.
Peroxiredoxin II regulates effector and secondary memory CD8+ T cell responses
Journal of Virology
Full Author Byline: Michalek RD, Crump KE, Weant AE, Hiltbold EM, Juneau DG, Moon EY, Yum DY, Poole LB, and Grayson JM Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H(2)O(2) on an antigen-specific CD8(+) T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8(+) T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8(+) T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8(+) T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8(+) T cell pool. During chronic viral infection, increased antigen-specific CD8(+) T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8(+) T cell expansion, secondary memory generation, and immunopathology.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis
Journal Of Biological Chemistry
Full Author Byline: Rhodes DV, Crump KE, Makhlynets O, Snyder M, Ge X, Xu P, Stubbe J, Kitten T. Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a Mn transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y*) in vitro whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y*) cofactor requires NrdI, and that MnIII2-Y* is more active than FeIII2-Y* with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent, adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y*-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why Mn is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR, and suggests NrdF and NrdI may serve as promising new antimicrobial targets.
Peroxiredoxin II regulates effector and secondary memory CD8+ T cell responses
Journal of Virology
Full Author Byline: Michalek RD, Crump KE, Weant AE, Hiltbold EM, Juneau DG, Moon EY, Yum DY, Poole LB, and Grayson JM Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H(2)O(2) on an antigen-specific CD8(+) T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8(+) T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8(+) T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8(+) T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8(+) T cell pool. During chronic viral infection, increased antigen-specific CD8(+) T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8(+) T cell expansion, secondary memory generation, and immunopathology.
Defects in apoptosis increase memory CD8+ T cells following infection of Bim-/-Faslpr/lpr mice.
Cellular Immunology
Full Author Byline: Weant AE, Michalek RD, Crump KE, Liu C, Konopitski AP, Grayson JM. During many infections, large numbers of effector CD8(+) T cells are generated. After pathogen clearance, the majority of these cells undergo apoptosis, while the survivors differentiate into memory CD8(+) T cells. Although loss of both Bim and Fas function dramatically increased antigen-specific CD8(+) T cells in the lymph nodes following acute lymphocytic choriomeningitis virus (LCMV) infection, it was unclear whether they were pardoned effector or true memory CD8(+) T cells. In this study, we demonstrate they are bona fide memory T cells as characterized by surface marker expression, cytokine production, homeostatic proliferation, and ability to clear a secondary challenge of pathogen. Loss of both Bim and Fas also increased the number of virus-specific CD4(+) T cells found in the lymph nodes compared to the parental genotypes or wildtype mice. These studies illustrate that decreasing apoptosis increases the number of memory T cells and therefore could increase the efficacy of vaccines.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis
Journal Of Biological Chemistry
Full Author Byline: Rhodes DV, Crump KE, Makhlynets O, Snyder M, Ge X, Xu P, Stubbe J, Kitten T. Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a Mn transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y*) in vitro whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y*) cofactor requires NrdI, and that MnIII2-Y* is more active than FeIII2-Y* with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent, adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y*-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why Mn is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR, and suggests NrdF and NrdI may serve as promising new antimicrobial targets.
Peroxiredoxin II regulates effector and secondary memory CD8+ T cell responses
Journal of Virology
Full Author Byline: Michalek RD, Crump KE, Weant AE, Hiltbold EM, Juneau DG, Moon EY, Yum DY, Poole LB, and Grayson JM Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H(2)O(2) on an antigen-specific CD8(+) T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8(+) T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8(+) T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8(+) T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8(+) T cell pool. During chronic viral infection, increased antigen-specific CD8(+) T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8(+) T cell expansion, secondary memory generation, and immunopathology.
Defects in apoptosis increase memory CD8+ T cells following infection of Bim-/-Faslpr/lpr mice.
Cellular Immunology
Full Author Byline: Weant AE, Michalek RD, Crump KE, Liu C, Konopitski AP, Grayson JM. During many infections, large numbers of effector CD8(+) T cells are generated. After pathogen clearance, the majority of these cells undergo apoptosis, while the survivors differentiate into memory CD8(+) T cells. Although loss of both Bim and Fas function dramatically increased antigen-specific CD8(+) T cells in the lymph nodes following acute lymphocytic choriomeningitis virus (LCMV) infection, it was unclear whether they were pardoned effector or true memory CD8(+) T cells. In this study, we demonstrate they are bona fide memory T cells as characterized by surface marker expression, cytokine production, homeostatic proliferation, and ability to clear a secondary challenge of pathogen. Loss of both Bim and Fas also increased the number of virus-specific CD4(+) T cells found in the lymph nodes compared to the parental genotypes or wildtype mice. These studies illustrate that decreasing apoptosis increases the number of memory T cells and therefore could increase the efficacy of vaccines.
Microbial Nucleic Acid Sensing in Oral and Systemic Diseases
Journal of Dental Research
Full Author Byline: Crump KE and Sahingur SE One challenge in studying chronic infectious and inflammatory disorders is understanding how host pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), sense and respond to pathogen- or damage-associated molecular patterns, their communication with each other and different components of the immune system, and their role in propagating inflammatory stages of disease. The discovery of innate immune activation through nucleic acid recognition by intracellular PRRs such as endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) and cytoplasmic proteins (absent in melanoma 2 and DNA-dependent activator of interferon regulatory factor) opened a new paradigm: Nucleic acid sensing is now implicated in multiple immune and inflammatory conditions (e.g., atherosclerosis, cancer), viral (e.g., human papillomavirus, herpes virus) and bacterial (e.g., Helicobacter pylori, pneumonia) diseases, and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis). Clinical investigations reveal the overexpression of specific nucleic acid sensors in diseased tissues. In vivo animal models show enhanced disease progression associated with receptor activation. The involvement of nucleic acid sensors in various systemic conditions is further supported by studies reporting receptor knockout mice being either protected from or prone to disease. TLR9-mediated inflammation is also implicated in periodontal diseases. Considering that persistent inflammation in the oral cavity is associated with systemic diseases and that oral microbial DNA is isolated at distal sites, nucleic acid sensing may potentially be a link between oral and systemic diseases. In this review, we discuss recent advances in how intracellular PRRs respond to microbial nucleic acids and emerging views on the role of nucleic acid sensors in various systemic diseases.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis
Journal Of Biological Chemistry
Full Author Byline: Rhodes DV, Crump KE, Makhlynets O, Snyder M, Ge X, Xu P, Stubbe J, Kitten T. Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a Mn transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y*) in vitro whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y*) cofactor requires NrdI, and that MnIII2-Y* is more active than FeIII2-Y* with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent, adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y*-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why Mn is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR, and suggests NrdF and NrdI may serve as promising new antimicrobial targets.
Peroxiredoxin II regulates effector and secondary memory CD8+ T cell responses
Journal of Virology
Full Author Byline: Michalek RD, Crump KE, Weant AE, Hiltbold EM, Juneau DG, Moon EY, Yum DY, Poole LB, and Grayson JM Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H(2)O(2) on an antigen-specific CD8(+) T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8(+) T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8(+) T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8(+) T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8(+) T cell pool. During chronic viral infection, increased antigen-specific CD8(+) T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8(+) T cell expansion, secondary memory generation, and immunopathology.
Defects in apoptosis increase memory CD8+ T cells following infection of Bim-/-Faslpr/lpr mice.
Cellular Immunology
Full Author Byline: Weant AE, Michalek RD, Crump KE, Liu C, Konopitski AP, Grayson JM. During many infections, large numbers of effector CD8(+) T cells are generated. After pathogen clearance, the majority of these cells undergo apoptosis, while the survivors differentiate into memory CD8(+) T cells. Although loss of both Bim and Fas function dramatically increased antigen-specific CD8(+) T cells in the lymph nodes following acute lymphocytic choriomeningitis virus (LCMV) infection, it was unclear whether they were pardoned effector or true memory CD8(+) T cells. In this study, we demonstrate they are bona fide memory T cells as characterized by surface marker expression, cytokine production, homeostatic proliferation, and ability to clear a secondary challenge of pathogen. Loss of both Bim and Fas also increased the number of virus-specific CD4(+) T cells found in the lymph nodes compared to the parental genotypes or wildtype mice. These studies illustrate that decreasing apoptosis increases the number of memory T cells and therefore could increase the efficacy of vaccines.
Microbial Nucleic Acid Sensing in Oral and Systemic Diseases
Journal of Dental Research
Full Author Byline: Crump KE and Sahingur SE One challenge in studying chronic infectious and inflammatory disorders is understanding how host pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), sense and respond to pathogen- or damage-associated molecular patterns, their communication with each other and different components of the immune system, and their role in propagating inflammatory stages of disease. The discovery of innate immune activation through nucleic acid recognition by intracellular PRRs such as endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) and cytoplasmic proteins (absent in melanoma 2 and DNA-dependent activator of interferon regulatory factor) opened a new paradigm: Nucleic acid sensing is now implicated in multiple immune and inflammatory conditions (e.g., atherosclerosis, cancer), viral (e.g., human papillomavirus, herpes virus) and bacterial (e.g., Helicobacter pylori, pneumonia) diseases, and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis). Clinical investigations reveal the overexpression of specific nucleic acid sensors in diseased tissues. In vivo animal models show enhanced disease progression associated with receptor activation. The involvement of nucleic acid sensors in various systemic conditions is further supported by studies reporting receptor knockout mice being either protected from or prone to disease. TLR9-mediated inflammation is also implicated in periodontal diseases. Considering that persistent inflammation in the oral cavity is associated with systemic diseases and that oral microbial DNA is isolated at distal sites, nucleic acid sensing may potentially be a link between oral and systemic diseases. In this review, we discuss recent advances in how intracellular PRRs respond to microbial nucleic acids and emerging views on the role of nucleic acid sensors in various systemic diseases.
Toll-Like Receptor 9-mediated inflammation triggers alveolar bone loss in experimental murine periodontitis
Infection and Immunity
Full Author Byline: Kim PD, Xia-Juan X, Crump KE, Abe T, Hajishengallis G, Sahingur SE Chronic periodontitis is a local inflammatory disease induced by a dysbiotic microbiota leading to destruction of the tooth-supporting structures. Microbial nucleic acids are abundantly present in the periodontium derived through release after phagocytic uptake of microbes and/or from biofilm-associated extracellular DNA. Binding of microbial DNA to its cognate receptors, such as Toll-like receptor 9 (TLR9), can trigger inflammation. In this study, we utilized TLR9 knockout (TLR9-/-) mice and wild-type (WT) controls in a murine model of Porphyromonas gingivalis-induced periodontitis and report first in vivo evidence that TLR9 signaling mediates induction of periodontal bone loss. P. gingivalis-infected WT mice exhibited significantly increased bone loss as compared to sham-infected WT mice or P. gingivalis-infected TLR9-/- mice, which were resistant to bone loss. Consistent with this, the expression of IL-6, TNF, and receptor-activator of nuclear factor kappa B-ligand (RANKL) was significantly elevated in the gingival tissues of the infected WT mice but not in infected TLR9-/- mice, as compared to controls. Ex vivo studies using splenocytes and bone marrow-derived macrophages revealed significantly diminished cytokine production in TLR9-/- cells relative to WT cells in response to P. gingivalis, thereby implicating TLR9 in inflammatory responses to this organism. Intriguingly, compared to WT cells, TLR9-/- cells exhibited significantly decreased pro-inflammatory cytokine production upon challenge with LPS (TLR4 agonist) or Pam3Cys (TLR2 agonist), suggesting a possible crosstalk between TLR9, TLR4, and TLR2. Collectively, our results provide first proof-of-concept evidence implicating TLR9-triggered inflammation in periodontal disease pathogenesis, thereby identifying a new potential therapeutic target to control periodontal inflammation.
Antioxidant treatment regulates the humoral immune response during acute viral infection
Journal of Virology
Full Author Byline: Crump KE, Langston PK, Rajkarnikar S, Grayson JM Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
The Reversible Formation of Cysteine Sulfenic Acid Promotes B-Cell Activation and Proliferation.
European Journal of Immunology
Full Author Byline: Crump KE, Juneau DG, Poole LB, Haas KM, Grayson JM B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 minutes. Increased ROI preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration dependent block in anti-IgM induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation
PLoS One
Full Author Byline: Pellom ST, Michalek RD, Crump KE, Langston PK, Juneau DG, Grayson JM. Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Genetic Characterization and Role in Virulence of the Ribonucleotide Reductases of Streptococcus sanguinis
Journal Of Biological Chemistry
Full Author Byline: Rhodes DV, Crump KE, Makhlynets O, Snyder M, Ge X, Xu P, Stubbe J, Kitten T. Streptococcus sanguinis is a cause of infective endocarditis and has been shown to require a Mn transporter called SsaB for virulence and O2 tolerance. Like certain other pathogens, S. sanguinis possesses aerobic class Ib (NrdEF) and anaerobic class III (NrdDG) ribonucleotide reductases (RNRs) that perform the essential function of reducing ribonucleotides to deoxyribonucleotides. The accompanying paper indicates that in the presence of O2, the S. sanguinis class Ib RNR self-assembles an essential diferric-tyrosyl radical (FeIII2-Y*) in vitro whereas assembly of a dimanganese-tyrosyl radical (MnIII2-Y*) cofactor requires NrdI, and that MnIII2-Y* is more active than FeIII2-Y* with the endogenous reducing system of NrdH and thioredoxin reductase (TrxR1). In this study, we have shown that deletion of either nrdHEKF or nrdI completely abolishes virulence in an animal model of endocarditis, whereas nrdD mutation has no effect. The nrdHEKF, nrdI, and trxR1 mutants fail to grow aerobically whereas anaerobic growth requires nrdD. The nrdJ gene encoding an O2-independent, adenosylcobalamin-cofactored RNR was introduced into the nrdHEKF, nrdI, and trxR1 mutants. Growth of the nrdHEKF and nrdI mutants in the presence of O2 was partially restored. The combined results suggest that MnIII2-Y*-cofactored NrdF is required for growth under aerobic conditions and in animals. This could explain in part why Mn is necessary for virulence and O2 tolerance in many bacterial pathogens possessing a class Ib RNR, and suggests NrdF and NrdI may serve as promising new antimicrobial targets.
Peroxiredoxin II regulates effector and secondary memory CD8+ T cell responses
Journal of Virology
Full Author Byline: Michalek RD, Crump KE, Weant AE, Hiltbold EM, Juneau DG, Moon EY, Yum DY, Poole LB, and Grayson JM Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H(2)O(2) on an antigen-specific CD8(+) T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8(+) T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8(+) T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8(+) T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8(+) T cell pool. During chronic viral infection, increased antigen-specific CD8(+) T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8(+) T cell expansion, secondary memory generation, and immunopathology.
Defects in apoptosis increase memory CD8+ T cells following infection of Bim-/-Faslpr/lpr mice.
Cellular Immunology
Full Author Byline: Weant AE, Michalek RD, Crump KE, Liu C, Konopitski AP, Grayson JM. During many infections, large numbers of effector CD8(+) T cells are generated. After pathogen clearance, the majority of these cells undergo apoptosis, while the survivors differentiate into memory CD8(+) T cells. Although loss of both Bim and Fas function dramatically increased antigen-specific CD8(+) T cells in the lymph nodes following acute lymphocytic choriomeningitis virus (LCMV) infection, it was unclear whether they were pardoned effector or true memory CD8(+) T cells. In this study, we demonstrate they are bona fide memory T cells as characterized by surface marker expression, cytokine production, homeostatic proliferation, and ability to clear a secondary challenge of pathogen. Loss of both Bim and Fas also increased the number of virus-specific CD4(+) T cells found in the lymph nodes compared to the parental genotypes or wildtype mice. These studies illustrate that decreasing apoptosis increases the number of memory T cells and therefore could increase the efficacy of vaccines.
Microbial Nucleic Acid Sensing in Oral and Systemic Diseases
Journal of Dental Research
Full Author Byline: Crump KE and Sahingur SE One challenge in studying chronic infectious and inflammatory disorders is understanding how host pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), sense and respond to pathogen- or damage-associated molecular patterns, their communication with each other and different components of the immune system, and their role in propagating inflammatory stages of disease. The discovery of innate immune activation through nucleic acid recognition by intracellular PRRs such as endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) and cytoplasmic proteins (absent in melanoma 2 and DNA-dependent activator of interferon regulatory factor) opened a new paradigm: Nucleic acid sensing is now implicated in multiple immune and inflammatory conditions (e.g., atherosclerosis, cancer), viral (e.g., human papillomavirus, herpes virus) and bacterial (e.g., Helicobacter pylori, pneumonia) diseases, and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis). Clinical investigations reveal the overexpression of specific nucleic acid sensors in diseased tissues. In vivo animal models show enhanced disease progression associated with receptor activation. The involvement of nucleic acid sensors in various systemic conditions is further supported by studies reporting receptor knockout mice being either protected from or prone to disease. TLR9-mediated inflammation is also implicated in periodontal diseases. Considering that persistent inflammation in the oral cavity is associated with systemic diseases and that oral microbial DNA is isolated at distal sites, nucleic acid sensing may potentially be a link between oral and systemic diseases. In this review, we discuss recent advances in how intracellular PRRs respond to microbial nucleic acids and emerging views on the role of nucleic acid sensors in various systemic diseases.
Toll-Like Receptor 9-mediated inflammation triggers alveolar bone loss in experimental murine periodontitis
Infection and Immunity
Full Author Byline: Kim PD, Xia-Juan X, Crump KE, Abe T, Hajishengallis G, Sahingur SE Chronic periodontitis is a local inflammatory disease induced by a dysbiotic microbiota leading to destruction of the tooth-supporting structures. Microbial nucleic acids are abundantly present in the periodontium derived through release after phagocytic uptake of microbes and/or from biofilm-associated extracellular DNA. Binding of microbial DNA to its cognate receptors, such as Toll-like receptor 9 (TLR9), can trigger inflammation. In this study, we utilized TLR9 knockout (TLR9-/-) mice and wild-type (WT) controls in a murine model of Porphyromonas gingivalis-induced periodontitis and report first in vivo evidence that TLR9 signaling mediates induction of periodontal bone loss. P. gingivalis-infected WT mice exhibited significantly increased bone loss as compared to sham-infected WT mice or P. gingivalis-infected TLR9-/- mice, which were resistant to bone loss. Consistent with this, the expression of IL-6, TNF, and receptor-activator of nuclear factor kappa B-ligand (RANKL) was significantly elevated in the gingival tissues of the infected WT mice but not in infected TLR9-/- mice, as compared to controls. Ex vivo studies using splenocytes and bone marrow-derived macrophages revealed significantly diminished cytokine production in TLR9-/- cells relative to WT cells in response to P. gingivalis, thereby implicating TLR9 in inflammatory responses to this organism. Intriguingly, compared to WT cells, TLR9-/- cells exhibited significantly decreased pro-inflammatory cytokine production upon challenge with LPS (TLR4 agonist) or Pam3Cys (TLR2 agonist), suggesting a possible crosstalk between TLR9, TLR4, and TLR2. Collectively, our results provide first proof-of-concept evidence implicating TLR9-triggered inflammation in periodontal disease pathogenesis, thereby identifying a new potential therapeutic target to control periodontal inflammation.
The Relationship of the Lipoprotein SsaB, Manganese, and Superoxide Dismutase in Streptococcus sanguinis Virulence for Endocarditis
Molecular Microbiology
Full Author Byline: Crump KE, Bainbridge B, Brusko S, Turner LS, Ge X, Stone V, Xu P, Kitten T Streptococcus sanguinis colonizes teeth and is an important cause of infective endocarditis. Our prior work showed that the lipoprotein SsaB is critical for S. sanguinis virulence for endocarditis and belongs to the LraI family of conserved metal transporters. In this study, we demonstrated that an ssaB mutant accumulates less manganese and iron than its parent. A mutant lacking the manganese-dependent superoxide dismutase, SodA, was significantly less virulent than wild-type in a rabbit model of endocarditis, but significantly more virulent than the ssaB mutant. Neither the ssaB nor the sodA mutation affected sensitivity to phagocytic killing or efficiency of heart valve colonization. Animal virulence results for all strains could be reproduced by growing bacteria in serum under physiological levels of O2 . SodA activity was reduced, but not eliminated in the ssaB mutant in serum and in rabbits. Growth of the ssaB mutant in serum was restored upon addition of Mn2+ or removal of O2 . Antioxidant supplementation experiments suggested that superoxide and hydroxyl radicals were together responsible for the ssaB mutant's growth defect. We conclude that manganese accumulation mediated by the SsaB transport system imparts virulence by enabling cell growth in oxygen through SodA-dependent and independent mechanisms.
